The analysis of trace quantities of pesticides utilizing thin layer chromatography and phosphorimetry.

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Title:
The analysis of trace quantities of pesticides utilizing thin layer chromatography and phosphorimetry.
Uncontrolled:
Thin layer chromatography and phosphorimetry
Physical Description:
ix, 125 l. : ill. ; 28 cm.
Language:
English
Creator:
Moye, H. Anson
Publisher:
s.n.
Place of Publication:
Gainesville
Publication Date:

Subjects

Subjects / Keywords:
Chromatographic analysis   ( lcsh )
Pesticides   ( lcsh )
Phosphorescence   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: l. 122-124.
Statement of Responsibility:
By H. Anson Moye.
General Note:
Manuscript copy.
General Note:
Vita.

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Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000423922
notis - ACH2327
oclc - 11031972
System ID:
AA00003568:00001

Full Text







THE ANALYSIS OF TRACE QUANTITIES OF

PESTICIDES UTILIZING THIN LAYER
CHROMATOGRAPHY AND PHOSPHORIMETRY
















By

HUGH ANSON MOYE


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











UNIVERSITY OF FLORIDA
April, 1965











ACiO, -VL:JJED- 3".i 1T


The author wishes to express his deepest appreciation

to Dr. Stratton H. Kerr of the Agricultural Experiment

Station who gave much of his time to the procurement of many

of the pesticides studied in this dissertation. He also

wishes to thank Dr. Charles Van Middelem for his helpful

advice and his donation of many of the pesticides that were

studied.

The author owes a special thanks to his research

director, Dr. James D. Winefordner, whose energies were

a continual inspiration and whose advice was invaluable.



















LIST OF TABLES . .

LIST OF FIL.. . .



Phosphorimetry. .

Thin-Layer Chromatography .

Choice of Pesticides for Study.


Chapter


Page

. ii



. vi

. 1

. .. 1



. 7


I. THE ANALYSIS OF NICOTINE, !CEJiCOTINE, AND
: IN TOBACCO . .

Introduction . .

Experimental Equipment . .

Experimental Procedure. . .

Results and Discussion . .

II. THE .XA.L'SIS OF p-::IRPE:TOL IN URIIE.. .

Introduction . .

Experimental 2Euipment. . .

Experimental Procedure . .

Discussion. . .... .

III. A PHOSPHORIIETRIC STUDY OF SOME COrLC-iiT
?P SlICIDES . ..

Introduction. . .


iii








Page

~7xcerimental. .... .......... .. 48

Results and Discussion. . 50

S . . 106

APPB~I DIX .... .... .......... 109

3i3L I HY. . . 122

3ILOGRAPHYICAL K... ..... ....... .125
BICGRAE~ICALSK~~E C .......,..........125











LIST OF Z 33


T2ble Page

1. Recoveries of Alkrloids From Aluminum Oxide G 25

2. Analysis of Tobacco Samples for Nicotine,
'ornicotine, and Anabasine . 26

3. -.2:: round Phosphorescence of Urine From Eight
Donors. . . 39

4. Recovery of p-Nitrophenol Added to Urine. .. 45

5. Phosphorescence Characteristics of 32
Pesticides and Related Compounds. 51










LIST OF FIGU?23


Figure Page

1. Phosphorescence excitation and emission
spectra of nicotine, nornicotine, and anabasine
in ethanol-H2SO4 at 770K. (2.84 x 10 H
Nornicotine with coarse sensitivity on 0.5.). 22

2. Analytical curves for nicotine, nornicotine,
and anabasine . . 25

3. Phosphorescence excitation and emission
spectra for urine background (dotted line) and
p-nitrophenol (solid line) in basic ethanol-
ether solution (coarse sensitivity on 0.003,
5.1 x 10-5 H p-nitrophenol) . 42

4. Analytical curve for p-nitrophenol in basic
ethanol . . 43

5. Phosphorescence excitation and emission spectra
of DDT (p,p') in ethanol at 770K. 58

6. Phosphorescence excitation and emission spectra
of DDD (p,p') in ethanol at 770K. ... 59

7. Phosphorescence excitation and emission spectra
of DDE (p,p') in ethanol at 770K. ... .. 60

8. Phosphorescence excitation and emission spectra
of :elthane in ethanol at 770K. .. 61

9. Phosphorescence excitation and emission spectra
of >.ethoxychlor in ethanol at 77K. 62

10. Phosohorescence excitation a:1- emission spectra
of Chlorobenzilate in ethanol at 77K 63

11. ?:osphorescence excitation and emission spectra
of :-.chene in ethanol at 77 . 64

12. Phosphorescence excitation and emission spectra
of Kponez in ethanol at 77K .. 65








i're Page

13. J.hosphrescence excitation and emission spectra
of Sulfenone in ethanol at 770K .. .. 66

14. Phosphorescence excitation a"! emission spectra
of Ie:lon in ethanol at 77T:. . 67

15. Phosphorescence excitation and emission spectra
of Orthotran in ethanol at 770K 68

16. --osphorescence excitation and emission spectra
of Parathion in ethanol at 770K .. .. 69

17. ?hosphorescence excitation and emission spectra
of Ronnel in ethanol at 770K. . 70

18. Phosphorescence excitation and emission spectra
of Co-Ral in ethanol at 770K. ... .. 71

19. Phosphorescence excitation and emission spectra
of Diazinon in ethanol at 770K. .. 72

20. Phosphorescence excitation and emission spectra
of Guthion in ethanol at 770K .. 73

21. Phosphorescence excitation and emission spectra
of :rithion in ethanol at 770K. . 74

22. Phosphorescence excitation and emission spectra
of Aramite in ethanol at 770 . 75

25. Phosphorescence excitation and emission spectra
of Isolan in ethanol at 770K. .. .. 76

24. Phosphorescence excitation and emission spectra
of Sevin in ethanol at 770K . 77

25. Phosphorescence excitation and emission spectra
of Zectran in ethanol at 770K . 78

26. Phosphorescence excitation and emission spectra
of Bayer 44646 in ethanol at 770K 79

27. Phosphorescence excitation and emission spectra
of 3ayer 37344 in ethanol at 770K .. ... 80

28. Phosphorescence excitation and emission spectra
of NIA 10242 in ethanol at 770K .. 81


vii







Page


29. Phosphorescence excitation and emission soectra
of U.C. 10854 in ethanol at 770K. .... .. 82

30. Phosphorescence excitation and emission spectra
of Imidan in ethanol at 770K. ...... 83

31. .hosphorescence excitation and emission spectra
of 2,4,5 ,:richlorophenoxy acetic acid in
ethanol at 7701 .. .. .. 84

32. >hosphorescence excitation and emission spectra
of 2,4 Dichlorophenoxy acetic acid in ethanol
at 77 . . 85

33. Phosphorescence excitation and emission spectra
of p-Chlorophenol in ethanol at 770K. 86

34. .hosphorescence excitation and emission spectra
of 2,4,5 Trichlorophenol in ethanol at 770K 87

35. Phosphorescence excitation and emission spectra
of op-itrophenol in ethanol at 770K 88

36. Phosphorescence excitation and emission spectra
of a-2aphthol in ethanol at 770K. 89

37. Analytical curves (relative intensity versus
moles per liter) for Chlorobenzilate (AD),
D.D.D. (p,p') (0), U.C. 10854 (AZ) and p-
Chlorophenol (AO) in ethanol at 770K. 91

38. Analytical curves (relative intensity versus
moles per liter) for Sevin (AC), Bayer 37344
(AT), Guthion (3), Isolan (N) and Kepone (AB)
in ethanol at 77.. 93

39. Analytical curves (relative intensity versus
moles per liter) for Aramite (Y), Ronnel (V),
Diazinon (2) and Toxaphene (K) in ethanol at
770K ... .. . 95

40. Anal "':ical curves (relative intensity versus
moles pr liter) for Sulfenone (?), 2,4,5-
-:ichlorophenoxy acetic acid (A), D.D.T.
(pn,') (A), 2,4-Dichlorophenoxy acetic acid
(Ai:) and Parathion (J) in ethanol at 77K 97








fiure Page
41. Analytical curves (relative intensity versus
moles per liter) for Co-Ral (LA), Tedion (T),
Trithion (U), I ieth::.-hlor (3), D.D.E. (p,p')
(AJ) and 2,4,5-Trichlorophenol (AN) in
ethanol at 770K .. . 99

42. Analytical curves (relative intensity versus
moles per liter) for p-fitrophenol (A') in
ethanol-diethylamine and for Kelthane (X), a-
.aphthol (AP) and Orthotran (W) in ethanol at
770K . . .. 101
43. Analytical curves (relative intensity versus
moles per liter) for NIA 10242 (AU) and Bayer
44646 (AS) in ethanol at 770K . 103

44. Analytical curves (relative intensity versus
moles per liter) for Inidan (AZA) and Zectran
(AR) in ethanol at 770K ... .. 105
















Spectrometry is that branch of physical science

treating the measurement of spectra. Phosphorimetry can

be defined as that branch of spectrometry treating the

measurement of phosphorescent spectra. For organic

molecules, phosphorescence is best described as lumines-

cence resulting from a radiational transition from the

first excited triplet state to the ground singlet state.

The population of the lowest energy triplet state is

produced by a quantum c~zhanically forbidden crossover in

the molecule from the first excited singlet state to the

first triplet state. In practice this crossover can be

realized because the molecule is distorted and perturbed

by its environment, even at liquid nitrogen temperatures.

efficiency of the crossover is greatly increased if the

molecule contains a he.-.' atom such as Cl, Br, I, etc. To

prevent radiationless deactivation of the triplet state the

phosphorescing molecule is generally frozen into a clear

solid medium at low temperatures, usually 770H, the

temperature of liquid nitrogen. 2,cause there is not enough

thermal ener-- available at the te-.* rature of liquid








nitrogen, the possibility of the molecule crossing back to

the i-ig-r energy first excited singlet state has been

diminished hy use of the solid medium. Then, in many

molecules a radiational tr.z-ition to the ground state may

become the major rate-controlling step of energy conversion

for molecules in the triplet state. In some molecules the

rate-controlling step for energy conversion in the triplet

state is non-ralistional quenching. In molecules in which

the radiational deactivation process is significant, the

phosphorescent yield is also appreciable, and therefore the

molecule will most likely give phosphorescence at low

concentration levels. This has been discussed at greater

length by Jablonski (18) and Lewis and Kasha (25).

Phosliphor-ecence of or-r.ic molecules is characterized

by a number of features. The time required for decay of the

;I.sphorescence is from 10- seconds up to tens of seconds.

Ie decay time is characteristic of the molecule, however

it becomes shorter with an increase in temperature. The

phosphorescence intensity-time relationship (41)

I = 1 e-t/ (1)

gives the phosphorescence intensity, I, at a time, t, after

which the exciting radiation, producing a phosphorescence

intensity Io, has been cut off. The decay time,r-, is de-

fined by this equation as that time required for Io to drop






3

to 1/e (i.e. 0.37) of original intensity. The phosphores-

cence excitation spectrum is also characteristic of the

molecule. This is obtained by varying the wavelength of

the band of radiation used for excitation and plotting this

versus emission intensity signal, which is -enerally measured

at the emission maximum. The emission spectrum, which is

also characteristic of the molecule, is obtained by exciting

at a wavelength that gives maximum emission intensity and

plotting emission intensity signal versus wavelength of the

radiation band emitted. These spectral measurements are

normally performed with a source-monochromator-detector

system which will give as linear a response as possible

throu -out the wavelength regions of interest.

The work to be described in this dissertation is in

part concerned with a specific application of phosphorimetry

to a quantitative analysis of a pesticide and in part

concerned with a study performed to indicate the general

applicability of phosphorimetry to a quantitative analysis

of a pesticide.

In any analysis it is essential that the measured

parameter is described by a relationship which gives the

concentration of hosphorescent molecules, or even the total

number of ~hosphorescent molecules, as a function of

o f: ~zC i ensl it. For a discussion of factors, and

:... .--.ss., which affect tLe phoo. horescent








intensity see the Ph.D. dissertation of Latz (22). He

presents an expression derived by Fletcher (9) for the

fluorescence intensity signal of one fluorescing component,

which is:

t = Io Kf (1-10-) (2)

and if the absorbance, A, is less than 0.01, then

Ft = 2.28 I b'-:a + 1 per cent (3)

where A is the absorbance, b is the cell width, a is the

molar absorptivity coefficient, I is the intensity of the

incident radiation, Ft is the instrumental signal due to

fluorescence, and K. is the -eneral constant that includes

both the fluorescent quantum efficiency and instrumental

factors. A similar e:::res3sion for phosphorescence can be

written as

Pt = 2.28 I bK ac + 1 per cent (4)

where the terms are analogous to those for fluorescence (9).

In this equation it is assumed that quenching effects

are small or at least constant with variation in concentra-

tion, that the instrumental response is linear with

intensity, and that only one phosphorescing species exists.

Consequently a linear relationship between concentration

&:i phosphorescence intensity signal is to be expected.

This is usually observed for nearly all molecules over an

intermediate ra'ne of concentrations.








Other instrumental factors must also be controlled

for equation 4 to be valid. It is necessary to minimize

reflections and scattering within the rigid solution being

studied. --is, in fact, results in maximizing K To do

this a rigid media is used which will not crack or snow

upon being subjected to liquid nitrogen temperatures.

Vinefordner and St. John (44) performed a study on phos-

phorimetric solvents. They found that ethyl alcohol was

one of the few which gave clear glasses at liquid nitrogen

temperature. Therefore ethyl alcohol was used for all work

described in this dissertation because of its ease of

purification, low volatility and favorable solvolytic

properties for a wide range of compounds. Also, ethyl

alcohol has no double bonds and so does not phosphoresce.

Therefore, by using phosphorimetry it is not only

possible to characterize molecules by excitation spectra,

emission spectra, and dec ~ time, but also to make quantita-

tive measurements on molecules over a very large range of

concentrations. Phosphorimetry is a flexible and unique

analytical tool as well as a very sensitive analytical tool.

The use of phosphorimetry as a means of chemical analysis

was first pointed out by Keirs, Britt and Wentworth (21).

Soon after, Parker and Hatchard reviewed some possible

applications of phosphorimetry to chemical analysis (30).

Yineforadnr and Latz (43) built a phos.:zrimeter from








co-.onents and used it to analyze for aspirin in blood

serum and plasma. Winefordner and Tin (45,46) analyzed for

conmounds of _;*-.colog-ical interest in blood and urine.

Sawicki, Stanley, Pfaff and Elbert (34) applied phospho-

rimetry to the problem of identification of small quantities

of air pollutants. It is evident that phosphorimetry in

just the last few years has made its way from a curiosity

to a useful analytical technique.




h-in-layer chromato raphy is a chromato-raphic

technique which combines the technical simplicity of paper

chromatogra-:y with the speed of gas chromatography. The

first reported use of a thin film of solid adsorbent

deposited on a glass plate with a mobile liquid developing

phase was that of Izmailov and S'.-aiber (17). But it was

not until the extensive work of Stahl (35) on many kinds of

adsorbents, solvent systems, activation techniques, develop-

ment techniques and spotting techniques that thin-layer

chromatography became practical for routine diagnosis and

analysis. His work stimulated the production of commercial

thin-layer adsorbents, spreaders and accessories.

Since the work described in this dissertation is

concerned with the development of rapid routine analyses of

-esticides and their metabolites, thin-layer chromatography








appeared to e the ideal eechni ue for separating the

compound of i-terest from interfering; compounds. Not only

does it possess the features mentioned previously, but also

the inorganic nature of the coraon adsorbents, unlike paper,

elimmiates the possibility of the introduction of inter-

.erin co=_ounds via the adsorbent. Also, by the use of

very thick thin-layers it is possible to separate large

quartities of interferi- =ateri als froa very snall

quantities of pesticide. This is especially advantageous

for urine analyses (see Capter II). For tobacco, the use

of thinner thin-layers is possible due to the absence of

lare uantit.i.es of interfere n materialss (see Chapter I).


lCoice of Pesticides for '.-


The number of pesticides presently marketedd in

co...rcial preoarations is quFte large. It is certainly

no i practical to sive a co-plete list of pesticides because

of tohe Lar-e ouber of such materials and because new oesti-

c~.s are continually being authorized for public sale.

v-in a liStln-: of -:-s of u-esticides available would

' 1 fiouL tu o erf orm because of ov erappi..: of different

1 3)0 215er ac bi ui tio. h -- choice of pesticides for

st as siplified, however, after reviewing the foals

;C o' set dow:: for the research lhat was to be done.









The objectives for the work reported in this dis-

sertation were: (1) to show that phos:h`rinetry and thin-

layer chronatography could be utilized for developing a

routine method of analysis for sticide residues on crops,

(2) to show that phosphorimetry and thin-layer chromatography

could be utilized for developing a routine method of analysis

for pesticides in biological fluids, (3) to illustrate the

innate sensitivity, accuracy, precision, rapidity and

specificity of phosphorimetry and thin-layer chromatography

when used together, (4) to illustrate the advantages of

using phosphorimetry and thin-layer chromato-raphy over

presently used analytical methods, and finally, (5) to

determine, by a study of the phosphorescence properties of

promising pesticides, whether phosphorimetry might advan-

tageously be applied to other pesticide analysis problems

in favor of some less sensitive methods that are at present

being employed in many residue laboratories.

?':. pesticides, nicotine, nornicotine and anabasine,

are commonly present in all tobacco samples and, therefore,

this eliminated the need to add quantitative amounts of

these pesticides to the tobacco. Therefore sampling prob-

lems were considerably simnler than if the pesticides had

been added or sprayed on the material to be analyzed. Also,

in tobacco the pesticides to be analyzed are tightly bound

within the plant cells. 31ends of commercial tobacco








contain large quantities, up to 8 per cent by weight, of

nicotine and somewhat less amounts of nornicotine and

an"basine (28). nicotine was used as early as 1763 as a

pesticide and as recently as in 1944 there were 1,197,000

pounds of free nicotine consumed as a pesticide (28).

Jornicotine, which is more toxic than nicotine, can be

easily obtained from various strains of plants that produce

it in abundance. Anabasine is even more toxic than nor-

nicotine, and it, too, can be found as the predominating

alkaloid in some l)ants from which it is extracted. Its

hijh content in a plant indigenous to the U.3.3.R. makes

it a favorite insecticide in that country (28). A method

for the analysis of these three pesticides in various

commercial tobacco preparations is discussed in Chapter I.

moost sensitive routine method for organo-

phosphate pesticide analysis is the electron capture

detector when used with gas chromato rap:hic separation.

Parathicn, probably zhe most widely used organo-phosphate,

is detected by the electron capture detector in quantities

as low as four picograms. Zas sensitivity is lost, how-

ever, when parathion uptake of a mammalian biological system

is to be measured, because the phosh.orous moiety can no

longer be recovered. It is the phosphorous atom which

responds so well to the electron capture detector. Para-

thion is metabolized to p-nitrophenol in hi1h yield by









mamnals (8). Because electron capture is insensitive to

this compound, there has been a need for a method of analysis

capable of .li n extremely low concentrations. The

compound p-nitrophenol appears mainly in urine, after para-

thion has been metabolized. In C'.ater II a complete

routine method for the analysis of extremely low concentra-

tions of p-nitrophenol in human urine is described.

Certainly the lack of phosphorimetric data in the

literature has discoura. :d many analysts from using

phosphorimetry as a means of analysis (22,39). To provide

a basis for future work on pesticides, a study was under-

taken to obtain phosphorimetric data for nearly all the

commonly used pesticides. Also included are compounds that

are metabolites or degradation products of many of the

pesticides. Among the types studied are organo-phozphates,

D.D.T. analogs, sulfones, sulfonates, chlorinated terpenes,

carbamates and chlorinated aromatics. Phosphorimetric data

for fifty-two selected compounds, including appropriate

discussion, a;pears in Chapter III.












THE A.".1-: C' CO ZICOTI:, ..-CC -i-T-, A2TD ANABASINE


Introduction


numerous methods have been reported for the analysis

of tobacco alkaloids. Methods vary from the complete and

lengthy procedure of Jeffrey and 2so (19,20), to the non-

specific and rapid method of Bowen and Barthel (6). Several

papers have dealt with the specific analysis of nornicotine

in the presence of other alkaloids (13,14,16,36) while

other papers have been primarily concerned with the non-

speciec analysis of total alkaloids in tobacco (2,27,42).

In this chapter a method is presented for the specific,

rapid and accurate analysis of each of the three major

lalokids present in cigaret, ciar, and pipe tobaccos.

The method utilizes t:e method of phosphorimetry for the

final measurement. Because of the sensitivity and selec-

tivity of phosphorimetry, only small amounts of samples are

n;ess o easurenent. -.erefore, it is Dossible to

-use tin-layer chr omtotraphy (,L) as the rapid means of

searation of small amounts of o he alkaloids from each other

and fro-: otr ir-uritices in the tobacco, to remove th.

o;s v:i a suita n solvent and to measure the resultant

11









extract pho:-.:rimetrically. .:1 entire procedure requires

loss than 90 minutes, which is considerably less than the

time for any other reported technique.

? --r chromator:.p--hy has long been favored for the

separation and identification of alkaloids in tobaccos

(19,20,24), but this method presents a number of diffi-

culties when making cuantitative measurements. For example,

the background absorption from the paper is high, the amount

of sample which can be applied is small, and the developme-nt

step is slow. A gas chromatographic method devised by quin

and Pappas (j2) requires frequent replottin- of analytical

curves, high concentrations of alkaloids in the tobacco

leaves and -i. concentrations of alkaloids in the extracts.

:lo:!ever, if the concentrations of nicotine were too hi

this resulted in so-called "oulin-up of the column." A

column elution neth o (15) required considerable time, was

intricate and resulted only in the separation of nicotine

and nornicotine.

Several workers (12,15,51,37,38) have shown the

icablit of TC for uanitative separation prior to

r as.ureoent. 3ird and co-w.orkers (5) have shown that TLC

should be sufficiently accurate and precise to allow its

use in control laboratories. 1illett, Moore and Saeman (29)

developed efficient collecting aind sotti techniques which

are :-_-:nifican iaprovements over the techniques listed by








-'i__ and Meyer (55). '::o comprehensive mono-rachs

co nrni 91 C have recently been written (4,40). Both of

these books give aoe discussion on the application of TLC

o quantitative analysis.

The possible application of phosphorimetry to

;chemical analysis vas first indicated 'c: 'eirs, 3ritt and

ycntwort (21). !inefordner and Latz (43) first applied

phosphorimetry to the analysis of trace amounts of drugs

in biological systems. In this chapter the great sensitivity

a1n selectiviey of vhosphorimeric analysis and small amount

of sa:.le required for anal.-sis is utilized for the rapid

and accurate -uantitative analysis of small amounts of

tobacco alkaloids separated from a tobacco extract by means

of iCtG,


:e'rimneatal Psui'ment


insTru':ncta. t in o rat. .4 ;-A1 phosphorimetric

;Su Q'e."mre s were taken with the Aminco 3oBovman Spectroohoto-

Zilor..,- ( -,_ '_-3202) with -The phosphoroscope attachment

c.,-n- -uPront Compan., Inc. ., Silver

`7, =,,:Jr1 .j). h 'e xenon la (o. was uso

oxzs a s, oe sor MsI-o I 7 -antit tVe measure

:o- and for record-in all soectra. -._ quantitative

.._ %;_-....... Ire made wxt :ith shlie s -i. >roram: A-3mm.,

- .. .... D- .. .7...,- .. -e s "stra with









the slit program: A-3mz. 3-0.5mm., C-0.5mm., D-7--., and

-. 35mm.

All :~ectra were recorded with a Moseley X-Y

recorder ('o. 135-A, F. _. :oseley Company, Pasadena,

California). The phosphorescence decay times (the time

for the intensity to drop from a given value to l/e of that

value) were obtained using the Moseley recorder and a manual

shutter arrn- e-.s'.t inserted in place of the fixed slit

holder in front of the xenon excitation source. Upon

closi-.- the shutter the Z-Y recorder was activated and the

ohosphorescence dec:; was observed.

The thin-layer chromatographic equipment consisted

of a rectangular Desaga developing tank and a model S-4

thin-layer applicator (Brinkman Instruments, Inc., Great

Teck, 'N.Y.). Tobacco extracts were applied to the thin-layer

by means of a cemented needle 100 pl. Hamilton syringe. The

tip of the hypodermic needle had a short length of poly-

ethylene tubing positioned so as to hold a piece of white

cotton thread (number 50). The thread extended about 0.5 mm.

below the point of the needle. A sample was then applied to

the thin-layer by placing the needle just above the thin-

layer until the thread just touched this surface. The

syringe was held in place by an ordinary laboratory clamp

and ring stand. 'Thfis procedure allowed samples to be

spotted slowly without damaging the thin-layer surface due








to the hypodermic needle scratching the surface. A Chaney

adaptor was also fitted on the syringe.

2eagents and chemicls.-hIloroform used in the ex-

traction of the alkaloids, and methanol, used in the elution

step were both spectrograde solvents. Brinkman aluminum

oxide type G was used as the thin-layer substrate. All

phosphorimetric analyses were carried out using ethanol

which had been distilled using a five foot vacuum jacketed

and silvered column. The column had a one inch inside

diameter and was packed with glass helices (Labglass, Inc.,

Vineland, _:ew Jersey). Using a reflux ratio of 15 to 1

only the center fraction was collected and checked for

phosphorescncce background. '.. tobacco alkaloids were

obtained from several chemical companies (nicotine and nor-

nicotine from E and Z Laboratories, Jamaica 33, '.Y., and

the anabasine from Aldrich Chemical Co., Inc., Milwaukee 10,

Vis.). All other reagents and chemicals were of reagent

gr'-e quality. Celf.e was used as an analytical filter aid

in the extraction procedure.

Stock solutions in ethanol of nicotine, nornicotine

and anabasine were oroeared. Solutions used to determine

the .::.lrtical curves were prepared from the stock solutions

of each of the above alkaloid.-. These solutions were stored

in a refrigerator maintained at 0C.








A stock solution containing exactly 220 m-. of ana-

basine in 10 ml. of chloroform was prepared. Accurate

volumes of this solution were added to several of the dry,

ground, tobacco samples. _'ese solutions were used to

study the recovery of anabasine and to increase the total

amount of anabasine so as to be on the linear region of the

analytical curve.

A stock solution of toluene in ethanol (0.2 ml.

toluene in 100 ml. of ethanol) was prepared. This solution

was used daily to check the sensitivity of the spectro-

phosphorimeter. If any change in the meter reading

occurred, the fine sensitivity adjustment of the photometer

circuit was changed to give the same meter reading as that

previously obtained. In this way the sensitivity of the

instrument could be checked each day. The analytical

curves of each of the alkaloids were found to never vary

more than the accuracy of measurement. The toluene solution

was stored at 0C in a refrigerator.


E: :T ri- :;nt^.l ?rcei ure


Extraction of tobacco.-The method used is similar to

that described by Cundiff and Karkunas (7). In this method

the free alkaloids are nearly quantitatively extracted into

chloroform from strongly basic aqueous solution. Five

grams of the dry tobacco were ground to a coarse power








with a rotary grinder and were then placed into a phos-

phorimetrically (43) clean 250 ml. Erlenmeyer flask. To

the ground tobacco one milliliter of the 220 mg./ml.

anabasine stock solution was added while shaking the flask.

A stream of dry air was passed through the tobacco to

evaporate the chloroform, and then 10 ml. of 1 M HC1 was

added with swirling. The resultant mixture was allowed to

stand for three minutes to insure co-plete wetting. Ten

milliliters of 36 per cent NaOH solution were then added

while the solution in the flask was swirled. After adding

60 ml. of chloroform, the flask was capped with a chloro-

form rinsed cork and was then clamped onto a platform

shaker and shaken for 20 minutes.

About 100 ml. of dried Celite was added to the flask

which was shaken for another three minutes. The sample was

filtered through a 125 ml. medium grade sintered glass

Buchner funnel and washed with enough chloroform to bring

the volume up to exactly 100 ml.

Thin-layer preparation and sample spotting.-A thin-

layer of aluminum oxide G (5 cm. x 20 cm. x 1 mm. thick) was

applied to a number of glass plates. The slurry of aluminum

oxide used for spreaidn7 was prepared and a-7lied as des-

cribed by the manufacturer. After spreading the aluminum

oxide, the plates were dried overnight at room temperature

ar. then activated at 1300C. for 30 minutes. They were then








stored at room temperature without desiccating or reacti-

vating prior to use. It was found convenient to prepare

20 to 50 plates at one time. A 100 pl. aliquot of the

extract was applied on the opposite lower corners of the

plate, 1 cm. from the sides and 2 cm. from the bottom of

the plate. The tobacco extracts were applied to the thin-

layer as previously described. The application of the

tobacco extracts was performed with care to maintain the

smallest possible spots (3 mm. diameter or less). It was

convenient to have a stream of warm air focused on the

point of application to allow a more rapid application of

the sample.

Spot development.-The sample plates were then placed

in a tank containing the solvent (100 ml. chloroform and

1.5 ml. methanol) and were developed for 30 minutes. After

30 minutes, the solvent front had moved about 10 cm. The

plates were then removed and dried after marking the solvent

front.

Spot detection.-One-half of each thin-layer was

covered while the other half was sprayed using Dragendorff's

reagent. The alkaloids showed up as orange spots on a

yellow background. An area corresponding to the location

and size of each spot on the sprayed side was scraped from

the unsprayed side into a polyethylene capped vial.








Spot analysis.-Five milliliters of ethanol were added

to each vial along with four drops of diethylamine. Each

vial was vigorously hand-shaken and decanted into a clean

vial containing 0.1 ml. of concentrated sulfuric acid. The

vial was shaken and a small volume of this solution was

used to clean and fill the lower end of a sample tube used

in the Aminco instrument. The excitation monochromator was

set at 270 mp and the emission monochromator at 390 mp.

The phosphoroscope speed was set to approximately one-third

maximum speed and liquid nitrogen was added to the Dewar

flask. The reading on the photometer meter was then re-

corded. In addition to this measurement, a solvent blank

(5 ml. ethanol with 0.1 ml. concentrated H2S04) was

measured in the same manner as the sample. The meter

reading for the blank was then subtracted from the meter

reading for the sample. The blank reading never exceeded

10 per cent on the most sensitive scale (0.001) when the

fine sensitivity was adjusted to one-half of its maximum

value. In most cases the phosphorescence signal due to

the sample was nearly 100 times the signal due to the blank.

Therefore, the accuracy of the blank signal did not greatly

influence the accuracy of the sample measurement. The

fluctuation in the meter reading due to the bubbling of

liquid nitro-en resulted in the greatest uncertainty in the

final meter read-in. (+ 2; of full scale). A series of






20

standard solutions of each of the alkaloids in ethanol were

also measured as described above. These results were used

to obtain the analytical curves, relative intensity or

meter reading versus concentration in moles/liter for each

of the alkaloids.

Instrument calibration.-At the beginning of a series

of determinations the photometer circuit of the phospho-

rimeter was adjusted to have exactly the same sensitivity

as on previous days. This was done by placing an Aminco

sample tube containing the standard toluene solution in the

phosphorimeter. The excitation and emission monochromators

were set at 270 mp and 385 mp, respectively, and the fine

sensitivity knob on the photometer was adjusted to give a

meter reading of 43 on the 0.3 coarse sensitivity setting.

The shutter speed was the same as described above. This

procedure allowed one analytical curve for each alkaloid

to be useable over a period of months.

Calculation of weight percent of alkaloid in tobacco.-

The percent by weight of each alkaloid in the tobacco can

be found using the equation below.

C F V
% Alkaloid = 100% ,

where C is the concentration in moles/liter corresponding

to the meter reading as read off the analytical curve, MW

is the molecular weight of the alkaloid in concern, F is








the factor to account for the dilution of the sample (in

this case 1,000 because only 0.1 ml. of the 100 ml. extract

is separated), V is the volume in liters of ethanol used to

remove the alkaloid from the thin-layer (in this case 0.005

1.) and W is the weight of tobacco sample being analyzed

(in this case 5 grams). Therefore, for the procedure des-

cribed in this chapter the above equation can be simplified

to

V% Alkaloid = C M 100% .

Results and Discussion


The experimentally measured phosphorescence excita-

tion and emission spectra for nicotine, nornicotine, and

anabasine were found to be identical within experimental

error, and so only one excitation and one emission spectra

are shown in Figure 1. The phosphorescence decay times

were also found to be nearly the same. The decay times for

nicotine, nornicotine, and anabasine were found to be 5.2,

5.3, and 6.2 seconds, respectively. Because of these

spectral similarities, it was necessary to use a physical

means of separating the alkaloids. Thin-layer chromatography

was used for the reasons discussed below. The analytical

curves for nicotine, nornicotine, and anabasine in the

ethanol-H2SO solvent are given in Figure 2.






































Z
0
C)



w


A.I1SN3.NI 3AIlV-13


O
0 0



*H 0
-P 0
0
0


0




0

-H0
co








Sd
ca
0 6








I- co




00
0 o0 c









-,0 C O



0 012 0


ri .i
o *
0 0' 1
0 00

o o

N
2*-




0 ^!











.D a.
CO


C\JLn


cz o.

w' V- .- co
Z -J F-
0 \LL
U0
0
-co 0



Z Z -
O0 \0
--
z a


00


z r'0 C ON 'A v
X 0 (D I 0 00 (D Q |
-^ \ 00
C1 O^\ '
^ \ ^









Thin-layer chromatography required only 30 minutes

for complete separation, was capable of handling fairly

large loads, gave no detectable phosphorescence background

even at the highest instrumental sensitivity and provided

a relatively simple means of removal of the spot. Silica

gel G with a variety of solvents was found to give a poor

separation under all conditions tested. The aluminum oxide

G with the chloroform-methanol solvent resulted in a nearly

perfect separation. The three major alkaloids in tobacco

(nicotine, nornicotine, and anabasine) were nearly equally

distributed over the solvent path. Recoveries from the

thin-layer for standard solutions of each of the three

tobacco alkaloids were found to be quite good as can be

seen from the data in Table 1. Over 50 different solvents

were tried for removing the alkaloid spots from the thin-

layer. Of these, the solution containing 5 ml. ethanol and

four drops diethylamine gave the most complete recovery of

all the alkaloids.

The method of applying the tobacco extract to the

thin-layer was fast and simple. By means of touching the

fiber to the thin-layer rather than the hypodermic needle

tip, the thin-layer surface was never damaged.

Six lesser known brands of tobacco were analyzed in

hopes of finding a brand containing more nornicotine than

nicotine. As can be seen from the data in Table 2, this was










TABLE 1

RECOVERIES OF ALKALOIDS FROM ALUI:IUI 1 OXIDE G*


Alkaloid Quantity Quantity % R Value
Added Recovered Recovery
(Micrograms) (Micrograms)

Nicotine 42.1 42.5 101 0.80

Nornicotine 67.6 60.9 90 0.26

Anabasine 81.0 78.6 97 0.48

The values listed are averages of six separate and
complete analyses in which the standard solutions were
applied to the thin layer, separated and phosphorimetrical-
ly analyzed.

The relative standard deviations for nicotine, nor-
nicotine and anabasine are 5.9%, 2.2% and 5.4%, respectively.










TABLE 2
ANALYSIS OF TOBACCO SAMPLES FOR NICOTINE, NORNICOTINE,
AND A:TABASIN3


Sample Weight % Found
Number Nicotine Nornicotine Anabasine*

1 4.96 0.055 0.65
2 1.46 0.016 0.49**

5 0.89 0.015 0.41
4 1.05 0.056 0.41

5 1.85 0.061 0.41
6 1.56 0.036 0.57

0.41% Anabasine was added to each 5 gram sample.
Complete determination was reported six times with
a relative standard deviation of 6.0%. All other results
in the above table are averages of triplicates.








not the case. Sample 1 is an American tobacco mixture.

Samples 2 and 5 are tobaccos obtained from Turkish

cigarettes. Samples 4, 5, and 6 are tobaccos obtained from

American non-filter cigarettes. The Rf values of nicotine,

nornicotine, and anabasine are given in Table 1. In addi-

tion to the thin-layer spots due to the above alkaloids,

three other spots with Rf values of 0.87, 0.65 and 0.00

were also found when separating the tobacco extracts on the

thin-layer. These spots were visible prior to color

development and so were attributed to three pigments present

in the tobaccos. If very large amounts of tobacco extracts

were applied, two other alkaloids with Rf values of 0.59

and 0.65 could be barely detected. One of these is probably

due to myosmine although the spot was so weak that the

exact identification of it was impossible. The value for

nicotine in sample number 1 in Table 2 appears abnormally

high. However, this value was checked a number of times

and found to be as listed in Table 2. Sample 2 was analyzed

six times for anabasine, and the relative standard deviation

was found to be 6.0 per cent. Each of the six analyses

involved the entire procedure consisting of extraction of

the alkaloids from the tobacco, thin-layer chromatographic

separation, dissolution of the spot with ethanol and phos-

phorimetric measurement. All other results in Table 2 are

the averages of triplicate determinations. Addition of






2S

known amounts of nicotine and nornicotine to several of the

tobacco samples resulted in recoveries similar to those

listed in Table 2 for anabasine.

Each of the measured solutions contained a small

volume (0.1 ml. concentrated H2S04 and 5.0 ml. ethanol) of

sulfuric acid. It was found that a ten-fold increase in

phosphorimetric sensitivity resulted when using the sulfuric

acid solution of ethanol. The reason for this is not known.

Ethanol and ethanol mixtures were previously (44) shown to

be good solvents for phosphorimetric measurements.

To check for the possibility of an interfering

species coinciding with an alkaloid spot, the following

was done. Several of the tobacco samples had their spots

measured in the phosphorimeter without, and then with,

sulfuric acid in the ethanol. In all cases there was at

least a ten-fold increase in intensity. Because this is

characteristic of the pure alkaloid, it appears indicative

that the intensities were due to the alkaloids alone.

Once analytical curves have been established for each

of the three alkaloids, an entire analysis of a dry tobacco

sample for nicotine, nornicotine, and anabasine can be com-

pleted in less than 90 minutes. It should be stressed that

the analytical curves were completely reproducible over a

period of four months as long as the sensitivity of the

photometer was adjusted according to the method described.








The time for the analysis is considerably shorter than any

previously described method in which each of the th

alkaloids were analyzed. The sensitivity of analysis is

better than any known absorption spectrophotometric method

for the pure alkaloids. Some visible absorption spectro-

photometric methods utilizing color reactions result in

comparable sensitivities. However, such methods require

even more tedious steps and chemical reagents and, there-

fore, are subject to even larger errors. In addition,

these methods have normally employed paper chromatography

as the means of separation, which is slow, utilizes only low

spot loads and requires large background corrections. In

addition, the sample cells used in.absorption spectropho-

tometry normally require five to ten times more solution

than those used in phosphorimetry. The method described

here appears to be the fastest, simplest method for the

simultaneous analysis of nicotine, nornicotine, and anabasine

in tobacco samples.

In order to apply the method presented here to the

analysis of the less concentrated alkaloids, it would have

been necessary for the extract of the tobacco samples in

concern to be further concentrated. In all samples analyzed

the alkaloids other than nicotine, nornicontine, and ana-

basine were over ten times less concentrated than the most

dilute of the three alkaloids analyzed. However, if any






30

other alkaloids were present in appreciable concentration,

it should be quite simple to perform a quantitative analysis

according to the procedures outlined in this chapter.










CHAPTER II


THE ANALYSIS OF p-NITROPHENOL IN URINE

Introduction

The most commonly used organo-phosphorous insecticide

is still parathion. Because of its widespread use and high

toxicity, sensitive and accurate analytical techniques are

needed in many agricultural and clinical laboratories in

order to protect agricultural workers and consumers. The

methods must not only be highly sensitive but also simple

enough to permit their use for routine analyses.

The widely used technique of measuring blood

cholinesterase activity has numerous faults when applied

to methods for monitoring human parathion exposure (11,26).

The direct measurement of p-nitrophenol excretion in urine

is preferred (8,11,26). This is normally done by a re-

duction of p-nitrophenol and coupling to a phenol to give

a colored azo compound which is then measured spectrophoto-

metrically (8,23). This method still requires about 10 pg.

of p-nitrophenol for an accurate analysis.

Freed and Salmre (10) and Winefordner and Latz (43)

showed that phosphorimetry could be used as a means of

analysis of constituents containing conjugated structures








in biological fluids. Winefordner and Latz (43) also made

a thorough study of the phosphorescent constituents in blood

and urine and applied phosphorimetry to the accurate analysis

of low concentrations of aspirin in blood. Winefordner and

Tin (46) obtained excellent sensitivities using phospho-

rimetry to analyze for cocaine and atropine in urine. How-

ever, since hydrolysis of the urine was not employed, the

problem of significant urine background was not encountered.

Since the measurement of low quantities of p-nitrophenol in

urine was performed in the studies described in this

chapter, it was necessary to include a hydrolysis step.


Experimental Equipment

Instrumentation and apparatus.-All phosphorimetric

measurements were made as described in Chapter I, except

that the mercury-xenon lamp (No. 416-993) was used as

excitation source for making all quantitative measurements

rather than the xenon lamp. The xenon lamp was used when

recording spectra, however. Slit arrangements were as those

described in Chapter I.

Reagents and materials.-Merck silica gel G (Brink-

mann Instrument Company, Cantiague Road, Westbury, N.Y.)

was used for all thin-layers. Pittsburgh microscope slides

(Fisher Scientific Company), 75 x 25 mm. were used for thin-

layer supports. Reagent grade oxalic acid, hydrochloric








acid, toluene and diethylamine were used. Technical grade

ethyl ether was re-distilled at a reflux ratio of 20:1

using a five foot vacuum-jacketed, helices packed column.

Absolute ethanol was purified as described in Chapter I.

The p-nitrophenol (Eastman) was recrystallized from water.

For calibration of the phosphorimeter a toluene

stock solution containing 0.2 ml. of toluene per 100 ml. of

ethanol solution was prepared. This stock solution was

diluted ten-fold with ethanol to prepare the toluene standard.

Both were stored at 00C. in a refrigerator in screw cap

bottles.


Experimental Procedure


Treatment of urine.-To 90 ml. of urine, 10 ml. of

concentrated HC1 was added. A minimum volume of 5 ml. of

urine was necessary in order to perform the following

studies. The urine was refluxed for one hour and then

stored in a refrigerator. At least 6 ml. of this urine

solution was put into a 12 ml. centrifuge tube and centri-

fuged at high speed (6,000 r.p.m.). Then exactly 5 ml. was

carefully drawn off with a pipette for analysis.

Preparation of thin-layer.-The silica gel G contained

organic contaminants which gave phosphorescent background.

To remove this background, it was heated at 7000C. for at

least twelve hours. This did not destroy the normal









chromatographic or physical properties of the silica gel,

although a slight pink coloration became evident.

Fourteen thin-layers were simultaneously made by

placing fourteen 75 x 25 mm. microscope slides on a 20 x 20

cm. glass plate. A 2 cm. deep dish was made by applying a

strip of masking tape to the edges of the plate. In a

graduated Erlenmeyer flask with an aluminum foil wrapped

cork, 150 ml. of the cleaned silica gel had enough 0.1 M

oxalic acid added to it to make a slurry of 150 ml. volume.

The slurry was shaken thoroughly to ensure that no dry

clumps of silica gel remained.

The slurry was immediately and slowly poured onto

the plate, care being taken to position the plate on a

perfectly level surface to ensure even settling of the

silica gel. The thin-layers were room dried, giving a layer

thickness of about 2 mm. when dry. The masking tape was

then stripped off, and the layers were activated at 1150C.

for at least one hour. They were then stored at room

temperature and humidity (60%) and were used as such with-

out reactivation. Care was taken to prevent contamination

of the activated plates.

To prepare the thin-layers for use, they were sepa-

rated with a razor blade. About 2 mm. was trimmed off

three edges and 4 mm. off the top. This allowed easier

handling.








Extraction of urine.-0nly one extraction of the

acidified urine was needed to recover essentially all of

the p-nitrophenol. Five ml. of urine was pipetted into a

snap-on polyethylene capped vial, and 6 ml. of ether was

added. The vial was shaken vigorously for five mi:.

The aqueous phase was removed with a hypodermic syringe

having an extra long needle. Water beads inside the vial

did not interfere. After evaporation of the ether solution

to about 2 ml. in vacuo, the ether solution was then applied

to the activated thin-layer.

Application of urine extract to thin-layer.-A capil-

lary pipette was made from eleven open-end capillary melting

point tubes by mounting them side by side on a small block

of polystyrene. Their ends were polished smooth to prevent

chipping of the surface of the thin-layer.

The ether was applied as a band 1 cm. from the bottom

of the thin-layer, using two 1 ml. portions of ether to

rinse the vial. Care was taken to avoid touching the pipette

to the sides of the vial where water might be picked up and

harmfully applied to the thin-layer. With a blast of hot

air from a hair dryer close to and focused on the area of

application, the time required to apply one sample was about

five minutes.

Development of thin-layer.-A circular jar 11 cm. high

and 8 cm. in diameter was used as a developing tank, with a








glass plate for a top. Filter paper was used to line the

tank. The thin-layer was developed by the ascending tech-

nique. A layer of ether, about 0.5 cm. in depth, was used

as the developing solvent. Development was continued until

the ether had moved exactly 5.5 cm. above the origin as was

noted by a scratch in the surface of the thin-layer. All

the thin-layer material was scraped away with a razor blade

except the area which was 3.4 to 4.2 cm. above the origin.

This was located directly above a strong blue fluorescent

band which showed up clearly on the thin-layer under a

black light.

Extraction of thin-layer.-The retained portion of

silica gel was scraped into a capped vial, and 5 ml. of

0.1 M HC1 was added. The vial was shaken vigorously for

ten minutes, and the slurry transferred quantitatively to

a 12 ml. centrifuge tube with several 1 ml. rinsings of

water. This was spun at high speed for several minutes.

The supernatant liquid was then decanted into another vial

by inverting the centrifuge tube and allowing a minute for

complete drainage. Five ml. of ether was added to the clear

liquid. This was shaken vigorously for five minutes. The

aqueous layer was removed with a hypodermic syringe with an

extra long needle, and the remaining ether transferred with

two 1 ml. washings to a 10 ml. volumetric flask. This was

diluted to the mark with ethanol. This gave an ethanol-ether








solution which was capable of being frozen at liquid

nitrogen temperatures to give a clear glass.

Phosphorimetric measurement.-Prior to any phosphori-

metric measurement the instrument was calibrated using the

standard toluene solution. The excitation wavelength was

set at 270 mp, the emission wavelength at 585 mp, and the

meter multiplier at 0.3. The fine sensitivity of the

photomultiplier photometer was adjusted so that this solu-

tion gave a reading of 84. This calibration was always

performed before each series of runs, although it rarely

required a change in fine sensitivity position. The wave-

lengths of the excitation and emission monochromator were

then set at 265 mp, and 525 mp, respectively, for the

quantitative measurements on urine.

To obtain the background phosphorescence due to

normal urine, that is, urine samples containing no p-nitro-

phenol, the ethanol-ether solutions were prepared, by the

same procedure previously described, for several urine

samples (from different subjects) containing no p-nitrophenol.

These solutions were slightly acidic since the ether was

saturated with 0.1 M HC1. The phosphorescence intensity of

the acidified ethanol-ether solution was then measured.

The phosphorescence intensity was obtained by multiplying

the phosphorescence signal read on the photomultiplier

photometer meter (read as %T) times the coarse meter








multiplier. All phosphorimetric readings were also cor-

rected for the thin-layer and solvent background. This

background na er exceeded 10 (that is, 10%) on the 0.001

scale, and was obtained by running 5 ml. of distilled water

through the entire procedure used for the urine samples.

The ethanol-ether solutions were then made slightly basic

by the addition of four small drops (0.1 ml.) of diethyl-

amine. Phosphorimetric intensity readings were obtained

for these solutions, and these readings were corrected for

the thin-layer and solvent background as described above.

The ratios of the corrected readings of the basic solutions

to the corrected readings of the acidic solutions gave an

average of 3.1 as can be seen from the data in Table 3.

Because p-nitrophenol showed negligible phosphorescence in

acidic solution, it was possible to obtain a urine blank

directly on the urine sample being measured by taking a

reading of an acidic ethanol-ether solution, correcting it

for thin-layer and solvent background, and multiplying it

by 3.1.

To obtain the p-nitrophenol concentration in urine,

the following procedure was then followed:

1. Obtain the phosphorescence intensity of the acidified

ethanol-ether solution.

2. Subtract the phosphorescence intensity of the thin-

layer and solvent blank.









TABLE 3
BACKGROUND PHOSPHORESCENCE OF URINE FROM EIGHT DONORS


Urine Acidic ab Basic Basic Reading Ac
Specimen Reading' Reading Acidic Reading
No.

1 21 68 3.24 0.15

2 34 110 3.24 0.15

5 26 81 5.12 0.05
4 62 200 3.22 0.15

5 54 170 3.14 0.05
6 51 85 2.75 0.54

7 40 110 2.76 0.33
8 32 105 5.30 0.21

Average ratio: 3.09. Standard deviation: 0.22. Relative
standard deviation: 7.1%. Average urinary background
expressed as equivalent p-nitrophenol concentration in
micrograms per 100 ml.: 1.5.
aThe photometer coarse sensitivity scale was 0.001.
Fine sensitivity was set as explained in calibration pro-
cedure but was always close to 40. Internal gain setting
of photometer was wide open.
Corrected for thin layer-solvent background.
CA is the deviation from the mean value.







3. Multiply the resultant phosphorescence intensity

by 3.1 to obtain the phosphorescence intensity of

the urine blank.

4. Make the ethanol-ether solution basic and measure

the phosphorescence intensity.

5. Subtract the phosphorescence intensity of the thin-
layer and solvent blank from the phosphorescence

intensity of the basic solution.

6. Subtract the phosphorescence intensity of the urine

blank obtained in item 3 from the phosphorescence

intensity obtained in item 5.

7. Using the analytical curve of phosphorescence

intensity versus p-nitrophenol concentration (see

Fig. 4), determine the p-nitrophenol concentration

corresponding to the phosphorescence intensity

obtained in item 6.

Calculations.-The p-nitrophenol concentration in the

original urine sample (expressed as micrograms per 100 ml.)

can then be calculated from the expression below.

C (pg./100 ml.) = 5.08 x 107 Y

where Y = concentration in moles per liter as read from the

analytical curve and the factor 3.08 x 107 accounts for the

dilution steps.









Precautions.-All glassware must be kept immaculately

clean and especially free from dust. Only the middle one-

third of both the ether and ethanol distillates was used.

This resulted in solvents of extremely high purity. As has

been previously emphasized (43) glassware should be cleaned

with Drene shampoo, and in no case should a detergent be

used.

Special attention should be given to the oven used

for thin-layer activation. If the thin-layers become con-

taminated in the oven, the oven should be cleaned and heated

to its maximum temperature while the door is periodically

opened.

The microscope slide thin-layers were best separated

with a sawing motion of the razor blade rather than with a

chopping motion.


Discussion


By comparing the phosphorimetric spectra of p-nitro-

phenol and the urine component, which appeared with it on

the thin-layer, it can be seen from the spectra in Figure 3

that nearly all of the background was eliminated by choosing

the correct excitation and emission wavelengths. Even so,

an average urine background equivalent to 1.3 pg/100 ml. of

p-nitrophenol resulted as can be seen from the data in

Table 3. This could be accounted for, however, with good
































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certainty by using the factor of 3.1. As can be seen from

the data in Table 4, good recoveries and reproducibilities

were obtained for extremely low p-nitrophenol concentra-

tions.

Various thin-layer materials were tried including

aluminum oxide G, polyamide and neutral silica gel. Neutral

silica gel gave the best separation of these but tended to

bind small quantities of p-nitrophenol uniformly over the

developed portion of the thin-layer surface. This gave

appreciable losses at low p-nitrophenol loads. By making

the thin-layer material slightly acidic, it was possible to

eliminate the sites causing holdup. Using the neutral

silica gel plates, it was only possible to recover about

70 per cent of 0.35 pg. of p-nitrophenol by scraping the

area 3.4-4.2 cm. However, with the silica gel treated with

oxalic acid, it was possible to recover 95 per cent of 0.35

pg. of p-nitrophenol by scraping the same portion. In

addition, the acidified silica gel thin-layer turned out to

be harder and more difficult to chip and break in handling.

This was especially important during sample application-

where the pipette comes in contact with the thin-layer sur-

face. The thin-layers were made unusually thick, that is,

2 mm., to prevent streaking of the exceptionally large

amounts of urinary materials.










TABLE 4

RECOVERY OF p-NITROPHENOL ADDED TO URINE


pg. p-Nitrophenol
Addeda to 5 ml. Urine


7.1

3.5

1.3


0.71
-b
0.35b
0.071

0.014


pg. p-Nitrophenol
Recovereda to 5 ml. Urine Recovery


6.4

3.1
1.2

0.61

0.34

0.057

0.019


136


Average % recovery (excluding last sample) = 88%.
aThree separate 5 ml. urine samples for each amount
were analyzed. The average of the three results is recorded.
Urine used was from the same specimen.
bThis amount was analyzed with a relative standard
deviation of 2.5%.








Since p-nitrophenol is much more soluble in aqueous

base than in aqueous acid, it would seem that base should

be used to remove the compound from the thin-layer material

rather than acid. On the contrary, recoveries were ex-

tremely low using 0.1 M NaOH. This probably result

because the base did not give as finely divided particles

in the washing as did the 0.1 M HC1. Apparently the base

does not hydrolyze the CaSO4 binder, and the acid does.

Neutral water performed nearly as well as did the acid.

No attempt was made to isolate and identify the

material causing the small urine background reading. It

appeared only after the hydrolysis step, however, and did

not possess any phosphorimetric characteristics similar to

those of a host of compounds that normally appear in urine.

Dialysis was attempted on the untreated urine in hopes that

the background compounds) might have come from a protein.

This brought no decrease in background as compared to urine

that was not dialyzed previous to hydrolysis.

A naturally decaying phosphorescing species should

follow the equation I = I1 e-t as discussed in the Intro-

duction of this dissertation. If a plot is made of log

intensity versus time for a decaying compound, a straight

line is obtained. A second compound of comparable intensity

but different-", if it is in solution with the first, will

produce a break in the log intensity versus time plot. To








obtain this plot, the phosphorescence signal due to the

urine background was measured on the X-Y recorder after

cut-off of the exciting radiation. The data from the decay

curve was then transposed to semilogarithm paper. Similar

semilogarithm plots were obtained at three different

emission wavelengths on the urine background and each re-

sulted in a straight line, indicating the probability that

the background was caused by only one compound.

The entire procedure, after hydrolysis, requires at

longest only 40 minutes. In addition, the sensitivity of

analysis was nearly one thousand times greater than any of

the previously used methods (8,11,23). High sensitivity,

along with good reproducibility and good accuracy was

attained throughout the range of concentrations. The

simplicity of the method should lead to the use of this

technique in routine analysis. It is hoped that this method

will be put to use as a routine procedure by researchers to

evaluate the symptoms of parathion poisoning.











CHAPTER III


A PHOSPHORIMETRIC STUDY OF SOME COMMON PESTICIDES

Introduction


To provide a basis for future work on crop residues

of pesticides a study was undertaken to determine if

phosphorimetry might be applicable to the analysis of these

residues. The data presented in this chapter is intended

to give the analyst as much aid as practicable while he is

considering phosphorimetry for his analytical method. In

this chapter a survey of the phosphorescence characteristics

of 52 pesticides (including several known degradation

products) is presented. Thirty-two of these phosphoresced

sufficiently such that excitation spectra, emission spectra,

excitation maxima, emission maxima, decay times, analytical

curves and limits of detection could be tabulated. The

other 20 compounds did not give detectable phosphorescence
-o
excitation and emission spectra for 102 M ethanolic

solutions.


Experimental


Apparatus.-All phosphorimetric measurements were made

as described in Chapter II.








Reagents.-All compounds were either analytical grade,

which were obtained from major pesticide manufacturers, or

were technical grade, which had been redistilled or recrystal-

lized until they appeared as one spot when chromatographed

on a thin-layer of silica gel. All compounds were stored

at near 00C. in a refrigerator before use.

Absolute ethanol, purified as previously described

in Chapter I, was used as the phosphorimetric solvent.

Stock ethanolic solutions of each compound were prepared.

Solutions of lower concentrations were prepared by succes-

sive dilution.

Procedure.-Prior to any series of measurements, the

phosphorimeter was calibrated as described in Chapter I.

Analytical curves of relative phosphorescence signal versus

compound concentration were obtained for all compounds

exhibiting phosphorescence. The phosphorescence intensity

signal due to the compound was obtained by subtracting out

the background due to the phosphorescing impurities in the

ethanol for each sample measured. From practical considera-

tions, it was found that the lowest concentration that

could be confidently measured was that which gave a reading

of one scale division (1% of full scale) over that of the

background. Because the ethanolic background was precise

to better than + one scale division this procedure was

possible. Therefore the limit'of detection was defined as









that concentration which gave a reading of one scale

division over the background on the most sensitive scale on

which the background could be measured.

The lifetime,T was measured by shutting off the

exciting radiation with a manual shutter and plotting the

phosphorescence signal versus time with the X-Y recorder.

The response of the recorder prevented any measurements of

'S shorter than 0.2 second.

Spectral measurements were made on solutions

approximately 10-2 M in ethanol, as were the lifetime

measurements.

Results and Discussion


In Table 5 the phosphorescence emission and excita-

tion peaks (uncorrected for instrumental response), the

phosphorescence lifetimes, the approximate ranges of

concentration over which near linear analytical curves

result, and the limits of detection of 32 pesticides (and

several metabolic products) are given. The low limits of

detection and the extensive range of concentrations over

which analytical curves can be used should be emphasized.

Those compounds which gave no detectable phosphorescence

and so are not listed in Table 5 were: chlordane, aldrin,

dieldrin, endrin, heptachlor, lindane, methyl parathion,

malathion, Thimet, Thiodan, Delnav, H.E.O.D., RH..D.N.,






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55

Telodrin, 3 amino-1,2,4 triazole, Phosphamidon, U.C. 21149,

dimethoate, dimethoate acid and dimethoate oxygen analog.

Often it is helpful not only to know the wavelengths

of maximum phosphorescence emission and excitation but also

to know the widths and shapes of these bands. Thus, if a

compound has a broad emission band and background phosphores-

cence interferes with the measurement of this emission at

the emission maximum, it is often possible to measure the

emission at another wavelength, where background is

negligible. Also there may be instances where the measure-

ment of an emission may be impaired because a second

compound, having a broad emission band, may interfere. The

same can be said about excitation bands, although these are

generally much sharper and well defined. In this case it

is frequently useful to know the relative intensities of

two excitation bands for one compound. Figures 5 through

36 show the excitation spectra and emission spectra of the

32 compounds that exhibited phosphorescence for the purpose

of showing band shape and relative intensities. Figures 37

through 44 show the analytical curves for the same compounds.

Two types of noise prevent the spectra from being

smooth curves. The uniform noise, present in nearly all of

the spectra, is a high frequency and low level nni- ; is

probably a combination of shot noise in the photomuluiplier

tube and noise generated by the rapidly swirling liquid








nitrogen in the Dewar flask. A type of noise evident in a

few of the spectra is caused by single bubbles of nitrogen.

This shows up as isolated downward spikes on a normally

smooth curve. The extensive amount of time required to

make an excitation or emission scan increases the difficulty

of preventing this infrequent bubbling. No spectra is

recorded in which bubble spikes can erroneously be inter-

preted as spectral minima.

Several of the compounds listed in Table 5 have
great phosphorimetric sensitivities but considerably poorer

sensitivities by other methods of analysis. In these cases,

the application of phosphorimetry seems particularly ideal.

'For example, the compounds Co-Ral and p-nitrophenol had
phosphorimetric detection limits less than 50 picograms per

milliliter. Other methods of analysis of Co-Ral and p-

nitrophenol are substantially less sensitive. The most

sensitive method of analysis of Co-Ral has been by gas

chromatography using the electron capture detector. For

example, Bonelli, Hartman and Demick (5) have detected Co-

Ral in amounts as low as 300 picograms using the electron

capture detector. Anderson, Adams and MacDougall (1) were

able to detect concentrations of Co-Ral in the microgram

per milliliter range in animal tissues using fluorometry.

Spectrophotometric methods are less sensitive. Because of
the great phosphorimetric sensitivity of p-nitrophenol it







should be possible to analyze for low concentrations of

parathion, which can be hydrolyzed to p-nitrophenol. It

has already been demonstrated in Chapter II that p-nitro-

phenol can be analyzed in urine, where it appears as a

metabolite from the detoxification of parathion. The

sensitivity of this analysis was limited by the high

phosphorescence background of hydrolyzed urine, which was

still appreciable after cleanup by thin-layer chromatography.

Residues on crops should be able to be analyzed with very

little background phosphorescence, as was the case in Chap-

ter I with tobacco. It should also be possible to determine

low concentrations of methyl parathion by hydrolysis to p-

nitrophenol. Methyl parathion itself shows no detectable

phosphorescence.

The phosphorimetric determination of the carbamates

(Sevin, Zectran, Bayer 44646, Bayer 37344, NIA 10242, U.C.

10854 and Imidan) also appears promising. All of these,

except possibly Imidan, should be relatively insensitive to

the electron capture detector. At the present time, colori-

metric methods are used for the determination of the

carbamates. The colorimetric limits of detection are always

in the microgram per milliliter range. The great sensitivi-

ties of many of the other pesticides and related compounds

in Table 5 should certainly result in many more agricultural

applications.





58

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Fig. 37.--Analytical curves (relative intensity versus
moles per liter) for Chlorobenzilate (AD),
D.D.D. (p,p') (0), U.C. 10854 (AZ) and p-
Chlorophenol (AO) in ethanol at 770K.








































AIISN31NI 3AIIV-13d