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Calorimetric measurements of the adsorption of collagen and other organics onto oxide surfaces

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Calorimetric measurements of the adsorption of collagen and other organics onto oxide surfaces
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Calorimetric measurements of the adsorption of collagen and other organics onto oxide surfaces
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Buscemi, Paul John
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Paul J. Buscemi
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Adsorption ( jstor )
Collagens ( jstor )
Enthalpy ( jstor )
Heat ( jstor )
Hydrogen ( jstor )
Ions ( jstor )
Molecules ( jstor )
Oxides ( jstor )
pH ( jstor )
Sulfates ( jstor )

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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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CALORIMETRIC MEASUREMENTS OF THE ADSORPTION OF COLLAGEN


AND OTHER ORGANIC ONTO OXIDE SURFACES









By



PAUL J. BUSCEMI


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMNETS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1978













TABLE OF CONTENTS



ACKNOWLEDGEMENTS .. . . . . . . .ii

ABSTRACT . . . . . ... . . v

CHAPTER Page

I INTRODUCTION . .. . . . . 1

Use of Protein Models . .. . . 2
Calorimetry as an Analytic Tool .. .. 4
Protein Adsorption . . .. . . 6
Surfaces. ............ . 10
Forces of Adsorption .. . . . . 15
Thermodynamics of Adsorption. . . ... 17
Heterogeneous Adsorption . . . ... 24
Experimental. . . . . . . ... 33

II ADSORPTION OF CARBOXYLIC ACIDS
ON ALUMINA AND HYDROXYAPATITE 41

Introduction. . . . . . . ... 41
Experimental . . . . . . ... 43
Results . . . . . . . . 44
Discussion. . . . . ... . . 51
Conclusions . . . . . .. .... 59

III ADSORPTION OF CHONDROITIN SULFATE
AND OTHER CARBOHYDRATES . . . .. 61

Introduction. . . . . . . ... 61
Experimental . . . . . . ... 62
Results . . . . . . . . 62
Discussion. . . . . . ... 70
Conclusions . . . . . .. . . 77

IV ADSORPTION OF POLYPEPTIDES. . . .. 79

Introduction. . . . . . . ... 79
Experimental. . . . . . . ... 81
Results . . . . . . . .. 81
Discussion. . . . . . ... 86
Conclusions . ... . . . . ... 92


iii







TABLE OF CONTENTS Continued



CHAPTER

V REACTION OF PEPTIDES AND CARBOHYDRATES
IN SOLUTION . . . . . .


Introduction. . . .
Experimental. . . .
Results . . . .
Discussion . ..
Conclusions .. ...

VI ADSORPTION OF COLLAGEN.


Introduction.
Experimental.
Results . .
Discussion. .
Conclusions .


VII BIOGLASS. . .


Introduction.
Experimental. .
Results . .
Discussion. .
Conclusions .


VIII SUMMARY .. ..

BIBLIOGRAPHY . . .

BIOGRAPHICAL SKETCH .


Page


. . . . . 93
. . . . 95
. . . . . 97
. . . . 108
. . . . . 115

. . . . 117

. . . . 117
. . . .. . 117
. . . . 117
. . . . 132
. . . . 138


. . . . . . 141

. . . . . . . 141
S . . . . . . 142
. . . . . . . 142
. . . . . . 148
. . . . . . . 150


. . 152


159

164







Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for'the Degree of Doctor of Philosophy


CALORIMETRIC MEASUREMENTS OF THE ADSORPTION OF COLLAGEN

AND OTHER ORGANIC ONTO OXIDE SURFACES


By

Paul J. Buscemi

March, 1978


Chairman; R. E. Loehman
Major Department: Materials Science and Engineering


The present work is the result of the application of

solution microcalorimetry to the problem of determining the

energies of adsorption of organic molecules onto ceramic

surfaces. The systems studied were chosen to model the

attachment of collagen to ceramics and to provide some expla-

nation for the observed bonding of ceramic implants to bone.

An aqueous solution of an organic molecule such as a

polyamino acid, polysaccharide, or smaller molecules with

similar functional groups was automatically mixed in a micro-

calorimeter with a slurry of a powdered oxide such as A1203,

SiO2, or a special glass composition and the heat evolved or

adsorbed was determined. Calorimetric measurements were

performed on increasing concentrations of reacting organic

molecules for a fixed weight of powder with known surface

area. Plots of the reaction heat, Q, versus the initial con-

centration of the organic, Co, yield thermometric titration








curves which were analyzed to give the enthalpy of the

reaction AH, the free energy change AG, and by difference

the entropy change for the reaction AS.

The systems were studied in order of increasing

structural complexity from simple carboxyls, amines, and

organic sulfates to amino acids, polyamino acids, polysaccha-

rides, and collagen. Changes in the aqueous solutions by

additions of salts or changes in pH and combinations of

organic molecules were also studied.

Results indicate that there are at least two forces

which contribute to the bonding of the collagen and other

organic to oxide surfaces, hydrogen bonding and ionic bond-

ing, the former releasing from 8 to 12 kcal/mole of functional

groups while the latter releases 4 to 6 kcal/mole of

functional groups depending on the relative polarities of

the adsorbing molecules and the surface. There are strong

indications of the denaturation of collagen at some surfaces

at which hydrogen bonding and ionic bonding act cooperatively.













CHAPTER I

INTRODUCTION


The study of adsorption and interaction of proteins and

other biological molecules onto non-biological surfaces is

important because of the increasing use of prosthetic materials

[1]. It is essential to know how each of these two distinctly

different components will interact in biological media. In

this study, a further understanding of the reaction between

the connective tissue protein, collagen, and various oxide

surfaces is sought.

Two properties of collagen adsorption are of major

concern: how well does the protein adhere to the oxide surface

and does the surface change the structure of the protein?

Protein adhesion relates to the binding of tissue to a prosthetic

material and can be approached by the determination of the

enthalpy AH of the adsorption reaction [2]. Changes induced

by the surface on the protein lead to denaturation. This

increases its vulnerability to enzyme attack and eventual

rejection from the host [3]. This question can be approached

by comparison of the enthalpy of reactions of model systems

with that of reactions which have been found not to be

disruptive to the structure of protein.

Three materials serve as substrates: silica, alumina,

and hydroxyapatite. Each of these materials is well character-







ized and has potential for use as a prosthetic material [4].

The calorimetric measurements made, therefore, are not for

the purpose of further characterizing these materials but to

study their influence on the adsorption of organic molecules.

The values of the thermodynamic parameters (AG, AH, and

AS), determined from the calorimetric measurements, do depend,

however, on the structural features of the surface as well as

those of the adsorbing molecule and on their mutual environment.

A'practical approach for studying complicated systems encountered

in actual application is to study simpler model systems [5],

which provide singular features for observation.

Within a series of model compounds, the structure of

the molecules, the solvent, and the surface can be systematically

varied and correlations can be made between the thermodynamic

data and the variations in experimental conditions. In this

study, extensive use is made of several types of molecular

model compounds including those representing collagen as well

as those representing carbohydrate and other physiologically

relevant organic structures.


Use of Protein Models


Past workers have used molecular model compounds designed

to study collagen. Specifically, the polyamino acids have

been well studied in this way. Poly-l-proline has been used

in conformational studies in CaC12 solution [6] showing that

disordering of the molecule is associated with its increasing

rotational ability. X-ray diffraction studies of poly-l-proline







have demonstrated that the backbone conformations of the

molecule are similar to native collagen [7]. Poly-l-lysine

and poly-l-arginine have been used as models for collagen in

relation to the structure of amorphous ground substance [8,9].

Poly-l-lysine and poly-l-glutamic acid have been used in

conformational studies using differential capacitance techniques

[10]. In other studies, workers have used combinations of

synthetically prepared amino acids to model collagen [11,12].

Smaller molecules representing isolated residues of

the collagen molecule have also been investigated but not as

frequently as the polyamino acids. Dyes containing amino

groups have been shown to selectively adsorb chondroitin sulfate

[13], an important carbohydrate in structural tissue [14].

Surface viscosity measurements using amines, amides, and

carboxylic acids as model proteins have been studied in relation

to bilayer film formation [15] in membrane studies.

In biological studies of proteins other than collagen,

molecular models have been widely studied. Enthalpies of

aqueous solution have been determined calorimetrically for

amines and carboxylic acids [16] as part of a quantitative

description of biological systems. Heat capacity measurements

[17] on several amino acid solutions have been made to help

explain protein structure. The binding of short amino acid

chains [18] in subunit studies of immunoglobulin has also been

investigated by calorimetry. Differential scanning calorimetry

has been used to study conformation changes of many polypeptides

used as models for collagen [19,20].








Molecular models in non-biological studies have also

been used. The adsorption of molecules containing the same

functional groups which proteins possess, amines [21], sulfates

[22], and organic acids [23], have been examined by various

methods. Calorimetry has not been extensively used for this

purpose. In general, the use of molecular models in biological

and non-biological systems appears widespread for the determina-

tion of various properties.



Calorimetry As An Analytic Tool


Calorimetry has long been used to measure the enthalpy

of adsorption (heat of wetting) of various liquids onto dry

oxide surfaces. Such measurements are made by immersing a

clean, dry solid powder into a liquid. The heat change is

measured in a calorimeter. For our purposes, the most

relevant liquid used in previous studies was water. Heats of

wetting of silica [24-26], of alumina [27-29], and of hydroxy-

apatite [29] have each been measured. The results of these

works show several consistent features. First, there are

differences in the heats of wetting with variation in the out-

gassing pressure and with temperature of evacuation, indicating

surface heterogeneities. Also there are differences in heats

of wetting with variation in particle size. Finally, the heats

of adsorption of water range from -10 to -20 kcal/mole of water

adsorbed and are attributed to hydrogen bonding of the water

to the surface [30].





5



Calorimetry has also been used in a similar manner

to measure the heat of adsorption of a second component from

an aqueous solvent [31]. The heat of adsorption of sodium

dodecyl sulfonate (SDS) was found by subtracting the heat of

wetting of alumina in pure solvent from that found with SDS

present. In this case the initial adsorption attraction was

attributed to coulombic forces between the surface and ion

and was calculated to be -12 kcal/mole.

Results for similar experiments have been confirmed by

other methods [32,33] using water and other polar and ionic

liquids as adsorbents. For example, the differential heat of

adsorption for the adsorption of octadecyl alcohol on alumina

from a benzene solution, calculated from the temperature

dependence of the adsorption coefficient was found to be -8.6

kcal/mole while that found directly from calorimetric measure-

ments was -8.68 kcal/mole [34].

The heat of adsorption of water on quartz determined

by adsorption measurements at several temperatures was found

to be between -11 and -14 kcal/mole of water adsorbed [25].

There were differences found when the quartz was ground and

exposed to water vapor prior to drying and evacuation. This

was attributed to the formation of an amorphous layer of silica

on the surface. These differences disappeared when the rough-

ened surfaces were annealed at 7000C. The heats of wetting

agree well with those found from calorimetric measurements [24].

Calorimetry has been extensively used in biochemical

applications [35]. However, there are few calorimetric data








on the adsorption of proteins onto oxide surfaces and there

is apparently none for the adsorption of collagen. The

calorimetric data most relevant to adsorption primarily

involve such globular serum proteins as serum albumin [36].

The remainder of this section will therefore be devoted to

previous studies of protein adsorption use with particular

emphasis on oxide substrates.



Protein Adsorption


The demonstration of molecular attachment of cell

proteins on foreign substances has been accomplished by various

methods. Multiple internal reflection spectroscopy has

been used to measure protein interaction using germanium [37]

as a substrate. A KRS-5 prism pressed against protein on a

hydroxyapatite substrate allowed protein-hydroxyapatite inter-

action to be studied [38]. Although energy calculations were

not carried out in these studies, the changes in adsorption

frequencies indicate chemical interaction with the surface of

the substrate.

Film compression studies [39] using collagen, gelatin,

and poly-l-alanine with silica gel showed adsorption hysterisis

indicating an irreversible process. The maximum interaction

of the silica gel and collagen occurred at pH 5.2. The iso-

electric point,where there is charge neutralization of the

protein, is 5.5. There was also interaction between alanine,

which has no ionic side groups, and the gel. The interaction









appeared to be of the same type as that of collagen. The

primary binding force was assumed to be hydrogen bonding [40].

Adsorption of bovine serum albumin (BSA) on hydro-

philic silica [41] exhibited a maximum surface density at

pH 5.5. The isoelectric point (IP) of this protein is 4.9.

The surface of the silica is negatively charged at this pH.

Desorption occurred readily at pH's away from the IP indicating,

as suggested by the author, that binding was due to hydrophobic

interaction. Other studies [42,43] showed that even after

extensive washing with water and EDTA that not all of the BSA

adsorbed onto pyrex glass could be recovered. Maximum

adsorption was near the IP of the protein and the free energy

change was estimated to be -2.5 kcal/mole of protein. The

enthalpy was not calculated.

Serum globulins have been shown to be preferentially

adsorbed by silicic acid [44] and silica [45] and by other

minerals [46]. Maximum adsorption took place at the isoelectric

point on these surfaces as well as on calcium phosphate gel

[47]. In none of these studies were determinations of the

enthalpy of the various adsorption reactions made.

The adsorption of albumin, fibrinogin, and globulin on

polyethylene has been determined by internal reflection spectro-

scopy [48]. The adsorption isotherms followed a Langmuir isotherm,

a common finding in which the quantity of solute adsorbed, X,

at the equilibrium concentration, C, is given by X = aC/(l + bC)

where a and.b are constants. The adsorption was assumed to be








due to hydrogen bonding because of the shift in the amide I

(C=O stretching) band at 1640 cm-1

Calorimetric measurements of the adsorption of human

serum albumin on negatively charged polystyrene (PS) [49]

were shown to be pH dependent. Maximum adsorption of the

protein occurred near the IP (4.9) where the enthalpy of the

reaction was positive. At pH values removed from the IP the

reaction was exothermic. It was suggested that at pH values

away from the IP the conformation of the adsorbed protein

changes for energetic reasons. Denaturation of the protein

is not surprising since it is known that the internal bonding

of serum albumin is weak [50]. The' enthalpy of the adsorption

reaction varied between zero and 8 kcal/mole as the surface

charge varied between -1.0 mpCcm-2 and -7.5 mpCcm-2. The

most negative enthalpy values were recorded for pH 3.8 and 9

and were near -8.4 kcal/mole.

Enthalpy values for the adsorption of albumin, gamma

globulin, and fibrinogin, were calculated from adsorption

measurements at several temperatures [51]. The results indicated

that the adsorption took place in two distinct ways. Both

types were apparently Langmuir and took place on separate

membrane sites. One type of adsorption was easily reversible

with a heat of adsorption in the neighborhood of -10 kcal/mole.

The other type of adsorption reaction was hydrophobic, endothermic,

and with heats of adsorption in the range of 5 to 20 kcal-

mole.








In another study [52], the forces involved in the

adsorption reactions between several globular proteins and

glass surfaces were determined to be primarily ionic amine-

silanol bonding and hydrogen bonding. Two rates of adsorption

were noted. The first appeared to be related to the number

of amines present on the surface of the protein. The second

was slower and seemed to be dependent on the molecular weight

of the protein. Hydrogen bonding was suspected since the

proteins could not be completely washed from the surface with

urea.

Ionic bonding of ribonuclease to glass was indicated

to be strong [53] since very little of the protein could be

removed by rinsing in several solvents. No enthalpy determina-

tions were made.' There was a decrease in adsorption with

an increase in ionic strength.

Further review of the literature reveals that the

various adsorption studies cannot be readily compared due to

the large number of experimental variables and to the random

manner in which they are controlled in each experiment. A

few common features in the study of protein adsorption do

emerge, however.

There is usually more than one type of interaction

present for any particular system and one of these is usually

hydrogen bonding. The observed enthalpy values are in the

range of -10 to +10 kcal/mole of protein. Finally, maximum

adsorption density appears to take place near the IP of the

protein. There are many exceptions to these general results,

however.








The effect of changes in ionic strength on adsorption

is also not well understood [54]. Dissolved salts disrupt

hydrogen bonds which proteins depend on for conformational

stability [55]. Changes in the ionic strength of the solvent

will also have effects on the adsorbing surface. For example,

phosphate, a common component in buffers, will change the surface

charge of alumina [56]. Generalizations are difficult to

make about the action of specific ions on adsorption unless the

specific system understudy is clearly defined.


Surfaces


Silica, alumina, and hydroxyapatite, the three materials

used in this study, are oxides. Hydroxyapatite is sparingly

soluble at neutral pH whereas silica and alumina are virtually

insoluble [57]. All are hydrophilic and each displays a

surface charge which varies with pH.

The surface charge results from exposed surface atoms

attempting to complete their coordination of nearest neighbors

[58]. Exposed cations do this by pulling an OH" ion or H20 from

solution and anions by attracting a proton from the aqueous

phase. The result is adsorbed H+ or OH- ions which assume their

respective charges on the surface.

Any other ion which can pass between the solid and

liquid phases may also help to establish the surface charge.

Such ions are called potential determining ions. Thus,
OH and H are potential determining ions for eachof the
OH and H are potential determining ions for each of the








+2 3
three surfaces used here. In addition Ca and P04- are

potential determining ions for hydroxyapatite. Certain

ions added as impurities may alter the surface charge such as

aluminum ions [59] or cobalt [60] on silica or phosphate on

alumina [61]. The wide variety of buffering systems used in

biological studies involving adsorption can thus lead to

differing results for proteins even if the same material is

used as a substrate.

The surface potential can be altered by a change in

pH. For each of the three oxide substrates in this study, there

exists a pH at which the surface charge is zero. This pH is

called the point of zero charge (pzc) and is listed in Table

1 [62,63] for the substances used as adsorbents.

As the pH varies from one side of the pzc to the

other, the sign of the surface charge will change as will the

adsorption properties of the protein.

Ions in solution which do not pass through both phases

but are attracted near the surface by electrostatic forces

are called counter ions. They will form a diffuse layer of

ions in solution near the surface and will tend to neutralize

the surface charge. The concentration of the ions generally

decreases exponentially with distance from the surface [64].

The.higher the concentration, the more compact the diffuse

layer will be. The thickness of the diffuse layer ranges from

about 10 A at .1 M solutions to about a few hundred A in .001 M

salt solutions [65].







12


Table I


PZC of Substances Used as Adsorbents



alumina 9

silica 2-4

hydroxyapatite 7.5









Due to the shape of the diffuse layer, the influence

of dissolved salts on proteins will be greater near a surface

than in bulk solution. Some proteins, because of their

large size, may extend entirely through a double layer. The

effect of dissolved salts on adsorbed proteins would then be

difficult to explain in detail.

The adsorption properties of the substrates are due

as much to adsorbed water as to their intrinsic stucture.

The -surface of silica in aqueous solution has been shown by

infrared spectroscopy to possess three types of surface ions

[36] as represented below:

Si 0-

Si 0-H

Si 0-H2

Unless the adsorbed water and associated ions are driven off

by heating, silicon ions cannot chemically react with organic

molecules arriving from solution. It has been shown that

ammonia will not react with hydroylated silica, although

chemisorption will occur if the silica has been subjected to
0
a prior vacuum degassing at temperatures in excess of 400 C

[66,67]. Silica treated with ammonium fluoride solution

showed evidence for Si-F bonding instead of silanol [68] but

this reaction occurred after heating the substrate to 4000C

in vacumm.* Trimethylsiloxane can be covalently bonded to

silica by refluxing them in acetone for 24 hours at 500C [69].

This gives some idea of the difficulty of penetrating the









adsorbed layers on the silica surface. It can be seen that

the reactions of aqueods solutions of proteins with silica may

occur with either surface oxygen or hydrogen, depending on

the compositional purity of the surface.

The same effect can be seen with alumina. Steric

acid was adsorbed from CC14 solvent after the alumina had been

evacuated at 8000C for one hour. Without the pretreatment,

steric acid would not covalently bind to the alumina surface

[70]. Methanol has been shown to adsorb on alumina [71] after

successive evacuation and heating at 4000C, heating in oxygen

to rid any hydrocarbons present and then heating again at

10-5 torr at not less than 3500C for 1/2 hour. A methoxide

surface is formed when the clean dry surface is exposed to

methanol vapor. From studies of adsorbed acetylene on alumina

it was concluded that the surface contains electron poor

and electron rich [71] sites (oxide ions, hydroxyl groups, and

aluminum ions) after the sample had been heated to 8000C.

Even at these temperatures not all the hydroxyl groups were

removed from the surface [72]. Hydrogen and hydroxyl ions on

alumina are exposed to the solution interface, yielding a

surface structure similar to that of silica.

Hydroxyapatite is assigned the formula Cal0(PO4)6(0H)2.

In solution the surface undergoes hydrolysis, yielding a surface

having the formula Ca2(HP04) (OH)2 [57]. It is different from

silica and alumina in that, in addition to surface OH- and H

ions, there are also Ca+2 ions which are capable of binding

adsorbing anions [73]. The multiple internal infrared spectra








of several synthetic and naturally occurring calcium phosphates

exposed to organic acids show shifts in the P-0 stretching

frequency [39] which were attributed to hydrogen bonding. Other

workers [30] have shown that H30+ ions are hydrogen bonded to

the calcium. There have been suggestions that there is some

covalent bonding between organic constituents and hydroxyapatite

in bone [74]. Binding of calcium ions by collagen has been

demonstrated by solution analysis [75]. It has not been shown

that collagen can attach to the surface of the hydroxyapatite

without an intervening water molecule or hydroxyl ion.


Forces of Adsorption

The relationship between the enthalpy of a reaction and

the total energy is H = E + PV. Most biological processes occur

in liquids rather than in the gas phase [76]. In this case

the changes in pressure and volume are small. To a good

approximation then, dE ~ dH.

The total energy of adsorption is affected by the type of

interaction between the surface and the molecule. This energy

is comprised of several components. They may be classified as

non-polar, ionic, hydrogen, and covalent bonding [77-79].

Non-polar dispersivee) forces are always present between

molecules. They arise because the time-averaged electron cloud

.interaction between uncharged atoms is attractive [80]. They

are moderately strong, producing energies in the range of 1 to

10 kcal/mole. Hydrophobic bonding is a result of dispersive









forces. This is a consequence of a decrease in displace-

ment of a polar medium when less polar components coalesce,

thus creating a lower energy state.

Ionic or electrostatic attraction can occur between

oxides and proteins both of which are normally charged in

aqueous solutions. Ionic bonding strength is decreased by

an increase in the ionic strength of the solvent because of

shielding, whereas dispersion forces are not affected. Ion

interaction is essentially independent of temperature [80,813.

Hydrogen bonding is partially ionic and partially

covalent [82,83]. It arises from the electrostatic force

acting between hydrogen and a lone-pair of electrons of

nitrogen, oxygen, or fluorine. The small size and the close

approach of the hydrogen atom accounts for the partial (20%)

covalent character [82]. Typical bond strengths are of the

order 1 to 10 kcal/mole. Hydrogen bonds are also weakened

by an increase in ionic strength of the solvent. Completely

covalent bonding rarely occurs in the adsorption phenomena

in which we are interested [62].

The classification of physical or chemical adsorption

is somewhat arbitrary [79,83]. If the adsorption is found

to be readily reversible and has an energy of the same order

of magnitude as the liquefaction of gas, it usually is classified

as physical [77 ]. Osipow states that Van der Waals forces

are responsible for physical adsorption whereas Fuerstenau

also includes coulombic attraction. Chemical adsorption is

irreversible and the magnitude of the energy change is of the

order of chemical reactions [78].









Thermodynamics of Adsorption


Complete reviews on the thermodynamics of adsorption

are given elsewhere [76-78]. In this section, only those

points needed to explain the following data will be presented.

Limited explanation of the ideas of earlier workers would

be in order, however.

Gibbs gave the first rigorous thermodynamic explanation

for why a given material should either adsorb or desorb at a

surface. He was able to predict the functional relationship

between surface tension and surface concentration and the bulk

concentrations of the surface-active solutes. His derivations

assume that substances tend to minimize the free energy of

the surface region by either becoming concentrated or depleted

there. As a result, it has been the free energy which has been

traditionally determined, and surface concentration measurements

are the most common method of doing this. Other methods, such

as the thermometric titration method [84], may be useful for

obtaining thermodynamic data.

The thermometric titration technique is an analytical

method in which the heat effect of a titration reaction is

used to measure the titer of a sample. It is applicable to

reactions of the type

RI + R2 P (1.1)

which entails a heat of reaction Q. 'In equation (1.1) Ri

refers to reactants i and P refers to the product. For single









step reactions an equilibrium constant, K, may be written

as

K = [P]
(1.2)
[R1] [R2]


where the brackets denote concentrations. Through knowledge

of equation (1.1) and the reaction heat, Q, the enthalpy change

of the reaction and the equilibrium constant may be determined.

The method by which this is done will be detailed for adsorption

measurements.

For an adsorption reaction equation (1.1) can be written

as

Su + R So

where R1 in equation 1.1 has been replaced by Su which is an

unoccupied site on a solid surface capable of adsorbing from

solution a reacting component R to produce an occupied site So.

The equilibrium constant is then written

K = [S ,

[Su] [R]

The concentration of occupied sites [Sol is equal to

the number of adsorbed molecules per unit area, Na/A, while

the concentration of unoccupied sites is equal to the total

number of sites N minus the number of occupied sites per unit

area, (N0 N )/A. The equilibrium constant is then

K = N

(No N ) ER]
s a








Dividing;- through by Ns gives
s
K = N /N
a (1.3)

(1-N /Ns) [R]
a s
or

K= 6
(1.4)

(1-0) [R]

where 0 = Na/N0 is the fraction of occupied sites. Equation
77
(1.4) is the Langmuir adsorption isotherm [ ] and will be

used later. Its use requires that each site is occupied by no

more than a single molecule and that no two sites interact.

These conditions are satisfied when the concentration of R is

low.

The concentration of reacting molecules is equal to the

number of moles of R in solution divided by the total volume

Nr/V. If No is the total number of moles of R on the surface

and in solution then N No Na. The equilibrium constant
r r a
can now be written as

K= Na V
(1.5)

(N Na) (N Na)
j r a

The enthalpy change for the adsorption reaction, AH, is

related to the reaction heat Q by

AH = -Q/Na (1.6)

where the minus sign denotes, by convention, that an exothermic

reaction (positive Q) will yield a negative enthalpy change.









Substituting equation (1.6) into (1.5), one obtains

-K = -QAHV (1.7)

(N AH + Q)(AHNo + Q)
s r

To determine K, at least two adsorption experiments are completed

in which Q is measured but a set of values is usually completed

to determine the endpoint of the titration. The original

number of moles of reacting molecules, No, is varied, and No

is held constant by keeping the surface area of the adsorbing

substrate constant. Values for Ql, Q2, N1, N, and sare

recorded where N1 and N are values for Nr. The value of K is
1 2 r
o
assumed to be nearly constant if N1 is not too different from

N2 Equation (1.7) can then be solved for AH by using the

quadratic equation (1.8)

AH2 Ns (Q1o 2 Q2N) + AH Q1Q2 (N2-N1) +Q1Q2(Q2-Ql)=O

for the two sets of values. The total number of surface sites

can be estimated by dividing the total surface area by the

known cross-sectional area of the adsorbing molecule. This

method is valid as long as the surface concentration of adsorbed

molecules is low and lateral interaction does not occur. Alterna-

tively three sets of values can be used to eliminate N from

equation 1.7. Both methods give results = 10% of each other,.

The volume V is held constant. The value of AH is substituted

into equation (1.7) to find K, and therefore AG by using the

relation


AGo = -RT In K


(1.9)








In order to complete the thermodynamic data (AGO =

AHo TASo) Ho must first be determined or approximated.

The number AH used in equations (1.6) to (1.8) is the total

enthalpy change for the reaction under experimental conditions

while AHo is the standard enthalpy change. Under suitable

conditions, AH can be shown to be = AH so that ASo can be

determined. Those conditions will now be explained.

The chemical potential /i or partial molar free energy

Gi of component i in a chemical reaction is defined by

'i = Gi = ('G/aNi)tp (1.10)
where G is the free energy change for all components in the

reaction. The chemical potential can be expressed as a function

of the activity ai of component i and of the chemical potential

in some reference state

^i = /iref + RTlnai (1.11)

The term RTlnai takes into account the energies of interaction

of component i with other components at a given concentration in

the mixture. The choice of reference state is quite arbitrary

and varies for experimental convenience. Generally, no matter

what reference state is chosen, the activity is expressed as a

function of the mole fraction component i, Xi, and a parameter

known as the activity coefficient fi

ai = xi fi
The activity coefficient approaches 1 in pure solutions for

the solvent while in dilute solutions of component i, fi

approaches a constant which may be greater or lesser than one.









The partial molar enthalpy is found to be

Hi = -RT2 ( lni/ T)pn (1.12)

or in view of equation (1.11)

H-i = -RT2(ln i ref/DT) RT2 ( n ai/DT)pn (1.13)

or = -RT2(OIn iref/T)n RT2 (~in fi/DT)p

Under the constant composition Xi does not vary with temperature.

The first term of equation (1.13) is the enthalpy

change for component i which would occur if the reaction was

held under reference conditions. The standard enthalpies, HO,

of pure compounds are the enthalpies of reaction of building

up those compounds from their elements under standard conditions

(P = 1 atm T = 298K). The chemical elements themselves have

zero standard enthalpies of formation. If the reaction is

carried out under standard conditions, the enthalpy change for

the reference state is equal to H?. Then
1
o 2
H. = H. RT (~ln f./ T) (1.14)

Knowledge of fi for the systems under study is lacking.

We therefore make the approximation that AHo is significantly

larger than the natural logarithm of the temperature variation

of fi. The validity of this approximation relies upon the non-

interaction of solute molecules. This condition is assumed to

hold for dilute solutions. Therefore

Hi = Hi (1.15)

To relate HO to AH it is noted that AH can be written

as the difference of the sums of the partial molar enthalpies

of products and reactants









AH = ZiHiX + kHkZk (116)

products reactants

where each sum is taken over each of the different components

for products and reactants and X. and Zk are the respective

mole fractions. In view of equation (1.15) the enthalpy

change for the adsorption reaction is

AH = H X + EkHk Zk (1.17)

or
AH = AH (1.18)

where AHo is the standard enthalpy change for the complete

reaction. Thus, under the rather ideal conditions in which

there is no interaction between solute molecules in solution or

on the surface at 1 atm and 2980K AH may substitute for AH.

The value of AH can be used with AGo to find at least an approximate

value for AS0.

The experimental conditions in this work meet the contraints

of pressure and temperature. The constraint of non-interaction

of solute molecules holds only for dilute solutions. In as

much as enthalpy values tended towards.constant rather than

steadily decreasing values,lateral interaction on the surface

between adsorbed molecules does not appear to have occurred.

Interaction of solute molecules in solution can only be assumed

to be small in the concentration ranges used, typically 10- to
-3
10 M.









Heterogeneous Adsorption


As mentioned earlier, from the standpoint of adsorption

studies, the surface of a substrate is often not uniform.

Heats of adsorption may vary at different positions on the

surface. If the condition is maintained that the different

sites are non-interacting, the adsorption onto a heterogenous

surface may be regarded as simultaneous independent reactions

of the type expressed by equation (1.3)

Sl + R Sol
S + R + So1

S2 + R So2
(1.19)


S + + R Soj

where the subscript j enumerates the different types of sites.

The concentration of a single type of solute molecules, R,

is common to all surface sites. Each adsorption reaction,

according to equation (1.19), would evolve a reaction heat

Qj. The total reaction heat, Qt, would be the sum of Qj for

each reaction on the different types of sites.

Qt Ql + Q2 + .. .Qj (1.20)
If AHi is the enthalpy change per mole of adsorbed molecules

on sites of type.j and Nj is the number of moles adsorbed then

equation (1.20) can be expressed according to equation (1.6) as

-Qt = AH1 N1 + AH2N2 + ....AHjNj (1.21)
In a calorimetric measurement it is the value of Qt

which is measured. If the solution is analyzed to determine










the total number of moles adsorbed, Nt a total apparent

enthalpy is found

AH = -Qt/Nt (1.22)

From equation (1.20) then

AHt = -Qt/Nt = 1/Nt (AH1 N1 + AH2 N2 +...AHj N )

or

AHt = AH1 Y + AH2 Y2 +....AH. yj (1.23)

where Yj is the fraction of the total number of moles of

molecules bound to sites of type j. Applying equation (1.18)

to each reaction

AHt = EjAH9 yj
(1.24)
= AHO

where AHO is the weighted sum of the standard enthalpy changes

for all adsorption processes. The standard free energy

change for each reaction expressed by equation (1.19) is given

by

AG = AH? TAS? (1.25)
3 1 1
Rewriting equation (1.25) for A and substituting into (1.24)
one obtains

AH = E.(AG9 + TAS9) y (1.26)
t i I I
= AC yj + TASjyj

or

AH = AGo + TASo
t t t
where


AGo = G. AG y.
L I 3 I


(1.27)







and

AS = ES? yj (1.28)

express the weighted sums of the standard free energy and

standard entropy changes for the complete adsorption reactions.

For each reaction expressed in equation (1.19) there

is an equilibrium constant Kj which can be written according to

equation (1.4) as

K. = 6. (1.29)


(1-06) [R]

or related to AGQ according to equation (1.9) as

AG? = -RT In K. (1.30)
J 1
Equations (1.29) and (1.30) express the fact that each site

carries on an equilibrium reaction independently of all others.

The fraction 6. = N./N? is the ratio of occupied sires of type

j to the total number of sites of type j.

For any equilibrium concentration [R] of solute molecules

the total fraction of occupied sites of all types, 8t, can be

written as

6 = N /No
t t s
or

S = X1 61 + X2 0 + ...X. (1.31)

where X. = N9/No is the fraction of sites of type j and is a
] 3 s
constant.

If equation (1.30) is substituted into equation (1.27)

one obtains


(1.32)


AGo = -E. (RT In Kj) Yi
t. J









or

= -RT'ln K' K^ ..... K (1.33)
12 2
Thus, even though each AG? for the individual.reactions is

constant, the overall free energy change will vary as each

fraction y. varies.

The net effect is that if any single type site adsorbs

a large percentage of all molecules adsorbed then

AGt approaches AG? as v 1

Generally, however, there is no simple number AGo which can

be expressed in terms of a single equilibrium constant Kt,

having the form

K = KJ K2 ........KY (1.34)
t 1 2 3
The value of Kt would vary as the fraction of occupied sites

varies.

The physical interpretation is that each site contributes

a specific amount of energy to the total energy change. At

very low concentrations only a very few sites react, presumably

those of higher energy. At higher concentrations a greater

number of sites react, but of overall lower energy. The result

is a lowering in the average energy change as the concentration

is increased.

Under the conditions of independently acting sites

evaluation of K would give AGo exactly. However, to do this,
t t
knowledge of each Ki and yi has to be available. In the absence

of such knowledge, the evaluation of K can be approximated by

evaluation of equation (1.29) by replacing 6j by Ot. The number

found from this method, K', could be used for determination of

AGO under suitable conditions.
t








Those conditions may be determined by estimated

values of Kj for calculation of K' and Kt and using each for

evaluation of AG The value of K' is found from

K' = Ot

(1.35)
(1-et) [R]

where 0t is defined by equation (1.31). The percent errors in

AGt is found from

% error AGt = n K In K' x 100


In K'

The percent error is calculated by setting values for K. and

Xj and directly calculating K' from equation (1.34) and Kt

from equation (1.40). If the percent error is acceptable, AGt

may be calculated.

An example for the case of two different types of

adsorption sites is given. The equations necessary to carry

out this calculation are given below for convenience

61 = K1 [R]/(l + K1 ER])

2 = K2 [R]/(l + K2 [R])

yl = 01No /(O1NO + 0 NO)
11 IN 1 2 2
y = 2N /(62No + 2NO)
Kt = K1 K2

X NO / (NO + NO)

X= N / (No + N
2 2 1 2
ot = X161 + X202
K' = t/(1 + t) [R]











In the example the values of [R] were carried over several

orders of magnitude while the total number of sites NO =
S
Nl + N2 was kept constant at 10-6 moles. The results are

shown in Figure 1

Two cases are presented. In (a) the fraction Ni/No

0.1 and K = 5000 are held constant, while K1 is given

values of 1000 and 5000. In (b) K1 and K2 are held constant

at 1000 and 2000 respectively while X varies over three

values; 0.002, 0.02, and 0.2.

While K2 is less than five times the value of K1 and

X1 is a good approximation of Kt with the error remaining

within 1%. As X1 approaches lthe error goes to zero. Also,

at very low concentrations, it is assumed that most molecules

would adsorb only onto the highest energy sites so all values

of y (equation 1.34) go to zero except Y and the error again

goes to zero. For oxides the overall fraction of highly

reactive sites is small [85,71]. Moreover, the hydrated

oxide surface will be of lower energy than a perfectly dry

surface, aiding in meeting the condition that K1 not be too

much larger than K2 [60,28]. Under these constraints and using

equations (1.8) and (1.7) with Q = Qt, AHt can be calculated.

For the calculations in the later chapters, the

difference between the values of NO for use in equation (1.8)
r
is kept small so that variation of AH in that concentration

interval is small. The values of AGo calculated tend to remain

within 20% of the highest to lowest values. This corresponds








S K2 = 5000


Sx = 0.1
10 K',Kt ( K1=50000) ,\ 4


9 (K = 50000)

( ) 8 -

7
(K1 = 10000)
6 0

5 K', Kt ( K1 = 10000) o
+

0
0
SK2 =2000 4
0 -x --"--" l =0 2 K1 =10000 0
.- ."' 2


2




(h) 6 0
Kt (.2)

4 Kt (.02)

Kt(.002)
2

I I I I I
7 6 5 4 3 2 1

-LOG (R)

Figure 1. Examples for the calculation of K' and Kt
for constant K2 and xl (a), and for constant K2 and K1 (b).








to a range of K. varying by about a factor of seven. Under

these conditions the maximum error in AGt is less than 2%.

The value of AS calculated from

ASo = AGo AH
t t

-T

can be given only simple interpretations. It is known that

values of AS will be between -20 and +20 cal/mole-deg [49,51].

A decrease in entropy is typically explained as a loss of

freedom of solute molecules as they adsorb. Increases in

entropy, generally found in experiments using macromolecules,

are explained as solvent molecules gaining additional freedom

as the largestructuring molecules are removed from solution.

Exceptions to this general rule are present.

The various plots of AG, AH, and AS presented in the

following chapters, in accordance with the previous discussion,

are to be understood as the composite values AGt, AHt, and

ASt. The values for these parameters are closer to single

values of AH?, AG?, and AS? in those regions of the curves

where they tend towards constant values. In these regions the

percent error is generally less than .5%.

The determination of the thermodynamic properties for

the adsorption of collagen on hydroxyapatite is presented as

an example of the calculations made in the following chapters.

The first step in the analytical procedure is to plot Q vs. C

(Figure 2). From this graph two values of Q and Co are chosen

for the sample calculation. In this case values of














7,5

Q2


5,0
SQ1I





c1 c22

I I I i

0,5 1,0 1,5 2.0

CO M

Figure 2. The first step in the calculation of the thermo-
dynamic functions is plotting the reaction heat, Q, vs the
original concentration Co. Here, two points are taken from
the measurements of the adsorption of collagen on alumina.









Q1 = 5.0 x 10"3 cal
-3
Q2 = 5.9 x 103 cal

C = 1 x 10-6 M

C = 1.33 x 10-6 M

are arbitrarily chosen. The values of CI and C2 correspond

to

N = 2 x 10-9 moles
1

N = 2.66 x 10-9 moles
2
The number of moles of surface sites, No, in this example
s
is taken as 2.5 x 10-9. This number was determined from

consideration of the surface area occupied by a collagen mole-

cule, the number of sites as calculated by the program, and

study of the reaction heat curve. Substitution of these

values into equation 1.8 yields -3.3 x 106 cal/mole for AH.

Substitution of AH, Q1, No and V (4 x 10 -3 l)into equation 1.7

gives 4.3 x 105 for K or -7.6 kcal/mole for AGo. This in turn

yields -1.1 x 104 e.u. for AS0.


Experimental


Calorimetric measurements were made using an LKB model

10700 batch microcalorimeter [86]. The basic calorimetric unit

consists of two identical gold cells situated in an aluminum

heat sink (see Figure 3). Each cell has two compartments

capable of holding 2 and 4 ml of fluid. Mixing of the fluids

in each compartment is accomplished by rotation of the entire

calorimeter. There is no stirring, and after rotation, the full









B D





















E
C A
























F




V A








S-----------


Figure-3. Schematic of the operation of an LKB model 10700
microcalorimiter. A-calorimeter, B-reaction cell, C-thermo-
pyle, D-heat sink, E-amplifier, F-chart recorder, G- reaction
heat curve. The reaction heat, Q, is proportional to the
area under the curve.









4 ml of fluid are contained in the forward compartment. In

all experiments, 2 ml of fluid were used in each compartment.

Measurement of the heat loss or gain incurred by the

mixing procedure is made through multiple thermopiles located

between the heat sink and cells on two sides of each cell.

The thermopiles are connected in opposition so that the signals

from reactions producing equal amounts of heat are cancelled

electronically. One cell is then arbitrarily chosen as a

reaction cell and the other as a reference cell in which

unwanted heats can be cancelled.

Determination of the reaction heat is made by manual

integration of the voltage vs. time curve produced during the

course of a reaction. The energy is calibrated against a

known heat produced in the reaction cell using a precision

resistor and a known current generated for a specific time

interval.

Each reaction in this work is of the type

rotation

A BI A + B

where A is a solution of organic molecules and B is a slurry

of powdered oxide used as a substrate. In the mixing operation

several reactions are possible, each contributing to the over-

all heat produced. They are due to 1) wetting of the cell

wall; 2) dilution of the organic molecule; 3) friction of

mixing; 4) chemical reaction. Only the last heat is desired.

The others must be eliminated.








The first three heats are accounted for in various

ways. The cells are first wet with the solvent being used

and emptied prior to filling with reacting components. This

eliminates the heat of wetting of the cell wall. The heat of

dilution of the organic molecules is accounted for in the

reference cell, while the heat of dilution of the oxide powders

and frictional heat are measured separately and subtracted

from the reaction heat.

The heat measured in this way gives a measure of the

reaction A + B C where C is a complex of solid particulates

and adsorbed organic molecules.

Calibration of the calorimeter was carried out as suggested

by the manufacturer and consisted of two procedures. The first

procedure determined the sensitivity of the thermopiles. The

manufacturer listed the sensitivity of the thermopiles as 28.0

and 30.0 microvolts for a constant current of 30 miliamps

through the calibration heaters. The measured values were

consistently within 28.0 L .05 microvolts and 30.0 1 .05 micro-

volts. The second procedure judged the accuracy of the unit's

calibration mechanism. The heat of dilution of a six percent

sucrose solution was measured periodically to be 6.36 .05

kcal/mole. The literature value is 6.36 t .03 kcal/mole.

No literature data could be found for the heats of

adsorption of surfactants from aqueous solution on prewetted

surfaces. However the heat of adsorption of sodium dodecyl

sulfonate (SDS) measured from the heat of immersion of dry

alumina (Linde A) was estimated to be 12 kcal/mole 1 kcal/









mole [23]. Using the method described in this work the

heat of adsorption of SDS on Linde A alumina was found to

be 10.2 kcal/mole of SDS. The discrepancy, -1 kcal/mole,

is thought to be due to the surface being wet prior to contact

with SDS. This would prevent any possibility of SDS coming

into contact with a drier, and presumably, higher energy

surface.

A Perkin-Elmer-Hitachi model 139 U.V. Vis spectro-

photometer was used for concentration determinations. Separa-

tion of particulates from supernatent solutions was carried

out by centrifuging or by filtering through micropore glass

filters. In those cases where filtering was used, the filter

was first saturated with a solution of the organic molecule to

be analyzed, then rinsed thoroughly. In all cases a standard

was used which had been put through identical procedures as the

unknown.

Protein and polypeptide determination was made through

the use of Biuret reagent [87 ] and measuring at 550 millimicrons.

Carbohydrates were determined using a phenol solution and

measuring at 490 millimicrons [88]. Amino acid concentrations

were determined by use of ninhydrin [87]. Carboxylic acids

were titrated with phenothalein as the indicator.

Distilled water, having an initial pH near 7, was used

for preparing solutions. The pH was varied by adding HC1 or

NaOH. Three solutions were used as solvents: a low ionic strength

solution (LISS) in which the only ions present were those added

by pH adjustment, a 0.165 M salt solution, and a buffered solution.








The concentration of NaCI in the low ionic strength solution

did not exceed 0.001 M., The 0.165 M salt (Ringers) solution

consisted of 9 gm/l of NaC1, 0.25 gm/1 of CaC12, and 0.42 gm/1

of KC1 and is known. The buffered solution was a 0.2 M solution

of mono- and di-basic phosphate. The phosphate buffer was

used only in Ringers solution bringing the total molarity to

0.365.

Except in those instances where solids were not used

at all, 0.1 gm solid particulates were added to the solution.

The solids were placed into suspension by mixing 1.0 gm of solid

powder with about 18 ml of distilled water, adjusting the pH

and then bringing final volume to 20 ml. One hour was allowed

for the pH to equilibrate. With stirring, 2 ml aloquots were

distributed into 10 tubes by pipetting. Using this method 0.1

gm 0.01 gm of solid was delivered to each tube.

The gold calorimeter reaction cells were washed daily

with detergent. The washing procedure included injecting a

solution of the surfactant into each compartment, rotating the

calorimeter and withdrawing the solution by aspiration. The cells

were then continually rinsed with distilled water while being

evacuated. By moving the tip of the aspirator tube from the

top of the cell to the bottom in one compartment, while filling

the other compartment, a good turbulent rinsing reaction

developed. Experience showed that five minutes of such procedure

cleaned the cell. Approximately once a week, the detergent

was left in the cells overnight to permit it to react more

thoroughly.









Between daily experiments, the same procedure was used

to clean the cells. The oxide powder slurry was removed for

analysis after mixing, however, and the detergent was not used.

Cleaning with 1 M HC1 or NaOH was found to be necessary only

rarely.
+
Organic constituents were weighed out to 1 -'.01 mg

and mixed volumetrically. Adjustment of pH was the same as

with solids. Incremental concentrations were made from standard

batches and measured volumetrically by pipet. Distribution of

organic, and solid, solutions into the reaction cell was made

by syringe. The lowest concentration was always used first.

The materials used as substrates include silica, alumina,

and tricalcium phosphate. The silica [89] was described by the

manufacturer as amorphous. It has. a specific surface area of

0.7 m2/gm and a pzc of 3. The alumina used [90] consisted of

two types: Linde A, a a-alumina of specific surface area 15 m2/gm

and a pzc of near 9 and Linde B, a mixture of y and a alumina

of specific surface area 82 m2/gm and a pzc near 9. The tri-

calcium phosphate, referred to as hydroxyapatite in the text,

consisted of 85 volume per cent hydroxyapatite and had a specific

surface area of 57 m2/gm. Surface measurements were made in

this laboratory using a multi-point B.E.T. nitrogen adsorption

isotherm. Measurements of pzc were also conducted in this

laboratory using a Zeta meterR [91].

All experiments were run at 250 C.

All solids were used without modification. Prior to

weighing, large (approximately 10 gm) batches of solids were








rinsed in distilled water, decanted and evacuated for 24

hours at 10'3 atm. Prepared powders were stored under vacuum

at room temperature.

All organic substances used were stored under refrigeration

prior to use. None was repurified or modified in any way.

Batch solutions were used within one week and were stored at

4C. Particular information on each substance used is given

in the appropriate chapter.

Reaction heat data were plotted against Co and fed into

a statistical analysis program available through the Northeast

Florida Regional Computing Center. The reaction heat, Q, was

held as the independent variable. The program generated an

approximating function which was used to calculate the thermo-

dynamic data. In all chapters, referral to "calculated thermo-

dynamic data" refers to this procedure. The graphs of Q versus

Co presented are original data.












CHAPTER II


ADSORPTION OF CARBOXYLIC ACIDS

ON ALUMINA AND HYDROXYAPATITE



Introduction


Several of the amino acids which make up collagen

possess charged side groups, principally amines and carboxyl.

The polysaccharide, chondroitin sulfate, often associated with

collagen, also possesses the carboxyl groups as well as the

sulfate group. Because each of these charged species has the

potential to interact with an aqueous oxide surface, knowledge

of the type and strength of the surface reaction is sought.

The thermodynamic and adsorption behavior of molecules

containing the sulfate group have been previously described.

The adsorption of sodium dodecyl sulfate on alumina has been

studied by calorimetric techniques [31] and by solution depletion

techniques [91]. Heats of adsorption for this molecule have

values near -6 kcal/mole at low concentrations when only ionic

forces drive the adsorption reaction. The free energy change

lies near -11 kcal/mole of ions. The adsorption of sulfate

ions onto solid barium sulfate from aqueous solution has been

measured by the thermometric titration technique. The heat of

precipitation was found to be -4.5 kcal/mole .[84].









The adsorption of alkyl ammonium acetate on quartz

has been followed as a-function of temperature and concen-

tration [92-93].It was found that at 25C at neutral pH the

isoteric heat of adsorption was between zero and 2 kcal/mole

of ions. The positive enthalpy was expected because of charge

repulsion of the quartz surface and the negatively charged

ions in solution. The free energy change at 250 remained near

-3.5 kcal/mole. In most cases the hydrocarbon chain of the

molecules caused abrupt changes in the thermodynamic properties

due to lateral interaction of the adsorbing molecules as the

equilibrium concentration increased.

Binding studies of sulfates, citrates, and amino acids

on calcium oxalate and calcium phosphate have been carried

out in relation to bone formation and kidney stone growth

[94,95]. Thermodynamic data are not readily available for these

reactions. Equilibrium constants of magnesium oxalate [94 ],

however, have been shown to be near 4000 which would correspond

to a free energy change near -5 kcal/mole for this precipitation

reaction.

The purpose of this chapter is to discuss the variation

of the heat of adsorption of simple and polymeric carboxlic

acids on alumina and hydroxyapatite in relation to surface charge,

molecular conformation and ionization and solution pH. Qualita-

tive results indicate that while electrostatic interactions

are required to initiate adsorption, other interactions such

as hydrogen bonding, take place.








The sodium salts of acetic acid, oxalic acid, citric

acid, and polyacrylic acid (PAA), containing one, two, three

and multiple carboxyl groups were selected for study. The

different number of charged groups, different degrees of

ionization and molecular structure of each of these molecules

should provide a sufficient variety of detectable changes in

the calorimetric measurements. Analysis of the various changes

should furnish a clear understanding of the adsorption

mechanism.

The structural: formulas of each of the molecules used

in the work described in this chapter are given below:


H3C-COOH HOOC-COOH

Acetic Acid Oxalic Acid


H OH H H H
I \ I I I
HOOC C C C COOH -4 C C-

H I H H COOH
COOH

Citric Acid Repeat Unit of
Polyacrylic Acid


Experimental


Sodium salts of the carboxlic acids were purchased

from Sigma Scientific, Inc. [96]. Poly(acrylic acid) in a

65% aqueous solution was purchased from Aldrich Chemical

Company [97] and was reported by them to have a molecular weight

of 2000. Linde A alumina, and hydroxyapatite were used as

substrates.








Titrations of the carboxlic acids were carried out

with HC1 or NaOH. Determination of the amount of acid

adsorbed was based on calibration against known concentrations.

Other methods and procedures were described earlier.


Results


Reaction heats for the adsorption of the three simple

carboxylic acids on alumina are presented in Figure 4. For

each acid, the maximum reaction heat was found to occur when

the solution was near pH 5. The reaction heats at pH 3 were

second and the lowest curves were recorded for pH 7. The

highest heat was recorded for sodium oxalate which also has

the lowest dissociation constant (see Table 2).

The enthalpy change upon adsorption was first found

by determining the amount of each acid adsorbed by titration.

The method was suitable only for pH 5. At pH 3 and 7 poor

precision resulted because of the small amount of each acid

adsorbed. Results are given below:

acetic oxalic citric

-AH (kcal/mole molec.) 4.2 4.8 4.6

(Co = .001M)

Thermodynamic data were calculated for pH 5 using a

smaller concentration range (C = 10'4 to 10-3 M) (see Figure

5). These results show that there are differences in the

enthalpy change for the three acids used. Each curve displays

a tendency towards more negative exothermicc) values at the

lower end of the concentration range. The heat of adsorption

























S/ /' 3

20- /5 / 5
20 -
o/ ./ 3

10/ /

S..7


I I I
4 3 2 1 0

-LOG C0

Figure 4. Reaction heats for the adsorption of three
carboxylid acids on alumina.




46



Table 2

Dissociation Constants
of Carboxylic Acids

Acetic Oxalic Citric

4.75 1.23 3.14
4.19 4.17
6.39





47








A ACETIC ACID

0 OXALIC ACID

30 C CITRIC ACID 60





25 5

C
S- 50





o 20 c C 0




c-

15 30
S- 30





o10 20

S20


-4- -. .- .


0.4 0.6


0.8


0. 2








lies between -6 and -24 kcal/mole. The free energy changes

are nearly equal at about -5.5 kcal/mole, while the entropy

changes are each negative and lie between -10 and -20 cal/mole-

deg (1 cal/mole'deg = 1 entropy unit or e.u.).

Calculated values for the thermodynamic functions for

the adsorption of the three carboxlyic acids on hydroxyapatite

are presented in Table 3. The values show less variation for

hydroxyapatite than they did for alumina. The enthalpy

change tends to be less negative than for alumina, while the

free energy and entropy changes are about the same.

Polyacrylic acid (PAA) had such a high affinity for

alumina and hydroxyapatite that a cotton-like gel formed at

pH 5 and 7, causing great difficulty in cleaning the gold

reaction cells. Three concentrations were run with alumina,

however, and were repeated several times to improve precision.

Titration determinations of the amount of PAA adsorbed leads

to an enthalpy of 82 cal/gm of PAA for the adsorption reaction.

Taking 72 gm/mole as the molecular weight of the monomer, the

determined enthalpy change is -5.7 kcal/mole of acrylic acid

monomer assuming all acid groups participate in the adsorption

reaction.

The calculated thermodynamic data for the adsorption

of PAA onto aluminaare presented in Figure 6. The enthalpy

change per residue lies between -7 and -12 kcal/mole, while

the .free energy change is concentration invarient at -5 kcal/

mole. The entropy change is negative as it is with the other

carboxylic acids used in this section.






Table 3


Themodynamic Variables for the Adsorption of
Carboxylic Acids on Hydroxyapatite at pH 5 and 7


Substance


Acetic
Oxalic
Citric

Acetic
Oxalic
Citric


-AG -AH AS
(kcal/mole) (kcal/mole) (cal/mole-deg)


5.4
5.1
5.5

6.1
5.2
5.1


3.3
9.0
11.0

5.4
9.8
17.0


-7.0
13.0
18.0

-2.3
15.0
39.0















15 -30

Ci)


(0


1 0




5 -- --"- .. ----- --.- i0







0.02 0.04 0.06 0.08

Co, mM

Figure 6. Thermodynamic data for the adsorption of poly-
acrylic acid on alumina at pH 7 in low ionic strength solu-
tion; AG----, AH-- AS.-.-









Discussion


As molecules are adsorbed from solution, other

molecules will ionize to try to maintain the original concen-

tration. At the same time the alumina or the hydroxyapatite

will act as a buffer to maintain the pH. So long as the pH

is constant, the degree ionization of the solute will remain

the same. This leads to a qualitative explanation for the

reaction heat curves of Figure 4.

The total ionic charge in solution is directly propor-

tional to the degree ionization, a, which is related to the

pH by [98]:

pH = pKo log [ (1 a)/a ] (2.1)

where pK is defined as
o
pK = -In [H+] [A-] (2.2)
0 [HAJ

This relation holds for single as well as polyelectrolytes

[99].

In the absence of any specific interaction between a

solid and electrolytes in solution, the surface potential is

given by [5b]:

S= RT In a/a (2.3)
SzF

where z is the valence (including sign) of the potential

ion, F is the Faraday constant, a is the activity of the

potential determining ion in solution and a is the activity

of the potential determining ion at the pzc. As mentioned

in the introduction, the potential determining ion for the








systems under consideration is H+. The surface potential

may then be written from equation (2.3) as

= 2.3 RT (log [H ] log [Ho]) (2.4)
o
where concentration are substituted for activities and

where [Ho] is the hydrogen ion concentration at the pzc.

The interaction due to the surface potential and the

charge, q, in solution gives rise to an interaction energy,

E [64]

E = o dq (2.5)
q 0
where integration is necessary since the charge concentration

is a differential process. The surface potential affecting

ions in solution will decrease as saturation of the surface by

charged ions is approached. The total interaction energy is

nearly equal to Poq in this model if the concentration of charged

ions in solution is low so that there is little interaction of

the adsorbed ions on the surface. In this case the surface

potential which each ion encounters will be the same. The

energy lost by the ions is transferred to other ions and solvent

in the form of kinetic energy and flows as heat out of the

system.

A plot of o from equation (2.4) and a from equation (2.1)
0
versus pH for a hypothetical monovalent acid with a pKo near 5

and an oxide with a pcz near 9 is given in Figure 7. At low

pH, a, and thus the charge in solution decreases. At high pH

the surface potential decreases. The highest interaction should

be expected to occur where o and a are not near zero.























2 4 6 8


Figure 7.






20


10-

10


Plot of 4o and a for a hypothetical acid.


ACETIC-
OXALIC -
CITRIC-*-

.001M
\ *CALC

\CALC
~/ ILEXP
/ XP'EXP
CALC
EXP


Figure 8. Values of the reaction heat calculated by use
of equation 2.6 and found by calorimetric experiments.


500


250


/


1.0




0,5


__









For example, at pH 5 equation (2.4) gives for a

monovalent electrolyte '(H+)

to = .059 (9 5) = .24 volts
The total charge in solution, using acetic acid as the mono-

valent acid at .001 M is

q = a Ane (2.6)

= 1.78x10-5(6xl023)(2xl0-6)(1.6x10-19)

2.78x10-5

= .12 coul.



where A is Avogadro's number, n is the total number of mole-

cules in solution, and e is the electric charge. The total

electronic interaction energy is

E = qo q

= (,24) (.12) = .028 j = 6.7 meal

The value of Q found from microcalorimetry is 4.7 meal. The

agreement is fair. Plots for other values of Q and E for other

acids at different pH values are shown in Figure 8 for C =

.001 M. Although the experimental and theoretical results do

not fit well for all cases, the simple model qualitatively

explains the experimental findings, which is what was sought.

Such factors, as treating the ions in solution as other than

point charges, and the potential at the Stern layer are not

taken into account. The most important result is that the

reaction heat and electronic interaction energy decrease as

either o or a decrease. The tentative conclusion develops that

electronic interaction will probably be the essential factor










in enthalpy changes measured for charged adsorbing molecules.

The enthalpy and free energy change values are within the

range expected from the investigations of other workers.

Some idea of the number of active groups of each

molecule actually on the surface can be obtained from the

following analysis. Using the data from Table 4and Table 5

the average number of ionized groups on acetic acid, oxalic

acid, or citric acid can be determined from their dissociation

constants. The numbers are given in Table 4. If we divide

the enthalpy change per molecule by the average number of

ionized groups. AH" is obtained. This assumes that the enthalpy

change for the adsorption of carboxyl groups is about the

same regardless of the molecule being considered. Results are

given in Table 5 for pH 5 and 7 at .001 M.

The values for AH* are fairly consistent in each case,

Considering the enthalpy change of the adsorption for acetate

as an arbitrary baseline, we can argue that if AH* for oxalate

or citrate had been much larger than that for acetate it

would have implied that more groups per molecule were participat-

ing in the surface reaction than expected from the degree of

ionization. Asis,AH* for oxalate is slightly lower and citrate is

slightly higher than the AH* for acetate at both pH values.

Oxalate is the most strongly dissociated of the three acids

implying, perhaps, a slightly stronger reaction with the surface.

It should be realized that a particular group cannot be partially

ionized at a particular instant in time. The invarience in




56




Table 4

Ionized Groups per Molecule
for Three Carboxylic Acids

pH Acetic Oxalic Citric

3 0.02 1.0 0.42
5 0.63 1.85 1.9
7 1.0 2.0 2.8







Table 5

Enthalpy Change per Mole, AH, and Enthalpy
Change per Mole of Ionized Groups, AH"


pH Substance
(acids)


Acetic
Oxalic
Citric

Acetic
Oxalic
Citric


-AH
(kcal/mole)

3.3
9.0
11.0

5.4
9.8
17.0


ionized
groups

0.63
1.85
1.90

1.00
2,00
2.80


-AH,
(kcal/mole)

5.20
4.86
5.90

5.40
4.90
6.10








AH* is evidence that only ionized groups participate in

adsorption. This supports the supposition that electro-

static attraction is primarily responsible for adsorption.

The same procedure applied to the data for the adsorp-

tion of the three acids on alumina does not give such consis-

tent results. At pH 5 AH* lies between -6 to -11 kcal/mole

for acetate, -2 to -7 kcal/mole for oxalate and -7 to -3 kcal/

mole for citrate.

Near Co = .4mM (see Figure 5), a medium concentration,

we find that AH* lies near -9.8 kcal/mole for acetate, -4.1

kcal/mole for oxalate and -8.4 kcal/mole for citrate. This

indicates that while acetate has one, and citrate two ionized

surface groups, oxalate has on the average only one of its

two ionized groups on the surface.

It was argued earlier that only ionized molecules are

attracted to the surface and that the number of such molecules

drops as the surface charge drops. It should be recalled

that the surface charge on alumina and hydroxyapatite is

produced by the adsorbed H+ ions. This suggests the possibility

that an approaching ionized carboxyl group and surface hydrogen

ion participate in hydrogen bonding.

Zeta potential measurements [100] show that citrate

changes the surface charge of hydroxyapatite at low concentra-

tions. This indicates that electrostatic attraction by itself

is not the only factor in adsorption. If it were, when the

surface charge had been neutralized by adsorption of a sufficient

amount of citrate, adsorption would have ceased and the surface

charge would not reverse.








The single charged acetate ion does not reverse the

surface charge of hydroxyapatite or alumina, this would

indicate the absence of other than electrostatic interaction.

It will be recalled from the introduction that .close approach

of hydrogen to an anion is required for hydrogen bonding to

occur. Since, at pH 7, acetate has one and citrate has three

ionized groups, it is thought that multiple groups are required

to pull the molecule close enough to the surface for hydrogen

bonding to occur. The interplay between surface charge,

molecular size and charge density, ionic and hydrogen bonding

becomes apparent in these situations.

Polyacrylic acid has such multiply-charged groups. It

is known to be a linear molecule which is fully ionized at pH 7

[ ]; therefore, there are no hydrogen bonds to be broken due

to an unfolding of the molecule upon adsorption. The AH between

-5.7 and -6.9 kcal/mole of residues of PAA is close to that

found for the adsorption of the carboxyl group of the other

molecules on alumina and hydroxyapatite. The similarity in all

these instances implies that the same type interaction occurs,

and is not a strict function of surface composition or of

molecular structure.


Conclusions


There appears to be no particular differences in the

enthalpy of adsorption of a carboxyl group onto alumina or

hydroxyapatite due to the number of groups on a molecule. In

each case the enthalpy change is near -6 kcal/mole and an








attracting force is required to initiate adsorption. Other-

wise a plot of E or Q versus pH would be similar in shape to

the a versus pH and not bell shaped.

Once adsorption occurs, the influence by multiply-

charged groups on the molecule was evidenced by a change in

surface charge. For this condition to arise, specific adsorp-

tion has to occur which requires forces other than electro-

static. The proximity of oxygen in the carboxyl groups and

H on the surface suggest hydrogen bonding.













CHAPTER III

ADSORPTION OF CHONDROITIN SULFATE

AND OTHER CARBOHYDRATES



Introduction


Polysaccharides, notably chondroitin sulfate (CS),

which contain carboxyl and sulfate groups, are present in

dentin and enamel [101] and in bone [102,103]. Under proper

conditions of pH and ionic strength, these polysaccharides

will complex with collagen [104]. In the presence of a

foreign surface, these polysaccharides, like other charged

molecules, will adsorb and react not only with collagen but

also with the surface. It is the purpose of the studies of

this chapter to explain the interaction of aqueous solutions

of chondroitin sulfate with alumina, hydroxyapatite, and

silica. A later chapter will discuss the interaction of CS

with collagen.

Chondroitin sulfate is a polysaccharide made up of

basic dimer units of glucoronic acid and galactosamine. Several

simpler carbohydrates were chosen to model CS: (a) galactose,

glucose, D-acetyl galactosamine; (b) glucose-6-sulfate (G6S),

D-galacturonic acid; (c) dextran, and (d) polygalacturonic

acid (PGA). Each carbohydrate was chosen because it possessed

a single feature of the CS molecule: (a) the carbohydrate








residue; (b) a charged carbohydrate; (c) the polymer back-

bone structure; and (d) the charged polymer. The structural

formulaefor these molecules are shown in Figure 9a and 9b.


Experimental


All carbohydrates were purchased from Sigma Chemical

Company [96] and used without further purification. The

chondroitin sulfate (sodium), dextran, and polygalacturonic

acid were reported to have molecular weights of 45,000, 60-

90,000, and 25,000 respectively. The chondroitin sulfate was

determined to be chondroitin-6-sulfate by infrared spectroscopy

using the KBr pellet technique, and had a molecular weight of

45-60,000. The materials used as substrates, Linde B alumina,

hydroxyapatite, and silica, and the low ionic strength solution

were described previously.


Results


The first set of experiments was conducted with

galactose, glucose, and dextran (see Figure 9b). None of these

molecules possesses charged groups. By measuring the heat of

adsorption of these molecules on the oxides, the contribution

to the total enthalpy change on adsorption of uncharged

carbohydrate monomer and polymer could be estimated. The initial

concentration, C was varied from .01 to .1 moles/liter. By

comparison with the reaction heats produced by the carboxylic

acids in this concentration range, it was estimated that 20 to

40.meal would be considered a significant reaction. The results







(ii)


oSO
OH / 3


NH H
H I
CO
I
CH3


(b)


H COO


.------ 28.7 A .

So S3


COO


Figure 9. Molecular structure of chondroitin sulfate (a)
( D-glucoronic acid N-acety galactosamine-6-sulfate)
(b) the three-fold helix of chondroitin sulfate.


(a)


(i)











HO- C H


HO- C H


Galactose


CH20H


Glucose


OSO3


CO
CH3


D-acetyl
Galactosamine


Glucose-6-Sulfate


Galacturonic acid


Figure 9b. Molecular structures of the carbohydrates
used in the experiments of this section.








are presented in Figure 10. The maximum reaction heat, Q,

produced was about .3 mcal at a concentration of .1 M on

alumina. The enthalpy change, as determined by solution

depletion, was approximately -100 cal/mole adsorbed for galactose

and glucose on alumina. For dextran, AH was about -20 cal/

mole of residues (.11 cal/gm) and -15 cal/mole of residues

(.8 cal/gm) on hydroxyapatite.

The enthalpy change for the adsorption of D-acetyl

galactosamine on alumina was found to be -430 cal/mole of

molecules. Using the thermometric titration method described

earlier, the free energy change was found to be -5.2 kcal/mole.

The enthalpy change for the adsorption of this molecule on

hydroxyapatite was found to be -400 cal/mole of molecules.

Calorimetric measurements for the adsorption of D-

galacturonic acid on alumina indicated a stronger reaction than

with the uncharged carbohydrates. The reaction heat, Q, reached

a maximum of 8.9 meal. The calculated enthalpy change and

that determined by solution analysis are presented in Figure 11

and lie near -10.5 kcal/mole. There is good agreement for the

enthalpy change using both methods except in the lower concentra-

tion range where the calculated values are more negative. The

free energy change varies between -4.6 and -5.5 kcal/mole

adsorbed. The entropy change is negative and lies between -8

and -30 cal/mole*deg per molecule adsorbed.

Measurements of the adsorption of D-galacturonic acid on

hydroxyapatite showed a similar enthalpy change to that on













r. 0.3






E-0.2



o I I I
0

<0.1
14 n0






.02 .04 .06 .08
Co, M

Figure 10. Reaction heats for the adsorption of dextran o ,
galactose ,, and glucose o, on alumina (closed symbols) and
hydroxyapatite ( open symbols) in low ionic strength solution
at pH 7.
























- -


- :- -- --Z --


- -


.02


20






10


.03
C, M
"0'


Figure 11. Thermodynamic data for the adsorption of D-galac-
turonic acid on alumina at pH7 in low ionic strength solution;
AG--- AH- AS---. Enthalpy change determined by solution
analysis -*-


10






5


__









alumina, as determined by solution depletion (see Figure 12).

Values for AH lie between -6 and -8 kcal/mole.

The adsorption of D-galacturonic acid on silica is

endothermic. At low concentration, the enthalpy is +700

cal/mole, becoming more positive at higher concentrations.

The amount adsorbed was determined by solution depletion. The

free energy change and entropy change were not calculated.

The change in enthalpy for the adsorption of glucose-6-

sulfate on alumina, hydroxyapatite, and silica was determined

to be -7.6, -5.4, and 0.3 kcal/mole of adsorbed molecules,

respectively.


PGA


Calorimetric measurements for the adsorption of poly-D-

galacturonic acid on alumina and hydroxyapatite were hampered

by agglomeration of the particles by the polymer. The thermo-

dynamic functions for this reaction were calculated and are

shown below.. In the concentration range used, .001 M to .01 M

of residues, these values were constant (1 0.1 kcal/mole).

AG AH AS
(kcal/mole) (kcal/mole) (kcal/mole/
deg)
alumina -3.4 -2.54 3.5

hydroxyapatite -4.2 -.31 15.0

In contrast to the decrease in entropy found for the

adsorption of PAA on alumina or hydroxyapatite, the entropy

change is positive for adsorption of poly-D-galacturonic acid

on both alumina and hydroxyapatite indicating an over-all

decrease of ordering.





69










2 -






0






o -2 -













-6






-8








C0 M

Figure 12. Heat of adsorption for D-galacturonic acid on
silica-e-- and hydroxyapatite -- in low ionic strength
solution at pH 7.








CS


The results of the calorimetric measurements of the

adsorption of chondroitin sulfate (CS) on alumina are

presented in Figure 13. Comparison of the enthalpy of

adsorption for CS on silica, alumina, and hydroxyapatite are

given below:

AH

Silica +2.46

Alumina -1.85

Hydroxyapatite -2.47

The enthalpy changes for the adsorption of CS on the

uncharged hydroxyapatite is found to be 15-30% more negative

than that for the positively charged alumina. The enthalpy

change for adsorption of CS on silica is found to be positive

endothermicc) and was determined by solution depletion, the

free energy change AG was not calculated.


Discussion


Glucose, galactose, and dextran are uncharged molecules.

Because of the availability of hydroxyl groups it is possible

that these molecules can undergo hydrogen bonding with an

oxide surface. The low value of the enthalpy change for the

adsorption of these molecules, however, does not indicate very

strong reaction with the oxide surface in comparison with the

charged molecules.





71







6-
6 30






S 4 .. 20
H










CD
0
S 0





4


H 3
o U
So
2


1


0 I I
0.2 0.4 0.6 0.8

CO, mM

Figure 13. Thermodynamic data for the adsorption of chon-
droitin sulfate on alumina in low ionic strength solution
at pH 7; AG----, AH- AS--.








The only difference in the experimental conditions

in using the uncharged versus charged molecules is

attributable to the functional groups. Therefore the large

differences in AH observed are seen as due to the presence of

the charged groups.

If it is assumed that, in the case of dextran, only

two or'three points of contact are made per molecule, then

the enthalpy change could be on the order of 3-4 kcal/mole

molecules. Comparison of dextran with poly-d-galacturonic

acid (PGA), however, still shows that dextran is much less

strongly bound than PGA.

In the concentration ranges used, 10-6 M dextran

molecules (not residues), there is little hydrogen bonding

of dextran chains to one another [76,98]. Therefore, the

breaking of interchain hydrogen bonds should not contribute

substantially to the low enthalpy change actually measured.

If the hydrogen bonding does take place between the oxide sur-

face and many dextran residues, it is not manifested in the

measured enthalpy change. In the absence of other interactions,

it is concluded that the charged groups of the carbohydrate

molecules are necessary to provide sufficient attraction of

the entire molecule to the surface.

The increase in the heat of adsorption of D-acetyl

galactosamine over that of galactose is attributed to the

presence of the NH2COCH3 side chain. The exact cause can only

be speculated. Perhaps the nonpolar methyl group is forced

from solution by more polar solvent ions, drawing the molecule









to the surface. Whatever the mechanism, if these uncharged

molecules are strongly bound to the surface, it is not

reflected in the enthalpy determination. The type of bond

which would occur would almost certainly be hydrogen.

In any event, the adsorption of D-galacturonic acid

on alumina and hydroxyapatite is much more energetic than that

of galactose or galactosamine. The similarity in the molecules

and in the adsorption experiment strongly suggests that the

charged carboxyl group is responsible for the higher enthalpy

change and that binding between solute molecules or desolvation

effects do not account for the noted change. The negative

entropy change indicates an overall increase in ordering. This

increase in ordering may be due to confinement of the carbo-

hydrate molecules to the surface and subsequent loss of freedom

[21].

Adsorption of D-galacturonic acid on silica produces a

positive enthalpy change. This can be accounted for by the

charge repulsion which exists between the surface and the molecule.

There was a finite amount of acid adsorbed, however. This

would indicate perhaps a second stronger force necessary to

overcome the charge repulsion or that the negatively charged

molecules are occupying the fewer positive siteS on the silica

surface, or reaction with high energy sites. Since the reaction

heat and adsorption measurements leveled off quickly, the last

two possibilities appear more likely; especially in view of the

finding that charge attraction appears necessary for strong

reaction.








Likewise the adsorption and calorimetric measurements

of glucose-6-sulfate suggest that the presence of a charged

group on the carbohydrate, opposite to that of the surface,

is required for stronger (more exothermic) reactions. The

negative sulfate group is attracted to the positive alumina

surface. As with'D-acetyl galacturonic acid on silica, the

adsorption of glucose-6-sulfate on silica is endothermic.

Poly-D galacturonic acid is obviously strongly attached

to the alumina and hydroxyapatite surfaces. The enthalpy

change is more negative for alumina, than for hydroxyapatite,

demonstrating the greater attraction for this surface. If

we consider that the enthalpy change is produced by the

charged groups bonding to the surface, then, using a figure

of -9kcal/mole as the enthalpy change found for the adsorption

of D-galacturonic acid on alumina, we can estimate that one

in three residues bonds to the surface. For hydroxyapatite,

this figure is perhaps one in twenty or thirty.


Chondroitin Sulfate


The chondroitin sulfate molecule is known to exist in

a threefold or eightfold helix [105,106] which is rigid in

solution [107] (see Figure 9). It possesses the ability to

change the conformation of positively charged polypeptides

from an extended coil to a helical structure (see Chapter 5).

The sulfate group extends further away from the carbohydrate

backbone than does the carboxyl group which is located on the

other side of the same dimer. The acetyl amine group extends








slightly further away from the backbone than does the

carboxyl and is located on the same side of the backbone.

From these considerations--helical structure, position of

the charged groups, and possible steric hinderance--it is

reasonable to assume that not all the charged groups participate

in bonding with the surface at the same time,

To help analyze the binding of CS to a surface, consider

that the interaction of a single dimer with the surface

permits interaction of both the carboxyl group and sulfate

group with the surface. Both groups would then contribute to

the enthalpy of the reaction. From the data in this chapter

and the previous one, it is seen that the change in enthalpy

is fairly constant for each type molecule, as it is between

carboxyl groups and that only charged groups contribute

significantly to the reaction heat, Q. The reaction heat due

to the adsorption of a carboxyl and sulfate group would be

between -12 and -16 kcal/mole. The measured enthalpy value is

about -2 kcal/mole of dimers for alumina and between -2.2 and

-2.8 kcal/mole for hydroxyapatite. Dividing the total enthalpy

possible by the measured value would indicate that between one

in three to one in seven dimers interact with the surface.

We may assume that only one of the charged groups inter-

acts per dimer. Using an enthalpy change between -6 and -12

kcal/mole of charged groups then one in three to one in five

groups would be indicated as interacting with the surface.

From the physical picture and the calorimetric data it seems

plausible to conclude that the.chondroitin sulfate molecule is








positioned horizontally on the surface with approximately

one out of every four dimers on the average coming into

contact with the oxide surface. In this instance, the charged

group interacting with the surface would be the sulfate group

since it extends further away from the CS backbone.

The entropy change due to adsorption of CS on alumina

is positive, indicating an increase in entropy or a decrease

in the order of the system. Since the molecule is rigid in

solution and is not likely to greatly change conformation on

the surface, no entropy contribution is attributable to a

change in shape. An increase in entropy can be attributed to

a release of solvent molecules from around the molecule or

from the surface into solution [106,107].

The adsorption of CS on silica can also be explained by

an increase in entropy. The surface charge on the silica is

the same as that on the carbohydrate. Overall, there is an

electrostatic repulsion between the surface and the charged

molecule. There must be some other energy supplied to over-

come this repulsion. Since it is the free energy change which

drives the reaction, and AH is positive, there must be at

least an equivalent positive entropy change so that TAS is

greater than AH. This entropy change, as suggested above,

can be supplied by the solvent ions.

It is difficult to speculate on the conformation of

CS on the silica surface as was done above with alumina. This

is so because the carbohydrate monomers are not attracted to

the surface of the silica as they are to alumina because of

charge repulsion.









Conclusions


In this section we have determined some of the thermo-

dynamic features of the adsorption of chondroitin sulfate on

alumina, hydroxyapatite, and silica by the use of model

carbohydrates. The results show that for positively charged

alumina the enthalpy change for the adsorption of charged

carbohydrates is about the same as that for the carboxylic

acids and lies between -7 and -9 kcal/mole of adsorbed species.

In these experiments the entropy change is negative and the

enthalpy change forms the major portion of the free energy

change. The enthalpy change for the adsorption of the charged

monomeric carboxylated carbohydrates on hydroxyapatite is close

to -7.6 kcal/mole. The adsorption of carbohydrates containing

the sulfate group on alumina or hydroxyapatite is about -5.4

kcal/mole. The similarity in enthalpy for the reaction suggests

a similar type reaction. The adsorption of charged carbohydrate

monomers on silica is weak and endothermic. The uncharged

monomers produce an enthalpy change only a fraction of that of

the charged monomers.

It was found that a model of the uncharged polymer backbone

of CS does not produce a large reaction heat or an enthalpy

change, suggesting that the presence of charged groups is

required to enhance the adsorption reaction. The data on

adsorption of polygalacturonic acid supports this conclusion.

Polygalacturonic acid adsorbed strongly, producing

agglomeration of the solid particles at high concentration (.1 M).








The enthalpy change per residue is lower than that found

for its monomeric counterpart. The conclusion drawn here,

as with polyacrylic acid in the previous chapter, is that

fewer points of contact are made, but that each point of

contact contributes essentially the same heat change as the

monomer. The entropy change was positive further increasing

the driving force.

Chondroitin sulfate was shown to adsorb to each of the

oxide powder substrates. The negative enthalpy change for

alumina and hydroxyapatite indicates that adsorption is strongly

enhanced by the opposite charges. Since the chondroitin sulfate

molecule is comparatively bulky relative to the models used,

fewer points of contact would be expected. The thermodynamic

calculations show, however, that perhaps as much as one-third

to one-fourth of possible bonding sites touch the surface.

The results for CS on silica are more speculative. The

enthalpy change is positive and it.is assumed that the major

contribution to the free energy change of adsorption is from

a positive entropy change related to the molecule size. Smaller

molecules were not found to produce a positive entropy change.

Relying on the results of the previous chapter, it

is assumed that once the molecules are attached to the surface,

that hydrogen bonding will take place.

In a subsequent chapter, we will investigate the type

interaction which CS and other molecules undergo with collagen

and collagen models. In the next chapter a study of the adsorp-

tion of molecules possessing amine side groups is discussed.













CHAPTER IV


ADSORPTION OF POLYPEPTIDES



Introduction


The calorimetric measurements discussed in the previous

chapters were related to the adsorption of molecules which

possessed a carboxyl group. The results showed that the mole-

cules on which the carboxyl group was ionized displayed greater

reaction heat and adsorption density than those molecules

which were not ionized. In this chapter, molecules containing

charged amine side groups are studied for the possible informa-

tion they can give on the adsorption of collagen onto silica,

alumina, and hydroxyapatite.

Several of the primary amino acids and their respective

polymers were investigated as collagen models. The molecules

used were alanine, poly-l-alanine (PA), proline, poly-l-

proline (PLP), poly-1-hydroxyproline (PLHP), lysine, poly-1-

lysine (PLP), and poly-l-arginine (PLA). Lysine and arginine

and their polymers possess basic side chains. The structural

formulaeof the monomers are given below. The polymers are linked

at the carboxyl and amino groups.







CH3CHCOO- ( COO-
I IN,
+NH3 H H

Alanine Proline


HO-
()coO- +H3N(CH2)4CHOO-

H H NH2

Hydroxyproline Lysine



H2NCNH(CH2)3CHOO-
II
+NH2 NH2

Arginine







The amino acids are dipolar ions. For the dibasic

amino acids, arginine and lysine, adsorption onto negative

surfaces will be enhanced. In polymeric form only poly-l-

lysine and poly-l-arginine retain any charge in neutral

solution.

The solubility in water of the other amino acids will

decrease as a result of their polymerization. Acidic amino

acids, aspartic acid and glutamic acid were not studied because

the carboxyl group has been discussed in the previous chapters.

Furthermore, interpretation of calorimetric data would be

difficult because of the presence of three charged groups.








Experimental


The amino acids used in this section were purchased

from Sigma Chemical Company. Both amino acid monomers and

polypeptides were chloride salts, except for poly-l-lysine,

which was a bromide salt. The molecular weights of the poly-

peptides were reported to be: 1,000-5,000, poly-l-alanine;

15,000-50,000, poly-1-arginine; 70,000, poly-l-lysine;

10,000-30,000, poly-l-proline; and 10,000-30,000, poly-l-

hydroxyproline.

The substrates and the solvent are the same as used

in the previous chapter. A few experiments were performed in

the .165 M salt solution (and will be indicated as such in

the text).

Mixing and calculation procedures were described

earlier.


Results



Alanine


Calorimetric and adsorption results for alanine and

PA on the three oxides are presented in Figure 14. The

amino acid monomer adsorbs strongly on all three surfaces at

this pH. It acts much as the carboxylic acids do. There is

no sharp endpoint in the Q vs. C curve for the monomer, but

the slope of the surve is greater than that for the polymer.

The enthalpy change for each surface tends towards -5 to -8

kcal/mole of monomers.





82










6

O









alanine TCP






3 alanine A1203


2




alanine
SiO2


1


PA Al 203

PA TCP
I p I

0.0 0.1 0.2 0.3 0.4 0.5

Co mM

Figure 14. Heats of adsorption for alanine and poly-l-alanine
(PA) onto alumina, silica, and hydroxyapatite (TCP) in low
ionic strength solution at pH 7. run in .165M salt solution.








When the dissolved salt (R-C1) concentration in the

solvent is increased to .16 M, the enthalpy change of the

adsorption of alanine on alumina decreases in the higher

concentrations range of alanine, but tends toward the -6

kcal/mole in the lower end.

The polymer, PA, exhibits much different behavior

showing no specific tendencies to adsorb. Reaction heats are

less than .5 kcal/mole and enthalpy changes are of magnitude

less than -1 kcal/mole.


Proline


Measurements made with proline and PLP and hydroxy-

proline and PLHP (see Figure 15) show results similar to

results for alanine and PA. The monomer again produces a

higher enthalpy change than the polymer (-4 to -8 kcal/mole).

However, PLP and PLHP are apparently more strongly attracted

than the poly-l-alanine with heats of adsorption lying between

-0.5 and -2.5 kcal/mole of residues. PLHP is somewhat more

strongly attracted on all three surfaces than is PLP. An

increase in ionic strength is noted by a decrease in the

enthalpy change for PLHP on alumina.


Lysine and Arginine


The addition of charged side groups causes a marked

change in the enthalpy curves, as shown in Figure 16. The

heat of adsorption of lysine on silica is, as expected,





84




1. P-A
2. HP-A
3. HP-S
1 4. HP-HA
5. PLP-S
6.PLHP-A
7.PLHP-HA
8.PLHP-S
2 9. PLHP-A*
10.PLP -S

3 0














5
6
2 7

8



*1 -
10
PLP-S



0
.02 .04 .06 .08
Co, mM
Figure 15., Heats of adsorption for proline (P), hydroxyproline
(HP), poly-1-hydroxyproline (PLHP), and poly-l-proline (PLP),
on alumina (A), silica (S), and on hydroxyapatite (HA) in low
ionic strength solution and Ringers solution (*).













12





10

PLA-A



8



HPLA-HA

~ 6



PLL-S PLL-HA


L-S




2






0.2 00.4 0.6 0.8
Co, mM

Figure 16. Heats of adsorption for lysine (L), poly-1-lysine
(PLL), and poly-1-arginine (PLA), on silica (S), alumina (A),
and hydroxyapatite (HA) in low ionic strength solution at
pH 7. Values determined by solution analysis -e- others
by calculation.









greater in magnitude than on alumina and is more pronounced

in the lower concentration end. The enthalpy change lies

in the range of -2 to -8 kcal/mole of monomers. The greatest

enthalpy change, however, for the polypeptides was recorded

for PLA on alumina. The second largest was on hydroxyapatite

and third on silica. This order also happens to be the order

of decreasing specific surface area. The enthalpy of adsorp-

tion of PLA was greater than that for PLL on hydroxyapatite.

The free energy change for PLA and PLL lie in the

range between -4 and -7 kcal/mole of residues (see Figure 17).

There is a sharp decrease in the free energy change of PLA

on hydroxyapatite at low concentrations. The entropy changes

for these molecules are small because of the similarity of

AH and AG.



Discussion


Alanine


The higher enthalpy change for the adsorption of

alanine, compared to poly-l-alanine, is due to the electro-

static attraction of the amine group or the carboxyl group

to the surface. PA, having no charge except for its terminal

groups is not strongly adsorbed despite its greater molecular

weight. PA is in a helical form [20], not coiled.

Since the molecule is uncharged,.the reaction heat of

the entire molecule due to adsorption only would be small in













8
PLA-HA




H 6
0

S/ PLL-SiO 2



4 PLL-Al /
203

PLL-HA



2





I I I I
0.0 0.2 0 .4 0.6 0.8 1.0

Co, mM

Figure 17. Free energy change for the adsorption of poly-1-
lysine (PLL) and poly-l-arginine (PLA) on silica, alumina and
hydroxyapatite at pH 7.








comparison with ionized molecules [50]. A large conformational

change would then cause the overall reaction to be endo-

thermic. The molecular concentrations are low. Therefore,

breaking of intermolecular hydrogen bonds should contribute

little to the enthalpy change.


Proline


The charged monomers of proline and hydroxyproline

are also more strongly attracted to the oxide surfaces than

their polymers (Figure 15). This is taken as a result of

electrostatic attraction. The heat of adsorption, measured

by solution depletion and found to be between -4 and -6 kcal/

mole for both monomers, results from reaction of the charged

groups with the oxide surface.

Calorimetric measurements for the adsorption of PLP

and PLHP did not show a specific pattern for any of the

surfaces. Both of these molecules have a helical conformation

in solution [20]. There was no attempt to determine whether

or not this structure was grossly disturbed upon adsorption,

or if it was, what contribution to the enthalpy change such

a disruption would make. Neither was there an attempt to

determine how many points of contact were made. The definite

conclusions which can be drawn from these data are relatively

few. There are some reasonable assumptions, however, that can

be made which, if accepted, will further explain the situation.







Since these molecules are in an extended conformation,

not coiled, there are no intramolecular hydrogen bonds. At

low concentrations (2 x 10-5 M of residues) there should be

little intermolecular hydrogen bonding [50]. The disruptions

which would primarily occur, then, would correspond to

rotational movement of the molecule [19], There is no

reason to suspect that these molecules should undergo grotesque

distortion on the surface since there are no strong attractive

forces. Therefore, the contribution to energetic changes due

to conformation alterations should be small.

Poly-l-proline and poly-l-hydroxyproline possess a

ring structure which is relatively nonpolar compared to the

polar solvent. Because of this, it is plausible to assume

that these less polar structures are in a lower energy state

on the surface, rather than in the solution. This is termed

hydrophobic bonding and could possibly account for the energetic

changes measured if most of the residues were near the surface

and not surrounded by the mobile polar ions in solution.

By comparison with the carbohydrates studied earlier

and in absence of detailed information on the geometric

smoothness of the surface, it would also be possible to suggest

that only a few points of contact are made on the surface [50].

Each of these contacts would assume a higher energy than

indicated by the average of 1-2 kcal/mole of residues measured.

If these contacts are hydrogen bonds made up of hydrogen

atoms on the ring structure and oxygen atoms on the surface,







then each bond would entail an energy change of about

-7kcal/mole. On the average then one out of 7 residues

would be in contact with the surface.

Comparison of the adsorption of these uncharged

molecules with those that possess charged functional groups

indicates that the role of PLP or PLHP would be minor in

comparison. Although the mechanism of adsorption has not

been fully explained in this case, there should be little

doubt that when positioned next to a charged molecule in a

peptide chain, the latter will play the dominant role in

adsorption to an oxide surface.


Lysine and Arginine


The adsorption heats of lysine on silica and alumina

are similar (Figure 16). Since this molecule possesses two

basic and one acidic group at neutral pH, it is reasonable

to assume that the charged carboxyl group is attracted to

the positive alumina surface, and that the positive amine

groups are attracted to the negative silica surface. Because

of the more basic properties of this molecule, it might be

suspected that the reaction with the silica surface would be

somewhat stronger than with the alumina surface. This appears

to be the case.

Poly-l-lysine and poly-l-arginine are known to exist

in an extended charged coil conformation in solution at

neutral pH [8,9]. If the coils, which are stabilized by







hydrogen bonding, were to break down, the enthalpy change

would be due to both the adsorption and the unfolding processes.

Enthalpy changes measured in this and the two previous chap-

ters indicate that enthalpy changes between -6 and -8

kcal/mole of single, charged groups are to be expected. The

magnitude of the unfolding process, the breaking of hydrogen

bonds, would lie between 5 and 7 kcal/mole [19,82], an

endothermic process. The resulting enthalpy changes for both

processes would be comparatively small with a value near 0

kcal/mole. Such a situation is encountered in experiments

presented in the next chapter where the coil-to-helix

transition is known to take place. Instead,the enthalpy

change is much more negative, decreasing in magnitude from

-14 kcal/mole to -6'kcal/mole. The variation is thought to

be due to adsorption on the fewer negatively charged sites

on the alumina and hydroxyapatite surface. These sites possess

a distribution of high to low energy within their own group.

Adsorption of PLL or PLA to neutral high energy sites

can be eliminated since PA, PLHP, and PLP each have heats of

adsorption which are smaller in magnitude. If the adsorption

had not depended on surface charge the neutral molecules

should have been just as strongly attracted to the surface.

The order of decreasing enthalpy change (alumina >

hydroxyapatite > silica) corresponds to the specific surface

area of the solids. The higher specific area of the alumina

(87m2/gm) provides more edges and peaks which are assumed to








form high energy sites. For equal amounts adsorbed, a

greater fraction of adsorbed molecules would be on these

sites for alumina, than for either hydroxyapatite or silica.

Negatively charged polysaccharides also displayed an increase

in enthalpy change at lower concentrations attributable to

high energy sites. More than a single species of these

specially adsorbing areas appears likely [72]. The free

energy changes are of the same order of magnitude as those

found for the carboxylic acids and polysaccharides.



Conclusion


As found in the previous chapters, molecules with

charged ionic side groups react more energetically with oxide

surfaces than those without. The enthalpy change per

charged group lies between -4 and -10 kcal/mole. For those

polyamino acids which possess no charged side groups, the

magnitude of the enthalpy change is found to be less, near

-1 kcal/mole of residues. Although the mechanism for uncharged

molecules is not clearly defined, it is believed that conforma-

tional deformation does not contribute significantly to the

enthalpy changes measured. It is possible than an uncharged

polyamino acid could be bound to an oxide surface by a few

relatively high energy contacts. Polyamino acids with charged

side groups, however, will play the dominant role in adsorption.













CHAPTER V


REACTION OF PEPTIDES AND

CARBOHYDRATES IN SOLUTION



Introduction


In the earlier chapters, an investigation of the

adsorption of molecules onto oxide surfaces has been discussed.

These calorimetric studies of compounds which are models for

collagen provided some information on the state of the adsorbed

molecules, the energy changes on adsorption and the type of

interaction they undergo.

In vivo, it is unlikely that a collagen molecule will

come into contact with a clean surface. In general, there

will be other substances present in the body fluids which

will adsorb first because of factors such as greater concentra-

tion. Also, collagen may not be present at all when the

hydrated oxide surface is first exposed to body fluids [102].

We should have some indication, therefore, of how these adsorbed

molecules will affect the adsorption of collagen. In order

to understand their interaction at a liquid-solid interface,

it would be helpful to first investigate their .interactions

in solution.







It is the purpose of this chapter to provide further

insight to the interaction of collagen, chondroitin sulfate,

and serum albumin in solution.

The same compounds which have been used previously

to model collagen and chondroitin sulfate have been used

here. For collagen they are poly-l-arginine (PLA), poly-l-

lysine (PLL), poly-lalanine (PA), and poly-l-proline (PLP).

For chondroitin sulfate they are dextran, galactose, galacturonic

acid, and polygalacturonic acid.

The reaction of chondroitin sulfate with collagen has

been studied by model systems, as mentioned in earlier

chapters. The reaction of collagen with dyes containing acidic

groups and with CS have been studied in regard to the under-

standing of the role of CS and collagen in connective tissue

[108]. It was found that the cationic groups of collagen bond

with the anions of the dyes and CS in a pH range of 1.5 to

7 with a sharp drop in the number of anions fixed below pH 2

and a more gradual decrease from maximum adsorption at pH 3

to zero at pH 7.

In experiments to determine the role of CS in the

calcifying mechanisms of bone [109], it was found that calcifi-

cation would not occur or would occur more slowly in an aqueous

collagen mixture when CS was not present. These experiments

were performed near pH 7. The authors suggested that binding

of CS to collagen at neutral pH would aid the natural calcifica-

tion process.








In another experiment with chondroitin sulfate [13]

and cationic dyes, it was concluded that aggregation of

dyes on the surface of the CS rather than ionic interaction

was mainly responsible for bonding. The thermodynamic functions

indicated an enthalpy change of -7 to -12 kcal/mole of dye

molecules.

The binding of cobalt hexammine (Co(NH3)6+3) to connective

tissue, micropolysaccharides, heparine, and sulfated chitosans

has been studied by a spectrophotometric procedure [110]. The

cobalt hexammine was used to represent amino functions of fibrin.

Ion pair formation was found to be the primary binding mechanism,

but was influenced by local binding factors, electrostatic attrac-

tion of neighboring charged groups, and competition with other

cations for binding sites.

Such previous work generally indicates that CS-collagen

or CS-polypeptide binding will be primarily ionic, pH, and

structure dependent. These previous results and the results

discussed in this chapter will serve as an aid in understanding

later calorimetric measurements.



Experimental


In these calorimetric measurements, a solid substrate

was not used. In the first set of experiments, the concentra-

tion of saccharides was held constant at a concentration Co

of 10-3 M of saccharide monomers or residues. In the case of

CS, this refers to dimer residues. Aquisition of the organic




Full Text
IIS
would then be easier for the molecule to denature. Consequently,
more extensive contact'between collagen and BSA would appear
to be possible than in the low ionic strength solution. This
increased contact would interfere in collagen-CS interaction.
Conclusions
There appears to be substantive evidence suggesting
that the attraction of PLA or PLL to chondroitin sulfate is
initiated by ionic forces. It was seen that uncharged mole
cules reacted much less strongly with PLA or PLL than CS.
The charged carbohydrate polymers also react more strongly
than the charged monomers. It is probable that the interaction
of the two macromolecules will include hydrogen bonding once
they come into contact. The reactions of CS and collagen also
appear to be ionically induced as mentioned in the beginning
of this chapter, with an enthalpy change between -500 cal/mole
and -700 cal/mole of CS dimers.
The interaction of BSA and collagen appears to be neg
ligible in comparison to that of CS and collagen. Although the
BSA may block isolated points on the collagen molecule, it
does not appear to inhibit the collagen-CS interaction. This
would suggest that binding to a surface in the presence of BSA.
By increasing the ionic strength of the solution, the
magnitude of the enthalpy changes of all interactions decreases.
In very high concentrations (1.0 M NaCl) of ions, binding of
CS by collagaen practically ceases [82]. In regard to surfaces


8
due to hydrogen bonding because of the shift in the amide I
(C = 0 stretching) band at 1640 cm'^.
Calorimetric measurements of the adsorption of human
serum albumin on negatively charged polystyrene (PS) [49]
were shown to be pH dependent. Maximum adsorption of the
protein occurred near the IP (4.9) where the enthalpy of the
reaction was positive. At pH values removed from the IP the
reaction was exothermic. It was suggested that at pH values
away from the IP the conformation of the adsorbed protein
changes for energetic reasons. Denaturation of the protein
is not surprising since it is known that the internal bonding
of serum albumin is weak [50]. The' enthalpy of the adsorption
reaction varied between zero and 8 kcal/mole as the surface
charge varied between -1.0 myCcm-^ and -7.5 myCcnf*2. The
most negative enthalpy values were recorded for pH 3.8 and 9
and were near -8.4 kcal/mole.
Enthalpy values for the adsorption of albumin, gamma
globulin, and fibrinogin, were calculated from adsorption
measurements at several temperatures [51]. The results indicated
that the adsorption took place in two distinct ways. Both
types were apparently Langmuir and took place on separate
membrane sites. One type of adsorption was easily reversible
with a heat of adsorption in the neighborhood of -10 kcal/mole.
The other type of adsorption reaction was hydrophobic, endothermic,
and with heats of adsorption in the range of 5 to 20 kcal*
mole.


114
mation [ 36 50 ]. The apparent lack of reaction for the
extended molecules seems to be due to the fact that they
are extended. The two coiled molecules are deformable [49]
and may adjust their conformation to increase surface area
contact with each other. For BSA and PLA, assuming from
previous data that each point of interaction contributes about
-7 kcal/mole and that these interaction points contribute
most to the enthalpy, there are about seven or eight points of
contact per molecule. Even though the1 reactions which take
place appear to be of an electrostatic nature, they occur
most extensively when the structure of the molecules are similar
The Mixture CS, BSA', and Collagen
In the presence of BSA, collagen and CS interact with
each other much in the same way as previously when the collagen
concentration, C is low, indicating that BSA will not strongly
affect the CS-collagen interaction. As CQ is increased, the
effect of the BSA becomes more prominent. It is suggested, in
explanation, that at low Cc only CS reacts with the relatively
few collagen molecules. At higher C CS is increasingly taken
from solution so BSA molecules may react, thus lowering the
overall average enthalpy change. The enthalpy change is. less
exothermic in the presence of BSA than in its absence when the
ionic strength is increased.
As the ionic strength of the solution is increased, the
intramolecular hydrogen bonds of!'BSA become weaker [82]. It


145
Curve (5) was produced in the same way as curve (4)
except.the bioglass was rinsed in distilled water several
times. After this, fresh buffer solution was added and
the collagen mixture was added separately.
Calorimetric measurements were performed on a sample of
bioglass which was permitted to undergo dissolution for 24
hours in buffered Ringers solution. The collagen was mixed
in a'solution of salts obtained from separate,samples of
bioglass which had also been allowed to undergo dissolution
for 24 hours. This corresponds to curve (4) above. The
results are shown in Figure 41.
This was the only situation in which the rate of
dissolution of bioglass had slowed enough so that dissolution
heats did not interfere with calorimetric measurements. In
all other situations the heat imbalance caused by the dissolving
glass did not allow adequate cancellation in the reference
cell. The enthalpy of adsorption of collagen on bioglass was
found to be between -100 and -150 cal/gm of collagen adsorbed,
an extremely high number. It was very difficult to place
the silica gel into the calorimeter and worse to remove, prevent
ing thermodynamic measurements.
The bioglass surface was identified by EDXA to have
formed a calcium phosphate film after 24 hours, which was not
removed by washing in distilled water.


meal.
10.7
Figure 24. Reaction heat, Q, for the mixing.of BSA with
FLA, PLA, and PA in low ionic strength solution at pH 7.
The BSA concentration was held constant at .7 gm/1.


kcal./mole
99
CQ, mM PLA
Figure 19. Reaction heats for the mixing of PLA with various
carbohydrates. Enthalpy change determined from solution
analysis Abbreviations are the same as in figure 18.


CHAPTER IV
ADSORPTION OF POLYPEPTIDES
Introduction
The calorimetric measurements discussed in the previous
chapters were related to the adsorption of molecules which
possessed a carboxyl group. The results showed that the mole
cules on which the carboxyl group was ionized displayed greater
reaction heat and adsorption density than those molecules
which were not ionized. In this chapter, molecules containing
charged amine side groups are studied for the possible informa
tion they can give on the adsorption of collagen onto silica,
alumina, and hydroxyapatite.
Several of the primary amino acids and their respective
polymers were investigated as collagen models. The molecules
used were alanine, poly-l-alanine (PA), proline, poly-1-
proline (PLP), poly-l-hydroxyproline (PLHP), lysine, poly-1-,
lysine (PLP), and poly-l-arginine (PLA). Lysine and arginine
and their polymers possess basic side chains. The structural
formulae of the monomers are given below. The polymers are linked
at the carboxyl and amino groups.
79


162
74. F.H. Reynolds, Biochemistry, 12(2) 359 (1973 )
75. L. Stryer, Biochemistry, 206, W.H. Freeman S Co. (1975).
76. K.E. Van Holde, Physical Biochemistry, 7, Prentice-Hall
(1971).
77. A.W. Adamson, Physical Chemistry of Surfaces, 398, Wiley-
Interscience (1976 ).
78. L.I. Osipow, Surface Chemistry, 60, R.E. Kriger (1970).
79. J.R. Dann, J. Col. Int. Sci., 32(2), 321 (1970).
80. W.J. Moore, Physical Chemistry, 914, Prentice-Hall (1971).
81. F.W. Billmeyer, Polymer Science, 95, Wiley-Interscience
(1971) : : :
82. G.C. Pimental., The Hydrogen Bond, 247 S. Freeman (1960).
83. S. Wu, J. Adhesion, 5, 39 (1973).
84. J. Barthel, Thermometric Titrations, ch. 1, John Wiley
6 Sons (1975).
85. K.J. Laidler, Chemical Kinetics, 256, McGraw-Hill (1965).
86. I. Wadso, Quart. Rev. Biophys, 3(4), 383 (1970).
87. F.D. Snell, C.T. Snell, Colorimetric. Methods of Analysis,
D. Van Nostran (1967).
88. H. Dubois. J. Anal. Chem. 28 3 (1956 K
89. Fisher Chemical Company, St. L5uis.
90. Union Carbide, Electronics Division, N.Y.
91. D.W. Fuerstenau, Soc. Min.. Eng. 252 276 (1972 ).
92. D.W. Fuerstenau, Disc. Faraday Soc., 52. 361 (1975).
93. A. Hatta, Bull. Chem. Soc. Jap., 48(12) 3441 (1975),
94. T. Watanabe, T. Tohuku, J. Exp. Med., 107 345 (1972).
95. E.P. Crematy, Aust. J. Chem., 21, 1067 (1968).
96. Sigma Chemical Company, St. Louis, Mo.
97. Aldrich Chemical Company, Mil., Wis.
98. J.H. Rai, W.G. Miller, Biopolymers, 12, 845 (1973).


PZC
Figure 37. Relationship between the point of zero charge,
pzc, and the heat of adsorption of collagen in the three
solutions used.


AH, kcal./mole
69
Figure 12. Heat of adsorption for D-galacturonic acid on
silicae and hydroxyapatite in low ionic strength
solution at pH 7.


25
the total number of moles adsorbed, a total apparent
enthalpy is found
AHt = -Qt/Nt (1.22)
From equation (1.20) then
AHt = -Qt/Nt '= 1/Nt (AH-l N1 + AH2 N2 +. . AHj Nj )
or
AHt = AH-l y1 + AH2 y2 +.... AH_. y^ (1.23)
where Yy is the fraction of the total number of moles of
molecules bound to sites of type j. Applying equation (1.18)
to each reaction
AHt = EoAHj y'j
= AH£
(1.24)
where AH£ is the weighted sum of the standard enthalpy changes
for all adsorption processes. The standard free energy
change for each reaction expressed by equation (1.19) is given
by
AG 9
3
AH? TAS?
(1.25)
Rewriting equation (1.25) for AH? and substituting into (1.24)
one obtains
AH = £^(AG9 + TAS9) y..
- Sj AG* TASjyj
(1.26)
or
where
AH
. t
AG + TASo
Ag = £. AG y.
t .3 3 3
(1.27)


MCAL.
45
| | I J
4 3 2 1 0
- LOG CQ
Figure 4._ Reaction heats for the adsorption of three
carboxylic acids' on alumina.


13 2
sudden rise followed by another gradual decrease. More
CS is adsorbed when collagen is not present. Cs is present
in the same solution as the alumina prior to mixing with the
collagen.
The heat of adsorption for collagen decreases in the
presence of CS. The step is still present but an overall
decrease of 5 to 10 cal/gm is seen.
In the buffered solvent the discontinuities disappear
(see Figure 36). As before, the amount of CS adsorbed decreases
in the presence of collagen even- though it was already in
solution with the alumina. Lesscollagen is adsorbed in the
presence of CS. The enthalpy change for the adsorption of
collagen is between -5 and -7 cal/gm.
Discussion
Collagen on Alumina, Hydroxyapatite, and Silica
The adsorption and calorimetric measurements produce
many findings. In agreement with the observations discussed in
previous chapters, there is a definite trend to an increase in
the exothermicity of the adsorption of collagen with an increase
in surface charge (see Figure 37). This result suggests that
the absorption of collagen on oxide surfaces is controlled by
ionic attractive forces.
The increase in dissolved salt concentration produces
an increase in enthalpy for equal amounts of collagen adsorbed.
One might expect a decrease in AH because the dielectric constant
of the medium is increased. One suggested reason for the increase


Table I
PZC of Substances Used as Adsorbent
alumina
9
2-4
silica
hydroxyapatite
7.5


CHAPTER V
REACTION OF PEPTIDES AND
CARBOHYDRATES IN SOLUTION
Introduction
In the earlier chapters, an investigation of the
adsorption of molecules onto oxide surfaces has been discussed.
These calorimetric studies of compounds which are models for
collagen provided some information on the state of the adsorbed
molecules, the energy changes on adsorption and the type of
interaction they undergo.
in vivo. it is unlikely that a collagen molecule will
come into contact with a clean surface. In general, there
will be other substances present in the body fluids which
will adsorb first because of factors such as greater concentra
tion. Also, collagen may not be present at all when the
hydrated oxide surface is first exposed to body fluids [102].
We should have some indication, therefore, of how these adsorbed
molecules will affect the adsorption of collagen. In order
to understand their interaction at a liquid-solid interface,
it would be helpful to first investigate their interactions
in solution.
93


13 3
C, gm/1 BSA
Figure 36. Adsorption of collagen onto alumina in the presence
of BSA and CS in buffered Ringers solution at pH 7.


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MCAL.
32
o
0,5 1.0 1.5 2.0
c0 y
Figure 2. The first step in the calculation of the thermo
dynamic functions is plotting the reaction heat, Q, vs the
original concentration CQ. Here, two points are taken from
the measurements of the adsorption of collagen on alumina.


17
Thermodynamics of Adsorption
Complete reviews on the thermodynamics of adsorption
are given elsewhere [76-78]. In this section, only those
points needed to explain the following data will be presented.
Limited explanation of the ideas of earlier workers would
be in order, however.
Gibbs gave the first rigorous thermodynamic explanation
for why a given material should either adsorb or desorb at a
surface. He was able to predict the functional relationship
between surface tension and surface concentration and the bulk
concentrations of the surface-active solutes. His derivations
assume that substances tend to minimize the free energy of
the surface region by either becoming concentrated or depleted
there. As a result, it has been the free energy which has been
traditionally determined, and surface concentration measurements
are the most common method of doing this. Other methods, such
as the thermometric titration method [84], may be useful for
obtaining thermodynamic data.
The thermometric titration technique is an analytical
method in which the heat effect of a titration reaction is
used to measure the titer of a sample. It is applicable to
reactions of the type
R1 + R2 + P (1.1)
which entails a heat of reaction Q. 'In equation (1.1)
refers to reactants i and P refers to the product. For single


15 6
In effect then, maximum protein adsorption will occur
near the IP of the protein in the absence of other influences.
Such contingencies, as denaturation, must be considered and
play an important role in determining adsorption density.
Protein denaturation has been suggested as being respon
sible for the decrease in protein adsorption sometimes noticed
when there is electrostatic attraction. In a calorimetric study
of the adsorption of BSA on polystyrene lattices of various
surface charged [49], it was shown that protein adsorption
was energetically favorable at pH values away from the IP. At
the Ip the adsorption reaction was endothermic. At neutral pH,
where the protein should maintain its globular shape, the heat
of adsorpion was near zero. The surface charge was produced
by sulfate groups and was unaffected by changes in pH. At high
or low pH, internal hydrogen bonding breaks down and conformation
al adaptability is made easier. Oppositely charged groups on
the protein occupies more space because of its unfolding and
adsorption decreases. This is the phenomenon found.
In this work it appears that an initiating force is
required to bond the macromolecules to the surfaces. These
studies find that inducement to be electrostatic attraction.
In other studies, for example, Minones [39] in film compression
experiments, suggested that alanine was hydrogen bonded to
silica. It was ignored, however, that the film compression
itself does work on the system and, in effect, replaces the
force of electrostatic attraction.


78
The enthalpy change per residue is lower than that found
for its monomeric counterpart. The conclusion drawn here,
as with polyacrylic acid in the previous chapter, is that
fewer points of contact are made, but that each point of
contact contributes essentially the same heat change as the
monomer. The entropy change was positive further increasing
the driving force.
Chondroitin sulfate was shown to adsorb to each of the
oxide powder substrates. The negative enthalpy change for
alumina and hydroxyapatite indicates that adsorption is strongly
enhanced by the opposite charges. Since the chondroitin sulfate
molecule is comparatively bulky relative to the models used,
fewer points of contact would be expected. The thermodynamic
calculations show, however, that perhaps as much as one-third
to one-fourth of possible bonding sites touch the surface.
The results for CS on silica are more speculative. The
enthalpy change is positive and it is assumed that the major
contribution to the free energy change of adsorption is from
a positive entropy change related to the molecule size. Smaller
molecules were not found to produce a positive entropy change.
Relying on the results of the previous chapter, it
is assumed that once the molecules are attached to the surface,
that hydrogen bonding will take place.
In a subsequent chapter, we will investigate the type
interaction which CS and other molecules undergo with collagen
and collagen models. In the next chapter a study of the adsorp
tion of molecules possessing amine side groups is discussed.


-LOG (R)
Figure 1. Examples for the calculation of K' and K-)-
for constant K2 and x-j_ (a), and for constant K2 and (b)
ERROR AG.


149
is available to do work on the system. Collagen does
exhibit a definite melting curve with T at 45C [75]. The
local temperature increase would depend on heat transfer away
from, and heat capacity of the interfacial region but a 10 to
20 local temperature change is not unreasonably large to
expect.
The alkali ion concentration of these solutions was'
not measured after the mixing process. It is doubtful that 1
the ion concentration of the solution of the last experiment
(Curve 5) would reach saturation in 10 minutes after having
been leached, for 24 hours and could account for the difference
observed.
The calorimeter measurements made for curve (4)
indicate that the collagen undergoes some degree of denaturation
The adsorption of collagen on hydroxyapatite produced an
enthalpy change of -7 cal/gm and for silica it was found to
be 0 to -1 cal/gm. An enthalpy change of -1 to -6 cal/gm of
collagen would be gratifying, indicating little conformational
change. However, this large AH reveals evidence for denatura
tion. The extremely high surface area may be a contributing
factor. It was suggested previously that high energy points
of contact are involved in protein denaturation. The high
surface area film of bioglass may also possess such points
of high surface energy, producing similar results.
It should be remembered that the collagen used in
these experiments is small compared to the fibrils which


7 0
CS
The results of the calorimetric measurements of the
adsorption of chondroitin sulfate (CS) on alumina are
presented in Figure 13. Comparison of the enthalpy of
adsorption for CS on silica, alumina, and hydroxyapatite are
given below:
AH
Silica
+ 2.46
Alumina
-1.85
Hydroxyapatite
-2.47
The enthalpy changes for the adsorption of CS on the
uncharged hydroxyapatite is found to be 15-30% more negative
than that for the positively charged alumina. The enthalpy
change for adsorption of CS on silica is found to be positive
(endothermic) and was determined by solution depletion, the
free energy change AG was not calculated.
Discussion
Glucose, galactose, and dextran are uncharged molecules
Because of the availability of hydroxyl groups it is possible
that these molecules can undergo hydrogen bonding with an
oxide surface. The low value of the enthalpy change for the
adsorption of these molecules, however, does not indicate very
strong reaction with the oxide surface in comparison with the
charged molecules.


. BIBLIOGRAPHY
1. L.L. Hench, Reports'1-7. An Investigation of Bonding
Mechanisms at the Interface of a Prosthetic Material
Contract no DA 17-70-C-001.
2. T. Allen, R. M. Patel, J. Apple. Chem.,20, 165 (1970).
3. I.R. Miller, Progress in Membrane Science, Academic
Press, (1971).
4. L.L: Hench, E. F.-. Ethridge, Adv. Biomed. Eng.", 5_, 35 (1975).
5. I. Wadso, Biochemical Calorimetry, 8.3, H.D. Brown, ed.
Academic Press (1970 )..
6. W.L. Mattice, L. Mandelkern, J. Am. Chem. Soc., 93. 7,
1770 (1971).
7. F.J. Padden, H.D. Kieth, J. Appl. Phys., 36, 2987 (1965).
8. R.A. Gelman, W.B. Rippon, Biopolymers, 12, 541 (1973).
9. R.A. Gelman, D. Glasser, Biopolymers, 12_, 1223 (1973).
10. 0. Pavolic, I.R. Miller, J. Polymer Sci., 34(C), 181 (1971).
11. B. Malcome, J. Polymer Sci., 34(C), 87 (1971).
12. A. Tonelli, J. Molecular Biol., 86, 627 (1974).
13. D.M. Power, J. Rad. Biol,. 20(2), 111 (1971). '
14. M. Mathews, Arch. Biochem. Biophys. 104 394 (1964).
15. W.B. Gratzer, G.H. Beaven, J. Phys. Chem. 23 2270 (1971).
16. M.G. Marenchek, J.M. Sturtevant, J. Phys. Chem., 77,
544 (1973) ;
17. G.C. Kresheck, L. Benjamin, J. Phys. Chem., 68, 2476 (1964).
18. G.E. Gajnos. F.E.Karasz, J. Phys. Chem., 77, 1139 (1973).
19. F.E. Karasz, J. Simon, J. Thermal. Anal., 9, 109 (1976).
20. G. Giacometti, Structural Chemistry and Molecular Biology
ch. 1, A. Rich, N.Davidson, ed., W.H. Freeman, (1968).
21. B.H. Bi j sterbosch, J. Col. Int. Sci. 47Q), 186 (1974),
159


86
greater in magnitude than on alumina and is more pronounced
in the lower concentration end. The enthalpy change lies
in the range of -2 to -8 kcal/mole of monomers. The greatest
enthalpy change, however, for the polypeptides was recorded
for PLA on alumina. The second largest was on hydroxyapatite
and third on silica. This order also happens to be the order
of decreasing specific surface area. The enthalpy of adsorp
tion of PLA was greater than that for PLL on hydroxyapatite.
The free energy change for PLA and PLL lie in the
range between -4 and -7 kcal/mole of residues (see Figure 17).
There is a sharp decrease in the free energy change of PLA
on hydroxyapatite at low concentrations. The entropy changes
for these molecules are small because of the similarity of
AH and AG.
Discussion
Alanine
The higher enthalpy change for the adsorption of
alanine, compared to poly-l-alanine, is due to the electro
static attraction of the amine group or the carboxyl group
to the surface. PA, having no charge except for its terminal
groups is not strongly adsorbed despite its greater molecular
weight. PA is in a helical form [20], not coiled.
Since the molecule is uncharged, the reaction heat of
the entire molecule due to adsorption only would be small in


The only difference in the experimental conditions
in using the uncharged versus charged molecules is
attributable to the functional groups. Therefore the large
differences in AH observed are seen as due to the presence of
the charged groups.
If it is assumed that, in the case of dextran, only
two or'three points of contact are made per molecule, then
the enthalpy change could be on the order of 3-4 kcal/mole
molecules. Comparison of dextran with poly-d-galacturonic
acid (PGA), however, still shows that dextran is much less
strongly bound than PGA.
In the concentration ranges used, 10 M dextran
molecules (not residues), there is little hydrogen bonding
of dextran chains to one another [76,98], Therefore, the
breaking of interchain hydrogen bonds should not contribute
substantially to the low enthalpy change actually measured.
If the hydrogen bonding does take place between the oxide sur
face and many dextran residues, it is not manifested in the
measured enthalpy change. In the absence of other interactions
it is concluded that the charged groups of the carbohydrate
molecules are necessary to provide sufficient attraction of
the entire molecule to the surface.
The increase in the heat of adsorption of D-acetyl
galactosamine over that of galactose is attributed to the
presence of the NH2COCH3 side chain. The exact cause can only
be speculated. Perhaps the nonpolar methyl group is forced
from solution by more polar solvent ions, drawing the molecule


28
Those conditions may be determined by estimated
values of Kj for calculation of K and and using each for
evaluation of AG .. The value of K' is found from
K = 0t.
(l-0t) CRl
where 0^ is defined by equation (1.31).
AG^ is found from
(1.35)
The percent errors in
% error AG_,
In Kt In K x 100
In K'
The percent error is calculated by setting values for and
Xj and directly calculating KT from equation (1.34) and
from equation (1.40). If the percent error is acceptable, AG^
may be calculated.
An example for the case of two different types of
adsorption sites is given. The equations necessary to carry
out this calculation are given below for convenience
el
II
[R]/(l
+
K1
[R])
0 2
= K2
[R]/(l
+
K2
CR])
yi
= 0-^
O
1
/(01
N
+
0 N)
2 2
y2
= 02N
o
2
/ (0 2
N
2
+
e2N)
Kt
= K1
k2
X1
O 11
s
II
/
(N
+
N)
X2
= N
/
(N
+
N
9t
= Xx0
1
+ x2
0 2
K =
et/(i + et) [R]


65
are presented in Figure 10. The maximum reaction heat, Q,
produced was about .3 meal at a concentration of .1 M on
alumina. The enthalpy change, as determined by solution
depletion, was approximately -100 cal/mole adsorbed for galactose
and glucose on alumina. For dextran, AH was about -20 cal/
mole of residues (.11 cal/gm) and -15 cal/mole of residues
(.8 cal/gm) on hydroxyapatite.
The enthalpy change for the adsorption of D-acetyl
galactosamine on alumina was found to be -430 cal/mole of
molecules. Using the thermometric titration method described
earlier, the free energy change was found to be -5.2 kcal/mole.
The enthalpy change for the adsorption of this molecule on
hydroxyapatite was found to be -400 cal/mole of molecules.
Calorimetric measurements for the adsorption of D-
galacturonic acid on alumina indicated a stronger reaction than
with the uncharged carbohydrates. The reaction heat, Q, reached
a maximum of 8.9 meal. The calculated enthalpy change and
that determined by solution analysis are presented in Figure 11
and lie near -10 . 5 -kcal/mole. There is good agreement for the
enthalpy change using both methods except in the lower concentra
tion range where the calculated values are more negative. The
free energy change varies between -4.6 and -5.5 kcal/mole
adsorbed. The entropy change is negative and lies between -8
and -30 cal/mole*deg per molecule adsorbed.
Measurements of the adsorption of D-galacturonic acid on
hydroxyapatite showed a similar enthalpy change to that on


62
residue; (b) a charged carbohydrate; (c) the polymer back
bone structure; and (d) the charged polymer. The structural
formulae for these molecules are shown in Figure 9a and 9b.
Experimental
All carbohydrates were purchased from Sigma Chemical
Company [96] and used without further purification. The
chondroitin sulfate (sodium), dextran, and polygalacturonic
acid were reported to have molecular weights of 45,000, 60-
90,000, and 25,000 respectively. The chondroitin sulfate was
determined to be chondroitin-6-sulfate by infrared spectroscopy
using the KBr pellet technique, and had a molecular weight of
45-60,000. The materials used as substrates, Linde B alumina,
hydroxyapatite, and silica, and the low ionic strength solution
were described previously.
Results
The first set of experiments was conducted with
galactose, glucose, and dextran (see Figure 9b). None of these
molecules possesses charged groups. By measuring the heat of
adsorption of these molecules on the oxides, the contribution
to the total enthalpy change on adsorption of uncharged
carbohydrate monomer and polymer could be estimated. The initial
concentration, CQ, was varied from .01 to .1 moles/liter. By
comparison with the reaction heats produced by the carboxylic
acids in this concentration range, it was estimated that 20 to
40.meal would be considered a significant reaction. The results


26
and
AS
E-s 3 3 J
(1.28)
express the weighted sums of the standard free energy and
standard entropy changes for the complete adsorption reactions.
For each reaction expressed in equation (1.19) there
is an equilibrium constant Kj which can be written according to
equation (1.4) as
K. =. 0. (1.29)
3 3
(1-0 j) [R]
or related to AG? according to equation (1.9) as
AG? = -RT In K. (1.30)
3 3
Equations (1.29) and (1.30) express the fact that each site
carries on an equilibrium reaction independently of all others.
The fraction 0. = N-/N? is the ratio of occupied sires of type
3 3 3
j to the total number of sites of type j.
For any equilibrium concentration [R] of solute molecules
the total fraction of occupied sites of all types, 0^., can be
written as
or
0 = N /N
t t s
0
t
Xx 0
where X. = N?/N
3 3 s
constant.
If equation
one obtains
, + X0 0O + ...X. 0. (1.31)
1.2 2 3 3
is the fraction of sites of type j and is a
(1.30) is substituted into equation (1.27)
AG = -E. (RT In K.) y.
t 3 3
(1.32)


mg. BSA adsorbed Q, meal.
126
Figure 32. Thermodynamic data for the adsorption of BSA
onto alumina at pH 7 in Ringers solution.
-AH, cal./gm BSA


Table 2
Acetic
4.75
Dissociation Constants
of Carboxylic Acids
Oxalic
1.23
4.19
Citric
3.14
4.17
6.39


76
positioned horizontally on the surface with approximately
one out of every four dimers on the average coming into
contact with the oxide surface. In this instance, the charged
group interacting with the surface would be the sulfate group
since it extends further away from the CS backbone.
The entropy change due to adsorption of CS on alumina
is positive, indicating an increase in entropy or a decrease
in the order of the system. Since the molecule is rigid in
solution and is not likely to greatly change conformation on
the surface, no entropy contribution is attributable to a
change in shape. An increase in entropy can be attributed to
a release of solvent molecules from around the molecule or
from the surface into solution [106,107].
The adsorption of CS on silica can also be explained by
an increase in entropy. The surface charge on the silica is
the same as that on the carbohydrate. Overall, there is an
electrostatic repulsion between the surface and the charged
molecule. There must be some other energy supplied to over
come this repulsion. Since it is the free energy change which
drives the reaction, and AH is positive, there must be at
least an equivalent positive entropy change so that TAS is
greater than AH. This entropy change, as suggested above,
can be supplied by the solvent ions.
It is difficult to speculate on the conformation of
CS on the silica surface as was done above with alumina. This
is so because the carbohydrate monomers are not attracted to
the surface of the silica as they are to alumina because of
charge repulsion.


135
in AH is that more of the collagen molecule comes into contact
with the surface. As the ionic strength of the solution is
increased, the hydrogen' bonds within the molecule weakens be
cause of their ionic character [50,82]. This would permit a
loosening of each strand permitting increased contact of the
molecule with the surface.
This loosening could also explain the change from an
endothermic reaction as the ionic strength is increased for
the adsorption of collagen on silica. This loosening would
perhaps allow the anionic carboxyl groups on the collagen mole
cule greater freedom either to move away from anionic, or move
to cationic sites on the silica surface. As a result, there
would be less charge repulsion and the heat of adsorption
would become exothermic.
In phosphate buffer, the high energy reaction for alumina
disappears. The simplest explanation is that the phosphate ions,
which are known to chemisorb to alumina [61], are preferentially
occupying high energy sites. It was suggested that these sites
cause most of the denaturation when collagen adsorbs to alumina.
The phosphate buffer has less of an effect on adsorption of
hydroxyapatite or silica since it is already present in the first
material whereas in the second it is repelled by the negatively
charged silica surface.
BSA
BSA exhibited an unusual calorimetric behavior upon
adsorption on alumina in Ringers solution. The calorimetric
behavior can be explained if the molecule denatures (unfolds)
and covers a relatively large area. Near .1 gm/1 the increased


CHAPTER VII
BIOGLASS
Introduction
The adsorption of collagen, through the study of
various models and of collagen itself, has been investigated
by calorimetry and has been discussed in previous chapters.
Competition with collagen for adsorption sites by other mole
cules has also been considered. The conclusions drawn are that,
on the three surfaces examined, ionic attraction is a prominent
force in the initial attraction of molecules and appears just
as important in the attraction of large molecules as it is in
small molecules. Collagen adsorption was also found to be
decreased by hinderance of BSA particularly at low concentrations.
In this chapter, use is made of this information to study
another material known as bioglass, which is of significance
among prosthetic materials. It is the purpose of the studies
described in this chapter to examine and to help explain the
interaction of collagen with bioglass.
Bioglass is the name of a series of glasses which are
known to be tissue compatible and have the unique property of
chemically bonding to bone [1]. The material has been extensive
ly studied by various methods. It is known that collagen
141


Likewise the adsorption and calorimetric measurements
of glucose-6-sulfate suggest that the presence of a charged
group on the carbohydrate, opposite to that of the surface,
is required for stronger (more exothermic) reactions. The
negative sulfate group is attracted to the positive alumina
surface. As with'D-acetyl galacturonic acid on silica, the
adsorption of glucose-6-sulfate on silica is endothermic. -
Poly-D galacturonic acid is obviously strongly attached
to the alumina and hydroxyapatite surfaces. The enthalpy
change is more negative for alumina, than for hydroxyapatite,
demonstrating the greater attraction for this surface. If
we consider that the enthalpy change is produced by the
charged groups bonding to the surface, then, using a figure
of -9kcal/mole as the enthalpy change found for the adsorption
of D-galacturonic acid on alumina, we can estimate that one
in three residues bonds to the surface. For hydroxyapatite,
this figure is perhaps one in twenty or thirty.
Chondroitin Sulfate
The chondroitin sulfate molecule is known to exist in
a threefold or eightfold helix [105,106] which is rigid in
solution [107] (see Figure 9). It possesses the ability to
change the conformation of positively charged polypeptides
from an extended coil to a helical structure (see Chapter 5).
The sulfate group extends further away from the carbohydrate
backbone than does the carboxyl group which is located on the
other side of the same dimer. The acetyl amine group extends


81
Experimental
The amino acids used in this section were purchased
from Sigma Chemical Company. Both amino acid monomers and
polypeptides were chloride salts, except for poly-l-lysine,
which was a bromide salt. The molecular weights of the poly
peptides were reported to be: 1,000-5,000, poly-l-alanine;
15,000-50,000, poly-l-arginine; 70,000, poly-l-lysine;
10,000-30,000, poly-l-proline; and 10,000-30,000, poly-1-
hydroxyproline. '
The substrates and the solvent are the same as used
in the previous chapter. A few experiments were performed in
the .165 M salt solution (and will be indicated as such in
the text).
Mixing and calculation procedures were described
earlier.
t
Results
Alanine
Calorimetric and adsorption results for alanine and
PA on the three oxides are presented in Figure 14. The
amino acid monomer adsorbs strongly on all three surfaces at
this pH. It acts much as the carboxylic acids do. There is
no sharp endpoint in the Q vs. Cq curve for the monomer, but
the slope of the surve is greater than that for the polymer.
The enthalpy change for each surface tends towards -5 to -8
kcal/mole of monomers.


9 5
In another experiment with chondroitin sulfate [13]
and cationic dyes, it was concluded that aggregation of
dyes on the surface of the CS rather than ionic interaction
was mainly responsible for bonding. The thermodynamic functions
indicated an enthalpy change of -7 to -12 kcal/mole of dye
molecules.
The binding of cobalt hexammine (Co(NHg)g+3) to connective
tissue, micropolysaccharides, heparine, and sulfated chitosans
has been studied by a spectrophotometric procedure [110]. The
cobalt hexammine was used to represent amino functions of fibrin.
Ion pair formation was found to be the primary binding mechanism,
but was influenced by local binding factors, electrostatic attrac
tion of neighboring charged groups, and competition with other
cations for binding sites.
Such previous work generally indicates that CS-collagen
or CS-polypeptide binding will be primarily ionic, pH, and
structure dependent. These previous results and the results
discussed in this chapter will serve as an aid in understanding
later calorimetric measurements.
Experimental
In these calorimetric measurements, a solid substrate
was not used. In the first set of experiments, the concentra
tion of saccharides was held constant at a concentration CQ
_ O
of 10 M of saccharide monomers or residues. In the case of
CS, this refers to dimer residues. Aquisition of the organic


TABLE OF CONTENTS Continued
CHAPTER Page
VREACTION OF PEPTIDES AND CARBOHYDRATES
IN SOLUTION 93
Introduction 9 3
Experimental 95
Results ..... 97
Discussion. 108
Conclusions 115
VI ADSORPTION OF COLLAGEN 117
Introduction 117
Experimental. v . 117
Results 117
Discussion 132
Conclusions 138
VII BIOGLASS 141
Introduction 141
Experimntal. 142
Results 142
Discussion 148
Conclusions 15Q
VIII SUMMARY 152
BIBLIOGRAPHY . 159
BIOGRAPHICAL SKETCH 164
IV


5
Calorimetry has also been used in a similar manner
to measure the heat of adsorption of-a second component from
an aqueous solvent [31]. The heat of adsorption of sodium-
dodecyl sulfonate (SDS) was found by subtracting the heat of
wetting of alumina in pure solvent from that found with SDS
present. In this case the initial adsorption attraction was
attributed to coulombic forces between the surface and ion
and was calculated to be -12 kcal/mole.
Results for similar experiments have been confirmed by
other methods [32,33] using water and other polar and ionic
\
liquids as adsorbents. For example5 the differential heat of
adsorption for the adsorption of octadecyl alcohol on alumina
from a benzene solution, calculated from the temperature
dependence of the adsorption coefficient was found to be -8.6
kcal/mole while that found directly from calorimetric measure
ments was -8.68 kcal/mole [34].
The heat of adsorption of water on quartz determined
by adsorption measurements at several temperatures was found
to be between -11 and -14 kcal/mole of water adsorbed[25].
There were differences found when the quartz was ground and
exposed to water vapor prior to drying and evacuation. This
was attributed to the formation of an amorphous layer of silica
on the surface. These differences disappeared when the rough
ened surfaces were annealed at 700C. The heats of wetting
agree well with those found from calorimetric measurements [24].
Calorimetry has been extensively used in biochemical
applications [35]. However, there are few calorimetric data


118
C0, mg/ml
Figure 26.
Reaction heat for the adsorption of collagen onto alumina in
three solutions at pH 7.


CHAPTER IT
ADSORPTION OF CARBOXYLIC ACIDS
ON ALUMINA AND HYDROXYAPATITE
Introduction
Several of the amino acids which make up collagen
possess charged side groups, principally amines and carboxyl.
The polysaccharide, chondroitin sulfate, often associated with
collagen, also possesses the carboxyl groups as well as the
sulfate group. Because each of these charged species has the
potential to interact with an aqueous oxide surface, knowledge
of the type and strength of the surface reaction is sought.
The thermodynamic and adsorption behavior of molecules
containing the sulfate group have been previously described.
The adsorption of sodium dodecyl sulfate on alumina has been
studied by calorimetric techniques [31] and by solution depletion
techniques [91]. Heats of adsorption for this molecule have
values near -6 kcal/mole at low concentrations when only ionic
forces drive the adsorption reaction. The free energy change
lies near -11 kcal/mole of ions. The adsorption of sulfate
ions onto solid barium sulfate from aqueous solution has been
measured by the thermometric titration technique. The heat of
precipitation was found to be -4.5 kcal/mole .[84].
41


163
99.S. Sugai, K. Nitta, Biopolymers, 12, 1363 (1973).
100. G.Y. Onoda, Report to Marion Labs. Inc. (1976).
101. P. Pincus, J. Calif. St. Dent, Assn., 26, 16 (1950).
102. A.W. Ham, Histology, Lippincott 388 (1969).
10 3. B. Sylvan, J. Bone Joint Surg. 2_9, 973 (1947).
104. R.A. Gelman, J. Blackwell, Biochem Biophys. Acta,
342, 254 (1974).
105. S. Arnott, J.M. Guss, Science, 180, 743 (1973).
106. D.H. Isaac, J. Mol. Biol. jHD, 773 (1973).
107. G. Nemethy, H. Scheraga, J. Chem. Phys. 3_6,3382 (1962 ).
108. H, Fleisli, Am. J Physiol., 200 (6 ) 1276 (961).
109. J. Einbinder, M Schubert, J Biol. Chem., 185, 725 (1950)
110. A.E. Sobel, M. Burger, Fed Proc., 13 300 (1954).
111.
112 .
I.C.N. Pharmaceuticals, Life Science Div.
A.J. Hopfinger, Biopolymers, 10, 1299 (1971).


CALORIMETRIC MEASUREMENTS OF THE ADSORPTION OF COLLAGEN
AND OTHER ORGANICS ONTO OXIDE SURFACES
By
PAUL J. BUSCEMI
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMNETS FOR THE
DEGREE OF DOCTOR OF PHILOSPPHY
UNIVERSITY OF FLORIDA
1978

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
ABSTRACT v
CHAPTER Page
IINTRODUCTION 1
Use of Protein Models .......... 2
Calorimetry as an Analytic Tool 4
Protein Adsorption. . 6
Surfaces 10
Forces of Adsorption 15
Thermodynamics of Adsorption 17
Heterogeneous Adsorption 24
Experimental 3 3
IIADSORPTION OF CARBOXYLIC ACIDS
ON ALUMINA AND HYDROXYAPATITE 41
Introduction. 41
Experimental. 4 3
Results 44
Discussion 51
Conclusions 59
IIIADSORPTION OF CHONDROITIN SULFATE
AND OTHER CARBOHYDRATES 61
Introduction. 61
Experimental. . 6 2
Results 62
Discussion 7 0
Conclusions ..... 77
IVADSORPTION OF POLYPEPTIDES 79
Introduction. 7 9
Experimental 81
Results 81
Discussion. .......... 86
Conclusions 92
iii

TABLE OF CONTENTS Continued
CHAPTER Page
VREACTION OF PEPTIDES AND CARBOHYDRATES
IN SOLUTION 93
Introduction 9 3
Experimental 95
Results ..... 97
Discussion. 108
Conclusions 115
VI ADSORPTION OF COLLAGEN 117
Introduction 117
Experimental. v . 117
Results 117
Discussion 132
Conclusions 138
VII BIOGLASS 141
Introduction 141
Experimntal. 142
Results 142
Discussion 148
Conclusions 15Q
VIII SUMMARY 152
BIBLIOGRAPHY . 159
BIOGRAPHICAL SKETCH 164
IV

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CALORIMETRIC MEASUREMENTS OF THE ADSORPTION OF COLLAGEN
AND OTHER ORGANICS ONTO OXIDE SURFACES
By
Paul J. Buscemi
March, 1978
Chairman; R. E. Loehman
Major Department: Materials Science and Engineering
The present work is the result of the application of
solution microcalorimetry to the problem of determining the
energies of adsorption of organic molecules onto ceramic
surfaces. The systems studied were chosen to model the
attachment of collagen to ceramics and to provide some expa
nation for the observed bonding of ceramic implants to bone.
An aqueous solution of an organic molecule such as a
polyamino acid, polysaccharide, or smaller molecules with
similar functional groups was automatically mixed in a micro
calorimeter with a slurry of a powdered oxide such as AI2O3,
SO2, or a special glass composition and the heat evolved or
adsorbed was determined. Calorimetric- measurements were
performed on increasing concentrations of reacting organic
molecules for a fixed weight of powder with known surface
area. Plots of the reaction heat, Q, versus the inital con
centration of the organic, CQ, yield thermometric titration
v

curves which were analyzed to give the enthalpy of the
reaction AH, the free energy change AG, and by difference
the entropy change for the reaction AS.
The systems were studied in order of increasing
structural complexity from simple carboxyls, amines, and
organic sulfates to amino acids, polyamino acids, polysaccha
rides, and collagen. Changes in the aqueous solutions by
additions of salts or changes in pH and combinations of
organic molecules were also studied.
Results indicate that there are at least two forces
which contribute to the bonding of the collagen and other
organics to oxide surfaces, hydrogen bonding and ionic bond
ing, the former releasing from 8 to 12 kcal/mole of functional
groups while the latter releases 4 to 6 kcal/mole of
functional groups depending on the relative polarities of
the adsorbing molecules and the surface. There are strong
indications of the denaturation of collagen at some surfaces
at which hydrogen bonding and ionic bonding act cooperatively.
vi

CHAPTER' I
INTRODUCTION
The study of adsorption and interaction of proteins and
other biological molecules onto non-biological surfaces is
important because of the increasing use of prosthetic materials
[1]. It is essential to know how each of these two distinctly
different components will interact in biological media. In
this study, a further understanding of the reaction between
the connective tissue protein, collagen, and various oxide
surfaces is sought.
Two properties of collagen adsorption are of major
concern: how well does the protein adhere to the oxide surface
and does the surface change the structure of the protein?
Protein adhesion relates to the binding of tissue to a prosthetic
material and can be approached by the determination of the
enthalpy AH of the adsorption reaction [2]. Changes induced
by the surface on the protein lead to denaturation. This
increases its vulnerability to enzyme attack and eventual
rejection from the host [3]. This question can be approached
by comparison of the enthalpy of reactions of model systems
with that of reactions which have been found not to be
disruptive to the structure of protein.
Three materials serve as substrates: silica, alumina,
and hydroxyapatite. Each of these materials is well character-
1

2
ized and has potential for use as a prosthetic material [4].
The calorimetric measurements made, therefore, are not for
the purpose of further characterizing these materials but to
study their influence on the adsorption of organic molecules.
The values of the thermodynamic parameters (AG, AH, and
AS), determined from the calorimetric measurements, do depend,
however, on the structural features of the surface as well as
those of the adsorbing molecule and on their mutual environment.
A practical approach for studying complicated systems encountered
in actual application is to study simpler model systems [5],
which provide singular features for observation.
Within a series of model compounds, the structure of
the molecules, the solvent, and the surface can be systematically
varied and correlations can be made between the thermodynamic
data and the variations in experimental conditions. In this
study, extensive use is made of several types of molecular
model compounds including those representing collagen as well
as those representing carbohydrate and other physiologically
relevant organic structures.
Use of Protein Models
Past workers have used molecular model compounds designed
to study collagen. Specifically, the polyamino acids have
been well studied in this way. Poly-l-proline has been used
in conformational studies in CaCl2 solution [6] showing that
disordering of the molecule is associated with its increasing
rotational ability. X-ray diffraction studies of poly-l-proline

3
have demonstrated that the backbone conformations of the
molecule are similar to native collagen [7]. Poly^-l^lysine
and poly-l-arginine have been used as models for collagen in
relation to the structure of amorphous ground substance [8,9].
Poly-l-lysine and poly-l-glutamic acid have been used in
conformational studies using differential capacitance techniques
[10]. In other studies, workers have used combinations of
synthetically prepared amino acids to model collagen [11,12].
Smaller molecules representing isolated residues of
the collagen molecule have also been investigated but not as
frequently as the polyamino acids. Dyes containing amino
groups have been shown to selectively adsorb chondroitin sulfate
[13], an important carbohydrate in structural tissue [14].
Surface viscosity measurements using amines, amides, and
carboxylic acids as model proteins have been studied in relation
to bilayer film formation [15] in membrane studies.
In biological studies of proteins other than collagen,
molecular models have been widely studied. Enthalpies of
aqueous solution have been determined calorimetrically for
amines and carboxylic acids [16] as part of a quantitative
description of biological systems. Heat capacity measurements
[17] on several amino acid solutions have been made to help
explain protein structure* The binding of short amino acid
chains [18] in subunit studies of immunoglobulin has also been
investigated by calorimetry. Differential scanning calorimetry
has been used to study conformation changes of many polypeptides
used as models for collagen [19,20],

4
Molecular models in non-biological studies have also
been used. The adsorption of molecules containing the same
functional groups which proteins possess, amines [21], sulfates
[22], and organic acids [23], have been examined by various
methods. Calorimetry has not been extensively used for this
purpose. In general, the use of molecular models in biological
and non-biological systems appears widespread for the determina
tion of various properties.
Calorimetry As An Analytic Tool
Calorimetry has long been used to measure the enthalpy
of adsorption (heat of wetting) of various liquids onto dry
oxide surfaces. Such measurements are made by immersing a
clean, dry solid powder into a liquid. The heat change is
measured in a calorimeter. For our purposes, the most
relevant liquid used in previous studies was water. Heats of
wetting of silica [24-26], of alumina [27-29], and of hydroxy
apatite [29] have each been measured. The results of these
works show several consistent features. First, there are
differences in the heats of wetting with variation in the out-
gassing pressure and with temperature of evacuation, indicating
surface heterogeneities. Also there are differences in heats
of wetting with variation in particle size. Finally, the heats
of adsorption of water range from -10 to -20 kcal/mole of water
adsorbed and are attributed to hydrogen bonding of the water
to the surface [30].

5
Calorimetry has also been used in a similar manner
to measure the heat of adsorption of-a second component from
an aqueous solvent [31]. The heat of adsorption of sodium-
dodecyl sulfonate (SDS) was found by subtracting the heat of
wetting of alumina in pure solvent from that found with SDS
present. In this case the initial adsorption attraction was
attributed to coulombic forces between the surface and ion
and was calculated to be -12 kcal/mole.
Results for similar experiments have been confirmed by
other methods [32,33] using water and other polar and ionic
\
liquids as adsorbents. For example5 the differential heat of
adsorption for the adsorption of octadecyl alcohol on alumina
from a benzene solution, calculated from the temperature
dependence of the adsorption coefficient was found to be -8.6
kcal/mole while that found directly from calorimetric measure
ments was -8.68 kcal/mole [34].
The heat of adsorption of water on quartz determined
by adsorption measurements at several temperatures was found
to be between -11 and -14 kcal/mole of water adsorbed[25].
There were differences found when the quartz was ground and
exposed to water vapor prior to drying and evacuation. This
was attributed to the formation of an amorphous layer of silica
on the surface. These differences disappeared when the rough
ened surfaces were annealed at 700C. The heats of wetting
agree well with those found from calorimetric measurements [24].
Calorimetry has been extensively used in biochemical
applications [35]. However, there are few calorimetric data

6
on the adsorption of proteins onto oxide surfaces and there
is apparently none for the adsorption of collagen. The
calorimetric data most relevant to adsorption primarily
involve such globular serum proteins as serum albumin [36].
The remainder of this section will therefore be devoted to
previous studies of protein adsorption use with particular
emphasis on oxide substrates.
Protein Adsorption
The demonstration of molecular attachment of cell
proteins on foreign substances has been accomplished by various
methods. Multiple internal reflection spectroscopy has
been used to measure protein interaction using germanium [37]
as a substrate. A KRS-5 prism pressed against protein on a
hydroxyapatite substrate allowed protein-hydroxyapatite inter
action to be studied [38]. Although energy calculations were
not carried out in these studies, the changes in adsorption
frequencies indicate chemical interaction with the surface of
the substrate.
Film compression studies [39] using collagen, gelatin,
and poly-l-alanine with silica gel showed adsorption hysteris.is
indicating an irreversible process. The maximum interaction
of the silica gel and collagen occurred at pH 5.2. The iso
electric point,where there is charge neutralization of the
protein, is 5.5. There was also interaction between alanine,
which has no ionic side groups, and the gel. The interaction

7
appreared to be of the same type as that of collagen. The
primary binding force was- assumed to be hydrogen bonding [40].
Adsorption of bovine serum albumin (BSA) on hydro
philic silica [41] exhibited a maximum surface density at
pH 5.5. The isoelectric point (IP) of this protein is 4.9.
The surface of the silica is negatively charged at this pH.
Desorption occurred readily at pHs away from the IP indicating,
as suggested by the author, that binding was due to hydrophobic
interaction. Other studies [42,43] showed that even after
extensive washing with water and EDTA that not all of the BSA
adsorbed onto pyrex glass could be recovered. Maximum
adsorption was near the IP of the protein and the free energy
change was estimated to be -2.5 kcal/mole of protein. The
enthalpy was not calculated.
Serum globulins have been shown to be preferentially
adsorbed by silicic acid [44] and silica [45] and by other
minerals [46]. Maximum adsorption took place at the isoelectric
point on these surfaces as well as on calcium phosphate gel
[47]. In none of these studies were determinations of the
enthalpy of the various adsorption reactions made.
The adsorption of albumin, fibrinogin, and globulin on
polyethylene has been determined by internal reflection spectro
scopy [48]. The adsorption isotherms followed a Langmuir isotherm,
a common finding in which the quantity of solute adsorbed, X,
at the equilibrium concentration, C, is given by X = aC/(l + bC)
where a and. b are constants. The adsorption was assumed to be

8
due to hydrogen bonding because of the shift in the amide I
(C = 0 stretching) band at 1640 cm'^.
Calorimetric measurements of the adsorption of human
serum albumin on negatively charged polystyrene (PS) [49]
were shown to be pH dependent. Maximum adsorption of the
protein occurred near the IP (4.9) where the enthalpy of the
reaction was positive. At pH values removed from the IP the
reaction was exothermic. It was suggested that at pH values
away from the IP the conformation of the adsorbed protein
changes for energetic reasons. Denaturation of the protein
is not surprising since it is known that the internal bonding
of serum albumin is weak [50]. The' enthalpy of the adsorption
reaction varied between zero and 8 kcal/mole as the surface
charge varied between -1.0 myCcm-^ and -7.5 myCcnf*2. The
most negative enthalpy values were recorded for pH 3.8 and 9
and were near -8.4 kcal/mole.
Enthalpy values for the adsorption of albumin, gamma
globulin, and fibrinogin, were calculated from adsorption
measurements at several temperatures [51]. The results indicated
that the adsorption took place in two distinct ways. Both
types were apparently Langmuir and took place on separate
membrane sites. One type of adsorption was easily reversible
with a heat of adsorption in the neighborhood of -10 kcal/mole.
The other type of adsorption reaction was hydrophobic, endothermic,
and with heats of adsorption in the range of 5 to 20 kcal*
mole.

9
In another study [52], the forces involved in the
adsorption reactions between several globular proteins and
glass surfaces were determined to be primarily ionic amine-
silanol bonding and hydrogen bonding. Two rates of adsorption
were noted. The first appeared to be related to the number
of amines present on the surface of the protein. The second
was slower and seemed to be dependent on the molecular weight
of the protein. Hydrogen bonding was suspected since the
proteins could not be completely washed from the surface with
urea.
Ionic bonding of ribonuclease to glass was indicated
to be strong [53] since very little of the protein could be
removed by rinsing in several solvents. No enthalpy determina
tions were made.' There was a decrease in adsorption with
an increase in ionic strength.
Further review of the literature reveals that the
various adsorption studies cannot be readily compared due to
the large number of experimental variables and to the random
manner in which they are controlled in each experiment. A
few common features in the study of protein adsorption do
emerge, however.
There is usually more than one type of interaction
present for any particular system and one of these is usually
hydrogen bonding. The observed enthalpy values are in the
range of -10 to +10 kcal/mole of protein. Finally, maximum
adsorption density appears to take place near the IP of the
protein. There are many exceptions to these general results,
however.

10
The effect of changes in ionic strength on adsorption
is also not well understood [54]. Dissolved salts disrupt
hydrogen bonds which proteins depend on for conformational
stability [55], Changes in the ionic strength of the solvent
will also have effects on the adsorbing surface. For example,
phosphate, a common component in buffers, will change the surface
charge of alumina [56]. Generalizations are difficult to
make about the action of specific ions on adsorption unless the
specific system under study is clearly defined.
Surfaces
Silica, alumina, and hydroxyapatite, the three materials
used in this study, are oxides. Hydroxyapatite is sparingly
soluble at neutral pH whereas silica and alumina are virtually
insoluble [57]. All are hydrophilic and each displays a
surface charge which varies with pH.
Thesurface charge results from exposed surface atoms
attempting to complete their coordination of nearest neighbors
[58]. Exposed cations do this by pulling an 0HT ion or H^O from
solution and anions by attracting a proton from the aqueous
phase. The result is adsorbed H+ or OH- ions which assume their
respective charges on the surface.
Any other ion which can pass between the solid and
liquid phases may also help to establish the surface charge.
Such ions are called potential determining ions. Thus,
OH- and H+ are potential determining ions for each of the

11
+ 2 o
three surfaces used here. In addition Ca and PO^-'3 are
potential determining ions for hydroxyapatite. Certain
ions added as impurities may alter the surface charge such as
aluminum ions [59] or cobalt [60] on silica or phosphate on
alumina [61]. The wide variety of buffering systems used in
biological studies involving adsorption can thus lead to
differing results for proteins even if the same material is
used as a substrate.
The surface potential can be altered by a change in
pH. For each of the three oxide substrates in this study, there
exists a pH at which the surface charge is zero. This pH is
called the point of zero charge (pzc) and is listed in Table
1 [62,63] for the substances used as adsorbents.
As the pH varies from one side of the pzc to the
other, the sign of the surface charge will change as will the
adsorption properties of the protein.
Ions in solution: which do not pass through both phases
but are attracted near the surface by electrostatic forces
are called counter ions. They will form a diffuse layer of
ions in solution near the surface and will tend to neutralize
the surface charge. The concentration of the ions generally
decreases exponentially with distance from the surface [64].
The.higher the concentration, the more compact the diffuse
layer will be. The thickness of the diffuse layer ranges from
about 10 A at .1 Msolutions to about a few hundred A in .001 M
salt solutions [65].

Table I
PZC of Substances Used as Adsorbent
alumina
9
2-4
silica
hydroxyapatite
7.5

13
Due to the shape of the diffuse layer, the influence
of dissolved salts on proteins will be greater near a surface
than in bulk solution. Some proteins, because of their
large size, may extend entirely through a double layer. The
effect of dissolved salts on adsorbed proteins would then be
difficult to explain in detail.
The adsorption properties of the substrates are due
as much to adsorbed water as to their intrinsic stucture.
The surface of silica in aqueous solution has been shown by
infrared spectroscopy to possess three types of surface ions
[36] as represented below:
Si 0"
Si 0-H
Si o~h2+
Unless the adsorbed water and ass.ociated ions are driven off
by heating, silicon ions cannot chemically react with organic
molecules arriving from solution. It has been shown that
ammonia will not react with hydroylated silica, although
chemisorption will occur if the silica has been subjected to
o
a prior vacuum degassing at temperatures in excess of 400 C
[66?67], Silica treated with ammonium fluoride solution
showed evidence for Si-F bonding instead of silanol [68] but
this reaction occurred after heating the substrate to 400C
in vacumm. Trimethylsiloxane can be covalently bonded to
silica by refluxing them in acetone for 24 hours at 50C [69]
This gives some idea of the difficulty of penetrating the

14
adsorbed layers on the silica surface. It can be seen that
the reactions of aqueous solutions of proteins with silica may
occur with either surface oxygen or hydrogen, depending on
the compositional purity of the surface.
The same effect can be seen with alumina. Steric
acid was adsorbed from CCl^ solvent after the alumina had been
evacuated at 800C for one hour. Without the pretreatment,
steric acid would not covalently bind to the alumina surface
[VO], Methanol has been shown to adsorb on alumina [71] after
successive evacuation and heating at 400C, heating in oxygen
to rid any hydrocarbons present and then heating again at
10^ torr at not less than 350C for 1/2 hour. A methoxide
surface is formed when the clean dry surface is exposed to
methanol vapor. From studies of adsorbed acetylene on alumina
it was concluded that the surface contains electron poor
and electron rich [71] sites (oxide ions, hydroxyl groups, and
aluminum ions) after the sample had been heated to 800C.
Even at these temperatures not all the hydroxyl groups were
removed from the surface [72]. Hydrogen and hydroxyl ions on
alumina are exposed to the solution interface, yielding a
surface structure similar to that of silica.
Hydroxyapatite is assigned the formula Ca-^Q (PO^) (OH)^
In solution the surface undergoes hydrolysis, yielding a surface
having the formula Ca2(HP04) (0H)2 [57]. It is different from
silica and alumina in that, in addition to surface OH" and H+
ions, there are also Ca ions which are capable of binding
adsorbing anions [73]. The multiple internal infrared spectra

15
of several synthetic and naturally occurring calcium phosphates
exposed to organic acids^ show shifts in the P-0 stretching
frequency [39] which were attributed to hydrogen bonding. Other
workers [30] have shown that HgO+ ions are hydrogen bonded to
the calcium. There have been suggestions that there is some
covalent bonding between organic constituents and hydroxyapatite
in bone [74]. Binding of calcium ions by collagen has been
demonstrated by solution analysis [75]. It has not been shown
that collagen can attach to the surface of the hydroxyapatite
without an intervening water molecule or hydroxyl ion.
Forces of Adsorption
The relationship between the enthalpy of a reaction and
the total energy is H = E + PV. Most biological processes occur
in liquids rather than in the gas phase [76]. In this case
the changes in pressure and volume are small. To a good
approximation then, dE ~ dH.
The total energy of adsorption is affected by the type of
interaction between the surface and the molecule. This energy
is comprised of several components. They may be classified as
non-polar, ionic, hydrogen, and covalent bonding [77-79].
Non-polar (dispersive) forces are always present between
molecules. They arise because the time-averaged electron cloud
interaction between uncharged atoms is attractive [80], They
are moderately strong, producing' energies in the range of 1 to
10 kcal/mole. Hydrophobic bonding is a result of dispersive

16
forces. This is a consequence of a decrease in displace
ment of a polar medium^ when less polar components coalesce,
thus creating a lower energy state.
Ionic or electrostatic attraction can occur between
oxides and proteins both of which are normally charged in
aqueous solutions. Ionic bonding strength is decreased by
an increase in the ionic strength of the solvent because of
shielding, whereas dispersion forces are not affected. Ion
interaction is essentially independent of temperature [80,813
Hydrogen bonding is partially ionic and partially
covalent [82^83], It arises from the electrostatic force
acting between hydrogen and a lone-pair of electrons of
nitrogen, oxygen, or fluorine. The small size and the close
approach of the hydrogen atom accounts for the partial (20%)
covalent character [82]. Typical bond strengths are of the
order 1 to 10 kcal/mole. Hydrogen bonds are also weakened
by an increase in ionic strength of the solvent. Completely
covalent bonding rarely occurs in the adsorption phenomena
in which we are interested [62].
The classification of physical or chemical adsorption
is somewhat arbitrary [7 9 ,83 ]. If the adsorption is found
to be readily reversible and has an energy of the same order
of magnitude as the liquefaction of gas, it usually is classified
as physical [77 ]. Osipow states that Van der Waals forces
are responsible for physical adsorption whereas Fuerstenau
also includes coulombic attraction. Chemical adsorption is
irreversible and the magnitude of the energy change is of the
order of chemical reactions [78].

17
Thermodynamics of Adsorption
Complete reviews on the thermodynamics of adsorption
are given elsewhere [76-78]. In this section, only those
points needed to explain the following data will be presented.
Limited explanation of the ideas of earlier workers would
be in order, however.
Gibbs gave the first rigorous thermodynamic explanation
for why a given material should either adsorb or desorb at a
surface. He was able to predict the functional relationship
between surface tension and surface concentration and the bulk
concentrations of the surface-active solutes. His derivations
assume that substances tend to minimize the free energy of
the surface region by either becoming concentrated or depleted
there. As a result, it has been the free energy which has been
traditionally determined, and surface concentration measurements
are the most common method of doing this. Other methods, such
as the thermometric titration method [84], may be useful for
obtaining thermodynamic data.
The thermometric titration technique is an analytical
method in which the heat effect of a titration reaction is
used to measure the titer of a sample. It is applicable to
reactions of the type
R1 + R2 + P (1.1)
which entails a heat of reaction Q. 'In equation (1.1)
refers to reactants i and P refers to the product. For single

18
step reactions an equilibrium constant, K, may be written
as .
K = [P]
[Rx] CR2]
(1.2)
where the brackets denote concentrations. Through knowledge
of equation (1.1) and the reaction heat, Q, the enthalpy change
of the reaction and the equilibrium constant may be determined.
The method by which this is done will be detailed for adsorption
measurements.
For an adsorption reaction equation (1.1) can be written
as
Su + R So
where R-^ in equation 1.1 has been replaced by Su which is an
unoccupied site on a solid surface capable of adsorbing from
solution a reacting component R to produce an occupied site SQ.
The equilibrium constant is then written
K = [SU
[su] [R]
The concentration of occupied sites [SQ] is equal to
the number of adsorbed molecules per unit area, Na/A, while
the concentration of unoccupied sites is equal to the total
number of sites N minus the number of occupied sites per unit
o
area, (N N )/A. The equilibrium constant is then
u d
. K =
(N N ) [R]
S cl

19
Dividing -
through by N gives
b
K = VNg
(1.3)
(1-N_/N)
d b
[R]
or
II
CD
(1.4)
(1-0)
[R]
...o .
where 0 = N-,/N is the fraction of occupied sites. Equation
s 7 7
(1.4) is the Langmuir adsorption isotherm [ ] and will be
used later. Its use requires that each site is occupied by no
more than a single molecule and that no two sites interact.
These conditions are satisfied when the concentration of R is
low.
The concentration of reacting molecules is equal to the
number of moles of R in solution divided by the total volume
Nr/V. If N is the total number of moles of R on the surface
and in solution then = N N The equilibrium constant
can now be written as
K = N V
Ct
(N? N ) (N N )
j d J- a.
(1.5)
The enthalpy change for the adsorption reaction, AH, is
related to the reaction heat Q by
AH = -Q/N (1.6)
a.
where the minus sign denotes, by convention, that an exothermic
reaction (positive Q) will yield a negative enthalpy change.

20
Substituting equation (1.6) into (1.5), one obtains
~K = -QAHV (1.7)
(N AH + Q)(AHN + Q)
To determine K, at least two adsorption experiments are completed
in which Q is measured but a set of values is usually completed
to determine the endpoint of the titration. The original
number of moles of reacting molecules, N, is varied, and Ng
is held constant by keeping the surface area of the adsorbing
substrate constant. Values for Qj, Q2, N, N, and N are
recorded where N and N are values for N. The value of K is
assumed to be nearly constant if N is not too different from
N. Equation (1.7) can then be solved for AH by using the
quadratic equation (1.8)
AH2 N (Q1 N Q2N) + AH Q-^ (Ng-N-^ +QiQ2 (Q2Qi> = 0
for the two sets of values. The total number of surface sites
can be estimated by dividing the total surface area by the
known cross-sectional area of the adsorbing molecule. This
method is valid as long as the surface concentration of adsorbed
molecules is low and lateral interaction does not occur. Alterna
tively three sets of values can be used to eliminate N from
o
equation 1.7. Both methods give results 10% of each other-.
The volume V is held constant. The value of AH is substituted
into equation (1.7) to find K, and therefore AG by using the
relation
. AG =
-RT In K
(1.9)

21
In order to complete the thermodynamic data (AG =
AH TAS) H must first be determined or approximated.
The number AH used in equations (1.6) to (1.8) is the total
enthalpy change for the reaction under experimental conditions
while. AH0 is the standard enthalpy change. Under suitable
conditions, AH can be shown to be AH so that AS can be
determined. Those conditions will now be explained.
The chemical potential /f^ or partial molar free energy
Gj_ of component i in a chemical reaction is defined by
/fi = G"i = (^G/c?Ni)tp (1.10)
where G is the free energy change for all components in the
reaction. The chemical potential can be expressed as a function
of the activity a^ of component i and of the chemical potential
in some reference state
Jil = y/Vef + RTlnai (1.11)
The term RTlna^ takes into account the energies of interaction
of component i with other components at a given concentration in
the mixture. The choice of reference state is quite arbitrary
and varies for experimental convenience. Generally, no matter
what reference state is chosen, the activity is expressed as a
function of the mole fraction component i, X-p, and a parameter
known as the activity coefficient fp
ai = xi fi
The activity coefficient approaches 1 in pure solutions for
the solvent while in dilute solutions of component i, fp
approaches a constant which may be greater or lesser than one.

22
The partial molar enthalpy is found to be
% =' -RT2 ( 9T)pn (1.12)
or in view of equation (1.11)
~Ui = -RT2(91n ^'iref/9T)pn RT2 (91n ai/9T)pn (1.13)
or = -RT2(9ln ^ef/9T)pn RT2 (9In f^T)
Under the constant composition does not vary with temperature
The first term of equation (1.13) is the enthalpy
change for component i which would occur if the reaction was
held under reference conditions. The standard enthalpies, ,
of pure compounds are the enthalpies of reaction of building
up those compounds from their elements under standard conditions
(P = 1 atm T = 298K). The chemical elements themselves have
zero standard enthalpies of formation. If the reaction is
carried out under standard conditions, the enthalpy change for
the reference state is equal to H?. Then
~Hi = H? RT2 ( 9In f/ 9T) (1.14)
Knowledge of f^ for the systems under study is lacking.
We therefore make the approximation that AH is significantly
larger than the natural logarithm of the temperature variation
of f. The validity of this approximation relies upon the non
interaction of solute molecules. This condition is assumed to
hold for dilute solutions. Therefore
Â¥i = Hp (1.15)
To relate to AH it is noted that AH can be written
as the difference of the sums of the partial molar enthalpies
of products and reactants

23
AH = SiHiXi + ^kHkZk (1,16)
products reactants
where each sum is taken over each of the different components
for products and reactants and X. and are the respective
1 i
mole fractions. In view of equation (1.15) the enthalpy
change for the adsorption reaction is
AH = Zi H? X + EkHg Zk (1.17)
or
AH = AH (1.18)
where AH0 is the standard enthalpy change for the complete
reaction. Thus, under the rather ideal conditions in which
there is no interaction between solute molecules in solution or
on. the surface at 1 atm and 298K AH may substitute for AH.
The value of AH can be used with AG to find at least an approximate
value for AS.
The experimental conditions in this work meet the contraints
of pressure and temperature. The constraint of non-interaction
of solute molecules holds only for dilute solutions. In as
much as enthalpy values tended towards.constant rather than
steadily decreasing values,lateral interaction on the surface
between adsorbed molecules does not appear to have occurred.
Interaction of solute molecules in solution can only be assumed
-4
to be small in the concentration ranges used, typically 10 to
10-3 M.

24
Heterogeneous Adsorption
As mentioned earlier, from the standpoint of adsorption
studies, the surface of a substrate is often not uniform.
Heats of adsorption may vary at different positions on the
surface. If the condition is maintained that the different
sites are non-interacting, the adsorption onto a heterogenous
surface may be regarded as simultaneous independent reactions
of the type expressed by equation (1.3)
+ R -y So-^
S2 + R -* SO2
(1.19)
Sj + R -* Soj
where the subscript j enumerates the different types of sites.
The concentration of a single type of solute molecules, R,
is common to all surface sites. Each adsorption reaction,
according to equation (1.19), would evolve a reaction heat
Qj. The total reaction heat, would be the sum of Qj for
each reaction on the different types of sites.
Qt = Qi + Q2 + Qj (1.20)
If AHj is'the enthalpy change per mole of adsorbed molecules
on sites of type, j and Nj is the number of moles adsorbed then
equation (1.20) can be expressed according to equation (1.6) as
-Q. = AH-i N, + AH2N2 + .... AH.N. (1.21)
In a calorimetric measurement it is the value of
which is measured. If the solution is analyzed to determine

25
the total number of moles adsorbed, a total apparent
enthalpy is found
AHt = -Qt/Nt (1.22)
From equation (1.20) then
AHt = -Qt/Nt '= 1/Nt (AH-l N1 + AH2 N2 +. . AHj Nj )
or
AHt = AH-l y1 + AH2 y2 +.... AH_. y^ (1.23)
where Yy is the fraction of the total number of moles of
molecules bound to sites of type j. Applying equation (1.18)
to each reaction
AHt = EoAHj y'j
= AH£
(1.24)
where AH£ is the weighted sum of the standard enthalpy changes
for all adsorption processes. The standard free energy
change for each reaction expressed by equation (1.19) is given
by
AG 9
3
AH? TAS?
(1.25)
Rewriting equation (1.25) for AH? and substituting into (1.24)
one obtains
AH = £^(AG9 + TAS9) y..
- Sj AG* TASjyj
(1.26)
or
where
AH
. t
AG + TASo
Ag = £. AG y.
t .3 3 3
(1.27)

26
and
AS
E-s 3 3 J
(1.28)
express the weighted sums of the standard free energy and
standard entropy changes for the complete adsorption reactions.
For each reaction expressed in equation (1.19) there
is an equilibrium constant Kj which can be written according to
equation (1.4) as
K. =. 0. (1.29)
3 3
(1-0 j) [R]
or related to AG? according to equation (1.9) as
AG? = -RT In K. (1.30)
3 3
Equations (1.29) and (1.30) express the fact that each site
carries on an equilibrium reaction independently of all others.
The fraction 0. = N-/N? is the ratio of occupied sires of type
3 3 3
j to the total number of sites of type j.
For any equilibrium concentration [R] of solute molecules
the total fraction of occupied sites of all types, 0^., can be
written as
or
0 = N /N
t t s
0
t
Xx 0
where X. = N?/N
3 3 s
constant.
If equation
one obtains
, + X0 0O + ...X. 0. (1.31)
1.2 2 3 3
is the fraction of sites of type j and is a
(1.30) is substituted into equation (1.27)
AG = -E. (RT In K.) y.
t 3 3
(1.32)

27
or
= -RT' In K^' K* .K# (1.33)
Thus, even though each AG? for the individual.reactions is
constant, the overall free energy change will vary as each
fraction y. varies.
1
The net effect is that if any single type site adsorbs
a large percentage of all molecules adsorbed then
AG| approaches AG? as y| + 1
Generally, howeiver, there is no simple number" AG which can
be expressed in terms of a single equilibrium constant ,
having the form
Kt = K1 K? V- -KjJ (1.34)
The value of K^_ would vary as the fraction of occupied sites
varies.
The physical interpretation is that each site contributes
a specific amount of energy to the total energy change. At
very low concentrations only a very few sites react, presumably
those of higher, energy. At higher concentrations a greater
number of sites react, but of overall lower energy. The result
is a lowering in the average energy change as the concentration
is increased.
Under the conditions of independently acting sites
evaluation of would give AG exactly. However, to do this,
knowledge of each and y. has to be available. In the absence
of such knowledge, the evaluation of K can be approximated by
evaluation of equation (1.29) by replacing 0j by 0^_. The number
found from this method, K, could be used for determination of
AG under suitable conditions.

28
Those conditions may be determined by estimated
values of Kj for calculation of K and and using each for
evaluation of AG .. The value of K' is found from
K = 0t.
(l-0t) CRl
where 0^ is defined by equation (1.31).
AG^ is found from
(1.35)
The percent errors in
% error AG_,
In Kt In K x 100
In K'
The percent error is calculated by setting values for and
Xj and directly calculating KT from equation (1.34) and
from equation (1.40). If the percent error is acceptable, AG^
may be calculated.
An example for the case of two different types of
adsorption sites is given. The equations necessary to carry
out this calculation are given below for convenience
el
II
[R]/(l
+
K1
[R])
0 2
= K2
[R]/(l
+
K2
CR])
yi
= 0-^
O
1
/(01
N
+
0 N)
2 2
y2
= 02N
o
2
/ (0 2
N
2
+
e2N)
Kt
= K1
k2
X1
O 11
s
II
/
(N
+
N)
X2
= N
/
(N
+
N
9t
= Xx0
1
+ x2
0 2
K =
et/(i + et) [R]

29
In the example the values of [R] were carried over several
orders of magnitude while the total number of sites N =
C
N| + was kept constant at 10 moles. The results are
shown in Figure 1 .
Two cases are presented. In (a) the fraction N-/N =
1 s
0.1 and 1<2 = 5000 are held constant, while K-^ is given
values of 1000 and 5000. In (b) and 1<2 are held constant
at 1000 and 2000 rspectively while X varies over three
values; 0.002, 0.02, and 0.2.
While 1<2 is less than five times the value of K-^ and
Xx is a good approximation of with the error remaining
within 1%. As approaches 1 the error goes to zero. Also,
at very low concentrations, it is assumed that most molecules
would adsorb only onto the highest energy sites so all values
of y (equation 1.34) go to zero except Y- and the error again
goes to zero. For oxides the overall fraction of highly
reactive sites is small [85,71]. Moreover, the hydrated
oxide surface will be of lower energy than a perfectly dry
surface, aiding in meeting the condition that K-^ not be too
much larger than K2 [60,28]. Under these constraints and using
equations (1.8) and (1.7) with Q = Q AH^_ can be calculated.
For the calculations in the later chapters, the
difference between the values of N for use in equation (1.8)
r
is kept small so that variation of AH in that concentration
interval is small. The values of AG calculated tend to remain
within 20% of the highest to lowest values. This corresponds

-LOG (R)
Figure 1. Examples for the calculation of K' and K-)-
for constant K2 and x-j_ (a), and for constant K2 and (b)
ERROR AG.

31
to a range of K_. varying by about a factor of seven. Under
these conditions the maximum error in AG^ is less than 2%.
The value of AS calculated from
AS0 '= AG AH
r-.T
can be given only simple interpretations. It is known that
values of AS will be between -20 and +20 cal/mole-deg
A decrease in entropy is typically explained as a loss of
freedom of solute molecules as they adsorb. Increases in
entropy, generally found in experiments using macromolecules,
are explained as solvent molecules gaining additional freedom
as the large structuring molecules are removed from solution.
Exceptions to this general rule are present.
The various plots of AG, AH, and AS presented in the
following chapters, in accordance with the previous discussion,
are to be understood as the composite values AG^, AH^, and
AS^. The values for these parameters are closer to single
values of AH?, AG?, and AS? in those regions of the curves
where they tend towards constant values. In these regions the
percent error is generally less than .5%.
The determination of the thermodynamic properties for
the adsorption of collagen on hydroxyapatite is presented as
n example of the calculations made in the following chapters.
The first step in the analytical procedure is to plot Q vs. CQ
(Figure 2). From this graph two values of Q and CQ are chosen
for the sample calculation. In this case values of

MCAL.
32
o
0,5 1.0 1.5 2.0
c0 y
Figure 2. The first step in the calculation of the thermo
dynamic functions is plotting the reaction heat, Q, vs the
original concentration CQ. Here, two points are taken from
the measurements of the adsorption of collagen on alumina.

33
are arbitrarily chosen. The
to
5.0 x 10r'^ cal
5.9 x 10-3 cal
1 x 10~6 M
1.33 x 10-6 M
values of and C2 correspond
N = 2 x 10^ moles
N = 2.66 x 10^ moles
2
The number of moles of surface sites, N, in this example
s
_ q ,
is taken as 2.5 x 10 This number was determined from
consideration of the surface area occupied by a collagen mole
cule, the number of sites as calculated by the program, and
study of the reaction heat curve. Substitution of these
c
values into equation 1.8 yields -3.3 x 10 cal/mole for AH.
Substitution of AH, Q-^, N and V (4 x 10 l)into equation 1.7
gives 4.3 x 10^ for K or -7.6 kcal/mole for AG. This in turn
yields -1.1 x 10^ e.u. for AS0.
Experimental
Calorimetric measurements were made using an LKB model
10700 batch microcalorimeter [86], The basic calorimetric unit
consists of two identical gold cells situated in an aluminum
heat sink (see Figure 3). Each cell has two compartments
capable of holding 2 and 4 ml of fluid. Mixing of the fluids
in each compartment is accomplished by rotation of the entire
calorimeter. There is no stirring, and after rotation, the full

34
B D
Figure-3. Schematic of the operation of an LKB model 10700
microcalorimiter. A-calorimeter, B-reaction cell, C-thermo-
pyle, D-heat sink, E-amplifier, F-chart recorder, G- reaction
heat curve. The reaction heat, Q, is proportional to the
area under the curve.

35
4 ml of fluid are contained in the forward compartment. In
all experiments, 2 ml of fluid were used in each compartment.
Measurement of the heat loss or gain incurred by the
mixing procedure is made through multiple thermopiles located
between the heat sink and cells on two sides of each cell.
The thermopiles are connected in opposition so that the signals
from reactions producing equal amounts of heat are cancelled
electronically. One cell Is then arbitrarily chosen as a
reaction cell and the other as a reference cell in which
unwanted heats can be cancelled.
Determination of the reaction heat is made by manual
integration of the voltage vs. time curve produced during the
course of a reaction. The energy is calibrated against a
known heat produced in the reaction cell using a precision
resistor and a known current generated for a specific time
interval.
Each reaction in this work is of the type
rotation
| A B 1 A + B
where A is a solution of organic molecules and B is a slurry
of powdered oxide used as a substrate. In the mixing operation
several reactions are possible, each contributing to the over
all heat produced. They are due to 1) wetting of the cell
wall; 2) dilution of the organic molecule; 3) friction of
mixing; 4) chemical reaction. Only the last heat is desired.
The others must be eliminated.

36
The first three heats are accounted for in various
ways. The cells are first wet with the solvent being used
and emptied prior to filling with reacting components. This
eliminates the heat of wetting of the cell wall. The heat of
dilution of the organic molecules is accounted for in the
reference cell, while the heat of dilution of the oxide powders
and frictional heat are measured separately and subtracted
from the reaction heat.
The heat measured in this way gives a measure of the
reaction A + B -t C where C is a complex of solid particulates
and adsorbed organic molecules.
Calibration of the calorimeter was carried out as suggested
by the manufacturer and consisted of two procedures. The first
procedure determined the sensitivity of the thermopiles. The
manufacturer listed the sensitivity of the thermopiles as 28.0
and 30.0 microvolts for a constant current of 30 miliamps
through the calibration heaters. The measured values were
consistently within 2 8.0 1 .05 microvolts and 30.0 t .05 micron-
volts. The second procedure judged the accuracy of the unit's
calibration mechanism. The heat of dilution of a six percent
sucrose solution was measured periodically to be 6.36 .05
kcal/mole. The literature value is 6.36 .03 kcal/mole.
No literature data could be found for the heats of
adsorption of surfactants from aqueous solution on prewetted
surfaces. However the heat of adsorption of sodium dodecyl
sulfonate (SDS) measured from the heat of immersion of dry
alumina (Linde A) was estimated to be 12 kcal/mole 1 kcal/

37
mole [23]. Using the method described in this work the
heat of adsorption of SDS on Linde A alumina was found to
be 10.2 kcal/mole of SDS. The discrepancy, al kcal/mole,
is thought to be due to the surface being wet prior to contact
with SDS. This would prevent any possibility of SDS coming
into contact with a drier, and presumably, higher energy
surface.
A Perkin-Elmer-Hitachi model 139 U.V. Vis spectro
photometer was used for concentration determinations. Separa
tion of particulates from supernatent solutions was carried
out by centrifuging or by filtering through micropore glass
filters. In those cases where filtering was used, the filter
was first saturated with a solution of the organic molecule to
be analyzed, then rinsed thoroughly. In all cases a standard
was used which had been put through identical procedures as the
unknown.
Protein and polypeptide determination was made through
the use of Biuret reagent [87 ] and measuring at 550 millimicrons.
Carbohydrates were determined using a phenol solution and
measuring at 490 millimicrons [88]. Amino acid concentrations
were determined by use of ninhydrin [873. Carboxylic acids
were titrated with phenothalein as the indicator.
Distilled water, having an initial pH near 7, was used
for preparing solutions. The pH was varied by adding HC1 or
NaOH. Three solutions were used as solvents: a low ionic strength
solution (LISS) in which the only ions present were those added
by pH adjustment, a 0.165 M salt solution, and a buffered solution

38
The concentration of NaCl in the low ionic strength solution
did not exceed 0.001 M.- The 0.165 M salt (Ringers) solution
consisted of. 9 gm/1 of NaCl, 0.25 gm/1 of CaCl2, and 0.42 gm/1
of KC1 and is known. The buffered solution was a 0.2 H solution
of mono- and di-basic phosphate. The phosphate buffer was
used only in Ringers solution bringing the total molarity to
*
0.365.
Except in those instances where solids were not used
at all, 0.1 gm solid particulates were added to the solution.
The solids were placed into suspension by mixing 1.0 gm of solid
powder with about 18 ml of distilled water, adjusting the pH
and then bringing final volume to 20 ml. One hour was allowed
for the pH to equilibrate. With stirring, 2 ml aloquots were
distributed into 10 tubes by pipetting. Using this method 0.1
gm 0.01 gm of solid was delivered to each tube.
The gold calorimeter reaction cells were washed daily
with detergent. The washing procedure included injecting a
solution of the surfactant into each compartment, rotating the
calorimeter and withdrawing the solution by aspiration. The cells
were then continually rinsed with distilled water while being
evacuated. By moving the tip of the aspirator tube from the
top of the cell to the bottom in one compartment, while filling
the other compartment, a good turbulent rinsing reaction
developed. Experience showed that five minutes of such procedure
cleaned the cell. Approximately once a week, the detergent
was left in the cells overnight to permit it to react more
thoroughly.

39
Between daily experiments, the same procedure was used
to clean the cells. The oxide powder slurry was removed for
analysis after mixing, however, and the detergent was not used.
Cleaning with 1 M HC1 or NaOH was found to be necessary only
rarely.
Organic constituents were weighed out to 1 .01 mg
and mixed volumetrically. Adjustment of pH was the same as
with solids. Incremental concentrations were made from standard
batches and measured volumetrically by pipet. Distribution of
organic, and solid, solutions into the reaction cell was made
by syringe. The lowest concentration was always used first.
The materials used as substrates include silica, alumina,
and tricalcium phosphate. The silica [89] was described by the
manufacturer as amorphous. It has. a specific surface area of
o
0.7 m /gm and a pzc of 3. The alumina used [90] consisted of
two types: Linde A, a a-alumina of specific surface area 15 m /gm
and a pzc of near 9 and Linde B, a mixture of y and a alumina
of specific surface area 82 m /gm and a pzc near 9. The tn-
calcium phosphate, referred to as hydroxyapatite in the text,
consisted of 85 volume per cent hydroxyapatite and had a specific
surface area of 57 m /gm. Surface measurements were made in
this laboratory using a multi-point B.E.T. nitrogen adsorption
isotherm. Measurements of pzc were also conducted in this
laboratory using a Zeta meterR [91],
All experiments were run at 25 C.
All solids were used without modification. Prior to
weighing, large (approximately 10 gm) batches of solids were

40
rinsed in distilled water, decanted and evacuated for 24
hours at 10 atm. Prepared powders were stored under vacuum
at room temperature.
All organic substances used were stored under refrigeration
prior to use. None was repurified or modified in any way.
Batch solutions were used within one week and were stored at
4C. Particular information on each substance used is given
in the appropriate chapter.
Reaction heat data were plotted against Co and fed into
a'statistical analysis program available through the Northeast
Florida Regional Computing Center. The reaction heat, Q, was
held as the independent variable. The program generated an
approximating function which was used to calculate the thermo
dynamic data. In all chapters, referral to "calculated thermo
dynamic data" refers to this procedure. The graphs of Q versus
CQ presented are original data.

CHAPTER IT
ADSORPTION OF CARBOXYLIC ACIDS
ON ALUMINA AND HYDROXYAPATITE
Introduction
Several of the amino acids which make up collagen
possess charged side groups, principally amines and carboxyl.
The polysaccharide, chondroitin sulfate, often associated with
collagen, also possesses the carboxyl groups as well as the
sulfate group. Because each of these charged species has the
potential to interact with an aqueous oxide surface, knowledge
of the type and strength of the surface reaction is sought.
The thermodynamic and adsorption behavior of molecules
containing the sulfate group have been previously described.
The adsorption of sodium dodecyl sulfate on alumina has been
studied by calorimetric techniques [31] and by solution depletion
techniques [91]. Heats of adsorption for this molecule have
values near -6 kcal/mole at low concentrations when only ionic
forces drive the adsorption reaction. The free energy change
lies near -11 kcal/mole of ions. The adsorption of sulfate
ions onto solid barium sulfate from aqueous solution has been
measured by the thermometric titration technique. The heat of
precipitation was found to be -4.5 kcal/mole .[84].
41

42
The adsorption of alkyl ammonium acetate on quartz
has been followed as a'function of temperature and concen
tration [92-93].it was found that at 25C at neutral pH the
isoteric heat of adsorption was between zero and 2 kcal/mole
of ions. The positive enthalpy was expected because of charge
repulsion of the quartz surface and the negatively charged
ions in solution. The free energy change at 25 remained near
-3.5 kcal/mole. In most cases the hydrocarbon chain of the
molecules caused abrupt changes in the thermodynamic properties
due to lateral interaction of the adsorbing molecules as the
equilibrium concentration increased.
Binding studies of sulfates, citrates, and amino acids
on calcium oxalate and calcium phosphate have been carried
out in relation to bone formation and kidney stone growth
[94v95], Thermodynamic data are not readily available for these
reactions. Equilibrium constants of magnesium oxalate [94 ],
however, have been shown to be near 4000 which would correspond
to a free energy change near -5 kcal/mole for this precipitation
reaction.
The purpose of this chapter is to discuss the variation
of the heat of adsorption of simple and polymeric carboxlic
acids on alumina and hydroxyapatite in relation to surface charge,
molecular conformation and ionization and solution pH. Qualita
tive results indicate that while electrostatic interactions
are required to initiate adsorption, other interactions such
as hydrogen bonding, take place.

43
The sodium salts of acetic acid, oxalic acid, citric
acid, and polyacrylic acid (PAA), containing one, two, three
and multiple carboxyl groups were selected for study. The
different number of charged groups, different degrees of
ionization and molecular structure of each of these molecules
should provide a sufficient variety of detectable changes in
the calorimetric measurements. Analysis of the various changes
should furnish a clear understanding of the adsorption
mechanism.
The structural; formulas of each of the molecules used
in the work described in this chapter are given below:
H3C-C00H
H00C-C00H
Acetic
Acid
Oxalic
Acid
H OH
1 \
HOOC C C -
i i
H
1
H
1
H
1
C COOH
1
1
(r C -
I
1
C -h
I
H H
COOH
i
H
1
COOH
Citric
Acid
Repeat
Unit
of
Polyacrylic
Acid
Experimental
Sodium salts of the carboxlic acids were purchased
from Sigma Scientific, Inc. [96]. Poly(acrylic acid) in a
65% aqueous solution was purchased from Aldrich Chemical
Company C97] and was reported by them to have a molecular weight
of 2000. Linde A alumina, and hydroxyapatite were used as
substrates.

44
Titrations of the carboxlic acids were carried out
with HC1 or NaOH. Determination of the amount of acid
adsorbed was based on. calibration against known concentrations.
Other methods and procedures were described earlier.
Results
Reaction heats for the adsorption of the three simple
carboxylic acids on alumina are presented in Figure 4. For
each acid, the maximum reaction heat was found to occur when
the solution was near pH 5. The reaction heats at pH 3 were
second and the lowest curves were recorded for pH 7. The
highest heat was recorded for sodium oxalate which also has
the lowest dissociation constant (see Table 2).
The enthalpy change upon adsorption was first found
by determining the amount of each acid adsorbed by titration.
The method was suitable only for pH 5. At pH 3 and 7 poor
precision resulted because of the small amount of each acid
adsorbed. Results are given below:
acetic oxalic
-AH (kcal/mole molec.) 4.2 4.8
(CQ .01M) -
Thermodynamic data were calculated for pH 5 using a
smaller concentration range (C = 10-L* to 10^ M) (see Figure
5).. These results show that there are differences in the
enthalpy change for the three acids used. Each curve displays
a tendency towards more negative (exothermic) values at the
lower end of the concentration range. The heat of adsorption
citric
4.6

MCAL.
45
| | I J
4 3 2 1 0
- LOG CQ
Figure 4._ Reaction heats for the adsorption of three
carboxylic acids' on alumina.

Table 2
Acetic
4.75
Dissociation Constants
of Carboxylic Acids
Oxalic
1.23
4.19
Citric
3.14
4.17
6.39

kcal./mole
47
0. 2
0.4
0.6
0.8

48
lies between -6 and -24 kcal/mole. The free energy changes
are nearly equal at about -5.5 kcal/mole, while the entropy
changes are each negative and lie between -10 and -20 cal/mole*
deg (1 cal/mole'deg = 1 entropy unit or e.u.).
Calculated values for the thermodynamic functions for
the adsorption of the three carboxlyic acids on hydroxyapatite
are presented in Table 3. The values show less variation for
hydroxyapatite than they did for alumina. The enthalpy
change tends to be less negative than for alumina, while the
free energy and entropy changes are about the same.
Polyacrylic acid (PAA) had such a high affinity for
alumina and hydroxyapatite that a cotton-like gel formed at
pH 5 and 7, causing great difficulty in cleaning the gold
reaction cells. Three concentrations were run with alumina,
however, and were repeated several times to improve precision.
Titration determinations of the amount of PAA adsorbed leads
to an enthalpy of 82 cal/gm of PAA for the adsorption reaction.
Taking 72 gm/mole as the molecular weight of the monomer, the
determined enthalpy change is -5.7 kcal/mole of acrylic acid
monomer assuming all acid groups participate in the-adsorption
reaction.
The'calculated thermodynamic data for the adsorption
of PAA onto alumina.are presented in Figure 6. The enthalpy
change per residue lies between -7 and -12 kcal/mole, while
the free energy change is concentration invarient at -5 kcal/
mole. The entropy change is negative as it is with the other
carboxylic acids used in this section.

LO LO LO
49
Table 3
pH
7
7
7
Themodynamic Variables for the Adsorption of
Carboxylic Acids on Hydroxyapatite at pH 5 and 7
Substance
-AG
(kcal/mole)
. -AH
(kcal/mole)
AS
(cal/moledeg)
Acetic
5.4
3.3
-7.0
Oxalic
5.1
9.0
13.0
Citric
5.5
11.0
18.0
Acetic
6.1
5.4
-2.3
Oxalic
5.2
9.8
15.0
Citric
5.1
17.0
39.0

-kcai./mol
50
0.02 0.04 0.06 0.08
CQ, mM
Figure 6. Thermodynamic data for the adsorption of poly
acrylic acid on alumina at pH 7 in low ionic strength solu
tion; AG AH AS. .
SV-

51
Discussion
As molecules are adsorbed from solution, other
molecules will ionize to try to maintain the original concen
tration. At the same time the alumina or the hydroxyapatite
will act as a buffer to maintain the pH. So long as the pH
is constant, the degree ionization of the solute will remain
the same. This leads to a qualitative explanation for the
reaction heat curves of Figure 4.
The total ionic charge in solution is directly propor
tional to the degree ionization, a, which is related to the
pH by [98]:
pH = pKQ log [ (1 a)/a ] (2.1)
where pK is defined as
o
pK = -In [H+] [A-] (2.2)
[HAT
This relation holds for single as well as polyelectrolytes
[99].
In the absence of any specific interaction between a
solid and electrolytes in solution, the surface potential is
given by [5 8] : '
i¡j = RT In a/a (2.3)
iF
where z is the valence (including sign) of the potential
ion, F is the Faraday constant, a is the activity of the
potential determining ion in solution and aQ is the activity
of the potential determining ion at the pzc. As mentioned
in the introduction, the potential determining ion for the

52
systems under consideration is H+. The surface potential
may then be written from equation (2.3) as
= 2.3 RT (log [H+] log [Hn]) (2.4)
where concentration are substituted for activities and
where [H ] is the hydrogen ion concentration at the pzc.
The interaction due.to the surface potential and the
charge, q, in solution gives rise to an interaction energy,
E [64]
E = f ijj dq (2.5)
where integration is necessary since the charge concentration
is a differential process. The surface potential affecting
ions in solution will decrease as saturation of the surface by
charged ions is approached. The total interaction energy is
nearly equal to ^Qq in this model if the concentration of charged
ions in solution is low so that there is little interaction of
the adsorbed ions on the surface. In this case the surface
potential which each ion encounters will be the same. The
energy lost by the ions is transferred to other ions and solvent
in the form of kinetic energy and flows as heat out of the
system.
A plot of \p from equation (2.4) and a from equation (2.1)
o
versus pH for a hypothetical monovalent acid with a pKQ near 5
and an oxide with a pcz near 9 is given in Figure 7. At low
pH, a, and thus the charge in solution decreases. At high pH
the surface potential decreases. The highest interaction should
be expected to occur where and a are not near zero.

pH
Figure' 7. Plot of and a for a hypothetical acid.
pH
Figure 8. Values of the reaction heat calculated by use
of equation 2.6 and found by calorimetric experiments.

54
For example, at pH 5 equation (2.4) gives for a
monovalent electrolyte '(H+)
ip0 = .059 (9 5) = .24 volts
The total charge in solution, using acetic acid as the mono
valent acid at .001 M is
q = a Ane (2.6)
= 1.78x10~5(6x1023)(2x10~6)(1.6xl0~19)
2.7 8xl0-5
= .12 coul.
where A is Avogadros number, n is the total number of mole
cules in solution, and e is the electric charge. The total
electronic interaction energy is
' E = q
= (,24) (.12) = .028 j = 6.7 meal
The value of Q found from microcalorimetry is 4.7 meal. The
agreement is fair. Plots for other values of Q and E for other
acids at different pH values are shown in Figure 8 for CQ =
.001 M. Although the experimental and theoretical results do
not fit well for all cases, the simple model qualitatively
explains the experimental findings, which is what was sought.
Such factors, as treating the ions in solution as other than
point charges, and the potential at the Stern layer are not
taken into account. The most important result is that the
reaction heat and electronic interaction energy decrease as
either \pQ or a decrease. The tenative conclusion develops that
electronic interaction will probably be the essential factor

55
in enthalpy changes measured for charged adsorbing molecules.
The enthalpy and free energy change values are within the
range expected from the investigations of other workers.
Some idea of the number of active groups of each
molecule actually on the surface can be obtained from the
following analysis. Using the data from Table 4.and Table 5
the average number of ionized groups on acetic acid, oxalic
acid, or citric acid can be determined from their dissociation
constants. The numbers are given in Table 4. If we divide
the enthalpy change per molecule by the average number of
ionized groups. AH" is obtained. This assumes that the enthalpy
change for the adsorption of carboxyl groups is about th
same regardless of the molecule being considered. Results are
given in Table 5 for pH 5 and 7 at .001 M.
The values for AH* are fairly consistent in each case.
Considering the enthalpy change of the adsorption for acetate
as an arbitrary baseline, we can argue that if AH* for oxalate
or citrate had been much larger than that for acetate it
would have implied that more groups per molecule were participat
ing in the surface reaction than expected from the degree of
ionization. As is,AH* for oxalate is slightly lower and citrate is
slightly higher than the AH* for acetate at both pH values.
Oxalate is the most strongly dissociated of the three acids
implying, perhaps, a slightly stronger reaction with the surface.
It should be realized that a particular group cannot be partially
ionized at a particular instant in time. The invarience in

en co
56
pH
Table 4
Ionized Groups per Molecule
for Three Carboxylic Acids
Acetic
0.02
0.63
1.0
Oxalic
1.0
1.85
2.0
Citric
0.42
1.9
2.8

5 7
Table 5
Enthalpy Change per Mole,
AH, and
Enthalpy
Change per
Mole of Ionized
Groups,
AH*
PH
Substance
-AH
ionized
-AH*
(acids)
(kcal/mole)
groups
(kcal/mole)
5
Acetic
3.3
0.63
5.20
5
Oxalic
9.0
1.85
4.86
5
Citric
11.0
1.90
5.90
7
Acetic
5.4
1,00
5.40
7
Oxalic
9.8
2,00
4.90
7
Citric
17.0
2.80
6.10

58
AH* Is evidence that only ionized groups participate in
adsorption. This supports the supposition that electro
static attraction is primarily responsible for adsorption.
The same procedure applied to the data for the adsorp
tion of the three acids on alumina does not give such consis
tent results. At pH 5 AH* lies between -6 to -11 kcal/mole
for acetate, -2 to -7 kcal/mole for oxalate and -7 to -3 kcal/
mole for citrate.
Near CQ = .4mM (see Figure 5), a medium concentration,
we find that AH* lies near -9.8 kcal/mole for acetate, -4.1
kcal/mole for oxalate and -8.4 kcal/mole for citrate. This
indicates that while acetate has one, and citrate two ionized
surface groups, oxalate has on the average only one of its
two ionized groups on the surface.
It- was argued earlier that only ionized molecules are
attracted to the surface and that the number of such molecules
drops as the surface charge drops. It should be recalled
that the surface charge on alumina and hydroxyapatite is
produced by the adsorbed H+ ions. This suggests the possibility
that an approaching ionized carboxyl group and surface hydrogen
ion participate in hydrogen bonding.
Zeta potential measurements [100] show that citrate
Changes the surface charge of hydroxyapatite at low concentra
tions. This indicates that electrostatic attraction by itself
is not the only factor in adsorption. If it were, when the
surface charge had been neutralized by adsorption of a sufficient
amount of citrate, adsorption would have ceased and the surface
charge would not reverse.

59
The single charged acetate ion does not reverse the
surface charge of hydroxyapatite or alumina, this would
indicate the absence of other than electrostatic interaction.
It will be recalled from the introduction that close approach
of hydrogen to an anion is required for hydrogen bonding to
occur. Since, at pH 7, acetate has one and citrate has three
ionized groups, it is thought that multiple groups are required
to pull the molecule close enough to the surface for hydrogen
bonding to occur. The interplay between surface charge,
molecular size and charge density, ionic and hydrogen bonding
becomes apparent in these situations.
Polyacrylic acid has such multiply-charged groups. It
is known to be a linear molecule which is fully ionized at pH 7
[ ]; therefore, there are no hydrogen bonds to be broken due
to an unfolding of the molecule upon adsorption. The AH between
-5.7 and -6.9 kcal/mole of residues of PAA is close to that
found for the adsorption of the carboxyl group of the other
molecules on alumina and hydroxyapatite. The similarity in all
these instances implies that the same type interaction occurs,
and is not a strict function of surface composition or of
molecular structure.
Conclusions
There appears to be no particular differences in the
enthalpy of adsorption of a carboxyl group onto alumina or
hydroxyapatite due to the number of groups on a molecule. In
each case the enthalpy change is near -6 kcal/mole and an

60
attracting force is required to initiate adsorption. Other
wise a plot of E or Q versus pH would be similar in shape to
the a versus pH and not bell shaped.
Once adsorption occurs, the influence by multiply-
charged groups on the molecule was evidenced by a change in
surface charge. For this condition to arise, specific adsorp
tion has to occur which requires forces other than electro
static. The proximity of oxygen in the carboxyl groups and
H+ on the surface suggest hydrogen bonding.

CHAPTER III
ADSORPTION OF CHONDROITIN SULFATE
AND OTHER CARBOHYDRATES
Introduction
Polysaccharides, notably chondroitin sulfate (CS),
which contain carboxyl and sulfate groups, are present in
dentin and enamel [101] and in bone [102,103]. Under proper
conditions of pH and ionic strength, these polysaccharides
will complex with collagen [104], In the presence of a
foreign surface, these polysaccharides, like other charged
molecules, will adsorb and react not only with collagen but
also with the surface. It is the purpose of the studies of
this chapter to explain the interaction of aqueous solutions
of chondroitin sulfate with alumina, hydroxyapatite, and
silica. A later chapter will discuss the interaction of CS
with collagen.
Chondroitin sulfate is a polysaccharide made up of
basic dimer units of glucoronic acid and galactosamine. Several
simpler carbohydrates were chosen to model CS: (a) galactose,
glucose, D-acetyl galactosamine; (b) glucose-6-sulfate (G6S) ,
D-galacturonic acid; (c) dextran, and (d) polygalacturonic
acid (PGA). Each carbohydrate was chosen because it possessed
a single feature of the CS molecule: (a) the carbohydrate
61

62
residue; (b) a charged carbohydrate; (c) the polymer back
bone structure; and (d) the charged polymer. The structural
formulae for these molecules are shown in Figure 9a and 9b.
Experimental
All carbohydrates were purchased from Sigma Chemical
Company [96] and used without further purification. The
chondroitin sulfate (sodium), dextran, and polygalacturonic
acid were reported to have molecular weights of 45,000, 60-
90,000, and 25,000 respectively. The chondroitin sulfate was
determined to be chondroitin-6-sulfate by infrared spectroscopy
using the KBr pellet technique, and had a molecular weight of
45-60,000. The materials used as substrates, Linde B alumina,
hydroxyapatite, and silica, and the low ionic strength solution
were described previously.
Results
The first set of experiments was conducted with
galactose, glucose, and dextran (see Figure 9b). None of these
molecules possesses charged groups. By measuring the heat of
adsorption of these molecules on the oxides, the contribution
to the total enthalpy change on adsorption of uncharged
carbohydrate monomer and polymer could be estimated. The initial
concentration, CQ, was varied from .01 to .1 moles/liter. By
comparison with the reaction heats produced by the carboxylic
acids in this concentration range, it was estimated that 20 to
40.meal would be considered a significant reaction. The results

o
Figure 9. Molecular structure of chondroitin sulfate (a)
( D-glucoronic acid N-acety galactosamine-6-sulfate)
(b) the three-fold helix of chondroitin sulfate.

64
?
HO C H
Galactose
CH20H
CO
CH3
D-acetyl
Galactosamine
COOH
OH
H
HO C ~ H
Glucose
0S0
I
ch2
OH
HO
)H
OH
Glucose-6-Sulfate
Galacturonic acid
Figure 9b. Molecular structures of the carbohydrates
used in the experiments of this section.

65
are presented in Figure 10. The maximum reaction heat, Q,
produced was about .3 meal at a concentration of .1 M on
alumina. The enthalpy change, as determined by solution
depletion, was approximately -100 cal/mole adsorbed for galactose
and glucose on alumina. For dextran, AH was about -20 cal/
mole of residues (.11 cal/gm) and -15 cal/mole of residues
(.8 cal/gm) on hydroxyapatite.
The enthalpy change for the adsorption of D-acetyl
galactosamine on alumina was found to be -430 cal/mole of
molecules. Using the thermometric titration method described
earlier, the free energy change was found to be -5.2 kcal/mole.
The enthalpy change for the adsorption of this molecule on
hydroxyapatite was found to be -400 cal/mole of molecules.
Calorimetric measurements for the adsorption of D-
galacturonic acid on alumina indicated a stronger reaction than
with the uncharged carbohydrates. The reaction heat, Q, reached
a maximum of 8.9 meal. The calculated enthalpy change and
that determined by solution analysis are presented in Figure 11
and lie near -10 . 5 -kcal/mole. There is good agreement for the
enthalpy change using both methods except in the lower concentra
tion range where the calculated values are more negative. The
free energy change varies between -4.6 and -5.5 kcal/mole
adsorbed. The entropy change is negative and lies between -8
and -30 cal/mole*deg per molecule adsorbed.
Measurements of the adsorption of D-galacturonic acid on
hydroxyapatite showed a similar enthalpy change to that on

66
Figure 10. Reaction heats for the adsorption of dextran a ,
galactose a, and glucose o, on alumina (closed symbols) and
hydroxyapatite ( open symbols) in low ionic strength solution
at pH 7.

meal. kcal./mole
67
O'
40
30
20
10
i
C
.01 .02 .03 .04
Figure 11. Thermodynamic data for the adsorption of D-galac-
turonic acid on alumina at pH7 in low ionic strength solution;
AG AH AS Enthalpy change determined by solution
analysis

68
almina, as determined by solution depletion (see Figure 12).
Values for AH lie between -6 and -8 kcal/mole.
The adsorption of D-galacturonic acid on silica is
endothermic. At low concentration, the enthalpy is +700
cal/mole, becoming more positive at higher concentrations.
The amount adsorbed was determined by solution depletion. The
free energy change and entropy change were not calculated.
The change in enthalpy for the adsorption of glucose-6-
sulfate on alumina, hydroxyapatite, and silica was determined
to be -7.6, -5.4, and 0.3 kcal/mole of adsorbed molecules,
respectively.
PGA
Calorimetric measurements for the adsorption of poly-D-
galacturonic acid on alumina and hydroxyapatite were hampered
by agglomeration of the particles by the polymer. The thermo
dynamic functions for this reaction were calculated and are
shown below.' In the concentration range used, .001 M to .01 M
of residues, these values were constant ( 0.1 kcal/mole).
AG
AH
AS
(kcal/mole)
(kcal/mole)
(kcal/mole/
deg)
alumina
-3.4
-2.54
3.5
hydroxyapatite
-4.2
-.31
15.0
In contrast to
the decrease in
entropy found
for the
adsorption of PAA on
alumina or hydroxyapatite, the
entropy
change is positive for adsorption of
poly-D-galacturonic acid
on both alumina and hydroxyapatite indicating an over-all
decrease of ordering.

AH, kcal./mole
69
Figure 12. Heat of adsorption for D-galacturonic acid on
silicae and hydroxyapatite in low ionic strength
solution at pH 7.

7 0
CS
The results of the calorimetric measurements of the
adsorption of chondroitin sulfate (CS) on alumina are
presented in Figure 13. Comparison of the enthalpy of
adsorption for CS on silica, alumina, and hydroxyapatite are
given below:
AH
Silica
+ 2.46
Alumina
-1.85
Hydroxyapatite
-2.47
The enthalpy changes for the adsorption of CS on the
uncharged hydroxyapatite is found to be 15-30% more negative
than that for the positively charged alumina. The enthalpy
change for adsorption of CS on silica is found to be positive
(endothermic) and was determined by solution depletion, the
free energy change AG was not calculated.
Discussion
Glucose, galactose, and dextran are uncharged molecules
Because of the availability of hydroxyl groups it is possible
that these molecules can undergo hydrogen bonding with an
oxide surface. The low value of the enthalpy change for the
adsorption of these molecules, however, does not indicate very
strong reaction with the oxide surface in comparison with the
charged molecules.

Figure 13. Thermodynamic data for the adsorption of chon-
droitin sulfate on alumina in low ionic strength solution
at pH 7; AG AH AS .

The only difference in the experimental conditions
in using the uncharged versus charged molecules is
attributable to the functional groups. Therefore the large
differences in AH observed are seen as due to the presence of
the charged groups.
If it is assumed that, in the case of dextran, only
two or'three points of contact are made per molecule, then
the enthalpy change could be on the order of 3-4 kcal/mole
molecules. Comparison of dextran with poly-d-galacturonic
acid (PGA), however, still shows that dextran is much less
strongly bound than PGA.
In the concentration ranges used, 10 M dextran
molecules (not residues), there is little hydrogen bonding
of dextran chains to one another [76,98], Therefore, the
breaking of interchain hydrogen bonds should not contribute
substantially to the low enthalpy change actually measured.
If the hydrogen bonding does take place between the oxide sur
face and many dextran residues, it is not manifested in the
measured enthalpy change. In the absence of other interactions
it is concluded that the charged groups of the carbohydrate
molecules are necessary to provide sufficient attraction of
the entire molecule to the surface.
The increase in the heat of adsorption of D-acetyl
galactosamine over that of galactose is attributed to the
presence of the NH2COCH3 side chain. The exact cause can only
be speculated. Perhaps the nonpolar methyl group is forced
from solution by more polar solvent ions, drawing the molecule

73
to the surface. Whatever the mechanism, if these uncharged
molecules are strongly bound to the surface, it is not
reflected in the enthalpy determination. The type of bond
which would occur would almost certainly be hydrogen.
In any event, the adsorption of D-galacturonic acid
on alumina and hydroxyapatite is much more energetic than that
of galactose or galactosamine. The similarity in the molecules
and in the adsorption experiment strongly suggests that the
charged carboxyl group is responsible for the higher enthalpy
change and that binding between solute molecules or desolvation
effects do not account for the noted change. The negative
entropy change indicates an overall increase in ordering. This
increase in ordering may be due to confinement of the carbo
hydrate molecules to the surface and subsequent loss of freedom
[21].
Adsorption of D-galacturonic acid on silica produces a
positive enthalpy change. This can be accounted for by the
charge repulsion which exists between the surface and the molecule
There was a finite amount of acid adsorbed, however. This
would indicate perhaps a second stronger force necessary to
overcome the charge repulsion or that the negatively charged
molecules are occupying the fewer positive sited on the silica
surface, or reaction with high energy sites. Since the reaction
heat and adsorption measurements leveled off quickly, the last
two possibilities appear more likely; especially in view of the
finding that charge attraction appears necessary for strong
reaction.

Likewise the adsorption and calorimetric measurements
of glucose-6-sulfate suggest that the presence of a charged
group on the carbohydrate, opposite to that of the surface,
is required for stronger (more exothermic) reactions. The
negative sulfate group is attracted to the positive alumina
surface. As with'D-acetyl galacturonic acid on silica, the
adsorption of glucose-6-sulfate on silica is endothermic. -
Poly-D galacturonic acid is obviously strongly attached
to the alumina and hydroxyapatite surfaces. The enthalpy
change is more negative for alumina, than for hydroxyapatite,
demonstrating the greater attraction for this surface. If
we consider that the enthalpy change is produced by the
charged groups bonding to the surface, then, using a figure
of -9kcal/mole as the enthalpy change found for the adsorption
of D-galacturonic acid on alumina, we can estimate that one
in three residues bonds to the surface. For hydroxyapatite,
this figure is perhaps one in twenty or thirty.
Chondroitin Sulfate
The chondroitin sulfate molecule is known to exist in
a threefold or eightfold helix [105,106] which is rigid in
solution [107] (see Figure 9). It possesses the ability to
change the conformation of positively charged polypeptides
from an extended coil to a helical structure (see Chapter 5).
The sulfate group extends further away from the carbohydrate
backbone than does the carboxyl group which is located on the
other side of the same dimer. The acetyl amine group extends

75
slightly further away from the backbone than does the
carboxyl and is located^ on the same side of the backbone.
From these considerations--helical structure, position of
the charged groups, and possible steric hinderanceit is
reasonable to assume that not all the charged groups participate
in bonding with the surface at the same time,
To help analyze the binding of CS to a surface, consider
that the interaction of a single dimer with the surface
permits interaction of both the carboxyl group and sulfate
group with the surface. Both groups would then contribute to
the enthalpy of the reaction. From the data in this chapter
and the previous one, it is seen that the change in enthalpy
is fairly constant for each type molecule, as it is between
carboxyl groups and that only charged groups contribute
significantly to the reaction heat, Q. The reaction heat due
to the adsorption of a carboxyl and sulfate group would be
between -12 and -16 kcal/mole. The measured enthalpy value is
about -2 kcal/mole of dimers for alumina and between -2.2 and
-2.8 kcal/mole for hydroxyapatite. Dividing the total enthalpy
possible by the measured value would indicate that between one
in three to one in seven dimers interact with the surface.
We may assume that only one of the charged groups inter
acts -per dimer. Using an enthalpy change between -6 and -12
kcal/mole of charged groups then one in three to one in five
groups would be indicated as interacting with the surface.
From the physical picture and the calorimetric data it seems
plausible to conclude that the chondroitin sulfate molecule is

76
positioned horizontally on the surface with approximately
one out of every four dimers on the average coming into
contact with the oxide surface. In this instance, the charged
group interacting with the surface would be the sulfate group
since it extends further away from the CS backbone.
The entropy change due to adsorption of CS on alumina
is positive, indicating an increase in entropy or a decrease
in the order of the system. Since the molecule is rigid in
solution and is not likely to greatly change conformation on
the surface, no entropy contribution is attributable to a
change in shape. An increase in entropy can be attributed to
a release of solvent molecules from around the molecule or
from the surface into solution [106,107].
The adsorption of CS on silica can also be explained by
an increase in entropy. The surface charge on the silica is
the same as that on the carbohydrate. Overall, there is an
electrostatic repulsion between the surface and the charged
molecule. There must be some other energy supplied to over
come this repulsion. Since it is the free energy change which
drives the reaction, and AH is positive, there must be at
least an equivalent positive entropy change so that TAS is
greater than AH. This entropy change, as suggested above,
can be supplied by the solvent ions.
It is difficult to speculate on the conformation of
CS on the silica surface as was done above with alumina. This
is so because the carbohydrate monomers are not attracted to
the surface of the silica as they are to alumina because of
charge repulsion.

77
Conclusions
In this section we have determined some of the thermo
dynamic features of the adsorption of chondroitin sulfate on
alumina, hydroxyapatite, and silica by the use of model
carbohydrates. The results show that for positively charged
alumina the enthalpy change for the adsorption of charged
carbohydrates is about the same as that for the carboxylic
acids and lies between -7 and -9 kcal/mole of adsorbed species.
In these experiments the entropy change is negative and the
enthalpy change forms the major portion of the free energy
change. The enthalpy change for the adsorption of the charged
monomeric carboxylated carbohydrates on hydroxyapatite is close
to -7.6 kcal/mole. The adsorption of carbohydrates containing
the sulfate group on alumina or hydroxyapatite is about -5.4
kcal/mole. The similarity in enthalpy for the reaction suggests
a similar type reaction. The adsorption of charged carbohydrate
monomers on silica is weak and endothermic. The uncharged
monomers produce an enthalpy change only a fraction of that of
the charged monomers.
It was found that a model of the uncharged polymer backbone
of CS does not produce a large reaction heat or an enthalpy
change, suggesting that the presence of charged groups is
required to enhance the adsorption reaction. The data on
adsorption of polygalacturonic acid supports this conclusion.
Polygalacturonic acid adsorbed strongly, producing
agglomeration of the solid particles at high concentration (.1 M).

78
The enthalpy change per residue is lower than that found
for its monomeric counterpart. The conclusion drawn here,
as with polyacrylic acid in the previous chapter, is that
fewer points of contact are made, but that each point of
contact contributes essentially the same heat change as the
monomer. The entropy change was positive further increasing
the driving force.
Chondroitin sulfate was shown to adsorb to each of the
oxide powder substrates. The negative enthalpy change for
alumina and hydroxyapatite indicates that adsorption is strongly
enhanced by the opposite charges. Since the chondroitin sulfate
molecule is comparatively bulky relative to the models used,
fewer points of contact would be expected. The thermodynamic
calculations show, however, that perhaps as much as one-third
to one-fourth of possible bonding sites touch the surface.
The results for CS on silica are more speculative. The
enthalpy change is positive and it is assumed that the major
contribution to the free energy change of adsorption is from
a positive entropy change related to the molecule size. Smaller
molecules were not found to produce a positive entropy change.
Relying on the results of the previous chapter, it
is assumed that once the molecules are attached to the surface,
that hydrogen bonding will take place.
In a subsequent chapter, we will investigate the type
interaction which CS and other molecules undergo with collagen
and collagen models. In the next chapter a study of the adsorp
tion of molecules possessing amine side groups is discussed.

CHAPTER IV
ADSORPTION OF POLYPEPTIDES
Introduction
The calorimetric measurements discussed in the previous
chapters were related to the adsorption of molecules which
possessed a carboxyl group. The results showed that the mole
cules on which the carboxyl group was ionized displayed greater
reaction heat and adsorption density than those molecules
which were not ionized. In this chapter, molecules containing
charged amine side groups are studied for the possible informa
tion they can give on the adsorption of collagen onto silica,
alumina, and hydroxyapatite.
Several of the primary amino acids and their respective
polymers were investigated as collagen models. The molecules
used were alanine, poly-l-alanine (PA), proline, poly-1-
proline (PLP), poly-l-hydroxyproline (PLHP), lysine, poly-1-,
lysine (PLP), and poly-l-arginine (PLA). Lysine and arginine
and their polymers possess basic side chains. The structural
formulae of the monomers are given below. The polymers are linked
at the carboxyl and amino groups.
79

80
CHoCHCOO"
+nh3
Alanine
+h3n(ch2)4choo
nh2
Lysine
H0NCNH(CH?)oCHOO"
2 ii 2 i
+nh2 nh2
Arginine
The amino acids are dipolar ions. for the dibasic
amino acids, arginine and lysine, adsorption onto negative
surfaces will be enhanced. In polymeric form only poly-1-
lysine and poly-l-arginine retain any charge in neutral
solution.
The solubility in water of the other amino acids will
decrease as a result of their polymerization. Acidic amino
acids, aspartic acid and glutamic acid were not studied because
the carboxyl group has been discussed in the previous chapters.
Furthermore, interpretation of calorimetric data would be
difficult because of the presence of three charged groups.
ho7\
^ )coo
N
h'-sh
Hydroxyproline
>C00
.N
h' nh
Proline

81
Experimental
The amino acids used in this section were purchased
from Sigma Chemical Company. Both amino acid monomers and
polypeptides were chloride salts, except for poly-l-lysine,
which was a bromide salt. The molecular weights of the poly
peptides were reported to be: 1,000-5,000, poly-l-alanine;
15,000-50,000, poly-l-arginine; 70,000, poly-l-lysine;
10,000-30,000, poly-l-proline; and 10,000-30,000, poly-1-
hydroxyproline. '
The substrates and the solvent are the same as used
in the previous chapter. A few experiments were performed in
the .165 M salt solution (and will be indicated as such in
the text).
Mixing and calculation procedures were described
earlier.
t
Results
Alanine
Calorimetric and adsorption results for alanine and
PA on the three oxides are presented in Figure 14. The
amino acid monomer adsorbs strongly on all three surfaces at
this pH. It acts much as the carboxylic acids do. There is
no sharp endpoint in the Q vs. Cq curve for the monomer, but
the slope of the surve is greater than that for the polymer.
The enthalpy change for each surface tends towards -5 to -8
kcal/mole of monomers.

ENTHALPY (-Kcal/mole)
82
C0 mM
Figure 14. Heats of adsorption for alanine and poly-l-alanine
(PA) onto alumina, silica, and hydroxyapatite (TCP) in low
ionic strength solution at pH 7. run in .165M salt solution.

83
When the dissolved salt (R-Cl) concentration in the
solvent is increased to .16 M, the enthalpy change of the
adsorption of alanine on alumina decreases in the higher
concentrations range of alanine, but tends toward the -6
kcal/mole in the lower end.
The polymer, PA, exhibits much different behavior
showing no specific tendencies to adsorb. Reaction heats are
less than .5 kcal/mole and enthalpy changes are of magnitude
less than -1 kcal/mole.'
Proline
Measurements made with proline and PLP and hydroxy-
proline and PLHP (see Figure 15) show results similiar to
results for alanine and PA. The monomer again produces a
higher enthalpy change than the polymer- (-4 to -8 kcal/mole).
However, PLP and PLHP are apparently more strongly attracted
than the poly-l-alanine with heats of adsorption lying between
-CL 5 and -2.5 kcal/mole of residues. PLHP is somewhat more
strongly attracted on all three surfaces than is PLP. An
increase in ionic strength is noted by a decrease in the
enthalpy change for PLHP on alumina.
Lysine and Arginine
The addition of charged side groups causes a marked
change in the enthalpy curves, as shown in Figure 16. The
heat of adsorption of lysine on silica is, as expected,

84
Figure 15. Heats of adsorption for proline (P), hydroxyproline
(HP), poly-1-hydroxyproline (PLHP), and poly-l-proline (PLP),
on alumina (A), silica (S), and on hydroxyapatite (HA) in low
ionic strength solution and Ringers solution (*).

-AH, kcal./mole
b b
Figure 16, Heats of adsorption for lysine (L), poly-l-lysine
(PLL), and poly-l-arginine (PLA), on silica (S), alumina (A),
and hydroxyapatite (HA) in low ionic strength solution at
pH 7. Values determined by solution analysis e others
by calculation.

86
greater in magnitude than on alumina and is more pronounced
in the lower concentration end. The enthalpy change lies
in the range of -2 to -8 kcal/mole of monomers. The greatest
enthalpy change, however, for the polypeptides was recorded
for PLA on alumina. The second largest was on hydroxyapatite
and third on silica. This order also happens to be the order
of decreasing specific surface area. The enthalpy of adsorp
tion of PLA was greater than that for PLL on hydroxyapatite.
The free energy change for PLA and PLL lie in the
range between -4 and -7 kcal/mole of residues (see Figure 17).
There is a sharp decrease in the free energy change of PLA
on hydroxyapatite at low concentrations. The entropy changes
for these molecules are small because of the similarity of
AH and AG.
Discussion
Alanine
The higher enthalpy change for the adsorption of
alanine, compared to poly-l-alanine, is due to the electro
static attraction of the amine group or the carboxyl group
to the surface. PA, having no charge except for its terminal
groups is not strongly adsorbed despite its greater molecular
weight. PA is in a helical form [20], not coiled.
Since the molecule is uncharged, the reaction heat of
the entire molecule due to adsorption only would be small in

8
H 6
O
e
ii
o
4
CD
<
I
PLA-HA
2
i i i i
0.0 0.2 0.4 0.6 0.8 1.0
C0, mM
Figure 17. Free energy change for the adsorption of poly-1-
lysine (PLL) and poly-l-arginine (PLA) on silica, alumina and
hydroxyapatite at pH 7.

88
comparison with ionized molecules [50]. A large conformational
change would then cause the overall reaction to be endo
thermic. The molecular concentrations are low. Therefore,
breaking of intermolecular hydrogen bonds should contribute
little to the enthalpy change.
Proline
The charged monomers of proline and hydroxyproline
are also more strongly attracted to the oxide surfaces than
their polymers (Figure 15). This is taken as a result of
electrostatic attraction. The heat of adsorption, measured
by solution depletion and found to be between -4 and -6 kcal/
mole for both monomers, results from reaction of the charged
groups with the oxide surface.
Calorimetric measurements for the adsorption of PLP
and PLHP did not show a specific pattern for any of the
surfaces. Both of these molecules have a helical conformation
in solution [20], There was no attempt to determine whether
or not this structure was grossly disturbed upon adsorption,
or if it was, what contribution to the enthalpy change such
a disruption would make. Neither was there an attempt to
determine how many points of contact were made. The definite
conclusions which can be drawn from these data are relatively
few. There are some reasonable assumptions, however, that can
be made which, if accepted, will further explain the situation.

Since these molecules are in an extended conformation,
not coiled, there are no intramolecular hydrogen bonds. At
low concentrations (2 x 10"5 M of residues) there should be
little intermolecular hydrogen bonding [50]. The disruptions
which would primarily occur, then, would correspond to
rotational movement of the molecule [19], There is no
reason to suspect that these molecules should undergo grotesque
distortion on the surface since there are no strong attractive
forces. Therefore, the contribution to energetic changes due
to conformation alterations should be small.
Poly-l-proline and poly-l-hydroxyproline possess a
ring structure which is relatively nonpolar compared to the
polar solvent. Because of this, it is plausible to assume
that these less polar structures are in a lower energy state
on the surface, rather than in the solution. This is termed
hydrophobic bonding and could possibly account for the energetic
changes measured if most of the residues were near the surface
and not surrounded by the mobile polar ions in solution.
By comparison with the carbohydrates studied earlier
and in absence of detailed information on the geometric
smoothness of the surface, it would also be possible to suggest
that only a few points of contact are made on the surface [50],
Each' of these contacts would assume higher energy than
indicated by the average of 1-2 kcal/mole of residues measured.
If these contacts are hydrogen bonds made up of hydrogen
atoms on the ring structure and oxygen atoms on the surface,

90
then each bond would entail an energy change of about
-7kcal/mole. On the average then one out of 7 residues
would be in contact with the surface.
Comparison of the dsorption of these uncharged
molecules with those that possess charged functional groups
indicates that the role of PLP or PLHP would be minor in
comparison. Although the mechanism of adsorption has not
been fully explained in this case, there should be little
doubt that when positioned next to a charged molecule in a
peptide chain, the latter will play the dominant role in
adsorption to an oxide surface.
Lysine and Arginine
The adsorption heats of lysine on silica and alumina
are similiar (Figure 16). Since this molecule possesses two
basic and one acidic group at neutral pH, it is reasonable
to assume that the charged carboxyl group is attracted to
the positive alumina surface, and that the positive amine
groups are attracted to the negative silica surface. Because
of the more basic properties of this molecule, it might be
suspected that the reaction with the silica surface would be
somewhat stronger than with the alumina surface. This appears
to be the. case.
Poly-l-lysine and poly-l-arginine are known to exist
in an extended charged coil conformation in solution at
neutral pH [8,9']. If the coils, which are stabilized by

91
hydrogen bonding, were to break down, the enthalpy change
would be due to both the adsorption and the unfolding processes
Enthalpy changes measured in this and the two previous chap
ters indicate that enthalpy changes between -6 and -8
kcal/mole of single, charged groups are to be expected. The
magnitude of the unfolding process, the breaking of hydrogen
bonds, would lie between 5 and 7 kcal/mole [19,82], an
endothermic process. The resulting enthalpy changes for both
processes would be comparatively small with a value near 0
kca.l/mole. Such a situation is encountered in experiments
presented in the next chapter where the coil-to-helix
transition is known to take place. Instead,the enthalpy
change is much more negative, decreasing in magnitude from
-14 kcal/mole to -6'kcal/mole. The variation is thought to
be due to adsorption on the fewer negatively charged sites
on the alumina and hydroxyapatite surface. These sites possess
a distribution of high to low energy within their own group.
Adsorption of PLL or PLA to neutral high energy sites
can be eliminated since PA, PLHP, and PLP each have heats of
adsorption which are smaller in magnitude. If the adsorption
had not depended on surface charge the neutral molecules
should have been just as strongly attracted to the surface.
The order of decreasing enthalpy change (alumina >
hydroxyapatite > silica) corresponds to the specific surface
area of the solids. The higher specific area of the alumina
(87m^/gm) provides more edges and peaks which are assumed to

form high energy sites. For equal amounts adsorbed, a
greater fraction of adsorbed molecules would be on these
sites for alumina, than for either hydroxyapatite or silica.
Negatively charged polysaccharides also displayed an increase
in enthalpy change at lower concentrations attributable to
high energy sites. More than a single species of these
specially adsorbing areas appears likely [72]. The free
energy changes are of the same order of magnitude as those
found for the carboxylic acids and polysaccharides.
Conclusion
As found in the previous chapters, molecules with
charged ionic side groups react more energetically with oxide
surfaces than those without. The enthalpy change per
charged group lies between -4 and -10 kcal/mole. For those
polyamino acids which possess no charged side groups, the
magnitude of the enthalpy change is found to be less, near
-1 kcal/mole of residues. Although the mechanism for uncharged
molecules is not clearly defined, it is believed that conforma
tional deformation does not contribute significantly to the
enthalpy changes measured. It is possible than an uncharged
polyamino acid could be bound to an oxide surface by a few
relatively high energy contacts. Polyamino acids with charged
side groups, however, will play the dominant role in adsorption

CHAPTER V
REACTION OF PEPTIDES AND
CARBOHYDRATES IN SOLUTION
Introduction
In the earlier chapters, an investigation of the
adsorption of molecules onto oxide surfaces has been discussed.
These calorimetric studies of compounds which are models for
collagen provided some information on the state of the adsorbed
molecules, the energy changes on adsorption and the type of
interaction they undergo.
in vivo. it is unlikely that a collagen molecule will
come into contact with a clean surface. In general, there
will be other substances present in the body fluids which
will adsorb first because of factors such as greater concentra
tion. Also, collagen may not be present at all when the
hydrated oxide surface is first exposed to body fluids [102].
We should have some indication, therefore, of how these adsorbed
molecules will affect the adsorption of collagen. In order
to understand their interaction at a liquid-solid interface,
it would be helpful to first investigate their interactions
in solution.
93

94
It is the purpose of this chapter to provide further
insight to the interaction of collagen, chondroitin sulfate,
and serum albumin in solution.
The same compounds which have been used previously
to model collagen and chondroitin sulfate have been used
here. For collagen they are poly-l-arginine (PLA), poly-.l-
lysine (PLL), poly-lalanine (PA), and poly-l^proline (PLP).
For chondroitin sulfate they are dextran, galactose, galacturonic
acid, and polygalacturonic acid.
The reaction of chondroitin sulfate with collagen has
been studied by model systems, as mentioned in earlier
chapters. The reaction of collagen with dyes containing acidic
groups and with CS have been studied in regard to the under
standing of the role of CS and collagen in connective tissue
[108]. it was found that the cationic groups of collagen bond
with the anions of the dyes and CS in a pH range of 1.5 to
7 with a sharp drop in the number of anions fixed below pH 2
and a more gradual decrease from maximum adsorption at pH 3
to zero at pH 7.
In experiments to determine the role of CS in the
calcifying mechanisms of bone [109], it was found that calcifi
cation would not occur or would occur more slowly in an aqueous
collagen mixture when CS was not present. These experiments
were performed near pH 7. The authors suggested that binding
of CS to collagen at neutral pH would aid the natural calcifica
tion process.

9 5
In another experiment with chondroitin sulfate [13]
and cationic dyes, it was concluded that aggregation of
dyes on the surface of the CS rather than ionic interaction
was mainly responsible for bonding. The thermodynamic functions
indicated an enthalpy change of -7 to -12 kcal/mole of dye
molecules.
The binding of cobalt hexammine (Co(NHg)g+3) to connective
tissue, micropolysaccharides, heparine, and sulfated chitosans
has been studied by a spectrophotometric procedure [110]. The
cobalt hexammine was used to represent amino functions of fibrin.
Ion pair formation was found to be the primary binding mechanism,
but was influenced by local binding factors, electrostatic attrac
tion of neighboring charged groups, and competition with other
cations for binding sites.
Such previous work generally indicates that CS-collagen
or CS-polypeptide binding will be primarily ionic, pH, and
structure dependent. These previous results and the results
discussed in this chapter will serve as an aid in understanding
later calorimetric measurements.
Experimental
In these calorimetric measurements, a solid substrate
was not used. In the first set of experiments, the concentra
tion of saccharides was held constant at a concentration CQ
_ O
of 10 M of saccharide monomers or residues. In the case of
CS, this refers to dimer residues. Aquisition of the organic

substances is as mentioned earlier. Variations in pH were
made through additions of HC1 or NaOH.
The thermometric titration technique was used to cal
culate the thermodynamic functions. Solution depletion was
also used to determine the enthalpy change and used as a check
on the calculations.
The collagen macromolecule used in this study is more
precisely termed tropocollagen. It is the precursor of the
large fibril seen in electron micrographs. It is synthesized
extracellularly by enzymatic reaction from procollagen [75].
Tropocollagen has a reported molecular weight of 285,000.
The molecular weight of the collagen used in these experiments
was found to be 290,000 as determined by gel chromotography
using BSA as a standard, and 300,000 as determined by the
manufacturer by request. The molecule consists of three similar
polypeptide chains which make up a rigid triple stranded helix
3,000 A long and 15 A in diameter. Each chain has about 1,000
amino acid, residues, one third of which are glycine, the
smallest of the amino acids. In human tendon, proline and hydroxy
proline make up about 22% of each molecular chain, whereas
lysine and arginine acid and glutamic acid make up 12%. The
greater percentage of acid groups brings the IP down to 5.5.
In this work, tropocollagen is referred to simply as collagen,
and was supplied by Aldrich Chemical Company [97] in an
aqueous solution.

97
Bovine serum albumin (BSA) supplied by ICN Life
Sciences Division [111] was a powdered preparation. It has
a molecular weight of 69,000 and an IP of 4.9.
Results
Poly-l-arginine
The results of the mixing of poly-l-arginine (PLA) and
various carbohydrates is shown in Figure 18. The mixing of
CS and PLA at pH 5 and 7 forms a precipitate in solution which
is filterable. Experiments for pH 5 and 7 show identical
results. There is a nearly constant increase in reaction
heat with an increase in concentration of PLA. Near CQ =
2 x 10-3 M, the curve levels and remains constant. The
enthalpy change for this reaction (Figure 19) was determined
to be -7.50 kcal/mole of PLA residues.
The mixing of the uncharged molecules presents a
different,picture. The mixing of galactose or dextran with
PLA showed little reaction heat and no precipitate formation.
Both showed a negative reaction heat between an initial mixing
concentration of zero and 2.0 M of PLA. The calculated enthalpy
change lies between +30 and -200 cal/mole of residues for
dextran and +50 and -30 cal/mole for monomers of galactose.
The variation is unexplained, but may be due to conformational
changes.
The results of the mixing of D-galacturonic acid or
glucose-6-sulfate with PLA are similar. Their reaction heats

meal.
98
4
3
2
O
-1
1.0 2.0 3.0 4-0
C mM PLA
Figure 18. Reaction heat, Q, for the mixing of PLA with
various carbohydrates:(PGA) polygalacturonic acid, (GAL)
galactose, (DEX) dextran, (DGA) D-galacturonic acid, (G6S)
glucose-6-sulfate, (CS) chondroitin sulfate. Run in low
ionic strength solution at pH 7.
J 1 i l

kcal./mole
99
CQ, mM PLA
Figure 19. Reaction heats for the mixing of PLA with various
carbohydrates. Enthalpy change determined from solution
analysis Abbreviations are the same as in figure 18.

100
are positive. The calculated enthalpies show strongly-
increasing trends at loyer concentrations and lie in the
range of -.4 to -1.0 kcal/mole of PLA residue.
Polygalacturonic acid (PGA) was mixed with PLA at two
pH values: 7 and 13. At pH 7, the mixing reaction appeared
to be endothermic. There was no precipitate formation as there
was with CS and PLA. If PGA is mixed with PLA at pH 13, a
precipitate will come out of solution. When the pH is lowered,
the precipitate dissolves. The reaction heat curve at pH 13
(Figure 18) of PLA and PGA reflects this interaction.
Poly-1-lysine
The reaction heats of mixing CS and poly-l-lysine (PLL)
(see Figure 20) are negative. The enthalpy changes for pH 5
and pH 7 are not the same as with PLA. At pH 5 the enthalpy
is near 200 cal/mole of PLL residues while at pH 7 it is near
400 cal/mole of PLL residues (see Figure 21). In both cases
a filterable precipitate forms. The reaction of PLL with
glucose-6-sulfate is exothermic and similar to that of glucose-
6-sulfate with PLA. There is no reaction evident between
dextran and PLL. The thermodynamic parameters associated with
the above reactions are given in Table 6 in terms of moles of
PLA or PLL residues.
Several experiments were run with PA and PLP with the
various carbohydrates (see Figure 22). The thermodynamic
functions were not calculated. The low magnitude and shape of
the reaction heat curves indicate little interaction for any

meal
101
J I i
1 2 3 4
C0 mM
Figure 20. Reaction heat, Q, for the mixing of PLL and various
carbohydrates in low ionic strength solution at pH 7 except as
noted.

1 2 3 4
CQ mM
Figure 21. Enthalpy change produced by the mixing of PLL
and CS at pH 5 and 7 in low ionic strength solution. Cal
culated values- determined by solution analysis-.

103
Table 6
Thermodyanmic Variables for the
Mixing of PLL and PLL with CS
PH
Substance
AG
(kcal/mole)
AH
(kcal/mole)
AS
(cal/mole
5
PLL
-8.9
0.20
21
7
PLL
-7.8
0.49
19
5,7
PLA
-8.6
-0.75
25

meal.
104
. 7 5
. 5 0.
.25
0.0
0 1.0 2.0 3.0 4.0 5.0
CQ, mM polypeptide
Figure 22. Reaction heats for the mixing of various poly
peptides with carbohydrates. The carbohydrate concentration
was held oenstant at .001M. Run at pH 7 in low ionic strength
solution; (PA) poly-lalanine, (PLP) poly-1-prline.
-
PLP-PGA
PLP-CS
-
PA-GAL
o PA- PGA
-
1 1 L
rA-Lo
^ e PA-PGA
i

IOS
of the combinations involved. There is no trend or break
in the reaction heat curves as there is for CS and PLA or
PLL.
Collagen
The thermodynamic data for the mixing of CS and collagen
are presented in Figure 23. The results for pH 5 and 7 are
identical. The date were calculated using the thermometric
titration methods. The enthalpy values appear high because
they are written in terms of moles of collagen. The free energy
change is always negative and shows an inflection near .06 M
of collagen. When the experiment was repeated in Ringers
solution, the reaction heat and enthalpy change did not show
significant deviation from the experiment run in low ionic
strength solution.
BSA .
The mixing of bovine serum albumin (BSA), PA, PLP, and
PLA are presented in Figure 24. The concentration of BSA was
held constant at .7 gm/1 which approximates the molar concentra
tion of the CS macromolecule. The reaction of PLA shows a
definite reaction pattern whereas the reaction heat curves of
BSA with PLP or PA are more linear. This enthalpy change for
the PLA-BSA interaction was calculated to be -50 kcal/mole of
BSA molecules with AG = -59 kcal/mole and AS = 30 cal/moledegree

AG, AH, cal./mole
106
to
I
o
I1
40
30
20
10
0
n>
h
x
hO
Figure 23. Thermodynamic data for the mixing of CS and col
lagen in low ionic strength solution at pH 7. Run in
Ringers solutions AG AH AS .
0T

meal.
10.7
Figure 24. Reaction heat, Q, for the mixing.of BSA with
FLA, PLA, and PA in low ionic strength solution at pH 7.
The BSA concentration was held constant at .7 gm/1.

108
Mixture of CS and Collagen in the Presence of BSA
Finally, the mixing of CS and collagen in the presence
of BSA in low ionic strength solution and in Ringers solution
is presented in Figure 25. The results for those mixing
reactions are similar to that of the mixing of CS and collagen
in low ionic strength solution without BSA present.
Discussion
Poly-l-lysine, Poly-l-arginine
In experiments discussed earlier in this chapter, where
the reaction heat was considered evidence for strong inter
action, it was agreed that electrostatic attraction was probable.
The mixing of PLA or PLL with CS formed a strong complex,
while the mixing of PLA or PLL with dextran did not. The pattern
fits those established in the previous chapters which is that
the charged polymers react strongly with oppositely charged
polymers (or surfaces).
The enthalpy change determined for the PLL or PLA-CS
reaction is low. It is known that PLL or PLA undergoes a
conformational change when mixed with CS [8,9]. Even though
calorimetric data are not available for these transitions,
similar unfolding reactions [19,20] indocate enthalpy changes,
which correspond to the breaking of the hydrogen bonds, on the
order of 2 to 7 kcal/mole of residues. It is assumed that the'
transitions of PLA or PLL would cause a similar enthalpy change.

-AH, cal./mole
109
0 O.14 0.8 1.2 1.6 2.0
C0, yM Collagen
Figure 25. Enthalpy change for the mixing of CS and collagen
in the presence of BSA: low ionic strength solution ,
Ringers solution without BSA in low ionic strength
solution .

110
If this were the only contribution to the enthalpy change,
the overall reaction would be endothermic. A negative
contribution can be provided by the binding of the polysaccharide,
CS, to PLA or PLL. The enthalpy change for the binding of
cationic dyes to CS has been shown to be near -8 kcal/mole
C13 3 The data presented earlier show that the mixing of
G6S or DGA with PLA or PLL have enthalpies, at low concentra
tion, near -1 kcal/mole. In the absence of any other major
contributions to the reaction, the mixing of CS with PLA or
PLL then consists of two components: a conformational change
of the polypeptide and the binding process. There is no
conformational change for CS [8,9]. The total enthalpy change
corresponding to these two processes would tend to cancel,
resulting in a low overall enthalpy change. In the case of PLL
and CS the enthalpy change is small and positive (endothermic).
The more exothermic reaction of PLA over PLL with CS is
attributed to the more basic guanidine group of PLA rather than
the E-amine groups of the PLL side chain.
D-gaiacturonic Acid
The large negative enthalpy change for D-galacturonic
acid and glucose-6-sulfate measured in the lower concentrations
of PLA used (Figure 18) indicate that strong interactions
are occuring in this range. The enthalpy change for CQ = .05 mM
to 1 mH are equivalent to that of CS and PLA. Since all the
sites for eac.h molecule are assumed to be identical, the
curvature in the AH curve cannot be due to different high

Ill
energy sites as claimed for solid surfaces. As stated,
however, the PLA molecule is folded, and whereas CS has a
directional stabilizing effect on the polypeptide, the
isolated monomer does not.
In the former case, the ionized groups of the poly
peptide would be exposed as the molecule is uncoiled by
relatively few CS molecules. This would allow a one to one
or two to one correspondence between basic polypeptide side
groups and the acid groups on CS [8,9]. In the latter case,
the single carbohydrate molecules at lower concentration,
without the advantage of a high linear charge density of CS,
can bind only to those basic groups on the polypeptide which
are exposed. At higher concentrations, more basic groups would
bind but other factors as charge repulsion and steric hinderance
of the charged carbohydrate molecule have increasing effect.
These factors have less of an effect on the CS molecule at
equivalent charge concentration. Therefore, the trend towards
higher energy at low concentrations is attributed to the
availability of positively charged groups on the polypeptides,
and steric and charge repulsion between monosaccharides.
Polygalacturonic Acid
The reaction of PLA with polygalacturonic acid (PGA) is
complex. PGA at pH 7 is in an extended helical structure [98].
At pH 7, no precipitate forms with PLA of PLL and the reaction
heat is lower than found with CS. It is known that CS that has

11.2
had the sulfate groups removed does not react with PLL or
PLA in the same manner as natural CS [8,9], It is also known
that the carboxyl groups of PGA (and CS) are charged at pH 7,
so that electrostatic attraction exists between PGA and PLA.
It was shown that if the pH of the PGA-PLL mixture is raised
above 11, that a precipitate does form. As pH increases, charge
neutralization of the polypeptide side chains increases, and
intramolecular hydrogen bonding of the polypeptide becomes
weaker [76]. One may speculate that PGA can then impose
directional constraints- on the conformation of the PLA molecule.
The calorimetric measurements of PGA and PLA at pH 13 (Figure
18) support this argument, since as the pH is adjusted from
7 to 11 the enthalpy change drops from +0.5 kcal/mole to -0.3
kcal/mole of P1A residues.
Poly-l-alanine, Poly-l-proline
The interaction of CS with PA or PLP (Figure 22) is
apparently of the same type as found with PLA and dextran. PA,
PLP, and CS are each found in a helical conformation at pH 7
[112]. There is no unfolding of the polypeptides as with
PLA to complicate the interaction. The lack of initial attraction
is what apparently prevents extensive reaction as found with
CS and PLA. It is as if, for these molecules, there is only a
single species in solution.
CS and Collagen
The isoelectric point of collagen is 5.5. At pH 7, it
maintains an overall negative charge. The CS-collagen mixing

113
is exothermic. In previous experiments negative enthalpy
changes were produced by the interaction of oppositely-
charged species and it is suggested then, that the anionic
groups of CS are binding to the cationic groups of collagen.
This result is in agreement with other workers who suggest
ionic interaction between CS and collagen [104 ,105 ]..
The enthalpy change found for the mixing of CS and
collagen in Ringer*s solution demonstrates the dielectric
effect of salt. This is apparent at the higher concentrations
of collagen used. At concentrations (CQ) below about .12
ymole of collagen, the enthalpy change is similar to that
found in the low ionic strength solution. Above this concen
tration the deviation becomes greater. This indicates that
those CS molecules which bind last are affected more by the
presence of the solvent ions. The enthalpy change per CS dimer
lies between -500 cal/mole and -700 cal/mole. If the binding
reaction is responsible for the greater portion of the enthalpy
change, then about one in seven to one in twenty dimers make
contact with the collagen molecule. This assumes an enthalpy
change of 5 kcal/mole to 10 kcal/mole per contact.
BSA
The BSA macromolecule is negatively charged at pH 7.
Therefore, both PLA and PLL would be elctrostatically attracted
PA, PLP, and collagen would be neutral or slightly affected.
Both BSA and PLA are in a coiled conformation in solution,
whereas the other polypeptides are in extended helical confor-

114
mation [ 36 50 ]. The apparent lack of reaction for the
extended molecules seems to be due to the fact that they
are extended. The two coiled molecules are deformable [49]
and may adjust their conformation to increase surface area
contact with each other. For BSA and PLA, assuming from
previous data that each point of interaction contributes about
-7 kcal/mole and that these interaction points contribute
most to the enthalpy, there are about seven or eight points of
contact per molecule. Even though the1 reactions which take
place appear to be of an electrostatic nature, they occur
most extensively when the structure of the molecules are similar
The Mixture CS, BSA', and Collagen
In the presence of BSA, collagen and CS interact with
each other much in the same way as previously when the collagen
concentration, C is low, indicating that BSA will not strongly
affect the CS-collagen interaction. As CQ is increased, the
effect of the BSA becomes more prominent. It is suggested, in
explanation, that at low Cc only CS reacts with the relatively
few collagen molecules. At higher C CS is increasingly taken
from solution so BSA molecules may react, thus lowering the
overall average enthalpy change. The enthalpy change is. less
exothermic in the presence of BSA than in its absence when the
ionic strength is increased.
As the ionic strength of the solution is increased, the
intramolecular hydrogen bonds of!'BSA become weaker [82]. It

IIS
would then be easier for the molecule to denature. Consequently,
more extensive contact'between collagen and BSA would appear
to be possible than in the low ionic strength solution. This
increased contact would interfere in collagen-CS interaction.
Conclusions
There appears to be substantive evidence suggesting
that the attraction of PLA or PLL to chondroitin sulfate is
initiated by ionic forces. It was seen that uncharged mole
cules reacted much less strongly with PLA or PLL than CS.
The charged carbohydrate polymers also react more strongly
than the charged monomers. It is probable that the interaction
of the two macromolecules will include hydrogen bonding once
they come into contact. The reactions of CS and collagen also
appear to be ionically induced as mentioned in the beginning
of this chapter, with an enthalpy change between -500 cal/mole
and -700 cal/mole of CS dimers.
The interaction of BSA and collagen appears to be neg
ligible in comparison to that of CS and collagen. Although the
BSA may block isolated points on the collagen molecule, it
does not appear to inhibit the collagen-CS interaction. This
would suggest that binding to a surface in the presence of BSA.
By increasing the ionic strength of the solution, the
magnitude of the enthalpy changes of all interactions decreases.
In very high concentrations (1.0 M NaCl) of ions, binding of
CS by collagaen practically ceases [82]. In regard to surfaces

116
which have high surface potential, attracting a high
concentration of counter ions, or surfaces which may release
ions into solution, this result has much significance. On
these surfaces protein denaturation would be at a maximum, while
protein-protein or protein-carbohydrate interaction would be
at a minimum. Such effects will be discussed in the following
chapters.

CHAPTER VI
ADSORPTION OF COLLAGEN
Introduction
In the previous chapter, it was determined that ionic
interactions are of fundamental importance in the initial
reactions between carbohydrates and polypeptides. The findings
also suggested that molecular conformation is of importance
when the two interacting molecules are of different shape. In
this chapter measurements of the adsorption of collagen onto
silica, alumina, and hydroxyapatite are discussed as a function
of concentration in relation to these findings. The effect of
adsorption of collagen on alumina in the presence of bovine
albumin and chondroitin sulfate is also presented.
Experimental
Procedures and sources of chemicals have been previously
described. Linde B alumina was used in these experiments.
Results
Alumina and Collagen
The results of the calorimetric measurements of the
adsorption of collagen onto alumina are shown in Figure 26.
117

118
C0, mg/ml
Figure 26.
Reaction heat for the adsorption of collagen onto alumina in
three solutions at pH 7.

119
The results are presented for runs made in low ionic strength
solution, in Ringers solution, and in Ringer's solution contain
ing a phosphate buffer. In low ionic strength solution the
reaction heat was lowest, in the presence of Ringer's solution
it was increased by a factor of six and in the presence of
phosphate buffer the reaction heat decreased to the value near
that of the experiment run in low salt solution.
The thermodynamic data are presented in Figure 27. The
values appear high because they are written in terms of moles
of collagen (Each molecule is a triple helix of molecular
weight 300,000). While the free energy changes and entropy
changes are similar for each solution, the enthalpy changes
reflect the change in solvents. All quantities are negative.
In a separate experiment, alumina was mixed as a slurry
in the phosphate buffer solution, permitted to sit for on
hour, centrifuged, and washed in distilled water three times.
The alumina was finally centrifuged and run against collagen
(see Figure 27) in pH 7 Ringer's solution. The enthalpy is
nearly the same as that found in the buffered solution.
Hydroxyapatite
The reaction heats and thermodynamic data of the adsorption
of hydroxyapatite are presented in Figure 28 and 29. The
reaction heats are of similar magnitude for each solvent, and
negative as they are with alumina. There are inflection points
noticed in the reaction heat curves for the reaction run in

AH, cal./mole
120
12
10
CO
'O
r
X
CD 4
<3
RINGERS
LOW IONIC.
STRENGTH
\
~ 2
0 d>
101
-fr
BUFFERED
AG = 10'
?
0
0
0.83
0.25
1.67
0.5
C
5
75
3.3
1.0
yjM
gm/1
Figure 27. Thermodynamic data for the adsorption of collagen
onto alumina at pH 7; AH AS; The free energy change
was approximately equal in each cas to-lO3 cal/mole. Enthal
py change determined by solution analysis --e;-

meal.
121
Figure 28. Reaction heat for the adsorption of collagen onto
hydroxyapatite at pH 7 in three solutions.

122
0.5 1.0 1.5 2.0
l
n>
C
X
H
O
I
-P
C0, uM collagen
Figure 29. Thermodyanmic data for the adsorption of collagen
onto hydroxyapatite at pH 7; AH AS The free energy
change in each case was near -104 cal/mole.

123
Ringers solution and buffered Ringers solution. The enthalpy
change for the experiment run in Ringers solution reflects
similar changes in slope.
Silica and Collagen
The mixing of collagen and silica in low ionic strength
solution is endothermic (Figure 30). When the dissolved salt
concentration is increased the reaction becomes exothermic.
In the buffered solution the reaction heat is increased again
over that found in Ringers solut-ion. The magnitudes of the
thermodynamic variables are generally smaller than those found
for alumina and hydroxyapatite (see Figure 31).
Alumina and BSA
BSA was mixed with alumina in increasing increments in
Ringers solution. Enthalpy was determined by solution depletion
measurements. There is a sharp break in the reaction heat
curve, but the adsorption curve is continuous (see Figure 32).
This causes a commensurate break in the enthalpy curve. When
BSA and alumina are mixed in buffered Ringers solution, the
break in the reaction heat curve disappears (see Figure 33).
Hydroxyapatite and BSA
When BSA is mixed with hydroxyapatite, the reaction heat,
adsorption curves, and enthalpy changes are smooth and without
discontinuities. The enthalpy change is about -5 cal/gm. The
results are the same for the buffered or unbuffered solutions.

meal
124
Figure 30. Reaction heat for the adsorption of collagen onto
silica at pH 7.

AH, cal./mole
-3
125
CD
I
O
H
-2
1
LOW IONIC STRENGTH
o~ ,
BUFFERED
~o
RINGERS
RINGERS
LOW IONIC STRENGTH
0.12
0.25
_L
0.38
0.5
CD
h
i
o,
CO
-2
-4
C0, pM collagen
Figure 31. Thermodynamic data for the adsorption of collagen
onto silica at pH 7; AH AS- . Enthalpy change determined
by solution analysis,----.
5

mg. BSA adsorbed Q, meal.
126
Figure 32. Thermodynamic data for the adsorption of BSA
onto alumina at pH 7 in Ringers solution.
-AH, cal./gm BSA

127
CQ, grn/1 BSA'
Figure 33. Heats of adsorption for the adsorption of BSA onto
alumina, silica, and hydroxyapatite in several solutions.

128
Silica and BSA
The heat of adsorption of BSA on silica was < 0.5 cal/gm.
The adsorption curve indicated that saturation was not
reached at the highest equilibrium concentration of 1 gm/1.
Only the buffered solution was used.
Alumina, BSA, and Collagen
The following mixture of alumina, BSA, and collagen was
performed' as indicated below:
reaction cell
( 1
compartment 1 compartment 2
alumina collagen + BSA
The concentration of the collagen was held constant at .3 gm/1.
The concentration of BSA was varied as before. The heat of
dilution of the collagen and of the BSA was nulled out in the
reference cell.
The reaction heat curve of the collagen-BSA mixture first
decreases from 12 meal to 7 meal, then rises. The turning
point coincides with the discontinuity described in the previous
discussion. If the reaction heat curve for the BSA alone is
subtracted from the reaction heat curve for the mixture, the
result (dotted line in Figure 34) is an approximate reaction
heat for the collagen adsorption. The amount of collagen
adsorbed could be determined by solution depletion. The amount
of BSA adsorbed was previously determined. The enthalpy change
for the adsorption of collagen in the presence of BSA could then
be estimated.

mg. collagen ads. Q., meal
129
Figure 34. Adsorption reaction parameters for the adsorption
of collagen in the presence of BSA at pH 7 in Ringers solution.
cal./gm. collagen adsorbed

130
This enthalpy (dashed line in Figure 34) decreases from
the region of 30 cal/gm to 7 cal/gm near .1 gm/1 BSA. Beyond
.1 gm/1 BSA, the enthalpy rises to about 15 cal/gm, decreasing
gradually as the BSA concentration increases.
Hydroxyapatite, Silica, BSA, and Collagen
The adsorption and reaction heat curves for the adsorption
of collagen on hydroxyapatite and silica in Ringers solution
show no discontinuities. The amount of collagen adsorbed
decreases slightly, but the enthalpy remains fairly constant
near -7 cal/gm and -1 cal/gm for hydroxyapatite and silica
respectively.
Alumina, BSA, CS, and Collagen
To determine the effect of BSA and CS on the adsorption
of collagen, the following measurement was made:
reaction cell
I : 1
compartment 1 compartment 2
alumina + CS + BSA Collagen + CS
The heat of dilution of the collagen and CS was accounted for
in the reference cell. The concentrations of the collagen
and CS were held constant at .3 gm/1 and 10 mole/1 respective
ly. The concentration of the BSA was varied in compartment'1
to yield the same final concentration from 0 to.l gm/1.
The adsorption and reaction heat curves for each species
of molecules follows the same pattern (see Figure 35). They
gradually decrease, then at about .1 gm/1 BSA, there is a

131
C gm/1 BSA
Figure 35. Heat of adsorption of collagen onto alumina in the
presence of BSA and CS ,; adsorption of collagen----.
Adsorption of CS in the presence of collagen and without
collagen presente.

13 2
sudden rise followed by another gradual decrease. More
CS is adsorbed when collagen is not present. Cs is present
in the same solution as the alumina prior to mixing with the
collagen.
The heat of adsorption for collagen decreases in the
presence of CS. The step is still present but an overall
decrease of 5 to 10 cal/gm is seen.
In the buffered solvent the discontinuities disappear
(see Figure 36). As before, the amount of CS adsorbed decreases
in the presence of collagen even- though it was already in
solution with the alumina. Lesscollagen is adsorbed in the
presence of CS. The enthalpy change for the adsorption of
collagen is between -5 and -7 cal/gm.
Discussion
Collagen on Alumina, Hydroxyapatite, and Silica
The adsorption and calorimetric measurements produce
many findings. In agreement with the observations discussed in
previous chapters, there is a definite trend to an increase in
the exothermicity of the adsorption of collagen with an increase
in surface charge (see Figure 37). This result suggests that
the absorption of collagen on oxide surfaces is controlled by
ionic attractive forces.
The increase in dissolved salt concentration produces
an increase in enthalpy for equal amounts of collagen adsorbed.
One might expect a decrease in AH because the dielectric constant
of the medium is increased. One suggested reason for the increase

13 3
C, gm/1 BSA
Figure 36. Adsorption of collagen onto alumina in the presence
of BSA and CS in buffered Ringers solution at pH 7.

PZC
Figure 37. Relationship between the point of zero charge,
pzc, and the heat of adsorption of collagen in the three
solutions used.

135
in AH is that more of the collagen molecule comes into contact
with the surface. As the ionic strength of the solution is
increased, the hydrogen' bonds within the molecule weakens be
cause of their ionic character [50,82]. This would permit a
loosening of each strand permitting increased contact of the
molecule with the surface.
This loosening could also explain the change from an
endothermic reaction as the ionic strength is increased for
the adsorption of collagen on silica. This loosening would
perhaps allow the anionic carboxyl groups on the collagen mole
cule greater freedom either to move away from anionic, or move
to cationic sites on the silica surface. As a result, there
would be less charge repulsion and the heat of adsorption
would become exothermic.
In phosphate buffer, the high energy reaction for alumina
disappears. The simplest explanation is that the phosphate ions,
which are known to chemisorb to alumina [61], are preferentially
occupying high energy sites. It was suggested that these sites
cause most of the denaturation when collagen adsorbs to alumina.
The phosphate buffer has less of an effect on adsorption of
hydroxyapatite or silica since it is already present in the first
material whereas in the second it is repelled by the negatively
charged silica surface.
BSA
BSA exhibited an unusual calorimetric behavior upon
adsorption on alumina in Ringers solution. The calorimetric
behavior can be explained if the molecule denatures (unfolds)
and covers a relatively large area. Near .1 gm/1 the increased

136
concentration prevents the molecules from unfolding. This
situation is depicted in Figure 38. Instead of being denatured,
the molecule is assumed to remain in a globular shape. The
slope of the Q vs. concentration curve remains smooth over the
range measured indicating that the change in reaction heat is
not due to a change in the number of molecules adsorbed.
BSA and Collagen
The adsorption of collagen on alumina in the presence of
BSA in Ringers solution follows a pattern which is the inverse of
the reaction heat curve for the adsorption of BSA on alumina.
It appears that the collagen is adsorbed on the remaining
free surface after BSA adsorption. It was also found that the
enthalpy change per molecule of adsorbed collagen in the presence
of BSA is fairly constant despite the shape of the adsorption
curve. It was shown earlier that the reaction of collagen and
BSA, assumed to be globular, is weak. These three facts combine
to predict that the collagen reacts only in a minimal way, if
at all, with adsorbed BSA. The step indicates some reaction
in this state. It is conjectured that it is the higher charge
density of the solid surface which attracts the collagen.
In the presence of BSA in the buffered solution, collagen
adsorbs onto alumina in a smooth pattern. The amount adsorbed
decreases with an increase in BSA concentration. This leads to
the same conclusion as before, that the phosphate adsorbs on
preferentially high energy sites and prevents denaturation of
the BSA. Thus, the BSA remains in globular form and occupies

13 7
Figure 38. Schematic representation of BSA adsorbing onto an
alumina surface; (A) a molecule in solution in its native
state., (B) adsorbed onto the surface in its native state, (C)
partially denatured on the surface, and (D) totally denatured
on the surface due to attractive forces.

138
fewer surface sites. The remaining area is available to
collagen. This result similarly applies to hydroxyapatite
and silica.
BSA, CS, and Collagen
When CS is added to the previous mixture, both it and
BSA affect the adsorption of collagen on alumina. Since the
CS adsorption has a similar concentration dependence as
collagen (see Figure 35), and was shown earlier not to react
strongly with BSA, it is suggested that the carbohydrate also
adsorbs between the BSA molecules. The amount of CS adsorbed
decreases in the presence of collagen. This indicates that,
even if CS is being replaced by collagen on the surface or
released from a collagen-CS complex as it adsorbs. This result
is based on the previous findings that CS and collagen bind to
each other [104], and because of the decrease in enthalpy
change of the adsorption of collagen measured in the presence
of CS.
Again in the buffered solution, the discontinuity dis
appears. There is a smooth decrease in the amount of collagen
adsorbed and the enthalpy of adsorption is lowered as before
in the case of the collagen-CS complex. Similar experiments
on hydroxyapatite and silica were not run.
Conclusions
Several effects of adsorption of mixtures of proteins
and the carbohydrates have been examined. The adsorption

139
phenomena of collagen on alumina, silica, and hydroxyapatite
appears to be partially induced by ionic attraction. The
enthalpy change for collagen on alumina as high as 70 cal/gm
indicates a strong reaction with the surface. This reaction,
although probably not entirely ionic, takes place through
the charged side groups of collagen.
The presence of BSA has a marked effect on the number of
collagen molecules adsorbed, but not the enthalpy change of
adsorption. The greatest change is apparently due to denatura- *
tion of the proteins. .The effect of the presence of BSA on
the adsorption of a large collagen fibril of molecular weight
many times higher than the triple helix used here, would probably
be greatest if the BSA denatured and spread over the surface.
In the globular form, BSA would simply be pushed aside.
There are several useful conclusions in regard to the
use of prosthetic materials. The effect of specific proteins
will depend on which ions and other proteins arrive at the
surface first. For example, if a clean alumina device of high
surface area is immersed in serum, a layer of denatured albumin
could be expected. If the device is conditioned in a phosphate
solution, less denaturation would occur. Spacial requirements
of each adsorbed moiety also become important. Denatured proteins
take up more room than their globular counterparts.
Secondly, a surface with an electric potential different
from zero will definitely enhance the adsorption process if
the protein is of opposite charge. Since collagen is negatively

140
charged at neutral pH, because the number of carboxyl groups
is greater than the number of amine groups, adsorption on
alumina is more favorable than on hydroxyapatite. Oppositely
charged reactants, however, also increase the chance for
denaturation of the protein, and tissue rejection. Systems
which allow tissue ingrowth without severe denaturation can
be predicted by the density and the sign of the surface
charge. Polymers which are neutral as polyethylene can be
expected not to denature collagen, and also to have low
adhesion properties.
The behavior of collagen as a function of solvent type,
moieties in solution, and surface composition have been
investigated. The behavior of a special glass in relation
to collagen adsorption will be discussed in the next chapter.

CHAPTER VII
BIOGLASS
Introduction
The adsorption of collagen, through the study of
various models and of collagen itself, has been investigated
by calorimetry and has been discussed in previous chapters.
Competition with collagen for adsorption sites by other mole
cules has also been considered. The conclusions drawn are that,
on the three surfaces examined, ionic attraction is a prominent
force in the initial attraction of molecules and appears just
as important in the attraction of large molecules as it is in
small molecules. Collagen adsorption was also found to be
decreased by hinderance of BSA particularly at low concentrations.
In this chapter, use is made of this information to study
another material known as bioglass, which is of significance
among prosthetic materials. It is the purpose of the studies
described in this chapter to examine and to help explain the
interaction of collagen with bioglass.
Bioglass is the name of a series of glasses which are
known to be tissue compatible and have the unique property of
chemically bonding to bone [1]. The material has been extensive
ly studied by various methods. It is known that collagen
141

142
is found in abundant quantities in the bioglass-bone inter
face which is partially comprised of a silica gel layer. The
reaction of collagen on the bioglass surface for short time
periods is less well understood.
Experimental
The bioglass used in this study, 45S5, has the composition
SiC^ CaO Na20
mole % 46.1 26.9 24.4 2.6
and was prepared in our laboratories. It had a surface area
2 2
of 0.44 m /gm before and near 400 m /gm after dissolution.
Silica gel was purchased from Fisher Scientific and
used without further purification. Its surface area was
9
measured to be 270 m /gm. Collagen concentrations were deter
mined using Biuret reagent and measuring optical adsorbance
at 540 nm.
Results
Figure 39 shows a plot of milligrams of collagen adsorbed
on 1.0 gm of silica gel at pH 7 as a function of the equilibrium
concentration: phospahte Ringers solution and a low ionic
strength solution. The gel was used in two conditions:
reprecipitated from a basic solution and without the reprecipi
tation procedure. When used without reprecipitation, the gel
would adsorb .05 mg of collagen or 1.85 x 10"^ mg/m^.
Two experiments were run in buffered solutions with the
gel: one with and one without reprecipitation. The former
adsorbed slightly more collagen than did the gel in the low

143
ionic strength solution. The reprecipitated gen, however,
adsorbed 30 times more collagen even though the specific
surface area of the two samples were measured to be the same
by nitrogen adsorption. Complete coverage (saturation) in
this instance was obtained at 1.4 mg or 0.06 mg/m^.
The variation in the amount of protein adsorbed on
bioglass under several mixing conditions is shown in Figure
40. Curve (1) shows the results of mixing collagen with bio
glass in Ringers solution before dissolution of the glass. In
2
this state the glass had a surface area of 0.44 m /gm. The
2
surface of the glass was saturated at 1.8 mg/m .
2
Curve (2) shows the results of mixing bioglass (.44 m^/gm)
with collagen in a salt solution. This solution had been
obtained from another identical sample of bioglass which had
been dissolved in buffered. Ringers solution for 24 hours. In
other words, this was a fresh surface with a concentrated
salt solution. In this experiment, the surface was saturated
at approximately 1.0 mg collagen.
Curve (3) the results produced by measuring the collagen
adsorbed after both collagen and bioglass had been mixed and
permitted to stand for 24 hours. Here the surface was saturated
at approximately 2.5 mg or 0.08 mg/m .
Curve (4) shows the results of mixing collagen with
samples of bioglass which had first been permitted to undergo
dissolution for 24 hours. The results show that less collagen
is adsorbed than by the previous case, reaching saturation
near 2.0 mg or 0.06 mg/m^.

Ceq, mg/ml
Figure 40. Adsorption of collagen onto 45S5 bioglass under
the conditions described in the text.

145
Curve (5) was produced in the same way as curve (4)
except.the bioglass was rinsed in distilled water several
times. After this, fresh buffer solution was added and
the collagen mixture was added separately.
Calorimetric measurements were performed on a sample of
bioglass which was permitted to undergo dissolution for 24
hours in buffered Ringers solution. The collagen was mixed
in a'solution of salts obtained from separate,samples of
bioglass which had also been allowed to undergo dissolution
for 24 hours. This corresponds to curve (4) above. The
results are shown in Figure 41.
This was the only situation in which the rate of
dissolution of bioglass had slowed enough so that dissolution
heats did not interfere with calorimetric measurements. In
all other situations the heat imbalance caused by the dissolving
glass did not allow adequate cancellation in the reference
cell. The enthalpy of adsorption of collagen on bioglass was
found to be between -100 and -150 cal/gm of collagen adsorbed,
an extremely high number. It was very difficult to place
the silica gel into the calorimeter and worse to remove, prevent
ing thermodynamic measurements.
The bioglass surface was identified by EDXA to have
formed a calcium phosphate film after 24 hours, which was not
removed by washing in distilled water.

mg. adsorbed
Figure 39. Adsorption of collagen onto silica gel.

cal.
147
o
O'
200
100
0 0.5 1.0 1.5 2.0
C0, gm/1
Figure 41. Reaction heat, Q, o and heat of adsorption
of collagen onto 45S5 bioglass corresponding to curve (4)
of Figure 40.
Heat of adsorption, cal./gm.

148
Discussion
The experiments with silica gel emphasize the finding
that the gel surface must be hydrated before adsorption
can easily occur. Since the bioglass is known to form a
gel in the dissolution process, it can be seen that bioglass
samples which have formed a gel and permitted to dehydrate
may react differently than a gel forming in solution. The
experiments using bioglass in a high and low surface area
state also emphasize this point.
Before the high surface area gel film formed on the
bioglass (Curve 1 and 2 in Figure 40), the amount of collagen
adsorbed was low compared to that adsorbed after 24 hours
dissolution (Curve 4). After dissolution, the amount adsorbed
increased tremendously in each case and most notably after
the glass was rinsed.
The differences between curves 3 and 4 can be explained
by speculating that the collagen adsorbed in the pre-mixed
situation (3) was incorporated into the film as it formed,
with new film formation developing on or with the surface of
collagen. Both samples (3) and (4) are of the same surface
area. The difference between curves (3) or (4) and curve (5)
is more difficult to explain.
The only difference which can be easily cited for curve
(5) is that the glass, upon rinsing, once again begins to
undergo dissolution. Associated with this is a heat of
dissolution of the dissolving ions. It is suggested that heat

149
is available to do work on the system. Collagen does
exhibit a definite melting curve with T at 45C [75]. The
local temperature increase would depend on heat transfer away
from, and heat capacity of the interfacial region but a 10 to
20 local temperature change is not unreasonably large to
expect.
The alkali ion concentration of these solutions was'
not measured after the mixing process. It is doubtful that 1
the ion concentration of the solution of the last experiment
(Curve 5) would reach saturation in 10 minutes after having
been leached, for 24 hours and could account for the difference
observed.
The calorimeter measurements made for curve (4)
indicate that the collagen undergoes some degree of denaturation
The adsorption of collagen on hydroxyapatite produced an
enthalpy change of -7 cal/gm and for silica it was found to
be 0 to -1 cal/gm. An enthalpy change of -1 to -6 cal/gm of
collagen would be gratifying, indicating little conformational
change. However, this large AH reveals evidence for denatura
tion. The extremely high surface area may be a contributing
factor. It was suggested previously that high energy points
of contact are involved in protein denaturation. The high
surface area film of bioglass may also possess such points
of high surface energy, producing similar results.
It should be remembered that the collagen used in
these experiments is small compared to the fibrils which

15.0
appear in electron micrographs. These large fibrils,
generally insoluble, may' not be affected in the same
manner since they are internally covelently cross-linked
[75].
Conclusions
The foremost conslusion which can be drawn is that
bioglass powders will not likely be useful as a prosthetic
material. This judgement is based on the finding that
denaturation of the collagen appears severe in small volumes.
Preliminary results using bioglass powder do not show-
great promise. Part of the reason for poor results lies in
the ability of small pieces of glass to release large amounts
of ions into solution.
It is known that large pieces of bioglass also form a
high surface area film. However, the release of ions per unit
volume of solvent would not be nearly as high for a large single
piece than of a powder of the same mass. This would eliminate
or at least reduce the potential hazard of very high alkali
concentration. The, controlled release of ions becomes important
in view of the high probability of protein denaturation shown
in this chapter.
It is known that this high surface area glass lso induces
P20^_ and Ca++ deposition [l] and binds tightly to bone. It
is also realized that materials which do not have or create a
high surface area film do not bond at all to bone [4]. There-

151
fore, it is concluded that the creation of the high surface
area to bind these materials is essential, not for bone
ingrowth, but for bone formation adjacent to a prosthetic
surface.
As an adjunct to the above conclusion is the hypothesis
that to produce soft tissue bonding to a prosthetic material
one must follow natures example somewhat. Collagen is not
glued to the surface of bone, rather bone grows around a fiber
[102,73]. In order to mimic this situation, the collagen
(tendon) must be placed between, not on, two surfaces which
can normally calcify the region between them. This implies
that the fiber, however large, must not be so large as to
preclude mineral junctions between the two prosthetic surfaces,
and that the surfaces must be far enough apart for dissolution
to occur. This may be achieved by fixing portions of collagen
fibers in position between bioglass and bone or bioglass
and bioglass surfaces which are set at a prefixed distance.

CHAPTER VIII
SUMMARY
There are several themes which have been developed through
out the dissertation the importance of ionic attraction be
tween surfaces and charged species in solution, and jointly, the
possibility of protein denaturation at the surface. Indirectly,
the application of the thermometric, titration technique to
adsorption studies has also been presented.
Although electrostatic interaction has been included
among the forces which can operate between a protein and a sur
face, it has not been emphasized as a force which can attract
and denature a protein. Part of the reason for this lies in
the use of protein models which cannot- denature, and part lies
with the inability of various methods to detect such changes.
Adsorption measurements, by far the most used method for
studying protein-solid interactions, can not distinguish between
the reaction of a surface with a protein in its native form or
in a denatured form. The area the protein occupies is only an
average value.
Erroneous results could arise from not recognizing the
possibility of such denaturation. For example, the amount of
denatured BSA which adsorbs onto alumina ( Chapter VI) decreases
with an increase in concentration. If.1 these: data were to be used
to. calculate the enthalpy change upon adsorption, it would be
152

found to be positive, whereas direct calorimetric measure
ments show the reaction to be exothermic.
Ionic interaction, as explained in the discussion section
of Chapter II, can account for the amount Of protein adsorbed
onto a surface in the same way that it accounts for the amount
of ions adsorbed. This is a function of the isoelectric point
Of the protein and the pcz of the surface. In that section
it was explained that the electrostatic interaction energy can
be described in terms of the amount of oppositely charged
species present in solution and on the surface.
If a scale is drawn (see Figure42) representing the
pzc of the surface and the IP of the protein, it can be seen
that as the IP increases the electrostatic attraction between
the protein and the surface increases. A plot of the predicted
amount of various proteins adsorbed vs. IP gives the correct
order as that found by Messing [52]. The curvature found in
Messing's adsorption curve can probably be attributed the differ
ences in the dissociation constants for each of the proteins
and also, as suggested by Messing, to the molecular weight of
the proteins.
In another experiment [42] the amount of BSA adsorbed
onto glass varied with pH as shown in Figure 42. The points
between the IP and the pzc would be predicted to display maximum
adsorption, and this is what is found. These results are similar
to those found for the carboxlic acids as described in Chapter
I, and the same explanation applies.

15 4
PEPSIN
PZC
i
TRYPSIN INHIBITOR
RIBONUCLEASE
CHYMOTRYPSIN
hr
CYTOCHROME C
2 4 6 8 10 12 14
.. I.P.
Figure 42a. The relationship between a glass surface with a
pzc near 3 and various proteins with increasing IP. The darkened
line indicates pH values where electrostatic attraction will '
occur.
2 4 6 8 10 12 14
I.P.
Figure 42b.Magnitude of molecular inclusion as a function of
the IP of various proteins, (from reference 52).

15 5
(a)
- GLASS
BSA +
J L
4 |
PH'
Figure 43. The relationship between pH and the area (dark
line) of unlike charge for pyrex glass and BSA. The relation
ship found from experiment (b) from reference 42.

15 6
In effect then, maximum protein adsorption will occur
near the IP of the protein in the absence of other influences.
Such contingencies, as denaturation, must be considered and
play an important role in determining adsorption density.
Protein denaturation has been suggested as being respon
sible for the decrease in protein adsorption sometimes noticed
when there is electrostatic attraction. In a calorimetric study
of the adsorption of BSA on polystyrene lattices of various
surface charged [49], it was shown that protein adsorption
was energetically favorable at pH values away from the IP. At
the Ip the adsorption reaction was endothermic. At neutral pH,
where the protein should maintain its globular shape, the heat
of adsorpion was near zero. The surface charge was produced
by sulfate groups and was unaffected by changes in pH. At high
or low pH, internal hydrogen bonding breaks down and conformation
al adaptability is made easier. Oppositely charged groups on
the protein occupies more space because of its unfolding and
adsorption decreases. This is the phenomenon found.
In this work it appears that an initiating force is
required to bond the macromolecules to the surfaces. These
studies find that inducement to be electrostatic attraction.
In other studies, for example, Minones [39] in film compression
experiments, suggested that alanine was hydrogen bonded to
silica. It was ignored, however, that the film compression
itself does work on the system and, in effect, replaces the
force of electrostatic attraction.

157
It is not suggested that hydrogen bonding or other
bonding forces are not present. It is reasoned that attraction
of a protein to a surface must be initiated by other than the
simultaneous presence in solution.
The study of protein adsorption should be accomplished
in relation to a specific surface in a specific solvent. The
difficulty in generalizing the adsorption of different proteins
thus arises from their varied experimental conditions.
If a generalized model must be made, then it should use
an amphoteric substance which is incapable of denaturation or
at least limited in conformational adaptability. The combination
of adsorption and calorimetric measurements is seen as the mini
mum in the type of experimental measurements to be made.
In a study of possible applications, however, emphasis
should be placed on the protein of interest, not models. Simplifi
cations should be derived, it is believed, from the solution,
not the protein, or the surface.
If there could be derived from this work criteria which
could serve as a guide for the successful application of a
prosthetic device in hard tissue they would include the following:
1) There should be tissue attachment accomplished
through charge attraction.
2) There should be ample room to accomodate the
natural quantities of protein found in the tissue
accomplished through high surface area.
3) Protein denaturation should be avoided by control
of the surface, surface area, and ionic strength
of the region.

15.8
It may be noted that metals which normally possess a
negative surface charge in neutral pH where collagen and 1 BSA
are also negatively charged do not fare well as bonding agents.
Bioglass and hydroxyapatite fit the above criteria and do enjoy
success as prosthetic materials. Alumina and other glass cer
amics falling outside the compositional range of bioglass
do not meet the above requirements and fail as a result.
Among the avenues which lead to future work in the .
study of adsorption of proteins by calorimetry are those
which will include detailed thermodynamic descriptions such
as those applied to conformational changes and to multipoint
equilibria in todays literature. Applied as an analytical
tool, calorimetry can offer a great amount of information
even if detailed mathematical description is ignored. There
is an enticing fundamental importance in such descriptions.
However, defining the variables in the adsorption of BSA in the
presence of CS and collagen would be a formidable task. In
lieu of such complexities, broader definitions and descriptions
have been shown to to yield plausible results and suggestions
for the advancement of studies in the field of prosthetic
devices.

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BIOGRAPHICAL SKETCH
Paul Buscemi was born in Brooklyn, New York,in 1946.
He attended high school in Jacksonville, Florida. He
received his Bachelor of Science in physics from the
University of Florida in 1968. He has taught high school,
trained in the Peace Corps, and served in the United States
Army as an infantry lieutenant. Since 1973 he has been
pursuing his doctorate at the University of Florida.
164

I cerify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Ronald E. Loehman ~
Professor of Material Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
X y"
L.L. Hench
Professor of Material Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
R'.T. DeHoff
Professor of Mq'xerial Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable satndards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
y .
Professor of Material Science
and Engineering

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy
^ ^ ^ -j'S-PjC
E,P, Goldberg
Professor of Material Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is full adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy
Q.B. Conklin
Professor of Physics

This dissertation was submitted to the Graduate Faculty
of the College of Engineering and to the Graduate Council,
and was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
March, 1978
Dean, College of Engineering
Dean, Graduate School

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DATE:
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94
It is the purpose of this chapter to provide further
insight to the interaction of collagen, chondroitin sulfate,
and serum albumin in solution.
The same compounds which have been used previously
to model collagen and chondroitin sulfate have been used
here. For collagen they are poly-l-arginine (PLA), poly-.l-
lysine (PLL), poly-lalanine (PA), and poly-l^proline (PLP).
For chondroitin sulfate they are dextran, galactose, galacturonic
acid, and polygalacturonic acid.
The reaction of chondroitin sulfate with collagen has
been studied by model systems, as mentioned in earlier
chapters. The reaction of collagen with dyes containing acidic
groups and with CS have been studied in regard to the under
standing of the role of CS and collagen in connective tissue
[108]. it was found that the cationic groups of collagen bond
with the anions of the dyes and CS in a pH range of 1.5 to
7 with a sharp drop in the number of anions fixed below pH 2
and a more gradual decrease from maximum adsorption at pH 3
to zero at pH 7.
In experiments to determine the role of CS in the
calcifying mechanisms of bone [109], it was found that calcifi
cation would not occur or would occur more slowly in an aqueous
collagen mixture when CS was not present. These experiments
were performed near pH 7. The authors suggested that binding
of CS to collagen at neutral pH would aid the natural calcifica
tion process.


35
4 ml of fluid are contained in the forward compartment. In
all experiments, 2 ml of fluid were used in each compartment.
Measurement of the heat loss or gain incurred by the
mixing procedure is made through multiple thermopiles located
between the heat sink and cells on two sides of each cell.
The thermopiles are connected in opposition so that the signals
from reactions producing equal amounts of heat are cancelled
electronically. One cell Is then arbitrarily chosen as a
reaction cell and the other as a reference cell in which
unwanted heats can be cancelled.
Determination of the reaction heat is made by manual
integration of the voltage vs. time curve produced during the
course of a reaction. The energy is calibrated against a
known heat produced in the reaction cell using a precision
resistor and a known current generated for a specific time
interval.
Each reaction in this work is of the type
rotation
| A B 1 A + B
where A is a solution of organic molecules and B is a slurry
of powdered oxide used as a substrate. In the mixing operation
several reactions are possible, each contributing to the over
all heat produced. They are due to 1) wetting of the cell
wall; 2) dilution of the organic molecule; 3) friction of
mixing; 4) chemical reaction. Only the last heat is desired.
The others must be eliminated.


CHAPTER VIII
SUMMARY
There are several themes which have been developed through
out the dissertation the importance of ionic attraction be
tween surfaces and charged species in solution, and jointly, the
possibility of protein denaturation at the surface. Indirectly,
the application of the thermometric, titration technique to
adsorption studies has also been presented.
Although electrostatic interaction has been included
among the forces which can operate between a protein and a sur
face, it has not been emphasized as a force which can attract
and denature a protein. Part of the reason for this lies in
the use of protein models which cannot- denature, and part lies
with the inability of various methods to detect such changes.
Adsorption measurements, by far the most used method for
studying protein-solid interactions, can not distinguish between
the reaction of a surface with a protein in its native form or
in a denatured form. The area the protein occupies is only an
average value.
Erroneous results could arise from not recognizing the
possibility of such denaturation. For example, the amount of
denatured BSA which adsorbs onto alumina ( Chapter VI) decreases
with an increase in concentration. If.1 these: data were to be used
to. calculate the enthalpy change upon adsorption, it would be
152


o
Figure 9. Molecular structure of chondroitin sulfate (a)
( D-glucoronic acid N-acety galactosamine-6-sulfate)
(b) the three-fold helix of chondroitin sulfate.


130
This enthalpy (dashed line in Figure 34) decreases from
the region of 30 cal/gm to 7 cal/gm near .1 gm/1 BSA. Beyond
.1 gm/1 BSA, the enthalpy rises to about 15 cal/gm, decreasing
gradually as the BSA concentration increases.
Hydroxyapatite, Silica, BSA, and Collagen
The adsorption and reaction heat curves for the adsorption
of collagen on hydroxyapatite and silica in Ringers solution
show no discontinuities. The amount of collagen adsorbed
decreases slightly, but the enthalpy remains fairly constant
near -7 cal/gm and -1 cal/gm for hydroxyapatite and silica
respectively.
Alumina, BSA, CS, and Collagen
To determine the effect of BSA and CS on the adsorption
of collagen, the following measurement was made:
reaction cell
I : 1
compartment 1 compartment 2
alumina + CS + BSA Collagen + CS
The heat of dilution of the collagen and CS was accounted for
in the reference cell. The concentrations of the collagen
and CS were held constant at .3 gm/1 and 10 mole/1 respective
ly. The concentration of the BSA was varied in compartment'1
to yield the same final concentration from 0 to.l gm/1.
The adsorption and reaction heat curves for each species
of molecules follows the same pattern (see Figure 35). They
gradually decrease, then at about .1 gm/1 BSA, there is a


38
The concentration of NaCl in the low ionic strength solution
did not exceed 0.001 M.- The 0.165 M salt (Ringers) solution
consisted of. 9 gm/1 of NaCl, 0.25 gm/1 of CaCl2, and 0.42 gm/1
of KC1 and is known. The buffered solution was a 0.2 H solution
of mono- and di-basic phosphate. The phosphate buffer was
used only in Ringers solution bringing the total molarity to
*
0.365.
Except in those instances where solids were not used
at all, 0.1 gm solid particulates were added to the solution.
The solids were placed into suspension by mixing 1.0 gm of solid
powder with about 18 ml of distilled water, adjusting the pH
and then bringing final volume to 20 ml. One hour was allowed
for the pH to equilibrate. With stirring, 2 ml aloquots were
distributed into 10 tubes by pipetting. Using this method 0.1
gm 0.01 gm of solid was delivered to each tube.
The gold calorimeter reaction cells were washed daily
with detergent. The washing procedure included injecting a
solution of the surfactant into each compartment, rotating the
calorimeter and withdrawing the solution by aspiration. The cells
were then continually rinsed with distilled water while being
evacuated. By moving the tip of the aspirator tube from the
top of the cell to the bottom in one compartment, while filling
the other compartment, a good turbulent rinsing reaction
developed. Experience showed that five minutes of such procedure
cleaned the cell. Approximately once a week, the detergent
was left in the cells overnight to permit it to react more
thoroughly.


BIOGRAPHICAL SKETCH
Paul Buscemi was born in Brooklyn, New York,in 1946.
He attended high school in Jacksonville, Florida. He
received his Bachelor of Science in physics from the
University of Florida in 1968. He has taught high school,
trained in the Peace Corps, and served in the United States
Army as an infantry lieutenant. Since 1973 he has been
pursuing his doctorate at the University of Florida.
164


16
forces. This is a consequence of a decrease in displace
ment of a polar medium^ when less polar components coalesce,
thus creating a lower energy state.
Ionic or electrostatic attraction can occur between
oxides and proteins both of which are normally charged in
aqueous solutions. Ionic bonding strength is decreased by
an increase in the ionic strength of the solvent because of
shielding, whereas dispersion forces are not affected. Ion
interaction is essentially independent of temperature [80,813
Hydrogen bonding is partially ionic and partially
covalent [82^83], It arises from the electrostatic force
acting between hydrogen and a lone-pair of electrons of
nitrogen, oxygen, or fluorine. The small size and the close
approach of the hydrogen atom accounts for the partial (20%)
covalent character [82]. Typical bond strengths are of the
order 1 to 10 kcal/mole. Hydrogen bonds are also weakened
by an increase in ionic strength of the solvent. Completely
covalent bonding rarely occurs in the adsorption phenomena
in which we are interested [62].
The classification of physical or chemical adsorption
is somewhat arbitrary [7 9 ,83 ]. If the adsorption is found
to be readily reversible and has an energy of the same order
of magnitude as the liquefaction of gas, it usually is classified
as physical [77 ]. Osipow states that Van der Waals forces
are responsible for physical adsorption whereas Fuerstenau
also includes coulombic attraction. Chemical adsorption is
irreversible and the magnitude of the energy change is of the
order of chemical reactions [78].


kcal./mole
47
0. 2
0.4
0.6
0.8


meal. kcal./mole
67
O'
40
30
20
10
i
C
.01 .02 .03 .04
Figure 11. Thermodynamic data for the adsorption of D-galac-
turonic acid on alumina at pH7 in low ionic strength solution;
AG AH AS Enthalpy change determined by solution
analysis


42
The adsorption of alkyl ammonium acetate on quartz
has been followed as a'function of temperature and concen
tration [92-93].it was found that at 25C at neutral pH the
isoteric heat of adsorption was between zero and 2 kcal/mole
of ions. The positive enthalpy was expected because of charge
repulsion of the quartz surface and the negatively charged
ions in solution. The free energy change at 25 remained near
-3.5 kcal/mole. In most cases the hydrocarbon chain of the
molecules caused abrupt changes in the thermodynamic properties
due to lateral interaction of the adsorbing molecules as the
equilibrium concentration increased.
Binding studies of sulfates, citrates, and amino acids
on calcium oxalate and calcium phosphate have been carried
out in relation to bone formation and kidney stone growth
[94v95], Thermodynamic data are not readily available for these
reactions. Equilibrium constants of magnesium oxalate [94 ],
however, have been shown to be near 4000 which would correspond
to a free energy change near -5 kcal/mole for this precipitation
reaction.
The purpose of this chapter is to discuss the variation
of the heat of adsorption of simple and polymeric carboxlic
acids on alumina and hydroxyapatite in relation to surface charge,
molecular conformation and ionization and solution pH. Qualita
tive results indicate that while electrostatic interactions
are required to initiate adsorption, other interactions such
as hydrogen bonding, take place.


64
?
HO C H
Galactose
CH20H
CO
CH3
D-acetyl
Galactosamine
COOH
OH
H
HO C ~ H
Glucose
0S0
I
ch2
OH
HO
)H
OH
Glucose-6-Sulfate
Galacturonic acid
Figure 9b. Molecular structures of the carbohydrates
used in the experiments of this section.


36
The first three heats are accounted for in various
ways. The cells are first wet with the solvent being used
and emptied prior to filling with reacting components. This
eliminates the heat of wetting of the cell wall. The heat of
dilution of the organic molecules is accounted for in the
reference cell, while the heat of dilution of the oxide powders
and frictional heat are measured separately and subtracted
from the reaction heat.
The heat measured in this way gives a measure of the
reaction A + B -t C where C is a complex of solid particulates
and adsorbed organic molecules.
Calibration of the calorimeter was carried out as suggested
by the manufacturer and consisted of two procedures. The first
procedure determined the sensitivity of the thermopiles. The
manufacturer listed the sensitivity of the thermopiles as 28.0
and 30.0 microvolts for a constant current of 30 miliamps
through the calibration heaters. The measured values were
consistently within 2 8.0 1 .05 microvolts and 30.0 t .05 micron-
volts. The second procedure judged the accuracy of the unit's
calibration mechanism. The heat of dilution of a six percent
sucrose solution was measured periodically to be 6.36 .05
kcal/mole. The literature value is 6.36 .03 kcal/mole.
No literature data could be found for the heats of
adsorption of surfactants from aqueous solution on prewetted
surfaces. However the heat of adsorption of sodium dodecyl
sulfonate (SDS) measured from the heat of immersion of dry
alumina (Linde A) was estimated to be 12 kcal/mole 1 kcal/


-kcai./mol
50
0.02 0.04 0.06 0.08
CQ, mM
Figure 6. Thermodynamic data for the adsorption of poly
acrylic acid on alumina at pH 7 in low ionic strength solu
tion; AG AH AS. .
SV-


34
B D
Figure-3. Schematic of the operation of an LKB model 10700
microcalorimiter. A-calorimeter, B-reaction cell, C-thermo-
pyle, D-heat sink, E-amplifier, F-chart recorder, G- reaction
heat curve. The reaction heat, Q, is proportional to the
area under the curve.


meal.
104
. 7 5
. 5 0.
.25
0.0
0 1.0 2.0 3.0 4.0 5.0
CQ, mM polypeptide
Figure 22. Reaction heats for the mixing of various poly
peptides with carbohydrates. The carbohydrate concentration
was held oenstant at .001M. Run at pH 7 in low ionic strength
solution; (PA) poly-lalanine, (PLP) poly-1-prline.
-
PLP-PGA
PLP-CS
-
PA-GAL
o PA- PGA
-
1 1 L
rA-Lo
^ e PA-PGA
i


116
which have high surface potential, attracting a high
concentration of counter ions, or surfaces which may release
ions into solution, this result has much significance. On
these surfaces protein denaturation would be at a maximum, while
protein-protein or protein-carbohydrate interaction would be
at a minimum. Such effects will be discussed in the following
chapters.


48
lies between -6 and -24 kcal/mole. The free energy changes
are nearly equal at about -5.5 kcal/mole, while the entropy
changes are each negative and lie between -10 and -20 cal/mole*
deg (1 cal/mole'deg = 1 entropy unit or e.u.).
Calculated values for the thermodynamic functions for
the adsorption of the three carboxlyic acids on hydroxyapatite
are presented in Table 3. The values show less variation for
hydroxyapatite than they did for alumina. The enthalpy
change tends to be less negative than for alumina, while the
free energy and entropy changes are about the same.
Polyacrylic acid (PAA) had such a high affinity for
alumina and hydroxyapatite that a cotton-like gel formed at
pH 5 and 7, causing great difficulty in cleaning the gold
reaction cells. Three concentrations were run with alumina,
however, and were repeated several times to improve precision.
Titration determinations of the amount of PAA adsorbed leads
to an enthalpy of 82 cal/gm of PAA for the adsorption reaction.
Taking 72 gm/mole as the molecular weight of the monomer, the
determined enthalpy change is -5.7 kcal/mole of acrylic acid
monomer assuming all acid groups participate in the-adsorption
reaction.
The'calculated thermodynamic data for the adsorption
of PAA onto alumina.are presented in Figure 6. The enthalpy
change per residue lies between -7 and -12 kcal/mole, while
the free energy change is concentration invarient at -5 kcal/
mole. The entropy change is negative as it is with the other
carboxylic acids used in this section.


151
fore, it is concluded that the creation of the high surface
area to bind these materials is essential, not for bone
ingrowth, but for bone formation adjacent to a prosthetic
surface.
As an adjunct to the above conclusion is the hypothesis
that to produce soft tissue bonding to a prosthetic material
one must follow natures example somewhat. Collagen is not
glued to the surface of bone, rather bone grows around a fiber
[102,73]. In order to mimic this situation, the collagen
(tendon) must be placed between, not on, two surfaces which
can normally calcify the region between them. This implies
that the fiber, however large, must not be so large as to
preclude mineral junctions between the two prosthetic surfaces,
and that the surfaces must be far enough apart for dissolution
to occur. This may be achieved by fixing portions of collagen
fibers in position between bioglass and bone or bioglass
and bioglass surfaces which are set at a prefixed distance.


8
H 6
O
e
ii
o
4
CD
<
I
PLA-HA
2
i i i i
0.0 0.2 0.4 0.6 0.8 1.0
C0, mM
Figure 17. Free energy change for the adsorption of poly-1-
lysine (PLL) and poly-l-arginine (PLA) on silica, alumina and
hydroxyapatite at pH 7.


pH
Figure' 7. Plot of and a for a hypothetical acid.
pH
Figure 8. Values of the reaction heat calculated by use
of equation 2.6 and found by calorimetric experiments.


80
CHoCHCOO"
+nh3
Alanine
+h3n(ch2)4choo
nh2
Lysine
H0NCNH(CH?)oCHOO"
2 ii 2 i
+nh2 nh2
Arginine
The amino acids are dipolar ions. for the dibasic
amino acids, arginine and lysine, adsorption onto negative
surfaces will be enhanced. In polymeric form only poly-1-
lysine and poly-l-arginine retain any charge in neutral
solution.
The solubility in water of the other amino acids will
decrease as a result of their polymerization. Acidic amino
acids, aspartic acid and glutamic acid were not studied because
the carboxyl group has been discussed in the previous chapters.
Furthermore, interpretation of calorimetric data would be
difficult because of the presence of three charged groups.
ho7\
^ )coo
N
h'-sh
Hydroxyproline
>C00
.N
h' nh
Proline


113
is exothermic. In previous experiments negative enthalpy
changes were produced by the interaction of oppositely-
charged species and it is suggested then, that the anionic
groups of CS are binding to the cationic groups of collagen.
This result is in agreement with other workers who suggest
ionic interaction between CS and collagen [104 ,105 ]..
The enthalpy change found for the mixing of CS and
collagen in Ringer*s solution demonstrates the dielectric
effect of salt. This is apparent at the higher concentrations
of collagen used. At concentrations (CQ) below about .12
ymole of collagen, the enthalpy change is similar to that
found in the low ionic strength solution. Above this concen
tration the deviation becomes greater. This indicates that
those CS molecules which bind last are affected more by the
presence of the solvent ions. The enthalpy change per CS dimer
lies between -500 cal/mole and -700 cal/mole. If the binding
reaction is responsible for the greater portion of the enthalpy
change, then about one in seven to one in twenty dimers make
contact with the collagen molecule. This assumes an enthalpy
change of 5 kcal/mole to 10 kcal/mole per contact.
BSA
The BSA macromolecule is negatively charged at pH 7.
Therefore, both PLA and PLL would be elctrostatically attracted
PA, PLP, and collagen would be neutral or slightly affected.
Both BSA and PLA are in a coiled conformation in solution,
whereas the other polypeptides are in extended helical confor-


CALORIMETRIC MEASUREMENTS OF THE ADSORPTION OF COLLAGEN
AND OTHER ORGANICS ONTO OXIDE SURFACES
By
PAUL J. BUSCEMI
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMNETS FOR THE
DEGREE OF DOCTOR OF PHILOSPPHY
UNIVERSITY OF FLORIDA
1978


18
step reactions an equilibrium constant, K, may be written
as .
K = [P]
[Rx] CR2]
(1.2)
where the brackets denote concentrations. Through knowledge
of equation (1.1) and the reaction heat, Q, the enthalpy change
of the reaction and the equilibrium constant may be determined.
The method by which this is done will be detailed for adsorption
measurements.
For an adsorption reaction equation (1.1) can be written
as
Su + R So
where R-^ in equation 1.1 has been replaced by Su which is an
unoccupied site on a solid surface capable of adsorbing from
solution a reacting component R to produce an occupied site SQ.
The equilibrium constant is then written
K = [SU
[su] [R]
The concentration of occupied sites [SQ] is equal to
the number of adsorbed molecules per unit area, Na/A, while
the concentration of unoccupied sites is equal to the total
number of sites N minus the number of occupied sites per unit
o
area, (N N )/A. The equilibrium constant is then
u d
. K =
(N N ) [R]
S cl


143
ionic strength solution. The reprecipitated gen, however,
adsorbed 30 times more collagen even though the specific
surface area of the two samples were measured to be the same
by nitrogen adsorption. Complete coverage (saturation) in
this instance was obtained at 1.4 mg or 0.06 mg/m^.
The variation in the amount of protein adsorbed on
bioglass under several mixing conditions is shown in Figure
40. Curve (1) shows the results of mixing collagen with bio
glass in Ringers solution before dissolution of the glass. In
2
this state the glass had a surface area of 0.44 m /gm. The
2
surface of the glass was saturated at 1.8 mg/m .
2
Curve (2) shows the results of mixing bioglass (.44 m^/gm)
with collagen in a salt solution. This solution had been
obtained from another identical sample of bioglass which had
been dissolved in buffered. Ringers solution for 24 hours. In
other words, this was a fresh surface with a concentrated
salt solution. In this experiment, the surface was saturated
at approximately 1.0 mg collagen.
Curve (3) the results produced by measuring the collagen
adsorbed after both collagen and bioglass had been mixed and
permitted to stand for 24 hours. Here the surface was saturated
at approximately 2.5 mg or 0.08 mg/m .
Curve (4) shows the results of mixing collagen with
samples of bioglass which had first been permitted to undergo
dissolution for 24 hours. The results show that less collagen
is adsorbed than by the previous case, reaching saturation
near 2.0 mg or 0.06 mg/m^.


33
are arbitrarily chosen. The
to
5.0 x 10r'^ cal
5.9 x 10-3 cal
1 x 10~6 M
1.33 x 10-6 M
values of and C2 correspond
N = 2 x 10^ moles
N = 2.66 x 10^ moles
2
The number of moles of surface sites, N, in this example
s
_ q ,
is taken as 2.5 x 10 This number was determined from
consideration of the surface area occupied by a collagen mole
cule, the number of sites as calculated by the program, and
study of the reaction heat curve. Substitution of these
c
values into equation 1.8 yields -3.3 x 10 cal/mole for AH.
Substitution of AH, Q-^, N and V (4 x 10 l)into equation 1.7
gives 4.3 x 10^ for K or -7.6 kcal/mole for AG. This in turn
yields -1.1 x 10^ e.u. for AS0.
Experimental
Calorimetric measurements were made using an LKB model
10700 batch microcalorimeter [86], The basic calorimetric unit
consists of two identical gold cells situated in an aluminum
heat sink (see Figure 3). Each cell has two compartments
capable of holding 2 and 4 ml of fluid. Mixing of the fluids
in each compartment is accomplished by rotation of the entire
calorimeter. There is no stirring, and after rotation, the full


2
ized and has potential for use as a prosthetic material [4].
The calorimetric measurements made, therefore, are not for
the purpose of further characterizing these materials but to
study their influence on the adsorption of organic molecules.
The values of the thermodynamic parameters (AG, AH, and
AS), determined from the calorimetric measurements, do depend,
however, on the structural features of the surface as well as
those of the adsorbing molecule and on their mutual environment.
A practical approach for studying complicated systems encountered
in actual application is to study simpler model systems [5],
which provide singular features for observation.
Within a series of model compounds, the structure of
the molecules, the solvent, and the surface can be systematically
varied and correlations can be made between the thermodynamic
data and the variations in experimental conditions. In this
study, extensive use is made of several types of molecular
model compounds including those representing collagen as well
as those representing carbohydrate and other physiologically
relevant organic structures.
Use of Protein Models
Past workers have used molecular model compounds designed
to study collagen. Specifically, the polyamino acids have
been well studied in this way. Poly-l-proline has been used
in conformational studies in CaCl2 solution [6] showing that
disordering of the molecule is associated with its increasing
rotational ability. X-ray diffraction studies of poly-l-proline


15 4
PEPSIN
PZC
i
TRYPSIN INHIBITOR
RIBONUCLEASE
CHYMOTRYPSIN
hr
CYTOCHROME C
2 4 6 8 10 12 14
.. I.P.
Figure 42a. The relationship between a glass surface with a
pzc near 3 and various proteins with increasing IP. The darkened
line indicates pH values where electrostatic attraction will '
occur.
2 4 6 8 10 12 14
I.P.
Figure 42b.Magnitude of molecular inclusion as a function of
the IP of various proteins, (from reference 52).


40
rinsed in distilled water, decanted and evacuated for 24
hours at 10 atm. Prepared powders were stored under vacuum
at room temperature.
All organic substances used were stored under refrigeration
prior to use. None was repurified or modified in any way.
Batch solutions were used within one week and were stored at
4C. Particular information on each substance used is given
in the appropriate chapter.
Reaction heat data were plotted against Co and fed into
a'statistical analysis program available through the Northeast
Florida Regional Computing Center. The reaction heat, Q, was
held as the independent variable. The program generated an
approximating function which was used to calculate the thermo
dynamic data. In all chapters, referral to "calculated thermo
dynamic data" refers to this procedure. The graphs of Q versus
CQ presented are original data.


19
Dividing -
through by N gives
b
K = VNg
(1.3)
(1-N_/N)
d b
[R]
or
II
CD
(1.4)
(1-0)
[R]
...o .
where 0 = N-,/N is the fraction of occupied sites. Equation
s 7 7
(1.4) is the Langmuir adsorption isotherm [ ] and will be
used later. Its use requires that each site is occupied by no
more than a single molecule and that no two sites interact.
These conditions are satisfied when the concentration of R is
low.
The concentration of reacting molecules is equal to the
number of moles of R in solution divided by the total volume
Nr/V. If N is the total number of moles of R on the surface
and in solution then = N N The equilibrium constant
can now be written as
K = N V
Ct
(N? N ) (N N )
j d J- a.
(1.5)
The enthalpy change for the adsorption reaction, AH, is
related to the reaction heat Q by
AH = -Q/N (1.6)
a.
where the minus sign denotes, by convention, that an exothermic
reaction (positive Q) will yield a negative enthalpy change.


83
When the dissolved salt (R-Cl) concentration in the
solvent is increased to .16 M, the enthalpy change of the
adsorption of alanine on alumina decreases in the higher
concentrations range of alanine, but tends toward the -6
kcal/mole in the lower end.
The polymer, PA, exhibits much different behavior
showing no specific tendencies to adsorb. Reaction heats are
less than .5 kcal/mole and enthalpy changes are of magnitude
less than -1 kcal/mole.'
Proline
Measurements made with proline and PLP and hydroxy-
proline and PLHP (see Figure 15) show results similiar to
results for alanine and PA. The monomer again produces a
higher enthalpy change than the polymer- (-4 to -8 kcal/mole).
However, PLP and PLHP are apparently more strongly attracted
than the poly-l-alanine with heats of adsorption lying between
-CL 5 and -2.5 kcal/mole of residues. PLHP is somewhat more
strongly attracted on all three surfaces than is PLP. An
increase in ionic strength is noted by a decrease in the
enthalpy change for PLHP on alumina.
Lysine and Arginine
The addition of charged side groups causes a marked
change in the enthalpy curves, as shown in Figure 16. The
heat of adsorption of lysine on silica is, as expected,


122
0.5 1.0 1.5 2.0
l
n>
C
X
H
O
I
-P
C0, uM collagen
Figure 29. Thermodyanmic data for the adsorption of collagen
onto hydroxyapatite at pH 7; AH AS The free energy
change in each case was near -104 cal/mole.


LO LO LO
49
Table 3
pH
7
7
7
Themodynamic Variables for the Adsorption of
Carboxylic Acids on Hydroxyapatite at pH 5 and 7
Substance
-AG
(kcal/mole)
. -AH
(kcal/mole)
AS
(cal/moledeg)
Acetic
5.4
3.3
-7.0
Oxalic
5.1
9.0
13.0
Citric
5.5
11.0
18.0
Acetic
6.1
5.4
-2.3
Oxalic
5.2
9.8
15.0
Citric
5.1
17.0
39.0


100
are positive. The calculated enthalpies show strongly-
increasing trends at loyer concentrations and lie in the
range of -.4 to -1.0 kcal/mole of PLA residue.
Polygalacturonic acid (PGA) was mixed with PLA at two
pH values: 7 and 13. At pH 7, the mixing reaction appeared
to be endothermic. There was no precipitate formation as there
was with CS and PLA. If PGA is mixed with PLA at pH 13, a
precipitate will come out of solution. When the pH is lowered,
the precipitate dissolves. The reaction heat curve at pH 13
(Figure 18) of PLA and PGA reflects this interaction.
Poly-1-lysine
The reaction heats of mixing CS and poly-l-lysine (PLL)
(see Figure 20) are negative. The enthalpy changes for pH 5
and pH 7 are not the same as with PLA. At pH 5 the enthalpy
is near 200 cal/mole of PLL residues while at pH 7 it is near
400 cal/mole of PLL residues (see Figure 21). In both cases
a filterable precipitate forms. The reaction of PLL with
glucose-6-sulfate is exothermic and similar to that of glucose-
6-sulfate with PLA. There is no reaction evident between
dextran and PLL. The thermodynamic parameters associated with
the above reactions are given in Table 6 in terms of moles of
PLA or PLL residues.
Several experiments were run with PA and PLP with the
various carbohydrates (see Figure 22). The thermodynamic
functions were not calculated. The low magnitude and shape of
the reaction heat curves indicate little interaction for any


Since these molecules are in an extended conformation,
not coiled, there are no intramolecular hydrogen bonds. At
low concentrations (2 x 10"5 M of residues) there should be
little intermolecular hydrogen bonding [50]. The disruptions
which would primarily occur, then, would correspond to
rotational movement of the molecule [19], There is no
reason to suspect that these molecules should undergo grotesque
distortion on the surface since there are no strong attractive
forces. Therefore, the contribution to energetic changes due
to conformation alterations should be small.
Poly-l-proline and poly-l-hydroxyproline possess a
ring structure which is relatively nonpolar compared to the
polar solvent. Because of this, it is plausible to assume
that these less polar structures are in a lower energy state
on the surface, rather than in the solution. This is termed
hydrophobic bonding and could possibly account for the energetic
changes measured if most of the residues were near the surface
and not surrounded by the mobile polar ions in solution.
By comparison with the carbohydrates studied earlier
and in absence of detailed information on the geometric
smoothness of the surface, it would also be possible to suggest
that only a few points of contact are made on the surface [50],
Each' of these contacts would assume higher energy than
indicated by the average of 1-2 kcal/mole of residues measured.
If these contacts are hydrogen bonds made up of hydrogen
atoms on the ring structure and oxygen atoms on the surface,


21
In order to complete the thermodynamic data (AG =
AH TAS) H must first be determined or approximated.
The number AH used in equations (1.6) to (1.8) is the total
enthalpy change for the reaction under experimental conditions
while. AH0 is the standard enthalpy change. Under suitable
conditions, AH can be shown to be AH so that AS can be
determined. Those conditions will now be explained.
The chemical potential /f^ or partial molar free energy
Gj_ of component i in a chemical reaction is defined by
/fi = G"i = (^G/c?Ni)tp (1.10)
where G is the free energy change for all components in the
reaction. The chemical potential can be expressed as a function
of the activity a^ of component i and of the chemical potential
in some reference state
Jil = y/Vef + RTlnai (1.11)
The term RTlna^ takes into account the energies of interaction
of component i with other components at a given concentration in
the mixture. The choice of reference state is quite arbitrary
and varies for experimental convenience. Generally, no matter
what reference state is chosen, the activity is expressed as a
function of the mole fraction component i, X-p, and a parameter
known as the activity coefficient fp
ai = xi fi
The activity coefficient approaches 1 in pure solutions for
the solvent while in dilute solutions of component i, fp
approaches a constant which may be greater or lesser than one.


meal
101
J I i
1 2 3 4
C0 mM
Figure 20. Reaction heat, Q, for the mixing of PLL and various
carbohydrates in low ionic strength solution at pH 7 except as
noted.


CHAPTER VI
ADSORPTION OF COLLAGEN
Introduction
In the previous chapter, it was determined that ionic
interactions are of fundamental importance in the initial
reactions between carbohydrates and polypeptides. The findings
also suggested that molecular conformation is of importance
when the two interacting molecules are of different shape. In
this chapter measurements of the adsorption of collagen onto
silica, alumina, and hydroxyapatite are discussed as a function
of concentration in relation to these findings. The effect of
adsorption of collagen on alumina in the presence of bovine
albumin and chondroitin sulfate is also presented.
Experimental
Procedures and sources of chemicals have been previously
described. Linde B alumina was used in these experiments.
Results
Alumina and Collagen
The results of the calorimetric measurements of the
adsorption of collagen onto alumina are shown in Figure 26.
117


1 2 3 4
CQ mM
Figure 21. Enthalpy change produced by the mixing of PLL
and CS at pH 5 and 7 in low ionic strength solution. Cal
culated values- determined by solution analysis-.


meal.
98
4
3
2
O
-1
1.0 2.0 3.0 4-0
C mM PLA
Figure 18. Reaction heat, Q, for the mixing of PLA with
various carbohydrates:(PGA) polygalacturonic acid, (GAL)
galactose, (DEX) dextran, (DGA) D-galacturonic acid, (G6S)
glucose-6-sulfate, (CS) chondroitin sulfate. Run in low
ionic strength solution at pH 7.
J 1 i l


meal.
121
Figure 28. Reaction heat for the adsorption of collagen onto
hydroxyapatite at pH 7 in three solutions.


TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
ABSTRACT v
CHAPTER Page
IINTRODUCTION 1
Use of Protein Models .......... 2
Calorimetry as an Analytic Tool 4
Protein Adsorption. . 6
Surfaces 10
Forces of Adsorption 15
Thermodynamics of Adsorption 17
Heterogeneous Adsorption 24
Experimental 3 3
IIADSORPTION OF CARBOXYLIC ACIDS
ON ALUMINA AND HYDROXYAPATITE 41
Introduction. 41
Experimental. 4 3
Results 44
Discussion 51
Conclusions 59
IIIADSORPTION OF CHONDROITIN SULFATE
AND OTHER CARBOHYDRATES 61
Introduction. 61
Experimental. . 6 2
Results 62
Discussion 7 0
Conclusions ..... 77
IVADSORPTION OF POLYPEPTIDES 79
Introduction. 7 9
Experimental 81
Results 81
Discussion. .......... 86
Conclusions 92
iii


AH, cal./mole
-3
125
CD
I
O
H
-2
1
LOW IONIC STRENGTH
o~ ,
BUFFERED
~o
RINGERS
RINGERS
LOW IONIC STRENGTH
0.12
0.25
_L
0.38
0.5
CD
h
i
o,
CO
-2
-4
C0, pM collagen
Figure 31. Thermodynamic data for the adsorption of collagen
onto silica at pH 7; AH AS- . Enthalpy change determined
by solution analysis,----.
5


11
+ 2 o
three surfaces used here. In addition Ca and PO^-'3 are
potential determining ions for hydroxyapatite. Certain
ions added as impurities may alter the surface charge such as
aluminum ions [59] or cobalt [60] on silica or phosphate on
alumina [61]. The wide variety of buffering systems used in
biological studies involving adsorption can thus lead to
differing results for proteins even if the same material is
used as a substrate.
The surface potential can be altered by a change in
pH. For each of the three oxide substrates in this study, there
exists a pH at which the surface charge is zero. This pH is
called the point of zero charge (pzc) and is listed in Table
1 [62,63] for the substances used as adsorbents.
As the pH varies from one side of the pzc to the
other, the sign of the surface charge will change as will the
adsorption properties of the protein.
Ions in solution: which do not pass through both phases
but are attracted near the surface by electrostatic forces
are called counter ions. They will form a diffuse layer of
ions in solution near the surface and will tend to neutralize
the surface charge. The concentration of the ions generally
decreases exponentially with distance from the surface [64].
The.higher the concentration, the more compact the diffuse
layer will be. The thickness of the diffuse layer ranges from
about 10 A at .1 Msolutions to about a few hundred A in .001 M
salt solutions [65].


39
Between daily experiments, the same procedure was used
to clean the cells. The oxide powder slurry was removed for
analysis after mixing, however, and the detergent was not used.
Cleaning with 1 M HC1 or NaOH was found to be necessary only
rarely.
Organic constituents were weighed out to 1 .01 mg
and mixed volumetrically. Adjustment of pH was the same as
with solids. Incremental concentrations were made from standard
batches and measured volumetrically by pipet. Distribution of
organic, and solid, solutions into the reaction cell was made
by syringe. The lowest concentration was always used first.
The materials used as substrates include silica, alumina,
and tricalcium phosphate. The silica [89] was described by the
manufacturer as amorphous. It has. a specific surface area of
o
0.7 m /gm and a pzc of 3. The alumina used [90] consisted of
two types: Linde A, a a-alumina of specific surface area 15 m /gm
and a pzc of near 9 and Linde B, a mixture of y and a alumina
of specific surface area 82 m /gm and a pzc near 9. The tn-
calcium phosphate, referred to as hydroxyapatite in the text,
consisted of 85 volume per cent hydroxyapatite and had a specific
surface area of 57 m /gm. Surface measurements were made in
this laboratory using a multi-point B.E.T. nitrogen adsorption
isotherm. Measurements of pzc were also conducted in this
laboratory using a Zeta meterR [91],
All experiments were run at 25 C.
All solids were used without modification. Prior to
weighing, large (approximately 10 gm) batches of solids were


51
Discussion
As molecules are adsorbed from solution, other
molecules will ionize to try to maintain the original concen
tration. At the same time the alumina or the hydroxyapatite
will act as a buffer to maintain the pH. So long as the pH
is constant, the degree ionization of the solute will remain
the same. This leads to a qualitative explanation for the
reaction heat curves of Figure 4.
The total ionic charge in solution is directly propor
tional to the degree ionization, a, which is related to the
pH by [98]:
pH = pKQ log [ (1 a)/a ] (2.1)
where pK is defined as
o
pK = -In [H+] [A-] (2.2)
[HAT
This relation holds for single as well as polyelectrolytes
[99].
In the absence of any specific interaction between a
solid and electrolytes in solution, the surface potential is
given by [5 8] : '
i¡j = RT In a/a (2.3)
iF
where z is the valence (including sign) of the potential
ion, F is the Faraday constant, a is the activity of the
potential determining ion in solution and aQ is the activity
of the potential determining ion at the pzc. As mentioned
in the introduction, the potential determining ion for the


4
Molecular models in non-biological studies have also
been used. The adsorption of molecules containing the same
functional groups which proteins possess, amines [21], sulfates
[22], and organic acids [23], have been examined by various
methods. Calorimetry has not been extensively used for this
purpose. In general, the use of molecular models in biological
and non-biological systems appears widespread for the determina
tion of various properties.
Calorimetry As An Analytic Tool
Calorimetry has long been used to measure the enthalpy
of adsorption (heat of wetting) of various liquids onto dry
oxide surfaces. Such measurements are made by immersing a
clean, dry solid powder into a liquid. The heat change is
measured in a calorimeter. For our purposes, the most
relevant liquid used in previous studies was water. Heats of
wetting of silica [24-26], of alumina [27-29], and of hydroxy
apatite [29] have each been measured. The results of these
works show several consistent features. First, there are
differences in the heats of wetting with variation in the out-
gassing pressure and with temperature of evacuation, indicating
surface heterogeneities. Also there are differences in heats
of wetting with variation in particle size. Finally, the heats
of adsorption of water range from -10 to -20 kcal/mole of water
adsorbed and are attributed to hydrogen bonding of the water
to the surface [30].


This dissertation was submitted to the Graduate Faculty
of the College of Engineering and to the Graduate Council,
and was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
March, 1978
Dean, College of Engineering
Dean, Graduate School


60
attracting force is required to initiate adsorption. Other
wise a plot of E or Q versus pH would be similar in shape to
the a versus pH and not bell shaped.
Once adsorption occurs, the influence by multiply-
charged groups on the molecule was evidenced by a change in
surface charge. For this condition to arise, specific adsorp
tion has to occur which requires forces other than electro
static. The proximity of oxygen in the carboxyl groups and
H+ on the surface suggest hydrogen bonding.


23
AH = SiHiXi + ^kHkZk (1,16)
products reactants
where each sum is taken over each of the different components
for products and reactants and X. and are the respective
1 i
mole fractions. In view of equation (1.15) the enthalpy
change for the adsorption reaction is
AH = Zi H? X + EkHg Zk (1.17)
or
AH = AH (1.18)
where AH0 is the standard enthalpy change for the complete
reaction. Thus, under the rather ideal conditions in which
there is no interaction between solute molecules in solution or
on. the surface at 1 atm and 298K AH may substitute for AH.
The value of AH can be used with AG to find at least an approximate
value for AS.
The experimental conditions in this work meet the contraints
of pressure and temperature. The constraint of non-interaction
of solute molecules holds only for dilute solutions. In as
much as enthalpy values tended towards.constant rather than
steadily decreasing values,lateral interaction on the surface
between adsorbed molecules does not appear to have occurred.
Interaction of solute molecules in solution can only be assumed
-4
to be small in the concentration ranges used, typically 10 to
10-3 M.


IOS
of the combinations involved. There is no trend or break
in the reaction heat curves as there is for CS and PLA or
PLL.
Collagen
The thermodynamic data for the mixing of CS and collagen
are presented in Figure 23. The results for pH 5 and 7 are
identical. The date were calculated using the thermometric
titration methods. The enthalpy values appear high because
they are written in terms of moles of collagen. The free energy
change is always negative and shows an inflection near .06 M
of collagen. When the experiment was repeated in Ringers
solution, the reaction heat and enthalpy change did not show
significant deviation from the experiment run in low ionic
strength solution.
BSA .
The mixing of bovine serum albumin (BSA), PA, PLP, and
PLA are presented in Figure 24. The concentration of BSA was
held constant at .7 gm/1 which approximates the molar concentra
tion of the CS macromolecule. The reaction of PLA shows a
definite reaction pattern whereas the reaction heat curves of
BSA with PLP or PA are more linear. This enthalpy change for
the PLA-BSA interaction was calculated to be -50 kcal/mole of
BSA molecules with AG = -59 kcal/mole and AS = 30 cal/moledegree


91
hydrogen bonding, were to break down, the enthalpy change
would be due to both the adsorption and the unfolding processes
Enthalpy changes measured in this and the two previous chap
ters indicate that enthalpy changes between -6 and -8
kcal/mole of single, charged groups are to be expected. The
magnitude of the unfolding process, the breaking of hydrogen
bonds, would lie between 5 and 7 kcal/mole [19,82], an
endothermic process. The resulting enthalpy changes for both
processes would be comparatively small with a value near 0
kca.l/mole. Such a situation is encountered in experiments
presented in the next chapter where the coil-to-helix
transition is known to take place. Instead,the enthalpy
change is much more negative, decreasing in magnitude from
-14 kcal/mole to -6'kcal/mole. The variation is thought to
be due to adsorption on the fewer negatively charged sites
on the alumina and hydroxyapatite surface. These sites possess
a distribution of high to low energy within their own group.
Adsorption of PLL or PLA to neutral high energy sites
can be eliminated since PA, PLHP, and PLP each have heats of
adsorption which are smaller in magnitude. If the adsorption
had not depended on surface charge the neutral molecules
should have been just as strongly attracted to the surface.
The order of decreasing enthalpy change (alumina >
hydroxyapatite > silica) corresponds to the specific surface
area of the solids. The higher specific area of the alumina
(87m^/gm) provides more edges and peaks which are assumed to


103
Table 6
Thermodyanmic Variables for the
Mixing of PLL and PLL with CS
PH
Substance
AG
(kcal/mole)
AH
(kcal/mole)
AS
(cal/mole
5
PLL
-8.9
0.20
21
7
PLL
-7.8
0.49
19
5,7
PLA
-8.6
-0.75
25


73
to the surface. Whatever the mechanism, if these uncharged
molecules are strongly bound to the surface, it is not
reflected in the enthalpy determination. The type of bond
which would occur would almost certainly be hydrogen.
In any event, the adsorption of D-galacturonic acid
on alumina and hydroxyapatite is much more energetic than that
of galactose or galactosamine. The similarity in the molecules
and in the adsorption experiment strongly suggests that the
charged carboxyl group is responsible for the higher enthalpy
change and that binding between solute molecules or desolvation
effects do not account for the noted change. The negative
entropy change indicates an overall increase in ordering. This
increase in ordering may be due to confinement of the carbo
hydrate molecules to the surface and subsequent loss of freedom
[21].
Adsorption of D-galacturonic acid on silica produces a
positive enthalpy change. This can be accounted for by the
charge repulsion which exists between the surface and the molecule
There was a finite amount of acid adsorbed, however. This
would indicate perhaps a second stronger force necessary to
overcome the charge repulsion or that the negatively charged
molecules are occupying the fewer positive sited on the silica
surface, or reaction with high energy sites. Since the reaction
heat and adsorption measurements leveled off quickly, the last
two possibilities appear more likely; especially in view of the
finding that charge attraction appears necessary for strong
reaction.


15 5
(a)
- GLASS
BSA +
J L
4 |
PH'
Figure 43. The relationship between pH and the area (dark
line) of unlike charge for pyrex glass and BSA. The relation
ship found from experiment (b) from reference 42.


-AH, cal./mole
109
0 O.14 0.8 1.2 1.6 2.0
C0, yM Collagen
Figure 25. Enthalpy change for the mixing of CS and collagen
in the presence of BSA: low ionic strength solution ,
Ringers solution without BSA in low ionic strength
solution .


6
on the adsorption of proteins onto oxide surfaces and there
is apparently none for the adsorption of collagen. The
calorimetric data most relevant to adsorption primarily
involve such globular serum proteins as serum albumin [36].
The remainder of this section will therefore be devoted to
previous studies of protein adsorption use with particular
emphasis on oxide substrates.
Protein Adsorption
The demonstration of molecular attachment of cell
proteins on foreign substances has been accomplished by various
methods. Multiple internal reflection spectroscopy has
been used to measure protein interaction using germanium [37]
as a substrate. A KRS-5 prism pressed against protein on a
hydroxyapatite substrate allowed protein-hydroxyapatite inter
action to be studied [38]. Although energy calculations were
not carried out in these studies, the changes in adsorption
frequencies indicate chemical interaction with the surface of
the substrate.
Film compression studies [39] using collagen, gelatin,
and poly-l-alanine with silica gel showed adsorption hysteris.is
indicating an irreversible process. The maximum interaction
of the silica gel and collagen occurred at pH 5.2. The iso
electric point,where there is charge neutralization of the
protein, is 5.5. There was also interaction between alanine,
which has no ionic side groups, and the gel. The interaction


15.0
appear in electron micrographs. These large fibrils,
generally insoluble, may' not be affected in the same
manner since they are internally covelently cross-linked
[75].
Conclusions
The foremost conslusion which can be drawn is that
bioglass powders will not likely be useful as a prosthetic
material. This judgement is based on the finding that
denaturation of the collagen appears severe in small volumes.
Preliminary results using bioglass powder do not show-
great promise. Part of the reason for poor results lies in
the ability of small pieces of glass to release large amounts
of ions into solution.
It is known that large pieces of bioglass also form a
high surface area film. However, the release of ions per unit
volume of solvent would not be nearly as high for a large single
piece than of a powder of the same mass. This would eliminate
or at least reduce the potential hazard of very high alkali
concentration. The, controlled release of ions becomes important
in view of the high probability of protein denaturation shown
in this chapter.
It is known that this high surface area glass lso induces
P20^_ and Ca++ deposition [l] and binds tightly to bone. It
is also realized that materials which do not have or create a
high surface area film do not bond at all to bone [4]. There-


curves which were analyzed to give the enthalpy of the
reaction AH, the free energy change AG, and by difference
the entropy change for the reaction AS.
The systems were studied in order of increasing
structural complexity from simple carboxyls, amines, and
organic sulfates to amino acids, polyamino acids, polysaccha
rides, and collagen. Changes in the aqueous solutions by
additions of salts or changes in pH and combinations of
organic molecules were also studied.
Results indicate that there are at least two forces
which contribute to the bonding of the collagen and other
organics to oxide surfaces, hydrogen bonding and ionic bond
ing, the former releasing from 8 to 12 kcal/mole of functional
groups while the latter releases 4 to 6 kcal/mole of
functional groups depending on the relative polarities of
the adsorbing molecules and the surface. There are strong
indications of the denaturation of collagen at some surfaces
at which hydrogen bonding and ionic bonding act cooperatively.
vi


66
Figure 10. Reaction heats for the adsorption of dextran a ,
galactose a, and glucose o, on alumina (closed symbols) and
hydroxyapatite ( open symbols) in low ionic strength solution
at pH 7.


24
Heterogeneous Adsorption
As mentioned earlier, from the standpoint of adsorption
studies, the surface of a substrate is often not uniform.
Heats of adsorption may vary at different positions on the
surface. If the condition is maintained that the different
sites are non-interacting, the adsorption onto a heterogenous
surface may be regarded as simultaneous independent reactions
of the type expressed by equation (1.3)
+ R -y So-^
S2 + R -* SO2
(1.19)
Sj + R -* Soj
where the subscript j enumerates the different types of sites.
The concentration of a single type of solute molecules, R,
is common to all surface sites. Each adsorption reaction,
according to equation (1.19), would evolve a reaction heat
Qj. The total reaction heat, would be the sum of Qj for
each reaction on the different types of sites.
Qt = Qi + Q2 + Qj (1.20)
If AHj is'the enthalpy change per mole of adsorbed molecules
on sites of type, j and Nj is the number of moles adsorbed then
equation (1.20) can be expressed according to equation (1.6) as
-Q. = AH-i N, + AH2N2 + .... AH.N. (1.21)
In a calorimetric measurement it is the value of
which is measured. If the solution is analyzed to determine


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy
^ ^ ^ -j'S-PjC
E,P, Goldberg
Professor of Material Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is full adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy
Q.B. Conklin
Professor of Physics


37
mole [23]. Using the method described in this work the
heat of adsorption of SDS on Linde A alumina was found to
be 10.2 kcal/mole of SDS. The discrepancy, al kcal/mole,
is thought to be due to the surface being wet prior to contact
with SDS. This would prevent any possibility of SDS coming
into contact with a drier, and presumably, higher energy
surface.
A Perkin-Elmer-Hitachi model 139 U.V. Vis spectro
photometer was used for concentration determinations. Separa
tion of particulates from supernatent solutions was carried
out by centrifuging or by filtering through micropore glass
filters. In those cases where filtering was used, the filter
was first saturated with a solution of the organic molecule to
be analyzed, then rinsed thoroughly. In all cases a standard
was used which had been put through identical procedures as the
unknown.
Protein and polypeptide determination was made through
the use of Biuret reagent [87 ] and measuring at 550 millimicrons.
Carbohydrates were determined using a phenol solution and
measuring at 490 millimicrons [88]. Amino acid concentrations
were determined by use of ninhydrin [873. Carboxylic acids
were titrated with phenothalein as the indicator.
Distilled water, having an initial pH near 7, was used
for preparing solutions. The pH was varied by adding HC1 or
NaOH. Three solutions were used as solvents: a low ionic strength
solution (LISS) in which the only ions present were those added
by pH adjustment, a 0.165 M salt solution, and a buffered solution


90
then each bond would entail an energy change of about
-7kcal/mole. On the average then one out of 7 residues
would be in contact with the surface.
Comparison of the dsorption of these uncharged
molecules with those that possess charged functional groups
indicates that the role of PLP or PLHP would be minor in
comparison. Although the mechanism of adsorption has not
been fully explained in this case, there should be little
doubt that when positioned next to a charged molecule in a
peptide chain, the latter will play the dominant role in
adsorption to an oxide surface.
Lysine and Arginine
The adsorption heats of lysine on silica and alumina
are similiar (Figure 16). Since this molecule possesses two
basic and one acidic group at neutral pH, it is reasonable
to assume that the charged carboxyl group is attracted to
the positive alumina surface, and that the positive amine
groups are attracted to the negative silica surface. Because
of the more basic properties of this molecule, it might be
suspected that the reaction with the silica surface would be
somewhat stronger than with the alumina surface. This appears
to be the. case.
Poly-l-lysine and poly-l-arginine are known to exist
in an extended charged coil conformation in solution at
neutral pH [8,9']. If the coils, which are stabilized by


123
Ringers solution and buffered Ringers solution. The enthalpy
change for the experiment run in Ringers solution reflects
similar changes in slope.
Silica and Collagen
The mixing of collagen and silica in low ionic strength
solution is endothermic (Figure 30). When the dissolved salt
concentration is increased the reaction becomes exothermic.
In the buffered solution the reaction heat is increased again
over that found in Ringers solut-ion. The magnitudes of the
thermodynamic variables are generally smaller than those found
for alumina and hydroxyapatite (see Figure 31).
Alumina and BSA
BSA was mixed with alumina in increasing increments in
Ringers solution. Enthalpy was determined by solution depletion
measurements. There is a sharp break in the reaction heat
curve, but the adsorption curve is continuous (see Figure 32).
This causes a commensurate break in the enthalpy curve. When
BSA and alumina are mixed in buffered Ringers solution, the
break in the reaction heat curve disappears (see Figure 33).
Hydroxyapatite and BSA
When BSA is mixed with hydroxyapatite, the reaction heat,
adsorption curves, and enthalpy changes are smooth and without
discontinuities. The enthalpy change is about -5 cal/gm. The
results are the same for the buffered or unbuffered solutions.


108
Mixture of CS and Collagen in the Presence of BSA
Finally, the mixing of CS and collagen in the presence
of BSA in low ionic strength solution and in Ringers solution
is presented in Figure 25. The results for those mixing
reactions are similar to that of the mixing of CS and collagen
in low ionic strength solution without BSA present.
Discussion
Poly-l-lysine, Poly-l-arginine
In experiments discussed earlier in this chapter, where
the reaction heat was considered evidence for strong inter
action, it was agreed that electrostatic attraction was probable.
The mixing of PLA or PLL with CS formed a strong complex,
while the mixing of PLA or PLL with dextran did not. The pattern
fits those established in the previous chapters which is that
the charged polymers react strongly with oppositely charged
polymers (or surfaces).
The enthalpy change determined for the PLL or PLA-CS
reaction is low. It is known that PLL or PLA undergoes a
conformational change when mixed with CS [8,9]. Even though
calorimetric data are not available for these transitions,
similar unfolding reactions [19,20] indocate enthalpy changes,
which correspond to the breaking of the hydrogen bonds, on the
order of 2 to 7 kcal/mole of residues. It is assumed that the'
transitions of PLA or PLL would cause a similar enthalpy change.


54
For example, at pH 5 equation (2.4) gives for a
monovalent electrolyte '(H+)
ip0 = .059 (9 5) = .24 volts
The total charge in solution, using acetic acid as the mono
valent acid at .001 M is
q = a Ane (2.6)
= 1.78x10~5(6x1023)(2x10~6)(1.6xl0~19)
2.7 8xl0-5
= .12 coul.
where A is Avogadros number, n is the total number of mole
cules in solution, and e is the electric charge. The total
electronic interaction energy is
' E = q
= (,24) (.12) = .028 j = 6.7 meal
The value of Q found from microcalorimetry is 4.7 meal. The
agreement is fair. Plots for other values of Q and E for other
acids at different pH values are shown in Figure 8 for CQ =
.001 M. Although the experimental and theoretical results do
not fit well for all cases, the simple model qualitatively
explains the experimental findings, which is what was sought.
Such factors, as treating the ions in solution as other than
point charges, and the potential at the Stern layer are not
taken into account. The most important result is that the
reaction heat and electronic interaction energy decrease as
either \pQ or a decrease. The tenative conclusion develops that
electronic interaction will probably be the essential factor


CHAPTER III
ADSORPTION OF CHONDROITIN SULFATE
AND OTHER CARBOHYDRATES
Introduction
Polysaccharides, notably chondroitin sulfate (CS),
which contain carboxyl and sulfate groups, are present in
dentin and enamel [101] and in bone [102,103]. Under proper
conditions of pH and ionic strength, these polysaccharides
will complex with collagen [104], In the presence of a
foreign surface, these polysaccharides, like other charged
molecules, will adsorb and react not only with collagen but
also with the surface. It is the purpose of the studies of
this chapter to explain the interaction of aqueous solutions
of chondroitin sulfate with alumina, hydroxyapatite, and
silica. A later chapter will discuss the interaction of CS
with collagen.
Chondroitin sulfate is a polysaccharide made up of
basic dimer units of glucoronic acid and galactosamine. Several
simpler carbohydrates were chosen to model CS: (a) galactose,
glucose, D-acetyl galactosamine; (b) glucose-6-sulfate (G6S) ,
D-galacturonic acid; (c) dextran, and (d) polygalacturonic
acid (PGA). Each carbohydrate was chosen because it possessed
a single feature of the CS molecule: (a) the carbohydrate
61


58
AH* Is evidence that only ionized groups participate in
adsorption. This supports the supposition that electro
static attraction is primarily responsible for adsorption.
The same procedure applied to the data for the adsorp
tion of the three acids on alumina does not give such consis
tent results. At pH 5 AH* lies between -6 to -11 kcal/mole
for acetate, -2 to -7 kcal/mole for oxalate and -7 to -3 kcal/
mole for citrate.
Near CQ = .4mM (see Figure 5), a medium concentration,
we find that AH* lies near -9.8 kcal/mole for acetate, -4.1
kcal/mole for oxalate and -8.4 kcal/mole for citrate. This
indicates that while acetate has one, and citrate two ionized
surface groups, oxalate has on the average only one of its
two ionized groups on the surface.
It- was argued earlier that only ionized molecules are
attracted to the surface and that the number of such molecules
drops as the surface charge drops. It should be recalled
that the surface charge on alumina and hydroxyapatite is
produced by the adsorbed H+ ions. This suggests the possibility
that an approaching ionized carboxyl group and surface hydrogen
ion participate in hydrogen bonding.
Zeta potential measurements [100] show that citrate
Changes the surface charge of hydroxyapatite at low concentra
tions. This indicates that electrostatic attraction by itself
is not the only factor in adsorption. If it were, when the
surface charge had been neutralized by adsorption of a sufficient
amount of citrate, adsorption would have ceased and the surface
charge would not reverse.


128
Silica and BSA
The heat of adsorption of BSA on silica was < 0.5 cal/gm.
The adsorption curve indicated that saturation was not
reached at the highest equilibrium concentration of 1 gm/1.
Only the buffered solution was used.
Alumina, BSA, and Collagen
The following mixture of alumina, BSA, and collagen was
performed' as indicated below:
reaction cell
( 1
compartment 1 compartment 2
alumina collagen + BSA
The concentration of the collagen was held constant at .3 gm/1.
The concentration of BSA was varied as before. The heat of
dilution of the collagen and of the BSA was nulled out in the
reference cell.
The reaction heat curve of the collagen-BSA mixture first
decreases from 12 meal to 7 meal, then rises. The turning
point coincides with the discontinuity described in the previous
discussion. If the reaction heat curve for the BSA alone is
subtracted from the reaction heat curve for the mixture, the
result (dotted line in Figure 34) is an approximate reaction
heat for the collagen adsorption. The amount of collagen
adsorbed could be determined by solution depletion. The amount
of BSA adsorbed was previously determined. The enthalpy change
for the adsorption of collagen in the presence of BSA could then
be estimated.


27
or
= -RT' In K^' K* .K# (1.33)
Thus, even though each AG? for the individual.reactions is
constant, the overall free energy change will vary as each
fraction y. varies.
1
The net effect is that if any single type site adsorbs
a large percentage of all molecules adsorbed then
AG| approaches AG? as y| + 1
Generally, howeiver, there is no simple number" AG which can
be expressed in terms of a single equilibrium constant ,
having the form
Kt = K1 K? V- -KjJ (1.34)
The value of K^_ would vary as the fraction of occupied sites
varies.
The physical interpretation is that each site contributes
a specific amount of energy to the total energy change. At
very low concentrations only a very few sites react, presumably
those of higher, energy. At higher concentrations a greater
number of sites react, but of overall lower energy. The result
is a lowering in the average energy change as the concentration
is increased.
Under the conditions of independently acting sites
evaluation of would give AG exactly. However, to do this,
knowledge of each and y. has to be available. In the absence
of such knowledge, the evaluation of K can be approximated by
evaluation of equation (1.29) by replacing 0j by 0^_. The number
found from this method, K, could be used for determination of
AG under suitable conditions.


119
The results are presented for runs made in low ionic strength
solution, in Ringers solution, and in Ringer's solution contain
ing a phosphate buffer. In low ionic strength solution the
reaction heat was lowest, in the presence of Ringer's solution
it was increased by a factor of six and in the presence of
phosphate buffer the reaction heat decreased to the value near
that of the experiment run in low salt solution.
The thermodynamic data are presented in Figure 27. The
values appear high because they are written in terms of moles
of collagen (Each molecule is a triple helix of molecular
weight 300,000). While the free energy changes and entropy
changes are similar for each solution, the enthalpy changes
reflect the change in solvents. All quantities are negative.
In a separate experiment, alumina was mixed as a slurry
in the phosphate buffer solution, permitted to sit for on
hour, centrifuged, and washed in distilled water three times.
The alumina was finally centrifuged and run against collagen
(see Figure 27) in pH 7 Ringer's solution. The enthalpy is
nearly the same as that found in the buffered solution.
Hydroxyapatite
The reaction heats and thermodynamic data of the adsorption
of hydroxyapatite are presented in Figure 28 and 29. The
reaction heats are of similar magnitude for each solvent, and
negative as they are with alumina. There are inflection points
noticed in the reaction heat curves for the reaction run in


84
Figure 15. Heats of adsorption for proline (P), hydroxyproline
(HP), poly-1-hydroxyproline (PLHP), and poly-l-proline (PLP),
on alumina (A), silica (S), and on hydroxyapatite (HA) in low
ionic strength solution and Ringers solution (*).


138
fewer surface sites. The remaining area is available to
collagen. This result similarly applies to hydroxyapatite
and silica.
BSA, CS, and Collagen
When CS is added to the previous mixture, both it and
BSA affect the adsorption of collagen on alumina. Since the
CS adsorption has a similar concentration dependence as
collagen (see Figure 35), and was shown earlier not to react
strongly with BSA, it is suggested that the carbohydrate also
adsorbs between the BSA molecules. The amount of CS adsorbed
decreases in the presence of collagen. This indicates that,
even if CS is being replaced by collagen on the surface or
released from a collagen-CS complex as it adsorbs. This result
is based on the previous findings that CS and collagen bind to
each other [104], and because of the decrease in enthalpy
change of the adsorption of collagen measured in the presence
of CS.
Again in the buffered solution, the discontinuity dis
appears. There is a smooth decrease in the amount of collagen
adsorbed and the enthalpy of adsorption is lowered as before
in the case of the collagen-CS complex. Similar experiments
on hydroxyapatite and silica were not run.
Conclusions
Several effects of adsorption of mixtures of proteins
and the carbohydrates have been examined. The adsorption


20
Substituting equation (1.6) into (1.5), one obtains
~K = -QAHV (1.7)
(N AH + Q)(AHN + Q)
To determine K, at least two adsorption experiments are completed
in which Q is measured but a set of values is usually completed
to determine the endpoint of the titration. The original
number of moles of reacting molecules, N, is varied, and Ng
is held constant by keeping the surface area of the adsorbing
substrate constant. Values for Qj, Q2, N, N, and N are
recorded where N and N are values for N. The value of K is
assumed to be nearly constant if N is not too different from
N. Equation (1.7) can then be solved for AH by using the
quadratic equation (1.8)
AH2 N (Q1 N Q2N) + AH Q-^ (Ng-N-^ +QiQ2 (Q2Qi> = 0
for the two sets of values. The total number of surface sites
can be estimated by dividing the total surface area by the
known cross-sectional area of the adsorbing molecule. This
method is valid as long as the surface concentration of adsorbed
molecules is low and lateral interaction does not occur. Alterna
tively three sets of values can be used to eliminate N from
o
equation 1.7. Both methods give results 10% of each other-.
The volume V is held constant. The value of AH is substituted
into equation (1.7) to find K, and therefore AG by using the
relation
. AG =
-RT In K
(1.9)


Ill
energy sites as claimed for solid surfaces. As stated,
however, the PLA molecule is folded, and whereas CS has a
directional stabilizing effect on the polypeptide, the
isolated monomer does not.
In the former case, the ionized groups of the poly
peptide would be exposed as the molecule is uncoiled by
relatively few CS molecules. This would allow a one to one
or two to one correspondence between basic polypeptide side
groups and the acid groups on CS [8,9]. In the latter case,
the single carbohydrate molecules at lower concentration,
without the advantage of a high linear charge density of CS,
can bind only to those basic groups on the polypeptide which
are exposed. At higher concentrations, more basic groups would
bind but other factors as charge repulsion and steric hinderance
of the charged carbohydrate molecule have increasing effect.
These factors have less of an effect on the CS molecule at
equivalent charge concentration. Therefore, the trend towards
higher energy at low concentrations is attributed to the
availability of positively charged groups on the polypeptides,
and steric and charge repulsion between monosaccharides.
Polygalacturonic Acid
The reaction of PLA with polygalacturonic acid (PGA) is
complex. PGA at pH 7 is in an extended helical structure [98].
At pH 7, no precipitate forms with PLA of PLL and the reaction
heat is lower than found with CS. It is known that CS that has


29
In the example the values of [R] were carried over several
orders of magnitude while the total number of sites N =
C
N| + was kept constant at 10 moles. The results are
shown in Figure 1 .
Two cases are presented. In (a) the fraction N-/N =
1 s
0.1 and 1<2 = 5000 are held constant, while K-^ is given
values of 1000 and 5000. In (b) and 1<2 are held constant
at 1000 and 2000 rspectively while X varies over three
values; 0.002, 0.02, and 0.2.
While 1<2 is less than five times the value of K-^ and
Xx is a good approximation of with the error remaining
within 1%. As approaches 1 the error goes to zero. Also,
at very low concentrations, it is assumed that most molecules
would adsorb only onto the highest energy sites so all values
of y (equation 1.34) go to zero except Y- and the error again
goes to zero. For oxides the overall fraction of highly
reactive sites is small [85,71]. Moreover, the hydrated
oxide surface will be of lower energy than a perfectly dry
surface, aiding in meeting the condition that K-^ not be too
much larger than K2 [60,28]. Under these constraints and using
equations (1.8) and (1.7) with Q = Q AH^_ can be calculated.
For the calculations in the later chapters, the
difference between the values of N for use in equation (1.8)
r
is kept small so that variation of AH in that concentration
interval is small. The values of AG calculated tend to remain
within 20% of the highest to lowest values. This corresponds


substances is as mentioned earlier. Variations in pH were
made through additions of HC1 or NaOH.
The thermometric titration technique was used to cal
culate the thermodynamic functions. Solution depletion was
also used to determine the enthalpy change and used as a check
on the calculations.
The collagen macromolecule used in this study is more
precisely termed tropocollagen. It is the precursor of the
large fibril seen in electron micrographs. It is synthesized
extracellularly by enzymatic reaction from procollagen [75].
Tropocollagen has a reported molecular weight of 285,000.
The molecular weight of the collagen used in these experiments
was found to be 290,000 as determined by gel chromotography
using BSA as a standard, and 300,000 as determined by the
manufacturer by request. The molecule consists of three similar
polypeptide chains which make up a rigid triple stranded helix
3,000 A long and 15 A in diameter. Each chain has about 1,000
amino acid, residues, one third of which are glycine, the
smallest of the amino acids. In human tendon, proline and hydroxy
proline make up about 22% of each molecular chain, whereas
lysine and arginine acid and glutamic acid make up 12%. The
greater percentage of acid groups brings the IP down to 5.5.
In this work, tropocollagen is referred to simply as collagen,
and was supplied by Aldrich Chemical Company [97] in an
aqueous solution.


43
The sodium salts of acetic acid, oxalic acid, citric
acid, and polyacrylic acid (PAA), containing one, two, three
and multiple carboxyl groups were selected for study. The
different number of charged groups, different degrees of
ionization and molecular structure of each of these molecules
should provide a sufficient variety of detectable changes in
the calorimetric measurements. Analysis of the various changes
should furnish a clear understanding of the adsorption
mechanism.
The structural; formulas of each of the molecules used
in the work described in this chapter are given below:
H3C-C00H
H00C-C00H
Acetic
Acid
Oxalic
Acid
H OH
1 \
HOOC C C -
i i
H
1
H
1
H
1
C COOH
1
1
(r C -
I
1
C -h
I
H H
COOH
i
H
1
COOH
Citric
Acid
Repeat
Unit
of
Polyacrylic
Acid
Experimental
Sodium salts of the carboxlic acids were purchased
from Sigma Scientific, Inc. [96]. Poly(acrylic acid) in a
65% aqueous solution was purchased from Aldrich Chemical
Company C97] and was reported by them to have a molecular weight
of 2000. Linde A alumina, and hydroxyapatite were used as
substrates.


13 7
Figure 38. Schematic representation of BSA adsorbing onto an
alumina surface; (A) a molecule in solution in its native
state., (B) adsorbed onto the surface in its native state, (C)
partially denatured on the surface, and (D) totally denatured
on the surface due to attractive forces.


I cerify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Ronald E. Loehman ~
Professor of Material Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
X y"
L.L. Hench
Professor of Material Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
R'.T. DeHoff
Professor of Mq'xerial Science
and Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable satndards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
y .
Professor of Material Science
and Engineering


52
systems under consideration is H+. The surface potential
may then be written from equation (2.3) as
= 2.3 RT (log [H+] log [Hn]) (2.4)
where concentration are substituted for activities and
where [H ] is the hydrogen ion concentration at the pzc.
The interaction due.to the surface potential and the
charge, q, in solution gives rise to an interaction energy,
E [64]
E = f ijj dq (2.5)
where integration is necessary since the charge concentration
is a differential process. The surface potential affecting
ions in solution will decrease as saturation of the surface by
charged ions is approached. The total interaction energy is
nearly equal to ^Qq in this model if the concentration of charged
ions in solution is low so that there is little interaction of
the adsorbed ions on the surface. In this case the surface
potential which each ion encounters will be the same. The
energy lost by the ions is transferred to other ions and solvent
in the form of kinetic energy and flows as heat out of the
system.
A plot of \p from equation (2.4) and a from equation (2.1)
o
versus pH for a hypothetical monovalent acid with a pKQ near 5
and an oxide with a pcz near 9 is given in Figure 7. At low
pH, a, and thus the charge in solution decreases. At high pH
the surface potential decreases. The highest interaction should
be expected to occur where and a are not near zero.


meal
124
Figure 30. Reaction heat for the adsorption of collagen onto
silica at pH 7.


148
Discussion
The experiments with silica gel emphasize the finding
that the gel surface must be hydrated before adsorption
can easily occur. Since the bioglass is known to form a
gel in the dissolution process, it can be seen that bioglass
samples which have formed a gel and permitted to dehydrate
may react differently than a gel forming in solution. The
experiments using bioglass in a high and low surface area
state also emphasize this point.
Before the high surface area gel film formed on the
bioglass (Curve 1 and 2 in Figure 40), the amount of collagen
adsorbed was low compared to that adsorbed after 24 hours
dissolution (Curve 4). After dissolution, the amount adsorbed
increased tremendously in each case and most notably after
the glass was rinsed.
The differences between curves 3 and 4 can be explained
by speculating that the collagen adsorbed in the pre-mixed
situation (3) was incorporated into the film as it formed,
with new film formation developing on or with the surface of
collagen. Both samples (3) and (4) are of the same surface
area. The difference between curves (3) or (4) and curve (5)
is more difficult to explain.
The only difference which can be easily cited for curve
(5) is that the glass, upon rinsing, once again begins to
undergo dissolution. Associated with this is a heat of
dissolution of the dissolving ions. It is suggested that heat


mg. collagen ads. Q., meal
129
Figure 34. Adsorption reaction parameters for the adsorption
of collagen in the presence of BSA at pH 7 in Ringers solution.
cal./gm. collagen adsorbed


cal.
147
o
O'
200
100
0 0.5 1.0 1.5 2.0
C0, gm/1
Figure 41. Reaction heat, Q, o and heat of adsorption
of collagen onto 45S5 bioglass corresponding to curve (4)
of Figure 40.
Heat of adsorption, cal./gm.


form high energy sites. For equal amounts adsorbed, a
greater fraction of adsorbed molecules would be on these
sites for alumina, than for either hydroxyapatite or silica.
Negatively charged polysaccharides also displayed an increase
in enthalpy change at lower concentrations attributable to
high energy sites. More than a single species of these
specially adsorbing areas appears likely [72]. The free
energy changes are of the same order of magnitude as those
found for the carboxylic acids and polysaccharides.
Conclusion
As found in the previous chapters, molecules with
charged ionic side groups react more energetically with oxide
surfaces than those without. The enthalpy change per
charged group lies between -4 and -10 kcal/mole. For those
polyamino acids which possess no charged side groups, the
magnitude of the enthalpy change is found to be less, near
-1 kcal/mole of residues. Although the mechanism for uncharged
molecules is not clearly defined, it is believed that conforma
tional deformation does not contribute significantly to the
enthalpy changes measured. It is possible than an uncharged
polyamino acid could be bound to an oxide surface by a few
relatively high energy contacts. Polyamino acids with charged
side groups, however, will play the dominant role in adsorption


44
Titrations of the carboxlic acids were carried out
with HC1 or NaOH. Determination of the amount of acid
adsorbed was based on. calibration against known concentrations.
Other methods and procedures were described earlier.
Results
Reaction heats for the adsorption of the three simple
carboxylic acids on alumina are presented in Figure 4. For
each acid, the maximum reaction heat was found to occur when
the solution was near pH 5. The reaction heats at pH 3 were
second and the lowest curves were recorded for pH 7. The
highest heat was recorded for sodium oxalate which also has
the lowest dissociation constant (see Table 2).
The enthalpy change upon adsorption was first found
by determining the amount of each acid adsorbed by titration.
The method was suitable only for pH 5. At pH 3 and 7 poor
precision resulted because of the small amount of each acid
adsorbed. Results are given below:
acetic oxalic
-AH (kcal/mole molec.) 4.2 4.8
(CQ .01M) -
Thermodynamic data were calculated for pH 5 using a
smaller concentration range (C = 10-L* to 10^ M) (see Figure
5).. These results show that there are differences in the
enthalpy change for the three acids used. Each curve displays
a tendency towards more negative (exothermic) values at the
lower end of the concentration range. The heat of adsorption
citric
4.6


68
almina, as determined by solution depletion (see Figure 12).
Values for AH lie between -6 and -8 kcal/mole.
The adsorption of D-galacturonic acid on silica is
endothermic. At low concentration, the enthalpy is +700
cal/mole, becoming more positive at higher concentrations.
The amount adsorbed was determined by solution depletion. The
free energy change and entropy change were not calculated.
The change in enthalpy for the adsorption of glucose-6-
sulfate on alumina, hydroxyapatite, and silica was determined
to be -7.6, -5.4, and 0.3 kcal/mole of adsorbed molecules,
respectively.
PGA
Calorimetric measurements for the adsorption of poly-D-
galacturonic acid on alumina and hydroxyapatite were hampered
by agglomeration of the particles by the polymer. The thermo
dynamic functions for this reaction were calculated and are
shown below.' In the concentration range used, .001 M to .01 M
of residues, these values were constant ( 0.1 kcal/mole).
AG
AH
AS
(kcal/mole)
(kcal/mole)
(kcal/mole/
deg)
alumina
-3.4
-2.54
3.5
hydroxyapatite
-4.2
-.31
15.0
In contrast to
the decrease in
entropy found
for the
adsorption of PAA on
alumina or hydroxyapatite, the
entropy
change is positive for adsorption of
poly-D-galacturonic acid
on both alumina and hydroxyapatite indicating an over-all
decrease of ordering.


55
in enthalpy changes measured for charged adsorbing molecules.
The enthalpy and free energy change values are within the
range expected from the investigations of other workers.
Some idea of the number of active groups of each
molecule actually on the surface can be obtained from the
following analysis. Using the data from Table 4.and Table 5
the average number of ionized groups on acetic acid, oxalic
acid, or citric acid can be determined from their dissociation
constants. The numbers are given in Table 4. If we divide
the enthalpy change per molecule by the average number of
ionized groups. AH" is obtained. This assumes that the enthalpy
change for the adsorption of carboxyl groups is about th
same regardless of the molecule being considered. Results are
given in Table 5 for pH 5 and 7 at .001 M.
The values for AH* are fairly consistent in each case.
Considering the enthalpy change of the adsorption for acetate
as an arbitrary baseline, we can argue that if AH* for oxalate
or citrate had been much larger than that for acetate it
would have implied that more groups per molecule were participat
ing in the surface reaction than expected from the degree of
ionization. As is,AH* for oxalate is slightly lower and citrate is
slightly higher than the AH* for acetate at both pH values.
Oxalate is the most strongly dissociated of the three acids
implying, perhaps, a slightly stronger reaction with the surface.
It should be realized that a particular group cannot be partially
ionized at a particular instant in time. The invarience in


10
The effect of changes in ionic strength on adsorption
is also not well understood [54]. Dissolved salts disrupt
hydrogen bonds which proteins depend on for conformational
stability [55], Changes in the ionic strength of the solvent
will also have effects on the adsorbing surface. For example,
phosphate, a common component in buffers, will change the surface
charge of alumina [56]. Generalizations are difficult to
make about the action of specific ions on adsorption unless the
specific system under study is clearly defined.
Surfaces
Silica, alumina, and hydroxyapatite, the three materials
used in this study, are oxides. Hydroxyapatite is sparingly
soluble at neutral pH whereas silica and alumina are virtually
insoluble [57]. All are hydrophilic and each displays a
surface charge which varies with pH.
Thesurface charge results from exposed surface atoms
attempting to complete their coordination of nearest neighbors
[58]. Exposed cations do this by pulling an 0HT ion or H^O from
solution and anions by attracting a proton from the aqueous
phase. The result is adsorbed H+ or OH- ions which assume their
respective charges on the surface.
Any other ion which can pass between the solid and
liquid phases may also help to establish the surface charge.
Such ions are called potential determining ions. Thus,
OH- and H+ are potential determining ions for each of the


AH, cal./mole
120
12
10
CO
'O
r
X
CD 4
<3
RINGERS
LOW IONIC.
STRENGTH
\
~ 2
0 d>
101
-fr
BUFFERED
AG = 10'
?
0
0
0.83
0.25
1.67
0.5
C
5
75
3.3
1.0
yjM
gm/1
Figure 27. Thermodynamic data for the adsorption of collagen
onto alumina at pH 7; AH AS; The free energy change
was approximately equal in each cas to-lO3 cal/mole. Enthal
py change determined by solution analysis --e;-


-AH, kcal./mole
b b
Figure 16, Heats of adsorption for lysine (L), poly-l-lysine
(PLL), and poly-l-arginine (PLA), on silica (S), alumina (A),
and hydroxyapatite (HA) in low ionic strength solution at
pH 7. Values determined by solution analysis e others
by calculation.


142
is found in abundant quantities in the bioglass-bone inter
face which is partially comprised of a silica gel layer. The
reaction of collagen on the bioglass surface for short time
periods is less well understood.
Experimental
The bioglass used in this study, 45S5, has the composition
SiC^ CaO Na20
mole % 46.1 26.9 24.4 2.6
and was prepared in our laboratories. It had a surface area
2 2
of 0.44 m /gm before and near 400 m /gm after dissolution.
Silica gel was purchased from Fisher Scientific and
used without further purification. Its surface area was
9
measured to be 270 m /gm. Collagen concentrations were deter
mined using Biuret reagent and measuring optical adsorbance
at 540 nm.
Results
Figure 39 shows a plot of milligrams of collagen adsorbed
on 1.0 gm of silica gel at pH 7 as a function of the equilibrium
concentration: phospahte Ringers solution and a low ionic
strength solution. The gel was used in two conditions:
reprecipitated from a basic solution and without the reprecipi
tation procedure. When used without reprecipitation, the gel
would adsorb .05 mg of collagen or 1.85 x 10"^ mg/m^.
Two experiments were run in buffered solutions with the
gel: one with and one without reprecipitation. The former
adsorbed slightly more collagen than did the gel in the low


Figure 13. Thermodynamic data for the adsorption of chon-
droitin sulfate on alumina in low ionic strength solution
at pH 7; AG AH AS .


ENTHALPY (-Kcal/mole)
82
C0 mM
Figure 14. Heats of adsorption for alanine and poly-l-alanine
(PA) onto alumina, silica, and hydroxyapatite (TCP) in low
ionic strength solution at pH 7. run in .165M salt solution.


97
Bovine serum albumin (BSA) supplied by ICN Life
Sciences Division [111] was a powdered preparation. It has
a molecular weight of 69,000 and an IP of 4.9.
Results
Poly-l-arginine
The results of the mixing of poly-l-arginine (PLA) and
various carbohydrates is shown in Figure 18. The mixing of
CS and PLA at pH 5 and 7 forms a precipitate in solution which
is filterable. Experiments for pH 5 and 7 show identical
results. There is a nearly constant increase in reaction
heat with an increase in concentration of PLA. Near CQ =
2 x 10-3 M, the curve levels and remains constant. The
enthalpy change for this reaction (Figure 19) was determined
to be -7.50 kcal/mole of PLA residues.
The mixing of the uncharged molecules presents a
different,picture. The mixing of galactose or dextran with
PLA showed little reaction heat and no precipitate formation.
Both showed a negative reaction heat between an initial mixing
concentration of zero and 2.0 M of PLA. The calculated enthalpy
change lies between +30 and -200 cal/mole of residues for
dextran and +50 and -30 cal/mole for monomers of galactose.
The variation is unexplained, but may be due to conformational
changes.
The results of the mixing of D-galacturonic acid or
glucose-6-sulfate with PLA are similar. Their reaction heats


127
CQ, grn/1 BSA'
Figure 33. Heats of adsorption for the adsorption of BSA onto
alumina, silica, and hydroxyapatite in several solutions.


mg. adsorbed
Figure 39. Adsorption of collagen onto silica gel.


15
of several synthetic and naturally occurring calcium phosphates
exposed to organic acids^ show shifts in the P-0 stretching
frequency [39] which were attributed to hydrogen bonding. Other
workers [30] have shown that HgO+ ions are hydrogen bonded to
the calcium. There have been suggestions that there is some
covalent bonding between organic constituents and hydroxyapatite
in bone [74]. Binding of calcium ions by collagen has been
demonstrated by solution analysis [75]. It has not been shown
that collagen can attach to the surface of the hydroxyapatite
without an intervening water molecule or hydroxyl ion.
Forces of Adsorption
The relationship between the enthalpy of a reaction and
the total energy is H = E + PV. Most biological processes occur
in liquids rather than in the gas phase [76]. In this case
the changes in pressure and volume are small. To a good
approximation then, dE ~ dH.
The total energy of adsorption is affected by the type of
interaction between the surface and the molecule. This energy
is comprised of several components. They may be classified as
non-polar, ionic, hydrogen, and covalent bonding [77-79].
Non-polar (dispersive) forces are always present between
molecules. They arise because the time-averaged electron cloud
interaction between uncharged atoms is attractive [80], They
are moderately strong, producing' energies in the range of 1 to
10 kcal/mole. Hydrophobic bonding is a result of dispersive


136
concentration prevents the molecules from unfolding. This
situation is depicted in Figure 38. Instead of being denatured,
the molecule is assumed to remain in a globular shape. The
slope of the Q vs. concentration curve remains smooth over the
range measured indicating that the change in reaction heat is
not due to a change in the number of molecules adsorbed.
BSA and Collagen
The adsorption of collagen on alumina in the presence of
BSA in Ringers solution follows a pattern which is the inverse of
the reaction heat curve for the adsorption of BSA on alumina.
It appears that the collagen is adsorbed on the remaining
free surface after BSA adsorption. It was also found that the
enthalpy change per molecule of adsorbed collagen in the presence
of BSA is fairly constant despite the shape of the adsorption
curve. It was shown earlier that the reaction of collagen and
BSA, assumed to be globular, is weak. These three facts combine
to predict that the collagen reacts only in a minimal way, if
at all, with adsorbed BSA. The step indicates some reaction
in this state. It is conjectured that it is the higher charge
density of the solid surface which attracts the collagen.
In the presence of BSA in the buffered solution, collagen
adsorbs onto alumina in a smooth pattern. The amount adsorbed
decreases with an increase in BSA concentration. This leads to
the same conclusion as before, that the phosphate adsorbs on
preferentially high energy sites and prevents denaturation of
the BSA. Thus, the BSA remains in globular form and occupies


160
22. S.G. Dick, J. Col. Int. Sci., 37(3) 595 (1971).
23. S. Raghavan, D.W. Fuerstenau, J. Col. Int. Sci.,
50(2) 319 (1975).
24. A.C. Zettlemoyer, J.T. Chessick, Adv. Chem. Ser., 5,
89 (1963).
25. N. Hackerman, A.C. Hall, J. Phys. Chem., 62, 1212 (1958).
26. A.C. Makrides, N. Hackerman, J. Phys. Chem., 63, 594
(1959). :
27. H. Eley, ed. Adhesion, Oxford University Press, (1961).
28. W.H. Wade, N. Hackerman, J. Phys.' Chem. 64 1196 (1960).
29. W.H. Wade, R.L. Every, J. Phys. Chem., 64, 355 (1960).
30 M.E. Dry, R.A. Beebe, J..Phys. Chem., 64, 1300 (1960).
31. D.W. Fuerstenau, P. Roy, J. Col Int. Sci., 26, 102 (1960)
32. W.K. Lowen, E.C. Broge, J. Phys. Chem., 64, 451 (1960).
33. D.S. Maclver, H.H. Tobin, J. Phys Chem., 65, 16 (1961).
34. D.J. Crisp, J. Col. Int. Sci., 11, 356,(1956).
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36. M. Hair, Chemistry of Biosurfaces, Marcel Decker (1971).
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38. M. Bertolucci, Adv. Chem. Ser., 9_, 125 (1963).
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40. G.A. Anslow, Disc. Far, Soc. 9_, 299 (1950).
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47. B. Sorbo, Acta. Chem. Scan., 15, 139 (1961)


77
Conclusions
In this section we have determined some of the thermo
dynamic features of the adsorption of chondroitin sulfate on
alumina, hydroxyapatite, and silica by the use of model
carbohydrates. The results show that for positively charged
alumina the enthalpy change for the adsorption of charged
carbohydrates is about the same as that for the carboxylic
acids and lies between -7 and -9 kcal/mole of adsorbed species.
In these experiments the entropy change is negative and the
enthalpy change forms the major portion of the free energy
change. The enthalpy change for the adsorption of the charged
monomeric carboxylated carbohydrates on hydroxyapatite is close
to -7.6 kcal/mole. The adsorption of carbohydrates containing
the sulfate group on alumina or hydroxyapatite is about -5.4
kcal/mole. The similarity in enthalpy for the reaction suggests
a similar type reaction. The adsorption of charged carbohydrate
monomers on silica is weak and endothermic. The uncharged
monomers produce an enthalpy change only a fraction of that of
the charged monomers.
It was found that a model of the uncharged polymer backbone
of CS does not produce a large reaction heat or an enthalpy
change, suggesting that the presence of charged groups is
required to enhance the adsorption reaction. The data on
adsorption of polygalacturonic acid supports this conclusion.
Polygalacturonic acid adsorbed strongly, producing
agglomeration of the solid particles at high concentration (.1 M).


AG, AH, cal./mole
106
to
I
o
I1
40
30
20
10
0
n>
h
x
hO
Figure 23. Thermodynamic data for the mixing of CS and col
lagen in low ionic strength solution at pH 7. Run in
Ringers solutions AG AH AS .
0T


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CALORIMETRIC MEASUREMENTS OF THE ADSORPTION OF COLLAGEN
AND OTHER ORGANICS ONTO OXIDE SURFACES
By
Paul J. Buscemi
March, 1978
Chairman; R. E. Loehman
Major Department: Materials Science and Engineering
The present work is the result of the application of
solution microcalorimetry to the problem of determining the
energies of adsorption of organic molecules onto ceramic
surfaces. The systems studied were chosen to model the
attachment of collagen to ceramics and to provide some expa
nation for the observed bonding of ceramic implants to bone.
An aqueous solution of an organic molecule such as a
polyamino acid, polysaccharide, or smaller molecules with
similar functional groups was automatically mixed in a micro
calorimeter with a slurry of a powdered oxide such as AI2O3,
SO2, or a special glass composition and the heat evolved or
adsorbed was determined. Calorimetric- measurements were
performed on increasing concentrations of reacting organic
molecules for a fixed weight of powder with known surface
area. Plots of the reaction heat, Q, versus the inital con
centration of the organic, CQ, yield thermometric titration
v


Ceq, mg/ml
Figure 40. Adsorption of collagen onto 45S5 bioglass under
the conditions described in the text.


22
The partial molar enthalpy is found to be
% =' -RT2 ( 9T)pn (1.12)
or in view of equation (1.11)
~Ui = -RT2(91n ^'iref/9T)pn RT2 (91n ai/9T)pn (1.13)
or = -RT2(9ln ^ef/9T)pn RT2 (9In f^T)
Under the constant composition does not vary with temperature
The first term of equation (1.13) is the enthalpy
change for component i which would occur if the reaction was
held under reference conditions. The standard enthalpies, ,
of pure compounds are the enthalpies of reaction of building
up those compounds from their elements under standard conditions
(P = 1 atm T = 298K). The chemical elements themselves have
zero standard enthalpies of formation. If the reaction is
carried out under standard conditions, the enthalpy change for
the reference state is equal to H?. Then
~Hi = H? RT2 ( 9In f/ 9T) (1.14)
Knowledge of f^ for the systems under study is lacking.
We therefore make the approximation that AH is significantly
larger than the natural logarithm of the temperature variation
of f. The validity of this approximation relies upon the non
interaction of solute molecules. This condition is assumed to
hold for dilute solutions. Therefore
Â¥i = Hp (1.15)
To relate to AH it is noted that AH can be written
as the difference of the sums of the partial molar enthalpies
of products and reactants


140
charged at neutral pH, because the number of carboxyl groups
is greater than the number of amine groups, adsorption on
alumina is more favorable than on hydroxyapatite. Oppositely
charged reactants, however, also increase the chance for
denaturation of the protein, and tissue rejection. Systems
which allow tissue ingrowth without severe denaturation can
be predicted by the density and the sign of the surface
charge. Polymers which are neutral as polyethylene can be
expected not to denature collagen, and also to have low
adhesion properties.
The behavior of collagen as a function of solvent type,
moieties in solution, and surface composition have been
investigated. The behavior of a special glass in relation
to collagen adsorption will be discussed in the next chapter.


15.8
It may be noted that metals which normally possess a
negative surface charge in neutral pH where collagen and 1 BSA
are also negatively charged do not fare well as bonding agents.
Bioglass and hydroxyapatite fit the above criteria and do enjoy
success as prosthetic materials. Alumina and other glass cer
amics falling outside the compositional range of bioglass
do not meet the above requirements and fail as a result.
Among the avenues which lead to future work in the .
study of adsorption of proteins by calorimetry are those
which will include detailed thermodynamic descriptions such
as those applied to conformational changes and to multipoint
equilibria in todays literature. Applied as an analytical
tool, calorimetry can offer a great amount of information
even if detailed mathematical description is ignored. There
is an enticing fundamental importance in such descriptions.
However, defining the variables in the adsorption of BSA in the
presence of CS and collagen would be a formidable task. In
lieu of such complexities, broader definitions and descriptions
have been shown to to yield plausible results and suggestions
for the advancement of studies in the field of prosthetic
devices.


en co
56
pH
Table 4
Ionized Groups per Molecule
for Three Carboxylic Acids
Acetic
0.02
0.63
1.0
Oxalic
1.0
1.85
2.0
Citric
0.42
1.9
2.8


found to be positive, whereas direct calorimetric measure
ments show the reaction to be exothermic.
Ionic interaction, as explained in the discussion section
of Chapter II, can account for the amount Of protein adsorbed
onto a surface in the same way that it accounts for the amount
of ions adsorbed. This is a function of the isoelectric point
Of the protein and the pcz of the surface. In that section
it was explained that the electrostatic interaction energy can
be described in terms of the amount of oppositely charged
species present in solution and on the surface.
If a scale is drawn (see Figure42) representing the
pzc of the surface and the IP of the protein, it can be seen
that as the IP increases the electrostatic attraction between
the protein and the surface increases. A plot of the predicted
amount of various proteins adsorbed vs. IP gives the correct
order as that found by Messing [52]. The curvature found in
Messing's adsorption curve can probably be attributed the differ
ences in the dissociation constants for each of the proteins
and also, as suggested by Messing, to the molecular weight of
the proteins.
In another experiment [42] the amount of BSA adsorbed
onto glass varied with pH as shown in Figure 42. The points
between the IP and the pzc would be predicted to display maximum
adsorption, and this is what is found. These results are similar
to those found for the carboxlic acids as described in Chapter
I, and the same explanation applies.


59
The single charged acetate ion does not reverse the
surface charge of hydroxyapatite or alumina, this would
indicate the absence of other than electrostatic interaction.
It will be recalled from the introduction that close approach
of hydrogen to an anion is required for hydrogen bonding to
occur. Since, at pH 7, acetate has one and citrate has three
ionized groups, it is thought that multiple groups are required
to pull the molecule close enough to the surface for hydrogen
bonding to occur. The interplay between surface charge,
molecular size and charge density, ionic and hydrogen bonding
becomes apparent in these situations.
Polyacrylic acid has such multiply-charged groups. It
is known to be a linear molecule which is fully ionized at pH 7
[ ]; therefore, there are no hydrogen bonds to be broken due
to an unfolding of the molecule upon adsorption. The AH between
-5.7 and -6.9 kcal/mole of residues of PAA is close to that
found for the adsorption of the carboxyl group of the other
molecules on alumina and hydroxyapatite. The similarity in all
these instances implies that the same type interaction occurs,
and is not a strict function of surface composition or of
molecular structure.
Conclusions
There appears to be no particular differences in the
enthalpy of adsorption of a carboxyl group onto alumina or
hydroxyapatite due to the number of groups on a molecule. In
each case the enthalpy change is near -6 kcal/mole and an


CHAPTER' I
INTRODUCTION
The study of adsorption and interaction of proteins and
other biological molecules onto non-biological surfaces is
important because of the increasing use of prosthetic materials
[1]. It is essential to know how each of these two distinctly
different components will interact in biological media. In
this study, a further understanding of the reaction between
the connective tissue protein, collagen, and various oxide
surfaces is sought.
Two properties of collagen adsorption are of major
concern: how well does the protein adhere to the oxide surface
and does the surface change the structure of the protein?
Protein adhesion relates to the binding of tissue to a prosthetic
material and can be approached by the determination of the
enthalpy AH of the adsorption reaction [2]. Changes induced
by the surface on the protein lead to denaturation. This
increases its vulnerability to enzyme attack and eventual
rejection from the host [3]. This question can be approached
by comparison of the enthalpy of reactions of model systems
with that of reactions which have been found not to be
disruptive to the structure of protein.
Three materials serve as substrates: silica, alumina,
and hydroxyapatite. Each of these materials is well character-
1


110
If this were the only contribution to the enthalpy change,
the overall reaction would be endothermic. A negative
contribution can be provided by the binding of the polysaccharide,
CS, to PLA or PLL. The enthalpy change for the binding of
cationic dyes to CS has been shown to be near -8 kcal/mole
C13 3 The data presented earlier show that the mixing of
G6S or DGA with PLA or PLL have enthalpies, at low concentra
tion, near -1 kcal/mole. In the absence of any other major
contributions to the reaction, the mixing of CS with PLA or
PLL then consists of two components: a conformational change
of the polypeptide and the binding process. There is no
conformational change for CS [8,9]. The total enthalpy change
corresponding to these two processes would tend to cancel,
resulting in a low overall enthalpy change. In the case of PLL
and CS the enthalpy change is small and positive (endothermic).
The more exothermic reaction of PLA over PLL with CS is
attributed to the more basic guanidine group of PLA rather than
the E-amine groups of the PLL side chain.
D-gaiacturonic Acid
The large negative enthalpy change for D-galacturonic
acid and glucose-6-sulfate measured in the lower concentrations
of PLA used (Figure 18) indicate that strong interactions
are occuring in this range. The enthalpy change for CQ = .05 mM
to 1 mH are equivalent to that of CS and PLA. Since all the
sites for eac.h molecule are assumed to be identical, the
curvature in the AH curve cannot be due to different high


161
48. H.L. Lee, J. Biomed. Mat. Res., 3_,- 349 (1966).
49 L, Nor de, Protides ^Bio'l: Fluids, 2 0", 467 (197Q)
50. C. Tanford, Physical Chemistry of Macromolecules, ch. 4,
J. Wiley-6 Sons, (1961).
51. W. Dilman, I Miller, J. Col. Int. Sci., 44(2) 221 (1973).
52. R.A. Messing, J. Non-Crystalline Sol., 19, 277 (1975)
53. J.P. Hummel, B.S. Amderson, Arch. Biochem. Biophys.,
112, 443 (1965) 3
54. E. Katchalski, I. Simon, Polyamino Acids Polypeptides
and Proteins, M.A. Stalhman, ed. U. Wis. Press. (1962).
55. A.D. McLaren, J. Phys. Chem., 58, 129 (1954).
56. Y.R.Chen, J. Col. Int. Sci., 43(2) 421 (1973)
57. V.K.LaMer, J. Phys. Chem., 66, 973 (1962).
58. G.A. Parks, D.L. DeBruyn, J. Phys. Chem., 66, 967 (1962).
59. R.K. Her, J. Electrochem. Soc., 117(1) 91 (1970).
60. R.O. James, T.W. Healy, J. Col. Int Sci., 40(1), 65 (1962)
61. C. Huang, W. Stumm, J. Col Int. Sci., 43(2), 409 (1972).
62. T.W. Healy, J. Macromolecular Sci., A8(3). 603 (1974).
63. J.A. Yopps, D.W. Fuers.tenau, J. Col. Int. Sci., 19,
61 (1964).
64. E.J.W. Verwey, J.T.G. Overbeek, Theory of the Stability
Lyophobic Colloids, Elsevier Publishing Co. Inc. (1948).
65. D.O. Shaw personal correspondence
66. M.J.P. Low, P.L. Lee, J. Col. Int. Sci., 45, 148 (1973).
67. K. Tsutsumi, H. Emori, Bui. Chem Soc. Jap., 48(10).
2613 (1975)
68. M.L. Hair, J. Non-Crystalline Sol., 19, 299 (1945).
69. H.H. Weetall, J. Biomed. Mat. Res. 3., 471.(1969).
70. -M. Hasegawa, M.J. Low, J. Col. Int. Sci ., 30 ( 3). 378 (1969 ).
71. R. Greenler, J. Chem. Phys. 37 (9 ), 209 4 (1962 )
72. M.M. Bhasin, C.Curran, J.. Phys. Chem., 74(22), 3973 (1970)
73. H. Schraer, Biological Calcification, Appleton-Century-
Crofts (1970).


3
have demonstrated that the backbone conformations of the
molecule are similar to native collagen [7]. Poly^-l^lysine
and poly-l-arginine have been used as models for collagen in
relation to the structure of amorphous ground substance [8,9].
Poly-l-lysine and poly-l-glutamic acid have been used in
conformational studies using differential capacitance techniques
[10]. In other studies, workers have used combinations of
synthetically prepared amino acids to model collagen [11,12].
Smaller molecules representing isolated residues of
the collagen molecule have also been investigated but not as
frequently as the polyamino acids. Dyes containing amino
groups have been shown to selectively adsorb chondroitin sulfate
[13], an important carbohydrate in structural tissue [14].
Surface viscosity measurements using amines, amides, and
carboxylic acids as model proteins have been studied in relation
to bilayer film formation [15] in membrane studies.
In biological studies of proteins other than collagen,
molecular models have been widely studied. Enthalpies of
aqueous solution have been determined calorimetrically for
amines and carboxylic acids [16] as part of a quantitative
description of biological systems. Heat capacity measurements
[17] on several amino acid solutions have been made to help
explain protein structure* The binding of short amino acid
chains [18] in subunit studies of immunoglobulin has also been
investigated by calorimetry. Differential scanning calorimetry
has been used to study conformation changes of many polypeptides
used as models for collagen [19,20],


131
C gm/1 BSA
Figure 35. Heat of adsorption of collagen onto alumina in the
presence of BSA and CS ,; adsorption of collagen----.
Adsorption of CS in the presence of collagen and without
collagen presente.


5 7
Table 5
Enthalpy Change per Mole,
AH, and
Enthalpy
Change per
Mole of Ionized
Groups,
AH*
PH
Substance
-AH
ionized
-AH*
(acids)
(kcal/mole)
groups
(kcal/mole)
5
Acetic
3.3
0.63
5.20
5
Oxalic
9.0
1.85
4.86
5
Citric
11.0
1.90
5.90
7
Acetic
5.4
1,00
5.40
7
Oxalic
9.8
2,00
4.90
7
Citric
17.0
2.80
6.10


11.2
had the sulfate groups removed does not react with PLL or
PLA in the same manner as natural CS [8,9], It is also known
that the carboxyl groups of PGA (and CS) are charged at pH 7,
so that electrostatic attraction exists between PGA and PLA.
It was shown that if the pH of the PGA-PLL mixture is raised
above 11, that a precipitate does form. As pH increases, charge
neutralization of the polypeptide side chains increases, and
intramolecular hydrogen bonding of the polypeptide becomes
weaker [76]. One may speculate that PGA can then impose
directional constraints- on the conformation of the PLA molecule.
The calorimetric measurements of PGA and PLA at pH 13 (Figure
18) support this argument, since as the pH is adjusted from
7 to 11 the enthalpy change drops from +0.5 kcal/mole to -0.3
kcal/mole of P1A residues.
Poly-l-alanine, Poly-l-proline
The interaction of CS with PA or PLP (Figure 22) is
apparently of the same type as found with PLA and dextran. PA,
PLP, and CS are each found in a helical conformation at pH 7
[112]. There is no unfolding of the polypeptides as with
PLA to complicate the interaction. The lack of initial attraction
is what apparently prevents extensive reaction as found with
CS and PLA. It is as if, for these molecules, there is only a
single species in solution.
CS and Collagen
The isoelectric point of collagen is 5.5. At pH 7, it
maintains an overall negative charge. The CS-collagen mixing


157
It is not suggested that hydrogen bonding or other
bonding forces are not present. It is reasoned that attraction
of a protein to a surface must be initiated by other than the
simultaneous presence in solution.
The study of protein adsorption should be accomplished
in relation to a specific surface in a specific solvent. The
difficulty in generalizing the adsorption of different proteins
thus arises from their varied experimental conditions.
If a generalized model must be made, then it should use
an amphoteric substance which is incapable of denaturation or
at least limited in conformational adaptability. The combination
of adsorption and calorimetric measurements is seen as the mini
mum in the type of experimental measurements to be made.
In a study of possible applications, however, emphasis
should be placed on the protein of interest, not models. Simplifi
cations should be derived, it is believed, from the solution,
not the protein, or the surface.
If there could be derived from this work criteria which
could serve as a guide for the successful application of a
prosthetic device in hard tissue they would include the following:
1) There should be tissue attachment accomplished
through charge attraction.
2) There should be ample room to accomodate the
natural quantities of protein found in the tissue
accomplished through high surface area.
3) Protein denaturation should be avoided by control
of the surface, surface area, and ionic strength
of the region.


9
In another study [52], the forces involved in the
adsorption reactions between several globular proteins and
glass surfaces were determined to be primarily ionic amine-
silanol bonding and hydrogen bonding. Two rates of adsorption
were noted. The first appeared to be related to the number
of amines present on the surface of the protein. The second
was slower and seemed to be dependent on the molecular weight
of the protein. Hydrogen bonding was suspected since the
proteins could not be completely washed from the surface with
urea.
Ionic bonding of ribonuclease to glass was indicated
to be strong [53] since very little of the protein could be
removed by rinsing in several solvents. No enthalpy determina
tions were made.' There was a decrease in adsorption with
an increase in ionic strength.
Further review of the literature reveals that the
various adsorption studies cannot be readily compared due to
the large number of experimental variables and to the random
manner in which they are controlled in each experiment. A
few common features in the study of protein adsorption do
emerge, however.
There is usually more than one type of interaction
present for any particular system and one of these is usually
hydrogen bonding. The observed enthalpy values are in the
range of -10 to +10 kcal/mole of protein. Finally, maximum
adsorption density appears to take place near the IP of the
protein. There are many exceptions to these general results,
however.


139
phenomena of collagen on alumina, silica, and hydroxyapatite
appears to be partially induced by ionic attraction. The
enthalpy change for collagen on alumina as high as 70 cal/gm
indicates a strong reaction with the surface. This reaction,
although probably not entirely ionic, takes place through
the charged side groups of collagen.
The presence of BSA has a marked effect on the number of
collagen molecules adsorbed, but not the enthalpy change of
adsorption. The greatest change is apparently due to denatura- *
tion of the proteins. .The effect of the presence of BSA on
the adsorption of a large collagen fibril of molecular weight
many times higher than the triple helix used here, would probably
be greatest if the BSA denatured and spread over the surface.
In the globular form, BSA would simply be pushed aside.
There are several useful conclusions in regard to the
use of prosthetic materials. The effect of specific proteins
will depend on which ions and other proteins arrive at the
surface first. For example, if a clean alumina device of high
surface area is immersed in serum, a layer of denatured albumin
could be expected. If the device is conditioned in a phosphate
solution, less denaturation would occur. Spacial requirements
of each adsorbed moiety also become important. Denatured proteins
take up more room than their globular counterparts.
Secondly, a surface with an electric potential different
from zero will definitely enhance the adsorption process if
the protein is of opposite charge. Since collagen is negatively


31
to a range of K_. varying by about a factor of seven. Under
these conditions the maximum error in AG^ is less than 2%.
The value of AS calculated from
AS0 '= AG AH
r-.T
can be given only simple interpretations. It is known that
values of AS will be between -20 and +20 cal/mole-deg
A decrease in entropy is typically explained as a loss of
freedom of solute molecules as they adsorb. Increases in
entropy, generally found in experiments using macromolecules,
are explained as solvent molecules gaining additional freedom
as the large structuring molecules are removed from solution.
Exceptions to this general rule are present.
The various plots of AG, AH, and AS presented in the
following chapters, in accordance with the previous discussion,
are to be understood as the composite values AG^, AH^, and
AS^. The values for these parameters are closer to single
values of AH?, AG?, and AS? in those regions of the curves
where they tend towards constant values. In these regions the
percent error is generally less than .5%.
The determination of the thermodynamic properties for
the adsorption of collagen on hydroxyapatite is presented as
n example of the calculations made in the following chapters.
The first step in the analytical procedure is to plot Q vs. CQ
(Figure 2). From this graph two values of Q and CQ are chosen
for the sample calculation. In this case values of


88
comparison with ionized molecules [50]. A large conformational
change would then cause the overall reaction to be endo
thermic. The molecular concentrations are low. Therefore,
breaking of intermolecular hydrogen bonds should contribute
little to the enthalpy change.
Proline
The charged monomers of proline and hydroxyproline
are also more strongly attracted to the oxide surfaces than
their polymers (Figure 15). This is taken as a result of
electrostatic attraction. The heat of adsorption, measured
by solution depletion and found to be between -4 and -6 kcal/
mole for both monomers, results from reaction of the charged
groups with the oxide surface.
Calorimetric measurements for the adsorption of PLP
and PLHP did not show a specific pattern for any of the
surfaces. Both of these molecules have a helical conformation
in solution [20], There was no attempt to determine whether
or not this structure was grossly disturbed upon adsorption,
or if it was, what contribution to the enthalpy change such
a disruption would make. Neither was there an attempt to
determine how many points of contact were made. The definite
conclusions which can be drawn from these data are relatively
few. There are some reasonable assumptions, however, that can
be made which, if accepted, will further explain the situation.


75
slightly further away from the backbone than does the
carboxyl and is located^ on the same side of the backbone.
From these considerations--helical structure, position of
the charged groups, and possible steric hinderanceit is
reasonable to assume that not all the charged groups participate
in bonding with the surface at the same time,
To help analyze the binding of CS to a surface, consider
that the interaction of a single dimer with the surface
permits interaction of both the carboxyl group and sulfate
group with the surface. Both groups would then contribute to
the enthalpy of the reaction. From the data in this chapter
and the previous one, it is seen that the change in enthalpy
is fairly constant for each type molecule, as it is between
carboxyl groups and that only charged groups contribute
significantly to the reaction heat, Q. The reaction heat due
to the adsorption of a carboxyl and sulfate group would be
between -12 and -16 kcal/mole. The measured enthalpy value is
about -2 kcal/mole of dimers for alumina and between -2.2 and
-2.8 kcal/mole for hydroxyapatite. Dividing the total enthalpy
possible by the measured value would indicate that between one
in three to one in seven dimers interact with the surface.
We may assume that only one of the charged groups inter
acts -per dimer. Using an enthalpy change between -6 and -12
kcal/mole of charged groups then one in three to one in five
groups would be indicated as interacting with the surface.
From the physical picture and the calorimetric data it seems
plausible to conclude that the chondroitin sulfate molecule is


14
adsorbed layers on the silica surface. It can be seen that
the reactions of aqueous solutions of proteins with silica may
occur with either surface oxygen or hydrogen, depending on
the compositional purity of the surface.
The same effect can be seen with alumina. Steric
acid was adsorbed from CCl^ solvent after the alumina had been
evacuated at 800C for one hour. Without the pretreatment,
steric acid would not covalently bind to the alumina surface
[VO], Methanol has been shown to adsorb on alumina [71] after
successive evacuation and heating at 400C, heating in oxygen
to rid any hydrocarbons present and then heating again at
10^ torr at not less than 350C for 1/2 hour. A methoxide
surface is formed when the clean dry surface is exposed to
methanol vapor. From studies of adsorbed acetylene on alumina
it was concluded that the surface contains electron poor
and electron rich [71] sites (oxide ions, hydroxyl groups, and
aluminum ions) after the sample had been heated to 800C.
Even at these temperatures not all the hydroxyl groups were
removed from the surface [72]. Hydrogen and hydroxyl ions on
alumina are exposed to the solution interface, yielding a
surface structure similar to that of silica.
Hydroxyapatite is assigned the formula Ca-^Q (PO^) (OH)^
In solution the surface undergoes hydrolysis, yielding a surface
having the formula Ca2(HP04) (0H)2 [57]. It is different from
silica and alumina in that, in addition to surface OH" and H+
ions, there are also Ca ions which are capable of binding
adsorbing anions [73]. The multiple internal infrared spectra


7
appreared to be of the same type as that of collagen. The
primary binding force was- assumed to be hydrogen bonding [40].
Adsorption of bovine serum albumin (BSA) on hydro
philic silica [41] exhibited a maximum surface density at
pH 5.5. The isoelectric point (IP) of this protein is 4.9.
The surface of the silica is negatively charged at this pH.
Desorption occurred readily at pHs away from the IP indicating,
as suggested by the author, that binding was due to hydrophobic
interaction. Other studies [42,43] showed that even after
extensive washing with water and EDTA that not all of the BSA
adsorbed onto pyrex glass could be recovered. Maximum
adsorption was near the IP of the protein and the free energy
change was estimated to be -2.5 kcal/mole of protein. The
enthalpy was not calculated.
Serum globulins have been shown to be preferentially
adsorbed by silicic acid [44] and silica [45] and by other
minerals [46]. Maximum adsorption took place at the isoelectric
point on these surfaces as well as on calcium phosphate gel
[47]. In none of these studies were determinations of the
enthalpy of the various adsorption reactions made.
The adsorption of albumin, fibrinogin, and globulin on
polyethylene has been determined by internal reflection spectro
scopy [48]. The adsorption isotherms followed a Langmuir isotherm,
a common finding in which the quantity of solute adsorbed, X,
at the equilibrium concentration, C, is given by X = aC/(l + bC)
where a and. b are constants. The adsorption was assumed to be


13
Due to the shape of the diffuse layer, the influence
of dissolved salts on proteins will be greater near a surface
than in bulk solution. Some proteins, because of their
large size, may extend entirely through a double layer. The
effect of dissolved salts on adsorbed proteins would then be
difficult to explain in detail.
The adsorption properties of the substrates are due
as much to adsorbed water as to their intrinsic stucture.
The surface of silica in aqueous solution has been shown by
infrared spectroscopy to possess three types of surface ions
[36] as represented below:
Si 0"
Si 0-H
Si o~h2+
Unless the adsorbed water and ass.ociated ions are driven off
by heating, silicon ions cannot chemically react with organic
molecules arriving from solution. It has been shown that
ammonia will not react with hydroylated silica, although
chemisorption will occur if the silica has been subjected to
o
a prior vacuum degassing at temperatures in excess of 400 C
[66?67], Silica treated with ammonium fluoride solution
showed evidence for Si-F bonding instead of silanol [68] but
this reaction occurred after heating the substrate to 400C
in vacumm. Trimethylsiloxane can be covalently bonded to
silica by refluxing them in acetone for 24 hours at 50C [69]
This gives some idea of the difficulty of penetrating the