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Design and Testing of an Apparatus to Measure Carbon Dioxide Solubility in Liquid Foods

Permanent Link: http://ufdc.ufl.edu/UFE0021868/00001

Material Information

Title: Design and Testing of an Apparatus to Measure Carbon Dioxide Solubility in Liquid Foods
Physical Description: 1 online resource (81 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: apparatus, carbon, dioxide, solubility, supercritical
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Carbon dioxide (CO2) is a nontoxic, inexpensive, chemically inert, nonflammable fluid and a safe solvent. It has multiple applications in the beverage industry, such as carbonation and dense phase carbon dioxide (DPCD) technology, a cold pasteurization process. The solubility of CO2 in water depends on pressure and temperature (Dodds and others 1956). Increasing pressure increases solubility of CO2 and increasing temperature decreases it. In addition, the presence of other dissolved substances could have a positive or negative effect in CO2 solubility (Meyssami and others 1991). DPCD is a continuous cold pasteurization method that affects microorganisms and enzymes under pressures below 50 MPa (7,250 psi) without the use of high temperature treatments. Currently, the use of CO2 in DPCD treatments is not optimized, since solubility of CO2 in liquid foods at different temperatures, pressures, and compositions is not known. Therefore, excess CO2 is used to assure saturation. If CO2 solubility could be predicted at any temperature, pressure and composition for a liquid food, then the amount of CO2 used would be slightly above this theoretical value, and the process would be more economical. The first step towards this type of prediction is the experimental measurement of CO2 solubility in model liquid food systems and its correlation with pressure, temperature and composition. Therefore, the objectives of this study were: 1) to design, build and test a high-pressure carbon dioxide solubility apparatus, 2) to compare the volume of CO2 that dissolves in pure water with the current literature; and finally 3) to determine the maximum amount of CO2 in solution with model liquid foods of known composition (binary solutions of fructose and malic acid). An experimental system to measure the CO2 solubility was designed and tested. The pressure and temperature were controlled in the system, and the design assured an accurate measurement of solubility. Experimental results of CO2 in solution in pure water were compared with the literature, and there was no significant difference at pressures of 7.56 MPa (1100 psi) and 10.3 MPa (1500 psi), at a constant temperature of 40 degrees C. At pressures of 7.58 and 10.34 MPa, solubilities of CO2 in water were 4.77 ?0.15 and 5.51?0.20 g/100 g of distilled water, respectively. By increasing the concentration of malic acid or fructose, solubility of CO2 decreased significantly. At 7.58 MPa of pressure, the amount of CO2 dissolved in the solutions of 0.5 and 5% of malic acid concentration was 4.47?0.03 and 4.47?0.02 g/100 g of solution. When the concentrations of fructose in the solution were 5 and 10%, solubilities of CO2 in the solutions were 4.55?0.11 and 4.12?0.07, respectively (at 7.58 MPa). At 10.34 MPa of pressure, solubilities of CO2 in solution with 0.5 and 5% of malic acid concentration were 4.90?0.08 and 4.79?0.07 g/100 g of solution. When the amount of fructose in the solution increased to 5 and 10 g/100 g of solution, solubilities of CO2 at 10.34 MPa were 4.78?0.14 and 4.19?0.17, respectively. This equipment design and method can experimentally measure CO2 solubility in real liquid foods, and therefore is a significant contribution to the DPCD field. This is the first step in the generation of experimental data for the thermodynamic modeling of CO2 solubility in liquid foods. The experimental apparatus could offer an advantage to the food industry, primarily to soft drink industry, and to the DPCD users, by eliminating the need to use excess CO2 and therefore reducing the production cost.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Balaban, Murat O.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021868:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021868/00001

Material Information

Title: Design and Testing of an Apparatus to Measure Carbon Dioxide Solubility in Liquid Foods
Physical Description: 1 online resource (81 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: apparatus, carbon, dioxide, solubility, supercritical
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Carbon dioxide (CO2) is a nontoxic, inexpensive, chemically inert, nonflammable fluid and a safe solvent. It has multiple applications in the beverage industry, such as carbonation and dense phase carbon dioxide (DPCD) technology, a cold pasteurization process. The solubility of CO2 in water depends on pressure and temperature (Dodds and others 1956). Increasing pressure increases solubility of CO2 and increasing temperature decreases it. In addition, the presence of other dissolved substances could have a positive or negative effect in CO2 solubility (Meyssami and others 1991). DPCD is a continuous cold pasteurization method that affects microorganisms and enzymes under pressures below 50 MPa (7,250 psi) without the use of high temperature treatments. Currently, the use of CO2 in DPCD treatments is not optimized, since solubility of CO2 in liquid foods at different temperatures, pressures, and compositions is not known. Therefore, excess CO2 is used to assure saturation. If CO2 solubility could be predicted at any temperature, pressure and composition for a liquid food, then the amount of CO2 used would be slightly above this theoretical value, and the process would be more economical. The first step towards this type of prediction is the experimental measurement of CO2 solubility in model liquid food systems and its correlation with pressure, temperature and composition. Therefore, the objectives of this study were: 1) to design, build and test a high-pressure carbon dioxide solubility apparatus, 2) to compare the volume of CO2 that dissolves in pure water with the current literature; and finally 3) to determine the maximum amount of CO2 in solution with model liquid foods of known composition (binary solutions of fructose and malic acid). An experimental system to measure the CO2 solubility was designed and tested. The pressure and temperature were controlled in the system, and the design assured an accurate measurement of solubility. Experimental results of CO2 in solution in pure water were compared with the literature, and there was no significant difference at pressures of 7.56 MPa (1100 psi) and 10.3 MPa (1500 psi), at a constant temperature of 40 degrees C. At pressures of 7.58 and 10.34 MPa, solubilities of CO2 in water were 4.77 ?0.15 and 5.51?0.20 g/100 g of distilled water, respectively. By increasing the concentration of malic acid or fructose, solubility of CO2 decreased significantly. At 7.58 MPa of pressure, the amount of CO2 dissolved in the solutions of 0.5 and 5% of malic acid concentration was 4.47?0.03 and 4.47?0.02 g/100 g of solution. When the concentrations of fructose in the solution were 5 and 10%, solubilities of CO2 in the solutions were 4.55?0.11 and 4.12?0.07, respectively (at 7.58 MPa). At 10.34 MPa of pressure, solubilities of CO2 in solution with 0.5 and 5% of malic acid concentration were 4.90?0.08 and 4.79?0.07 g/100 g of solution. When the amount of fructose in the solution increased to 5 and 10 g/100 g of solution, solubilities of CO2 at 10.34 MPa were 4.78?0.14 and 4.19?0.17, respectively. This equipment design and method can experimentally measure CO2 solubility in real liquid foods, and therefore is a significant contribution to the DPCD field. This is the first step in the generation of experimental data for the thermodynamic modeling of CO2 solubility in liquid foods. The experimental apparatus could offer an advantage to the food industry, primarily to soft drink industry, and to the DPCD users, by eliminating the need to use excess CO2 and therefore reducing the production cost.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Balaban, Murat O.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021868:00001


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DESIGN AND TESTING OF AN APPARATUS TO MEASURE CARBON DIOXIDE
SOLUBILITY INT LIQUID FOODS




















By

THELMA FRANCISCA CALIX LARA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008





































O 2008 Thelma Francisca Calix Lara





























To those that have guided and inspired me









ACKNOWLEDGMENTS

I wish to express my special gratitude to my maj or advisor, Dr. Murat O. Balaban, for his

valuable support, guidance and for being an example of motivation and hard work to us. I also

like to thank my advising committee, Dr. Charles A. Sims and Dr. Allen F. Wysocki, for their

guidance, assistance and time for my research.

I am very grateful for my lab partners and friends, Luis, Jose, Max, Milena, Alberto,

Zareena, Mutlu, Diana, Wendy, Yavuz, Maria and Giovanna for their assistance and for making

the work in the lab such an enjoyable experience.

I thank infinitely to my parents, Winston and Sagrario, my sister and my brother, Lourdes

and Winston, for their unconditional love and support during my entire life. They have been my

primary inspiration to pursue my goal.

Finally, my dearest thanks to Jorge, for all the support and happiness he brought to my life.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................. ...............7..____ .....


LIST OF FIGURES .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 1 1..


CHAPTER


1 INTRODUCTION ................. ...............14.......... ......


2 LITERATURE REVIEW ................. ...............17................


2.1 Carbonated Soft Drinks .............. ...............17....
2.2 Carbon Dioxide............... ...............19
2.2.1 General Information .............. ...............19....
2.2.2 Applications............... ..............2
2.3 Carbonation............... ..............2
2.3.1 D efinition................ .. .. .... ..................2
2.3.2 Methods and Apparatus for Carbonation .............. ...............29....
2.4 Supercritical Carbon Dioxide ................ ...............31................
2.4.1 Definition............... .. .................3
2.4.2 Dense Phase Carbon Dioxide ................ ...............33........... ...
2.4.3 Supercritical Fluid Extraction (SFE) ................. ........... .... ............ ... 3
2.6 Constituents that Affect Carbon Dioxide Solubility in Beverages ................. ................35
2.6.1 Carbonated Soft Drinks .............. ...............35....
2.6. 1. 1 Carbohydrate sweetener ............... ...............36...............
2.6.1.2 Acidulants............... ..............3
2.6.2 M ilk .............. ...............39....


3 MATERIALS AND METHODS .............. ...............40....


3.1 Design of the High Pressure Carbon Dioxide Solubility Apparatus .............. .... ............40
3.2 Preparation of the Samples .....__ ................ ...............46. ...
3.2.1 Distilled Water............... ...............46.
3.2.2 Fructose .............. ...............46....
3.2.3 Malic Acid ................. ...............46....___ .....
3.3 Experimental Procedure............... ...............4
3.3.1 Preliminary Procedure ............... ..... ......_. ...............47..
3.3.2 Pumping the Liquid Sample into the System .............. ...............48....
3.3.3 Carbonating the Sample .............. ...............48....
3.3.4 Measuring Solubility .............. ...............49....
3.3.5 Cleaning the Apparatus .............. ...............50....











3.4 Experimental Design .............. ...............50....
3.5 Data Analysis............... ...............51

4 RESULTS AND DISCUSSION............... ...............5


4. 1 Solubility of Carbon Dioxide in Distilled Water .....___._ ..... ... .__. ......_._........5
4.2 Solubility of Carbon Dioxide in Fructose Solutions. ......____ ..... .. ...............55
4.3 Solubility of Carbon Dioxide in Malic Acid Solutions .............. ..... ............... 5

5 SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR FUTURE STUDY ................61

5.1 Summary ........._.___..... ._ __ ...............61....
5.2 Conclusions............... ........ ..........6
5.3 Suggestions for Future Study ........._.___......___ ...............62..

APPENDIX

A DESIGN OF THE HIGH PRESSURE CARBON DIOXIDE SOLUBILITY
APPARATUS .............. ...............63....

B THE HIGH PRESSURE CARBON DIOXIDE SOLUBILITY APPARATUS
PROCEDURE S .............. ...............73....

LI ST OF REFERENCE S ................. ...............76................

BIOGRAPHICAL SKETCH .............. ...............8 1....











LIST OF TABLES


Table page

2-1 Physical properties associated with the phase diagram of CO2 .............. ....................20

2-2 Quality standard of carbon dioxide............... ...............21

2-3 Soft drink components and their typical use level ................ ...............36........... .

2-4 Properties of bulk sweeteners ................. ...............37.......... ....

2-5 Acidulants used in beverage formulation .............. ...............38....

3-1 Different levels of each variable in the two phases of the experiment .............. ..... ..........5 1

4-1 Experimental results of solubility of CO2 in pUTO Water ................. .........................53

4-2 Summary of the analysis of variance (ANOVA) of the solubility of CO2 in pUTO
water ................. ...............54.................

4-3 Summary of the least square mean (LS Mean) analysis of the solubility of CO2 in
pure water ................. ...............54.................

4-4 P-values from the least significant difference (LSD) test between experimental mean
data and literature data of solubility of CO2 in pUTO Water .............. ....................5

4-5 Experimental results of CO2 Solubility in solutions with different concentration of
fructose............... ...............55

4-6 Analysis of variance (ANOVA) of solubility of CO2 in fructose solutions of different
concentrations .............. ...............57....

4-7 Least Square (LS) Mean analysis of solubility of CO2 in fructose solutions of
different concentrations .............. ...............57....

4-8 P-values from the least Significant Difference (LSD) test of solubility of CO2 in
fructose solutions of different concentrations ................. ...............57........... ...

4-9 Experimental data of CO2 Solubility in solutions with different concentration of
m alic acid ................. ...............58.......... ......

4-10 Analysis of variance (ANOVA) of solubility of carbon dioxide in malic acid
solutions of different concentrations............... .............5

4-11 Least Square (LS) Mean analysis of solubility of carbon dioxide in malic acid
solutions of different concentrations............. .............5










4-12 P-values from the least Significant Difference (LSD) test of solubility of carbon
dioxide in malic acid solutions of different concentrations ................. ............ .........60










LIST OF FIGURES


Figure page

2-1 Beverage sectors and segments............... ...............17

2-2 Global beverage consumption by category, 1998-2003 .............. ....................1

2-3 Beverage consumption by countries (1998-2003) ......___ ... ....__ ........... .....18

2-4 Carbon dioxide phase diagram............... ...............19

2-5 Density-pressure isotherms for carbon dioxide .............. ...............20....

2-6 Carbon dioxide delivery process............... ...............22

2-7 Carbonation: CO2 chart in solution with pure water............... ...............24.

2-8 Solubility of carbon dioxide in water as a function of pressure and temperature.. ...........25

2-9. Carbonation methods .............. ...............29....

2-10 Measurement of carbonation ................. ...............30................

2-11 Phase equilibrium in the H20 and CO2 System ................. ...............32........... .

2-12 Variation of physico-chemical properties of CO2 at near-critical conditions ................... .34

2-13 Diagram of supercritical fluid extraction equipment ................. ............................35

3-1 Schematics of the high pressure CO2 Solubility apparatus ................. .......................41

4-1 Solubility of CO2 VeTSus concentration of fructose solutions, at 7.58 and 10.34 MPa,
and at a constant temperature: 40 oC. Vertical bars denote 0.95 confidence intervals.....56

4-2 Graph of solubility of carbon dioxide versus concentration of malic acid solutions, at
two different pressures: 7.58 and 10.34 MPa, and at a constant temperature: 40 oC.
Vertical bars denote 0.95 confidence intervals. .............. ...............58....

A-1 Pictures of the high pressure carbon dioxide solubility apparatus ........._.._.. .................. 63

A-2 Picture of the mobile part of the apparatus ........._._.. ....__.. ....._... ........6

A-3 Picture of the teflon tape used in every male connection ........._. ...... .. ..............64

A-4 Picture of the CO2 tank connections. The carbon dioxide tank, the three-way valve 1
(TV1), and the pressure gauge 1 (PGl) .............. ...............64....

A-5 Pictures of the high pressure pump (HHP) ................. ....___.....__ ..........6











A-6. Picture of the heat exchanger (HE) ................. ...............65........... ..

A-7 Back Pressure Regulator (BPR) diagram ...._. ......_._._ .......__. ...........6

A-8 Pictures of the back pressure regulator (BPR) used in the machine ................. ...............66

A-9 Picture of the heating tape (around the BPR) and the variable autotransformer ...............67

A-10 Picture of the Hart Scientific high precision water bath and the Masterfiex peristaltic
pum p .............. ...............67....

A-11 Picture of the thermocouple used inside the vessel 1 ........._._. ......._. ........_.......68

A-12 Picture of the metering valve (MV) ................. ...............68........... ..

A-13 Picture of the gas meter (GM) ................. ...............68.......... ...

A-14 Picture and diagram of the cross angles connector ................. ...............69........... .

A-15 Picture of the two way valve (V) ................ ...............69..............

A-16 Picture of the quick connect stem (female NPT) and body (male NPT) ................... ........69

A-17 Vessel 1.............. ...............70....

A-18 Diagram of vessel 2 .............. ...............70....

A-19 Picture of vessel 2 with the hole at the bottom ....__ ......_____ ..... ...__ .......7

A-20 Picture of the forged stainless steel gate valve ..........._ ..... ..__ ......_ ........7

A-21 Picture of the bottle and the stopper ..............._ ...............72.............

B-1 Picture of the mobile part of the equipment, before immersion into the cooler ................73

B-2 Pictures showing the sample being pumped .............. ...............73....

B-3 Picture of disconnecting QC6 .............. ...............74....

B-4 Picture showing when V4 was opened to collect the liquid sample ..............._ ...............74

B-5 Picture showing the measurement of the liquid sample collected .............. ...............75









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

DESIGN AND TESTING OF AN APPARATUS TO MEASURE CARBON DIOXIDE
SOLUBILITY IN LIQUID FOODS

By

Thelma Francisca Calix Lara

May 2008

Chair: Murat O. Balaban
Maj or: Food Science and Human Nutrition

Carbon dioxide (CO2) is a non-toxic, inexpensive, chemically inert, non-flammable fluid

and a safe solvent. It has multiple applications in the beverage industry, such as carbonation and

dense phase carbon dioxide (DPCD) technology, a cold pasteurization process.

The solubility of CO2 in water depends on pressure and temperature (Dodds and others

1956). Increasing pressure increases solubility of CO2 and increasing temperature decreases it.

In addition, the presence of other dissolved substances could have a positive or negative effect in

CO2 Solubility (Meyssami and others 1991). DPCD is a continuous cold pasteurization method

that affects microorganisms and enzymes under pressures below 50 MPa (7,250 psi) without the

use of high temperature treatments. Currently, the use of CO2 in DPCD treatments is not

optimized, since solubility of CO2 in liquid foods at different temperatures, pressures, and

compositions is not known. Therefore, excess CO2 is used to assure saturation. If CO2 Solubility

could be predicted at any temperature, pressure and composition for a liquid food, then the

amount of CO2 USed would be slightly above this theoretical value, and the process would be

more economical. The first step towards this type of prediction is the experimental measurement

of CO2 Solubility in model liquid food systems and its correlation with pressure, temperature and

composition. Therefore, the objectives of this study were: 1) to design, build and test a high-










pressure carbon dioxide solubility apparatus, 2) to compare the volume of CO2 that dissolves in

pure water with the current literature; and finally 3) to determine the maximum amount of CO2 in

solution with model liquid foods of known composition (binary solutions of fructose and malic

acid).

An experimental system to measure the CO2 Solubility was designed and tested. The

pressure and temperature were controlled in the system, and the design assured an accurate

measurement of solubility. Experimental results of CO2 in Solution in pure water were compared

with the literature, and there was no significant difference at pressures of 7.56 MPa (1 100 psi)

and 10.3 MPa (1500 psi), at a constant temperature of 40oC. At pressures of 7.58 and 10.34

MPa, solubilities of CO2 in water were 4.77 10. 15 and 5.5110.20 g/100 g of distilled water,

respectively .

By increasing the concentration of malic acid or fructose, solubility of CO2 decreased

significantly. At 7.58 MPa of pressure, the amount of CO2 dissolved in the solutions of 0.5 and

5% of malic acid concentration was 4.4710.03 and 4.4710.02 g/100 g of solution. When the

concentrations of fructose in the solution were 5 and 10%, solubilities of CO2 in the solutions

were 4.5510. 11 and 4. 1210.07, respectively (at 7.58 MPa). At 10.34 MPa of pressure,

solubilities of CO2 in Solution with 0.5 and 5% of malic acid concentration were 4.9010.08 and

4.7910.07 g/100 g of solution. When the amount of fructose in the solution increased to 5 and

10 g/100 g of solution, solubilities of CO2 at 10.34 MPa were 4.7810. 14 and 4. 1910. 17,

respectively.

This equipment design and method can experimentally measure CO2 Solubility in real

liquid foods, and therefore is a significant contribution to the DPCD Hield. This is the first step in

the generation of experimental data for the thermodynamic modeling of CO2 Solubility in liquid









foods. The experimental apparatus could offer an advantage to the food industry, primarily to

soft drink industry, and to the DPCD users, by eliminating the need to use excess CO2 and

therefore reducing the production cost.









CHAPTER 1
INTTRODUCTION

Carbon dioxide (CO2) has numerous food applications and many others are being

developed, providing simple and economic process alternatives to the beverage industry. CO2

gas is used in the carbonation of soft drinks, beers and wines because many studies have

demonstrated that CO2 prevents fungal and bacterial growth (Eastoe and others 1996; Del Pozo-

Insfran and others 2006b; Gunes and others. 2005). In addition, CO2 enhances the physical

properties and sensory attributes of carbonated beverages (Kappes and others 2007; Del Pozo-

Insfran and others 2006a)

In 1998 and 2003, the global beverage consumption volume reached an estimated 1.2

trillion liters and 1.4 trillion liters, respectively. Carbonated soft drinks represented 14% of the

total in both years, increasing by 3.0 million liters during that period (Roethenbaugh 2005). In

2003, the United States was the biggest consumer, with 30% of the total volume of carbonated

soft drinks, followed by Mexico with 9%. However, the overall compound annual growth

forecasts a growing concern for health and wellness. That forecast will drive the growth of

bottled water and still drinks consumption in the coming years; save for soft drinks, which have

increased in importance in recent years against other beverage categories (Roethenbaugh 2005).

Carbon dioxide is a non-toxic, chemically inert, non-flammable fluid and a safe solvent for

process applications (Eastoe and others 1996). Carbon dioxide has a specific gravity of 1.53

(based on air), and its molecular weight is 44.01 grams per mol and/or 22.4 liters per mol (Steen

2005; Subramaniam and Busch 2002). In food applications, CO2 can be present as a gas, liquid

or solid, depending on temperature and pressure. According to the phase diagram, CO2 has a

triple point (-56.6 oC at 0.52 MPa=5.11 atm), a sublimation range and a critical point

(temperature 31oC and pressure 7.4 MPa=73 atm). The triple point is the condition that CO2









simultaneously exist as a solid, liquid and gas. The sublimation range is when CO2 gaS can

become solid CO2 (dry ice), without passing through the liquid phase (and vice-versa). The

critical point is the temperature above which it is impossible to liquefy the gas (Steen 2005).

Most of the studies about supercritical CO2 have been mainly focused on supercritical

extractions. However, the purpose of this proj ect was focused on the area of dense phase carbon

dioxide (DPCD) pasteurization, to design and build an experimental apparatus to measure

solubility of CO2 in liquid foods. DPCD is a cold pasteurization method that kills

microorganisms, inactivates certain enzymes and viruses at pressures below 50 MPa without

applying high temperature treatments (Damar and Balaban 2006). The microbial inactivation

effect of DPCD is partly based on gas dissolution under pressure in a microbial cell that, when

decompressed to atmospheric pressure, causes fatal damage (Balaban and others 1991; Park and

others 2002). Furthermore, there is the theory that high pressure carbon dioxide dissolves in

water to produce carbonic acid, thus the pH will be temporarily lowered and therefore inactivates

enzymes (Damar and Balaban 2006).

Kincal and others (2005) tested a continuous high-pressure carbon dioxide system for

microbial reduction in orange juice, and Arreola and others (1991) studied the effects of CO2 On

some quality attributes. Results showed that the treatment was able to cause a 5-log reduction of

the natural flora in spoiled juice and could achieve a 5-log reduction of pathogenic Escherichia

coli 0157:H7, Salmonella tyiphimurium and Listeria monocytogenes. Balaban and others (1991)

stated that pectinesterase was inactivated under supercritical conditions, suggesting that its

inactivation was due to the combination effects of pressure, temperature, pH reduction and

process time. Dagan and Balaban (2006) measured the effect of DPCD pasteurization on beer

quality using a continuous system, and the process was able to produce a clear, consistently









fresh-tasting beer with extended shelf life.

In DPCD, the use of carbon dioxide is not optimized, since solubility is not known, and not

predicted. Thus, excess carbon dioxide is used to assure saturation. In addition, Ji and others

(2007) mentioned that the reduction of greenhouse gases emissions has become an issue of

worldwide concern (CO2 TepreSents about 64% of the enhanced greenhouse effect). If CO2

solubility could be measured at any temperature, pressure and composition of food, the amount

of CO2 USed would be slightly above this theoretical value, and the process would be more

economical. Therefore, the objectives of this study were:

* To design and build a high-pressure apparatus to measure CO2 Solubility in liquid foods.

* To test the accuracy and precision of the high-pressure CO2 Solubility apparatus, and
compare with the literature the amount (v/v) of CO2 dissolved in pure water.

* To determine the maximum amount (v/v) of CO2 in Solution with a model liquid food of
known composition (solution of sugar or acid).










CHAPTER 2
LITERATURE REVIEW

2.1 Carbonated Soft Drinks

According to Roethenbaugh (2005), soft drinks are sweetened water-based beverages,

usually balanced by acidity, and their primary function is hydration. Soft drinks are divided into

two large groups, the ready-to-drink (RTD), and the concentrated or diluted-to-taste products.

RTD products are subdivided into carbonated products. Figure 2.1 shows the beverage sectors

and segments.


All beverages


Hot drin ks Soft drinks Milk drinks Alcoholic drinks

Tea Bo~ltledi water White rnilk Beer

Coffee Carbonated drink Flavoured milk Wine

Other hot drinks Dilutables Spirits

Fruit juice/nectars Other alcoholic drinks


Figure 2-1. Beverage sectors and segments (Roethenbaugh 2005).

The dominant world players in the carbonated RTD soft drink industry are Coca Cola,

Pepsi and Cadbury-Schweppes. Together with their associated brand names, they constitute over

half the world' s consumption of such products. In 2003, carbonated soft drinks became the

world's second most popular beverage after tea. In 1998 and 2003, the global beverage

consumption volume reached an estimated 1.2 trillion liters and 1.4 trillion liters, respectively,

and approximately fourteen percent (14%) of the total were carbonated soft drinks (Figure 2-2)

(Roethenbaugh 2005).










% orf 1998 volume

Others
1400

Bottled water ;
t ,

Beer c :Tlir ;t


%,' of 2003 volumne

Others
14%b L


Tea
2P-o


Tea
25"3


Beer


Coffee '

Bottled water
11% Carbonates 147o Milk 14%
11%

Vo~Lume: 1,2 trillion litres Volume: 1 .4 trillion litres

Figure 2-2. Global beverage consumption by category, 1998-2003 (Roethenbaugh 2005).

Over the period 1998-2003, the global consumption of beverages was dominated by the

United States (15%), followed by India (10%) and China (9%) (Figure 2-3). In carbonated soft

drinks the United States represented thirty percent (30%) of global consumption, followed by

Mexico (9%). The fastest growing consuming countries between 1998-2003 were Bosnia-

Herzegovina (160% increase), Serbia (86%) and China (86%).


"a of 1998 volume

UnitedJ Stattes 5i%


% of 20033 volurne

SUnited States 15%


Others
54C.


Others
57%


Indra. 10%


SChina 1 041ni ~



Braindi5%

an 4%


ermranay So? ~a3s.
Japan 5% Jap;

~Volurne: 1.2 trillion ltres Volurne: 1.4 tril ion litres
Figure 2-3. Beverage consumption by countries (1998-2003).









2.2 Carbon Dioxide

2.2.1 General Information

Carbon dioxide (CO2) is a non-toxic, inexpensive (approximately 4.85 cents/kg in 2007),

chemically inert, non-flammable fluid and a safe solvent for process applications (Eastoe and

others 1996, Steen 2005). It has a specific gravity of 1.53 (based on air) under conditions of

room temperature and atmospheric pressure, and its molecular weight or molar mass is 44.0098

+ 0.0016 grams and/or 22.4 liters per mol (Steen 2005; Subramaniam and Busch 2002; Span and

Wagner 1996). The molecule of CO2 is linear with a double bond between the carbon and

oxygen atoms (O=C=0) (Song 2002).

The effects of temperature and pressure on the state of CO2 (physical properties) are shown

in Figure 2-4. The triple point (-56.6 oC at 0.52 MPa=5.11 atm) is the condition in which CO2

can be present as a solid, liquid or gas (the three phases coexist). The sublimation range is when

CO2 Solid (dry ice) directly becomes gas (and vice-versa), for example at -78.8 oC and 1 atm

(Song 2002; Steen 2005, Levelt 2000).

10000

1000~ CO, solicl

100C CO, Ilquicl

10Subllrnation range i as
Critical point


0_ l ~ Triple point
0.1 W -56.60C at 5.11 atrn

0 .0 1 CO, gas


-140 -120 -100 -80 -60 -40 -20 0 20 40 60 80 100
Tern pe rature (oC )
Figure 2-4. Carbon dioxide phase diagram (Steen 2005).










Above the critical point (temperature 31oC and pressure 7.4 MPa=73 atm) it is impossible

to liquefy the gas, and CO2 eXiSts as a fluid or dense gas (Song 2002). At atmospheric pressure

and room temperature (25 oC), CO2 CannOt be present as liquid. To liquefy CO2 it is necessary to

compress and cool between the triple point and the critical point conditions (Steen 2005). The

density, viscosity and diffusion coefficient associated with different phases of CO2 are Shown in

Table 2-1.

Table 2-1. Physical properties associated with the phase diagram of CO2 (Koutchma and
Murakami 2005; Buhler and others 1998).
States Density (g/ cm3) Viscosity (cP) Diffusion coefficient (cm /s)
Gas 0.002 0.014 0.01
Super Critical 0.467 0.02-0.12 0.0001
Liquid 1.0 1.0 0.00001

In CO2, the density (p) increases rapidly around the critical pressure (pe). When the

temperature decreases, the change in p is more remarkable and it does not move to higher

pressures as fast as at high temperatures; thus, p becomes difficult to control around the critical

temperature (Clifford and Williams 2000). Figure 2-5 shows four density-pressure isotherms.

0.9
31TI~
0.8 400C

0 7 50

0.6 -I ,60 C




S0.2




0.1 Y- Pc

0.0
50 75 100l 125 150
Pressure (lbar)
Figure 2-5. Density-pressure isotherms for carbon dioxide (Clifford and Williams 2000).










According to Steen (2005), the European Industrial Gases Association, the Compressed

Gases Association of America and the International Association of Beverage Technologist have

described the standard quality of CO2 for use in foods and beverages. Table 2-2 shows the

specifications for commercial liquid CO2.

Table 2-2. Quality standard of carbon dioxide (Steen 2005).
Component Specification
Assay 99.9% v/v min.
Moisture 50 ppm v/v max. (20 ppm w/w max)
Acidity To pass JECFA* test
Ammonia 2.5 ppm v/v max.
Oxygen 30 ppm v/v max.
Oxides of nitrogen (NO/NO2) 2/5 ppm v/v max. each
Non-volatile residue (particulates) 10 ppm w/w max.
Non-volatiles organic residue (oil and grease) 5 ppm w/w max.
Phosphene <0.3 ppm v/v
Total volatile hydrocarbons (calculated as 50 ppm v/v max. of which 20 ppm v/v max.
methane) non-methane hydrocarbons
Acetaldehyde 0.2 ppm v/v max.
Benzene 0.02 ppm v/v max.
Carbon monoxide 10 ppm v/v max.
Methanol 10 ppm v/v max.
Hydrogen cyanide <0.5 ppm v/v
Total sulphur 0.1 ppm v/v max
Taste and odor in water No foreign taste or odor
SJECFA: Joint FAO/WHO Expert Committee on Food Additives.

In commercial use, there are several methods of CO2 prOduction. Fermentation and direct

combustion are two typical processes. Fermentation consists of mixing a carbohydrate-based

solution with yeast and oxygen in a fermenter; CO2 and alcohol are obtained. The CO2 is passed

through a separator to eliminate the foam; then CO2 is compressed. Finally, CO2 is washed with

water to remove water-soluble impurities. Direct combustion consists of burning a hydrocarbon

fuel, such as light oil or natural gas, to produce a gas containing less than 0.5% oxygen by

volume. The gas is cooled, scrubbed (to remove impurities) and passed through an absorbent

tower that gets in contact with a CO2 absorbing solution. This solution is then pumped to a

striping tower, used to release the CO2 in vapor form (Steen 2005).










Carbon dioxide is delivered as a liquid to the customer, often by road tanker, and then it is

transferred to pressurized vessels (20.5 bars) of 5-50 tons capacity at -17oC; temperature is

controlled by a small refrigeration unit. To change CO2 liquid to CO2 gaS is necessary to use a

carbon dioxide vaporizer. Every batch supplied must have a certificate of conformance to ensure

that there are no dangerous residues in CO2, Such as benzene, and to assure the safety of the user.

A hazard analysis and critical control points (HACCP) survey of the installed system is required

(Steen 2005). Figure 2-6 shows how CO2 is delivered: A liquid and gas phases are connected in

both tanks (Steen 2005).

To carbonator
at around 15 bar pressure
C -- ~- - --- ------4 C3 arbon dioxide
road tanker

I-I





Steam vapreronsr-Isltdcro



Liquid Pump

Refrigeration
unit


Figure 2-6. Carbon dioxide delivery process (Steen 2005).

2.2.2 Applications

Carbon dioxide has numerous applications in the food and beverage industries. CO2 liquid

or solid is used for quick freezing, surface freezing, chilling and refrigeration in the

transportation of foods. Cold sterilization can be obtained with a mixture of 90% CO2 and 10%

ethylene oxide; CO2 has a stabilizing effect on the ethylene oxide and diminishes the hazard of

explosion. CO2 gaS is used in the processing of carbonated soft drinks, beers and wines. CO2










provides sensory characteristics. For consumers, CO2 can be a pleasurable and sought-after

sensation; while, for some people, CO2 can be an irritating or even painful sensation (Dessirier

and others 2000). CO2 bubbles are responsible for several sensations because they stimulate the

mechanoreceptors on the tong and because of chemogenic origin by formation of carbonic acid

(Descoins and others 2004). In addition, dense phase CO2 is used in beverages due to its

effective antimicrobial effects (Eastoe and othersl996).

During the last 20 to 30 years, considerable interest has centered on supercritical fluid

extraction (SFE) using CO2 (Bamberger and others 1999; Harris and Yung 1995). Isolation of

flavors, decaffeination of coffee beans, removal of hexane from soybean oil, extraction of

triglycerides, cedarwood oil, cedarleaf oil, carotenoids, and anthocyanins (a type of flavonoid or

a group of phenolic compounds (Netzel and others (2006)) are some examples of SFE

applications (Bamberger and others 1999). Identification of acetylated soybean lecithin

components was also performed to test the surfactant potential of supercritical CO2 (Nasir and

others 2007).

2.3 Carbonation

2.3.1 Definition

Carbonation is the impregnation of a liquid with CO2 gaS (Steen 2005) and its

effectiveness depends on the quantity of gas in solution with the soft drink (Mitchell 1978).

Solubility of gases in liquids has been studied by many investigators since the early nineteenth

century (Battino and Clever 1965; King and others 1992). Solubility of a solute (solid, liquid or

gas) is the analytical composition of a saturated solution, expressed in terms of the proportion of

the designated solute in a designated solvent (Karukstis and Van Hecke 2000). Dodds and others

(1956) state that solubility of CO2 in water depends on its pressure and temperature in

equilibrium with the solution. Pressure has a direct effect on the solubility of CO2 (preSSUTO










increases the solubility of CO2). On the other hand, with increasing temperature, solubility of

CO2 decreases. The presence of other substances could have a positive or negative effect in

solubility (Descoins and others 2006; Meyssami and others 1991). In addition, solubility of CO2

has an opposite behavior than solubility of solids (e.g. sugar or salt) in water. Most solids

dissolve more readily in hot water than in cold. Figure 2-7 shows how the relationship between

pressure and temperature affect the volume of CO2 in Solution with pure water at relatively low

pressures. Figure 2-8 shows the effect of temperature and pressure on the solubility of CO2 in

pure water.




25
1 Voil. 2Vols. 3 Vols. J4Vols. 5 Voils. 6 iVols.















Pressure (bar)
Figure 2-7. Carbonation: CO2 chart in solution with pure water (Steen 2005).
























II


o
.1" L~!LCZ







'8 $ L~aI
1 I



01 I


(Ddd an oter 95)









When CO2 is dissolved in water, it forms carbonic acid (Equation 2-1). Carbonic acid, in

combination with the product, produces the acidic and biting taste in carbonated beverages,

making the beverage more palatable and visually attractive (Steen 2005; Descoins and others

2006).

CO2 (gaS, liquid) t, CO2 (aq) (2-1)
CO2 (aq) + H20 t H2CO3 K = 1.6 x 10-3
H2CO3 (aq) + H20 t HCO3- (aq) + H30+ (aq) pKa = 6.35
HCO3- (aq) + H20 CO3-2 + H30+ pKa = 10.33
According to Steen (2005), Henry's law postulated by William Henry (1774-1 83 6) states

that "the amount of gas dissolved in a given volume of solvent is proportional to the pressure of

the gas with which the solvent is in equilibrium". The universal ideal gas law is:

pV = mR T (2-2)

p is the absolute pressure in atmospheres (atm), V is the volume of the gas (m3), m iS the number

of moles of gas, R is the specific gas constant (J kg moll K 1) and T is absolute temperature (in

Kelvins). CO2 mOlar mass (M)1 is 44.0098 & 0.0016 g mol-l and its molar gas constant (Rm) is

8.314510 + 0.000210 J kg mol lK- The specific gas constant for CO2 (R = Rm/M) is 0. 1889241

& 0.0000116 kJ kg-l K (~Span and Wagner 1996).

Diamond and Akinfiev (2003) state that solubility of CO2 in terms of the Henry's law can

be expressed as follows:

02o(PT YYI
xCO2 (aq) = (2-3)
kH(P,T) YCO2 tag

XCO2(aq) and y are the mole fractions of carbon dioxide in the aqueous and non-aqueous phase

respectively, kH(P,T) in MPa is the Henry's constant of carbon dioxide in pure water on a mole

fraction basis at specified pressure and temperature conditions, foCO2(PT) is the fugacity of pure









carbon dioxide (in MPa) at specified pressure and temperature conditions, yeo2(aq) is the

asymmetric activity coefficient carbon dioxide in Henry's law (yCO2(aq) 1 aS XCO2 (aq) 0)

and y, is symmetric (Raoult' s law) activity coefficient of carbon dioxide in the non-aqueous

phase (y, 1 as y -1).

To calculate solubilities using Eq. (3), it is necessary to assume that yCO2 (aq) and y, are

equal to unity and foCO2 (PT) for the CO2-rich vapor and for the CO2-rich liquid are calculated

from the fundamental equation of state, expressed in form of the Helmholtz energy (Span and

Wagner 1996):

AT= #(3, r)= #"(3, r)+ #" 3, ), (2-4)
(R T)

where A is the specific Helmholtz energy, p is mass density, Tis temperature, R is the gas

constant, #is the dimensionless Helmholtz energy (# = A/RT), 6 is the reduced density (6 = p/pe)

and z is the inverse reduced temperature (z = Te/T). The dimensionless Helmholtz energy (#) is

split into a part depending on the ideal gas behavior (# o) and a part which takes into account the

residual fluid behavior (# r). The critical density of carbon dioxide (pe) is 467.6 kg/m3 and

critical temperature (Te) is 304. 1282 K (Span and Wagner 1996).

The vapor pressure (ps) and the densities of saturated liquid (') and saturated vapor (") can

be determined from an equation explicit in # by simultaneous solution of the equations:


= 1 3'#,(3' r),(2-5)
RTp'


L7 = 1+ 3"#",r (3"l, ),
RTp"


RT p' "









which correspond to the equality of pressure, temperature and specific Gibbs energy in the

coexisting phases (Span and Wagner 1996). The Gibbs energy (GE) iS used to describe the non-

ideality of the H20-rich phase in the equation of state (Costa-Gomez 2007; Joidecke and others

2007; Krichevsky and Kasarnosky 1935). The Helmholtz energy of the ideal gas is given by:

Ao(p, T)= ho(T)-RXT- Tso(p, T), (2-6)

where the enthalpy ho of the ideal gas is a function of the temperature only, and the entropy so of

the ideal gas depends on temperature and density (Span and Wagner 1996).

Also, y (mole fraction of CO2 in the non-aqueous phase) is estimated according to the

Lewis-Randall rule (at low pressures):


y = 1 oer (2-7)
Ptotal

where Pwatersar is the pressure of liquid-vapor equilibrium of pure H20 (in MPa) and Ptotal is the

total pressure (in MPa), at a specified temperature. At high pressures, y is determined from the

following empirical equation:

y = 1 (0. 1256t 0.0212) x 10-3 P (0.065t + 1.121) x 10- (2-8)

where t is temperature in oC and P in MPa.

Henry's constant (kH) is calculated from the equation of state (Akinfiev and Diamond

2003):


In(kH O HO~i,~ P"+ 2p oa + b ,] (2-9)

where foH20 is the fugacity (in MPa) and poH20 is the density (in g cm-3) Of pure water at

determined P and T tin Kelvins), R is the gas constant (8.31441 cm3 MPa K^1 mol ), Mw is the

molar mass of H20 (18.0153 g mol- ), and 5 dimensionlesss), a (in cm3 -1l) and b (in cm3 Ko5g

1) are empirical parameters (Diamond and Akinfiev 2003; Akinfiev and Diamond 2003).










Many of the parameters and thermodynamic coefficients require experimental data for

accurate calculation. Such CO2 Solubility data for real liquid foods is rare or non-existent.

2.3.2 Methods and Apparatus for Carbonation

The complexity and cost of the equipment used to carbonate and to measure solubility of

CO2 in liquids are extended from mass spectrometer to the simple manometric van Slyke

apparatus, used to measure gas quantities released from blood (Battino and Clever 1965). Figure

2-9 shows different schemes of carbonation methods. The most common method is the

bubblerr" that consists of sparging CO2 into the liquid under pressure, to form small bubbles of

gas which can be easily absorbed by the liquid (Steen 2005). The waterfall method represents

the early carbonators which used to operate at around 4oC.

Product







CO2 CO



Product
-Carbonated-
product Carbonated

Static Wated~all C rdc
Bub~bler
(difficult to achieve (the product descends as a
efficient carbonation.I waterfall in the vessel)

Figure 2-9. Carbonation methods (Steen 2005).

According to Steen (2005), the rate of flow and the pressure of CO2 are critical to ensure

the correct carbonation level. When the pressure increases, the gas bubbles formed at the sparger

will be smaller and the gas bubbles' surface area will facilitate the gas absorption by the liquid.










In addition, the greater surface area of liquid exposed to CO2 Will inCreaSe the rate of absorption

of CO2 by the liquid.

Figure 2-10 shows an example of a typical apparatus to measure carbonation which

consists of a jig to restrain the container, and a piercer connected to a pressure gauge. The

container is shaken before the pressure is measured and the release valve is opened until the

pressure gauge reads zero. For a second time, the container is shaken and the pressure is retaken

and the temperature measured. The volume of carbonation is then obtained from a carbonation

chart by finding the point that relates the conditions of temperature and pressure previously

measured (Steen 2005).

Pressure
ga lge


Release
valve




-------- Piercer


SFrame






Figure 2-10. Measurement of carbonation (Steen 2005).

The purpose of releasing the pressure for the first time is to exclude other gases from the

head space of the can (container) or from the beverage. Air has approximately one fiftieth the

solubility of CO2 in a liquid. Air constituents are approximately 79% nitrogen and 21% oxygen.

Due to the differing solubility and proportion of oxygen and nitrogen, the dissolved air actually









contains 35% oxygen and 65% nitrogen (low solubility). The presence of oxygen could create

spoilage problems (Steen 2005). This method is not practical or safe under pressures used by the

DPCD method.

2.4 Supercritical Carbon Dioxide

2.4.1 Definition

According to Subramaniam and others (2002), supercritical phase reactors are gaining

acceptance in industry. DuPont has announced a US$ 40 million demonstration plant for

supercritical polymerization. Turner (2006) states that supercritical gas or liquid occurs when

the temperature increases over the critical point. In the CO2 phase diagram (Figure 2-4), the

curves represent coexistence between the phases. When the curve representing the liquid + gas

phase ends at the critical point, the distinction between vapor and liquid disappears (Levelt 2000)

and the densities of the two phases become identical (Clifford and Williams 2000).

In supercritical fluid mixtures, it is important to predict the phase equilibria to successfully

develop and operate the physical separation processes and solubility models (Stratmann and

Schwiger 2004). The fundamental equation of phase equilibria establishes that, at a given

temperature and pressure, the fugacity of a component i in the gas phase, J"g must be equal to the

fugacity of the same component in the solid phase /," (Valderrama and others 2006). Binary

fluid mixture phase diagrams are usually represented as two-dimensional proj section of P-T

(pressure-temperature) (Levelt 2000). Phase equilibria diagram between the binary mixture of

water and CO2 System is represented in Figure 2-11.









100.0


10.0 -
LCEP







0 0 2 0 09 0
Teprtue/"
Figure$ 2-11.~ Phs qulbim nteH2 n O2 ytm(Daodan knev20)
LCE deoeh owe criia n-on fth ytm udul pitQ niae h
eqiiru ewenH0ie O-lahaehdae O2baigwtran O-ao Ie
Cl-a-) Teqarpl on 2 hw h qulbimbewe O-cahaehyrtC 2
berigwae, O-lqid and CO2vpr(l-a-C2V (imn n kne 03

Logh 20)AtLElq id n a hssciial eg ofr igefudpaei











thgue rysta strctue of libice(Smey andt z other 2005 Fser dowsamnd Oa 205 Aknderso 2002).









The CO2 hydrate is a structure -I hydrate. Structure-I hydrate consists of 46 water molecules and

8 guest molecules at most per unit cell and forms two smaller cages of pentagonal dodecahedron

(512) and six larger cages of tetrakaidodecahedron (512612). CO2 hydrates consist of three parts:

interaction between water-water molecules, interaction between water-CO2 mOlecules and

interaction between CO2-CO2 mOlecules. According to Anderson (2002), once carbon dioxide

clathrate begins to form, it becomes nearly impossible to assess the partitioning of CO2 between

the liquid and solid phase. Regarding solubility of CO2, SeVeral studies state that solubility with

the hydrate would decrease with decreasing temperature and pressure (Ferdows and Ota 2005).

2.4.2 Dense Phase Carbon Dioxide

Dense phase carbon dioxide (DPCD) processing, which includes liquid and supercritical

CO2, iS a continuous cold pasteurization method that affects microorganisms and enzymes under

pressures below 50 MPa (7,252 psi) without applying high temperature treatments, based on the

hypothesis that high pressure CO2 dissolves in water to produce carbonic acid, thus the pH value

will be temporarily lowered and able to inactivate enzymes (Damar and Balaban 2006; Del Pozo-

Insfran and others 2006b; Ishikawa and others 1995; Shimoda and others 2001). Moreover,

DPCD system removes dissolved oxygen, preventing degradation of several compounds, such as

antioxidant phytochemicals; thus, DPCD process decreases phytochemical and antioxidant losses

that occur during storage of juices (Del Pozo-Insfran 2006a; Subramaniam and others 2001).

Fraser (1951) was the first to prove that DPCD technology can disrupt bacterial cells and some

years later, Taniguchi and others (1987) showed the effect that supercritical CO2 (20 MPa, 35 C,

2 hr) has on the enzyme activity. Furthermore, supercritical CO2 has been recognized for

sterilizing and inactivating viruses. Usually, supercritical reaction media have the potential to

increase reaction rates, selectivity of chemical reactions, and facilitate separation of reactants,

products and catalysts after reaction. In addition, principles for gas or liquid phase reactions may









be applied to supercritical phase reactions (Figure 2-12) (Subramaniam and Busch 2002;

Subramaniam and others 2002).













;AI I 0P

Prssr (a
Figure ~~~c'~, 2-2 aito fpyiochmclpoete fC2atna-rtclcniin
(Sbaaimad tes20)
2..3Suecriia Flui Exrcto (SE)

Exrcini n motn eh d toioaentrloo/lvrcmoet n te
naua poutssc s setilols h rodc oban d fr m dsilto/xtato vcu
ds illtoetato ihlqi ovnsadasrto a ovn eius(abre n
other 199) Hoevr sueciia O xrcinrslsinapoutwtotsletrsde

adwtabetr tat/aoa because it is5 a o-hra rcsM reuadohr 95
Palzogu nd alaan199).Accrdig o Sbraanam ndBush (00), O2 aslimte
rectviy ths ueciia O a ayceialapiain orpaecnetoa
orai ovns opromsprrtclfudetatosi is neesry opm O hog










a vessel previously filled with sample. After extraction, the supercritical fluid is depressurized

for collection of extracted compounds (Schneider and others 2000). According to Clifford

(1999), the factors that control supercritical fluid extraction are: the solute must be sufficiently

soluble in the supercritical fluid to be removed by solution in the fluid flow; the solute must be

transported sufficiently rapidly from the interior of the matrix in which it is contained and; the

solute must be released by the matrix. A supercritical fluid instrument includes a high-pressure

pump (one or two) to carry the supercritical fluid, a high pressure vessel to contain the sample, a

polar cosolvent (ethanol) if necessary, a restrictor and a collection vial (Turner 2006). Figure 2-

13 shows a schematic of SFE equipment.

Restrictolr
-Oven


Sarnple
High-pressure vessel
pum as
T Collection
COz Solvent
vial
pump pump

Figure 2-13. Diagram of supercritical fluid extraction equipment (Turner 2006).

2.6 Constituents that Affect Carbon Dioxide Solubility in Beverages

2.6.1 Carbonated Soft Drinks

Carbon dioxide solubility can be affected positively or negatively by different constituents

present in the formulation of different soft drinks. The term soft drink applies to beverages

containing flavors and/or fruit juices together with other constituents of technological or

nutritional value designed to enhance the appearance and stability of the product and to ensure

that its sensory properties remain intact during a reasonable shelf life (Taylor 2005). Some

categories of ingredients are discussed in more detail in the following sections, in terms of CO2










solubility. The functional constituents of soft drinks and their typical usage levels are listed in

Table 2-3.

Table 2-3. Soft drink components and their typical use level (Taylor 2005).
Component Typical use level
Water Up to 98% v/v when high-intensity sweeteners in use
Sugars 7-12% m/v when sole source of sweetener
Fruit juice Widely variable usage
High-intensity sweeteners Use based upon sucrose equivalent (e.g. aspartame might be
employed at 0.40-6% m/v as sole sweetener)
Carbon dioxide 0.30-6% m/v
Acids (e.g. citric) 0.05-0.03% m/v
Flavours Nature-identical and artificial: 0. 10%-28% m/m. Natural: up
to 0.5% m/m
Emulsifier 0.1% m/v
Colours (naturals or synthetic) 0-70 ppm
Preservatives Statutory limits apply (e.g. sorbic acid up to 250 ppm in EU)
Antioxidants (e.g. BHA) Less than 100 ppm, subject to user-country legislation
Quillaia extract (saponins) Up to 200 mg/1 (EU), up to 95 mg/1 (USA)
Hydrocolloids mucilaginouss gums) 0.1-0.2% per GMP, minimum amount required to create
desired effect
Vitamins/Minerals ADIa applies
aADI: acceptable daily intake.

2.6.1.1 Carbohydrate sweetener

According to Bessley (1990), the first association between sugars and carbonated soft

drinks occurred in the seventeenth century when lemon juices containing natural sugars were

added to spring water. Since Captain Cook, the use of carbohydrate sweeteners in juices and

drinks has increased, in order to preserve the juice for long periods (O'Donnell 2005; Bessley

1990). In the past 10-15 years, the profile of bulk and intense sweetener use in soft drinks has

changed (technical improvements in the manufacture of bulk sweetener with different

carbohydrate profiles that deliver equivalent sweetness and mouthfeel properties to sugar at

lower cost). In addition, due to an increasing consumer awareness of healthy eating and concern

about the growing incidence of obesity and Type 2 diabetes, interest in low calorie carbonated

soft drinks has increased considerably (O'Donnell 2005). An extensive number of carbohydrate

sweeteners are used in soft drink industry, providing sweetness, mouthfeel, stability and color (in










some cases). Table 2-4 illustrates the properties of some carbohydrate sweeteners used in soft

drinks.

Table 2-4. Properties of bulk sweeteners (O'Donnell 2005).
Sweetener/ Relative Solubility Insulin- Caloric Approval for use
carbohydrate sweetness (g/100g water dependent value in soft drinks
(sucrose=1) at 20oC) metabolism (kcal/g) EU USA
Sucrose 1 200 Yes 4 N/A N/A
Glucose syrup 0.6 Higha Yes 4 N/A N/A
HFGS 0.9-1.0 Higha Yes 4 N/A N/A
Fructose 1.2-1.8 374.83 No 4 N/A N/A
FOSb 0.3-0.6 75 No 2 N/A N/A
Inulin 0 10 No 1 N/A N/A
Polydextrose 0 80 No 1 Yes Yes
Trehalose 0.45 40.8 Yese 4 Yes Yes
Tagatose 0.92 62.0 No 1.5 No Yes
a Solubility good, but varies with products of different specifications. bFructo-oligosaccharides. "Glucose
and insulin responses lower than glucose.

In general, sugars diminish the average level of CO2 Solubility, at pressures from 1 to 6

bars and pH set at 3.1(Descoins and others 2006). In addition, sugars contribute sweetness and

body to the drink, act as synergist and give balance to flavor (Steen 2005). Carbohydrate sugars

used in carbonated soft drinks can be divided into those in a dry, granular form (e.g. granulated

sucrose) and those in a liquid or syrup form (e.g. liquid sugar (sucrose in solution with water and

glucose-type syrup produced from maize or wheat, e.g. glucose syrup or high-fructose glucose

syrup)). Granulated sugar is a dry, crystallized disaccharide extracted from sugar beet and sugar

cane called sucrose and liquid sugar consists of an aqueous solution of sucrose at a saturated

concentration of 67 oBrix at 20 oC (Bessley 1990).

In United States, high-fructose corn syrups dominate the carbohydrate-sweetened soft

drinks sector; however, in Europe the use of high-fructose glucose syrup (HFGS) is restricted by

production quotas. Fructose (levulose) is a sugar in crystalline or syrup form. It is produced by

hydrolyzing sucrose to a glucose-fructose mixture; the resulting monosaccharides (glucose and

fructose) are separated using chromatography and the fructose is crystallized. Its sweetener










capacity is higher than sucrose (Table 2-4); thus, it can be used at lower concentrations to

decrease the level of energy content in the formulations. The absorption and metabolic process

of fructose by the body is very slow and does not depend of insulin production, being appropriate

for diabetics (O'Donnell 2005).

2.6.1.2 Acidulants

The use of acidulants is an essential part of beverage formulation. Acidulants contribute to

sharpness, sourness, background to flavor and increase thirst-quenching effects in soft drinks

(Steen 2005). In addition, acidulants can indirectly prevent discoloration and rancidity, by acting

as a synergist to antioxidants (Taylor 2005). Table 2-5 shows the list of the most common

acidulants used in soft drinks.

Table 2-5. Acidulants used in beverage formulation (Taylor 2005).
Acidulants Molecular weight Melting point (oC)
Citric acid : 2-hydroxy-1,2,3-propane tricarboxylic acid 192.1 152-154
Tartaric acid (D-tartaric): 2,3-dibydroxy butanedioic acid 150.1 171-174
Phosphoric acid: orthophosphoric acid 98.0 42.35
Lactic acid (DL-lactic): 2-hydroxy propanoic acid 90.1 18
Malic acid (D-malic): 2-hydroxy butanedioic acid 134.1 98-102
Fumaric acid: trans-butenedioic acid 116.1 299-300
Acetic acid: ethanoic acid 60.0 16-18


Citric acid is the most widely used acid in fruit-flavored beverages. It used to be

commercially produced from lemons, limes or bergamots by concentrating and precipitating the

juice. Recently, it is produced using enzymes starting from glucose and other sugars. Tartaric

acid is naturally present in grapes, currants, blackberries and cranberries. Tartaric acid has a

sharper flavor than citric acid; thus, it is used in lower concentration to provide equivalent palate

acidity. However, tartaric acid has lower solubility than citric acid. Phosphoric acid is used in

soft drinks for its astringency to complement the dry and balsamic characteristic of cola drinks.

It has a drier and sharper flavor than citric and tartaric acid. Malic acid is closely associated with









apples and it is a preferred acidulants in low-calorie drinks. It is also used to mask the off-taste

of some sugar substitutes (Taylor 2005).

2.6.2 Milk

Skim milk contains about 9% total solid, casein (2.5%), whey proteins (0.5%) and lactose.

Solubility of CO2 in milk has had the same behavior as in water, at different conditions of

pressure and temperature. However at 25oC, solubility of CO2 in milk was less. At 5.5 MPa and

at 38 and 50 oC, solubility of CO2 in milk was very similar to the solubility of water because

casein was completely precipitated. This proved that casein was competing with CO2 to get in

solution (Tomasula and Boswell 1998).









CHAPTER 3
MATERIALS AND METHODS

3.1 Design of the High Pressure Carbon Dioxide Solubility Apparatus

The concept of the solubility of a gas in a liquid at high pressures is simple. After the gas is

saturated in the liquid, isolate a known amount of liquid at high pressure, reduce its pressure and

measure the amount of gas that comes out of the liquid. The ratio of the mass of gas to that of

liquid gives solubility. However, there are many design-related and practical challenges.

Designing, building and testing the high pressure CO2 Solubility apparatus took one year and a

half. The idea started from previous attempts to build an apparatus to measure CO2 Solubility,

under high pressures, without success. The principle of the machine was to assure that the CO2

measured was previously in solution in a liquid of known mass. Several versions of the machine

were tested with water. The main dilemma was compressed gas (CO2) trapped in the system,

during processing under high pressures, giving the impression that solubility of CO2 WAS three or

even four times higher than literature values. The solution was to generate a system able to

recirculate the liquid during processing; based on the hypothesis that the density of CO2 is l0WeT

than the density of water (or liquid food), at experimental conditions.

The experimental apparatus was designed to accurately measure the amount (v/v) of CO2

in solution with water or any other liquid. There are two vessels: vessel 2 is above vessel 1.

Vessel 1 has a capacity of approximately 200 ml and can be operated at temperatures between 0

to 45oC and pressure up to 24 MPa. Figure 3-1 shows the schematic drawing of the apparatus.

Figure 3-1 shows the CO2 tank, high pressure pump (HPP), heat exchanger (HE), pressure gauge

(PG), back pressure regulator (BPR), thermocouple (T), metering valve (MV), gas meter (GM),

water bath, bottle, stopper, fume hood (FH), two way valves (V), three way valves (TV), vessels

and quick connects (QC). Figure A-1 shows some pictures of the equipment. From the CO2










tank until QC3, the apparatus is fixed, and between QC3 and BPR the apparatus is mobile

(Figure A-2).

PG3






PG2 E
,HPP ,, topper





I~~ IIIn














Figure 3-1. Schematics of the high pressure CO2 Solubility apparatus.

To connect various pieces of the apparatus together, it was necessary to use Teflon tape in

the threads. The Teflon tape avoids leaks at high pressures. The Teflon tape has to be applied

clockwise to every male connection (Figure A-3). Leaks were detected using a soap solution,

from ACE Hardware Corp (Oak Brook, IL).

The CO2 tank was ordered from Airgas South, Inc (in Gainesville, FL). It had a siphon

tube, and contained 34 kg of liquid CO2 (99.91% of purity). The tank valve was labeled as Vl in

Figure 3-1. It was connected to TV1, a three-way valve to purge the line, followed by a pressure

gauge PG1 (Figure A-4), to control and regulate the flow of CO2 frOm the tank. TV1, TV2 and









TV3 were ordered from Swagelok Company (Jacksonville, FL, model SS-83XKF4). They were

made of stainless steel, and can hold 41.2 MPa of pressure at temperature of 3 8 oC. TV2 was

used to depressurize the line from the tank, in case of emergency. TV3 was also used to

depressurize the line from the tank, during processing.

The high pressure pump (HPP) is from Milton Roy Company in Ivyland, PA (model X-10,

serial No M229150-1). It was used to pump liquid CO2 through the system, to increase the

pressure up to 27.5 MPa (Figure A-5). To liquefy CO2, it was necessary to use a heat exchanger

(HE), containing a solution of water (50%) and antifreeze (50%), to reach very low temperatures

(approximately -15 oC). The heat exchanger (HE) was from Fisher Scientific, model 900 and

serial No 870075 (Figure A-6).

The back pressure regulator (BPR) was used to regulate the pressure in the system. It was

purchased from TESCOM Industrial Controls, Elk River (part number is 26-1762-24). Its body

and trim are made of stainless steel, the back-up ring is made of Teflon, the O-rings are made of

buna-N, the seal is made of CTFE and remaining parts are made of stainless steel. Its controlled

pressure range is from 0.34 to 41.37 MPa. Its inlet and outlet port type is NPT and the size is

1/"). Its weight is 5 lbs (2.2 kg) and the BPR ambient operating and fluid media temperature is -

40 oC to 73.8 oC. Figure A-7 shows the schematics of the high pressure/back pressure regulator

and Figure A-8 shows the BPR used in the machine.

To control the pressure, the back pressure regulator is turned clockwise (to increase

pressure) or counter-clockwise (to decrease it). When the system is depressurized using the

BPR, the exiting CO2 expands, cools and may turn to solid (dry ice), and the BPR may be

obstructed. To prevent this, it was necessary to use an electrical heating tape around the BPR,

and a variable autotransformer (Staco Energy Products, Dayton, OH) used to control the voltage










supplied to the belt. The percent used for the experiment varied between thirty and forty percent

(30-40%) of 110 V. This setup is shown in Figure A-9.

The "water bath" was a cooler with a capacity of about 60 L water. The water in the cooler

was recirculating through plastic tubes to a small high precision water bath (Hart Scientific,

Utah; model 603 5) that was set at 46-50 oC to increase and/or maintain a constant temperature in

the system. To recirculate the water, it was necessary to use a Masterfiex peristaltic pump (Cole

Farmer, Chicago; model 7553-50), set around 3.5 in the speed control dial (Figure A-10).

To control the temperature in the cooler and inside the system, it was necessary to use

thermocouples. The thermocouple used in the cooler was ordered from Omega@ Engineering,

part # JMQSS-125G-12. The thermocouple was made of stainless steel, 12" of length and

0. 125" of diameter. The thermocouple inside the system was also ordered from Omega@

Engineering (part # TQSS-116G-12 and QC# PLO40052). The thermocouple was made of

stainless steel, 1/16" of diameter, and 12" of length. It was designed to withstand vibration, high

temperature and high pressure (Figure A-11). To connect this thermocouple to the system, a

Swagelok tube fitting (part # SS-100-1-4) was used. It is a male connector 1/16" and male NPT

1/4".

The metering valve (MV) was ordered from Swagelok Company (Jacksonville, FL, model

# SS-31RF4). It is a medium-flow high pressure (34.47 MPa) metering valve. It was used to

control the flow of the CO2 COming out of the system (Figure A-12), to be measured using the

gas meter (GM). The GM was obtained from Alicat Scientific (Tucson, AZ), model number M-

1SLPM-D (CO2). It is calibrated to measure CO2 Specifically, at a maximum flow of 1 standard

liter per minute (Figure A-13). The GM is based on the accurate measurement of volumetric

flow at a standard temperature and pressure (0.1 MPa and 25 OC). The volumetric flow rate is









determined by creating a pressure drop across a unique internal restriction, name as Laminar

Flow Element (LFE), and measuring the difference on pressure across it. Its operating principle

is known as the Poiseuille Equation:

(P~ -p,);rr4
O = (3-1)
S8yL

where Q is the volumetric flow rate, Pi is the static pressure at the inlet, P2 is the static pressure

at the outlet, r is the radius of the restriction, rl is the (eta) absolute viscosity of the fluid, and L is

the length of the restriction. Since ~n, r (radius) and L (length) are constant; Equation 3-1 is

rewritten as:


S= K (3-2)


where K is a constant factor calculated from the restriction geometry, AP is the differential

pressure and rl the absolute viscosity. Viscosity of carbon dioxide at 0. 1 MPa and 25 o C is

149.33 g-cm -~sec^l. The volumetric flow rate units are SLPM (standard liters per minute).

The cross-angled connector between the MV, V4, T and Vessel 1 was designed and made

of stainless steel to withstand high pressures. Based on the buoyancy principle, the angle

connections allow any possible bubble in the system to move to the top of the equipment. This

reduces / eliminates the error of excess CO2 reading in the gas meter (GM) when the system is

depressurized (Figure A-14).

The two way valves (V), and quick connects (QC) were ordered from Swagelok Company

(Jacksonville, FL), and are made of stainless steel. The two way valves (straight ball valves) are

model SS-83KF4, 1/4" end connections size and female NPT end connection type. Their

pressure rating is 41.37 MPa at 38 oC (Figure A-15). The QC are also made of stainless steel,

their pressure rating is 41.37 MPa at 21.11 oC. The quick connect stems are model SS-QF4-S-









4PF (female NPT) and SS-QF4-S-4PM (male NPT). The quick connects bodies are model SS-

QF4-B-4PF (female NPT) and SS-QF4-B-4PM (male NPT) (Figure A-16).

Vessel 1 has a maximum allowable working pressure of 34.4 MPa at 121 oC. It was made

of stainless steel, its capacity is 150 ml, and its end size connections are 1/4" NPT (Figure A-17).

Vessel 2 has a maximum allowable working pressure of 68.95 MPa at 22 oC. Its body and cover

were made of stainless steel; the o-ring of Buna N; and the cap of alloy steel. Its capacity is 55

ml, and its end size connections are 1/4" NPT (Figure A-18 and Figure A-19). The main

difficulty was that the gas that remained in vessel 1 at the end of the saturation period had to

"bubble up" to vessel 2 through valve V4, and at the same time some liquid from vessel 2 had to

flow down to vessel 1 by gravity. Because of the small inner diameter of the valve V4, this did

not happen, and some gas was left trapped in vessel 1. Since this gas was under pressure, upon

de-pressurization its volume expanded greatly, and gave false readings about solubility. Valve

V4 was replaced with a larger diameter gate valve (a forged stainless steel gate valve, Figure A-

20, class 1500, 0.38" of opening diameter, v/2" NPT connection, model number 15GT-6L-U-G,

from NEWCO/OIC Valves, Jacksonville, FL) with the expectation of easier CO2-liquid

exchange between vessels 1 and 2. However, this valve leaked. Another solution had to be

found. To recirculate the liquid from the top vessel 2 to the bottom vessel 1, it was necessary to

drill an opening (1/4" NPT) in the bottom of vessel 2 (Figure A-19) and connect it to the bottom

of vessel 1. This assured that the liquid pressure at the top would force the liquid to flow from

vessel 2 to vessel 1 by gravity by the side arm, and therefore "push" any gas remaining in vessel

1 to bubble up to vessel 2 though valve V4. Therefore, it was assured that after the flow of CO2

from the bottom of vessel 1 (to reach saturation) was stopped, all gas remaining in vessel 1

would bubble up to vessel 2. The accuracy of the experimental solubility measurement depended









on no un-dissolved gas remaining in vessel 1. Please note that at pressures where the density of

CO2 becomes higher than that of the liquid, this system will not work properly.

The plastic bottle has a capacity of 5 gallons and it was made of high density polyethylene

(HDPE). The opening of the bottle was restricted by a stopper (number 1 1) with two holes. One

opening was for the incoming CO2 (with possible entrained liquid), and the other was for the

CO2 to go out to the gas meter. The tube attached in the outgoing opening was shorter, to allow

just the CO2 to exit, and be measured at the GM (Figure A-21)

3.2 Preparation of the Samples

3.2.1 Distilled Water

To verify that the high-pressure CO2 apparatuS worked properly, it was necessary to

compare its results with the literature values (v/v) of CO2 dissolved in pure water. The distilled

water used was obtained from the building source (triple filtration). The volume of distilled

water used was approximately 500 ml per run.

3.2.2 Fructose

The D-Fructose (reagent grade) was ordered from Fisher Scientific (Fair Lawn, New

Jersey; lot number 068019). The sample with 10% fructose and 90% water (200 grams of

fructose in a total solution of 2000 ml with distilled water) corresponds to 9.7 oBrix, and the

sample with 5% fructose and 95% water (100 grams of fructose in a total solution of 2000 ml

with distilled water) corresponds to 4.8 oBrix. The fructose was measured using an analytical

balance (model PG603-S), Mettler Toledo. The oBrix data was measured with a portable

refractometer, model 13-946-20, from Fisher.

3.2.3 Malic Acid

The malic acid (food grade, C4H20s) in powder was ordered from Presque Isle Wine

Cellars (Philadelphia, PA). The sample with 0.5% malic acid and 99.5% water (10 grams of









malic acid in a total solution of 2000 ml with distilled water) had a pH of 2.35. The sample with

5% malic acid and 95% water (100 grams of malic acid in a total solution of 2000 ml with

distilled water) had a pH of 1.79. The pH values were measured using an expandable ion

analyzer (EA 920, model Q027A), from Orion Research Incorporated (Cambridge, MA).

3.3 Experimental Procedure

3.3.1 Preliminary Procedure

First, the HE was turned on for about 20 minutes before starting the HPP (HE had to reach

approximately -15 oC). During that period, the cooling j acket of the HPP was getting covered

with a thin layer of ice. At the same time, the high precision water bath was turned on, to start

warming up the water inside of the cooler. Since the warming process could take about 1.5 to 2

hours, batches with water (approximately 2 liters per batch) were heated in the microwave and

added to the cooler, to speed up the process.

In the stationary part of the apparatus, it was important to verify that VI (that corresponds

to the tank) and V2 were closed, TV1 was turned to the right and TV2 was turned to the left.

The previous procedure was performed to prevent CO2 to escape out of the apparatus, at the time

to open Vl; therefore, to avoid gas inhalation. Then, before submerging the mobile part in the

cooler, it was important to confirm that V5 and MV were closed, and that V3 andV4 were

opened. TV3 should be turned to the left or facing up and the BPR should be slightly open

(clockwise), to hold a back-pressure of approximately 2.76 MPa. Also, QC4, QC5, QC6 and

QC7 should be already connected (Figure B-1).

When the complete apparatus was connected (stationary and mobile parts), and the plastic

tubes coming from TV3, BPR and GM were placed inside of the fume hood (FH), it was

necessary to ensure that between QC6 and GM there was only CO2. For that reason, Vl was

slightly opened until approximately 2.76 MPa was reached (pressure was confirmed in PG2).










Next, V2 was slowly opened and the BPR was regulated to increase the pressure up to 1.38 MPa

(pressure was confirmed in PG3); then, V2 was closed. The GM was zeroed and cleared (Alicat

Scientific 2007), and the stopper was verified to be in place. Then, the MV was opened slowly

and the CO2 COming out of the system was measured until read about 0.250 standard liters /min,

MV was closed. The system was depressurized, and when the pressure was 0 psig (pressure read

at PG3), QC3 was disconnected.

3.3.2 Pumping the Liquid Sample into the System

Before starting, QC8 should be disconnected and a plastic tube should be connected to

QC8 stem. The sample was held in a small container of approximately 2 liters. By using the

peristaltic pump, the sample was pumped through a plastic tube to the stem connection of QC3.

The liquid was pumped until both vessel 1 and 2 were filled. When the system was full, water

started coming from the plastic tube connected to the QC8 stem (Figure B-2). The peristaltic

pump was turned off. The plastic tube in the QC3 stem was removed, and the QC3 stem was

quickly connected to its QC3 body. The plastic tube in the QC8 stem was also removed, and

QC8 body was connected to its corresponding stem.

3.3.3 Carbonating the Sample

Before starting the carbonation process, it was necessary to verify that the temperature

inside of the system was the desired temperature. Then, Vl was opened until reaching

approximately 2.76 MPa (pressure was confirmed in PG2); then, it was closed. Next, V2 was

opened very slowly and the BPR was turned to increase the pressure, at the same time. The flow

rate of the CO2 with water coming from the BPR should be very slow. The previous step was

performed to ensure that the system was filled with CO2, and that there were no other gases. The

V2 was closed, the BPR was regulated to hold approximately 6.89 MPa and the HPP was set to

about 77% of flow rate using the micrometers (Figure A-5).









Vl was opened again and the HPP was turned on. The variable autotransformer was also

turned on. V2 was opened very slowly and, if some gas with water was coming out of the

system, the BPR was opened to increase the pressure, until around 1.03-1.38 MPa above the

desired pressure was reached. At that point, the CO2 flOw rate of the HPP was reduced to about

37%, by turning clockwise the micrometers. The HPP remained on during 1 hour and 10

minutes to reach saturation of CO2 in the liquid. During this time, CO2 COntinuously bubbled

through the liquid (entering from the bottom of vessel 1, and exiting off the top of vessel 2

through BPR), and saturated the liquid with CO2.

3.3.4 Measuring Solubility

After the saturation time, with CO2 paSsing through the system, V2 was closed.

Immediately after this, the HPP was turned off; and V1 was closed. Next, TV3 was very slowly

turned to the right (clockwise); and V2 was opened very slowly, while it was warmed up with

some hot water from the cooler. The variable transformer was turned off. When the pressure

was 0 MPa (pressure was confirmed in PG2), QC3 and QC4 were disconnected. Then, the

mobile part of the equipment was shaken from side to side, while still immersed in water (around

1 minute per side) and up/down (20 times). The purpose of this was to loosen any CO2 bubbles

attached to the inner surfaces of vessel 1, and allow them to bubble up to vessel 2. This

procedure took around 3-5 minutes. If the pressure (read in PG3) was just at the desired level,

first V3 and then V4 were closed. However, if the pressure was still higher than desired, it was

necessary to adjust it to the desired level.

The variable transformer was turned on and the BPR was turned very slowly, to

depressurize the top part of V4. The GM was again zeroed and cleared.

At this point, it was certain that there was only liquid phase in vessel 1, and the dissolved

CO2 in that liquid. The MV was slightly opened to start depressurization, and this continued









until all the dissolved gas came out of the system and the pressure read in PG3 was 0 MPa. The

maximum flow rate allowed in the GM was 0.350 slpm (standard liters per minute), to prevent

that the system cooled by expansion of CO2. To expand and read all the CO2 that was in solution

took around 20-25 minutes. When the volumetric flow rate in the GM was 0 slpm, QC8 was

disconnected and the rest of the mobile part was removed from the ice cooler. As soon as the

equipment was removed, QC5 was disconnected under a small container of 2 liters of volume, to

allow the liquid coming out of vessel 2 to be collected. Then QC7 was disconnected.

All the end connections were dried. QC6 was disconnected and all the liquid in the plastic

tube and in the bottle was collected in a container of 5 liters (Figure B-3). V4 was opened inside

of the same container of 5 liters (Figure B-4), subsequently TV3 was turned to the left and all the

liquid from vessel 1 was collected. At the end, the liquid collected was measured using a

graduated cylinder (Figure B-5). This amount was the basis of the solubility calculations.

To convert the volumetric flow (standard liters) to mass flow of CO2, it was necessary to

divided it by 22.4 slt-mol- and then, multiply it by 44.01 g mol- Solubility was calculated as

the rate of grams of CO2 in 100 grams of liquid.

3.3.5 Cleaning the Apparatus

The mobile part of the apparatus was separated by disconnecting the QC's. The various

parts were washed in a solution of water with soap. Then, the parts were rinsed with abundant

water, and consequently, they were rinsed with distilled water.

3.4 Experimental Design

The carbonation process variables were pressure, temperature and composition. The pH

and oBrix of the model liquid samples were also measured. Experimental conditions were

decided with the goal of measuring the solubility of CO2 above its critical point (temperature

31oC and pressure 7.4 MPa) and below high CO2 density levels at high pressures. The first










phase of the experiment consisted of testing the high pressure CO2 Solubility apparatus, and

comparing the experimental solubility values of CO2 in pUTO Water with literature data. Two

levels of pressure were used: 7.58 MPa (74.85 atm) and 10.34 MPa (102.07 atm) at 40 oC.

Carbon dioxide density at those pressures and temperature was approximately 0.2 g/ml and 0.65

g/ml, respectively (Clifford and Williams 2000).

The second phase of the experiment consisted in determining the solubility of CO2 in

model liquid food of known composition (different concentrations of fructose and malic acid).

Two levels of fructose concentration (5% and 10% w/v), and two levels of malic acid

concentration (0.5% and 5% w/v) were used. The two concentrations of fructose and malic acid

were selected according to the typical usage levels of functional constituents in soft drinks

(Taylor 2005). Every solubility measurement was performed in duplicate. Table 3-1 shows the

different levels of each carbonation variable.

Table 3-1. Different levels of each variable in the two phases of the experiment.
First Phase
Treatment
Temperature Pressure Model Liquid
1 40 oC 7.58 MPa Distilled water
2 40 oC 10.34 MPa Distilled water
Second Phase
Temperature Pressure Model Liquid
3 40 oC 7.58 MPa Fructose 5 % m/v
4 40 oC 7.58 MPa Fructose 10 %/m/v
5 40 oC 10.34 MPa Fructose 5 % m/v
6 40 oC 10.34 MPa Fructose 10 % m/v
7 40 oC 7.58 MPa Malic Acid 0.5 % m/v
8 40 oC 7.58 MPa Malic Acid 5 % m/v
9 40 oC 10.34 MPa Malic Acid 0.5 % m/v
10 40 oC 10.34 MPa Malic Acid 5 % m/v

3.5 Data Analysis

Statistical analysis consisted of performing an analysis of variance (ANOVA) and mean

separation to evaluate the difference between solubilities at the different conditions of pressure

and composition, at 40 oC. The analysis was divided into two phases. In the first phase









significant differences between the experimental and literature values were evaluated. The null

hypothesis (Ho) was: At least one of the experimental mean results is different from literature

results. The alternative hypothesis (H1) was: Means of the experimental results are equal to

literature results. The second phase consisted in determining if the reduction in the solubility of

CO2 in Solution with pure water was significant, by increasing the solute (fructose or malic acid)

concentration in the liquid. The null hypothesis (Ho) was: Means of the solute experimental

results are equal to the experimental mean reference. The alternative hypothesis (H1) was: There

was significant difference between solute experimental results and the mean of experimental

reference. The statistical data analysis was conducted by performing a Least Square Mean (LS-

Mean) analysis and the Least Significant Differences (LSD) test, with P< 0.05. The software

programs used were STATISTICA 97 and SAS 9.1.









CHAPTER 4
RESULTS AND DISCUSSION

4.1 Solubility of Carbon Dioxide in Distilled Water

To demonstrate the accuracy and precision of the high-pressure CO2 Solubility apparatus, it

was necessary to compare the experimental results of CO2 Solubility in pure water with the

literature values. The CO2 Solubility chart developed by Dodds and others (1956) was used as

reference. Also the chemical/thermodynamic simulation program Aspen PlusTM 12.1 (Schweiss

1997) was used as a simulation reference. The CO2 Solubility chart (Figure 2-8) shows the

solubility of CO2 in pUTO Water (kg/100 kg of water), at pressures from 1 to 70.93 MPa, and at

temperatures from 0 to 120 oC. According to this chart, solubility of CO2 at 1 10.34 MPa and 40

oC is 5.68 kg/100 kg of pure water. At conditions of 7.58 MPa and 40 oC, solubility of CO2 is

5.00 kg/100 kg of water. The Aspen PlusTM 12.1 was designed to predict the behavior of

chemical systems (Schweiss 1997). According to this program, at a constant temperature of 40

oC, solubilities of CO2 in pUTO Water at 7.58 MPa and 10.34 MPa of pressures are 4.6 and 5.7

kg/100 kg of liquid, respectively.

The experimental results of solubility of CO2 in pUTO Water obtained from the apparatus, at

a constant temperature of 40 oC, are displayed in Table 4-1.

Table 4-1. Experimental results of solubility of CO2 in pUTO Water.
Pressure Distilled CO2 CO2 Solubility Mean St. Literature Difference
(MPa) water (g) (slt') (g) (g/100g2) (g/100g2) Dev. (g/100g2) (g/100g2)
7.58 171 4.063 7.98 4.67
7.58 168 4. 176 8.20 4.88 4.77 0.15 5.00 0.23
10.34 169 4.625 9.08 5.38
10.34 169 4.863 9.55 5.65 5.51 0.20 5.68 0.17
'Standard liters. 2Grams of CO2 per 100 g of pure water. 3Standard Deviation in g of CO2 per 100 g of
pure water.

According to Table 4-1, the mean solubility of CO2 in water at pressure of 7.58 MPa is

0.23 g/100 g lower than the literature. Also, at pressure of 10.34 MPa, the mean of solubility of










CO2 is 0. 17 g/100g below the reference. Those differences could be attributed to the time of

saturation with CO2. If the time of passing CO2 through the system increases, solubility of CO2

in liquids would increase until it reaches saturation. This has been demonstrated in preliminary

experiments (data not shown). According to statistical analysis using Statistica 97 and SAS 9.1

programs, those differences were not significantly different. Tables 4-2, 4-3 and 4-4 show the

summary of the analysis of variance (ANOVA), summary of least square mean (LS Mean)

analysis and the least significant difference (LSD) tests, respectively.

Table 4-2. Summary of the analysis of variance (ANOVA) of the solubility of CO2 in pUTO
water.
Sum of Mean F Multiple Multiple Adjusted
Source DF Pr > F 2 2
squares square value R R R
Model 10.5462 0.5462 17.77 0.0519 0.9480 0.8988 0.8482
Error 2 0.0614 0.0307
Corrected Total 3 0.6076

Table 4-3. Summary of the least square mean (LS Mean) analysis of the solubility of CO2 in
pure water.
Pressure Solubility Mean Solubility Solubility Solubility Standard Coefficient
(MPa) (g/100g*) Standard Error -95.00% +95.00% deviation of variation
7.58 4.77 0.1239 4.24 5.30 0.15 0.03
10.34 5.51 0.1239 4.98 6.05 0.20 0.03
*Grams of CO2 per 100 g of pure water.

Table 4-4. P-values from the least significant difference (LSD) test between experimental mean
data and literature data of solubility of CO2 in pUTO Water.
TreatentsExperimental data Literature data
7.58 MPa 10.34 MPa 7.58 MPa 10.34 MPa
Experimental 7.58 MPa -0.0519 0.4045 0. 0499
data 10.34 MPa 0.0519 -0.1390 0.4776
7.58 MPa 0.4045 0.1390 -0.1059
Literature data
10.34 MPa 0. 0499 0.4776 0.1059
Note: P-values in italic font are lower than a = 0.05; thus, the correspondent groups are significantly
different.

Table 4-2 shows that the null hypothesis (Ho) was not rej ected, due to a P-value higher

than a = 0.05 (Pr>F = 0.0519); therefore, at least on the experimental mean results is different

from the literature. Table 4-4 shows significant difference between the pressure of 7.58 MPa,










from experimental mean data, and the pressure of 10.34 MPa, from literature results. However,

within the same conditions of pressure, there was no significant difference between the

experimental mean results and the literature data; differential values were higher than at = 0.05,

and the average coefficient of variation level was 0.03. In addition, the confidence interval for

pressures of 7.58 and 10.34 MPa were 5.5111.76 and 4.7711.37 g of CO2/100g of liquid,

respectively. The corresponding literature values for each pressure fits within the confidence

interval. Therefore, the experimental results, obtained from the high-pressure carbon dioxide

solubility apparatus are statistically acceptable and the apparatus is a functional instrument to

measure CO2 Solubility in liquids at the conditions of pressure and temperature tested in this

study .

4.2 Solubility of Carbon Dioxide in Fructose Solutions

Solubility of CO2 in fructose solutions was measured at a constant temperature of 40 oC

and at two levels of pressures (7.58 and 10.34 MPa). Table 4-5 shows the experimental results

of CO2 Solubility in the different fructose solution concentrations (5% and 10%).

Table 4-5. Experimental results of CO2 Solubility in solutions with different concentration of
fructose.

Pressure Fructose CO2 CO2 Solubility Mean St. DW4 Difference5
(MPa) solution (g) (slt') (g) (g/100g2) (g/100g2) dev3 (g/100g2) (g/100g2)
5% Fructose Concentration
7.58 169 3.983 7.82 4.63
7.58 172 3.913 7.69 4.47 4.55 0. 11 4.77 0.23
10.34 172 4.102 8.06 4.68
10.34 170 4.228 8.31 4.89 4.78 0. 14 5.51 0.73
10 % Fructose Concentration
7.58 170 3.608 7.09 4. 17
7.58 172 3.563 7.00 4.07 4. 12 0.07 4.77 0.66
10.34 170 3.523 6.92 4.07
10.34 172 3.774 7.41 4.31 4. 19 0. 17 5.51 1.32
'Standard liters. 2Grams of CO2 per 100 grams of solution. 3Standard Deviation in grams of CO2 per 100
grams of solution. 4Mean Solubility of CO2 in distilled water. 5Differences between DW and Mean.










According to Table 4-5, by increasing the fructose concentration in a liquid, solubility of

carbon dioxide decreases. Descoins and others (2004) stated that at conditions of 6 atm of

pressure and at pH of 3. 1 an increment of the concentration of sugar (0, 10 and 40 g/1 of sugar)

yields a decrease of the average level of dissolved CO2 due to different orientation of the water

molecules around the solute particles; therefore, water is less free to interact with other

compounds, in this case CO2. Figure 4-1 shows the change in solubility of CO2 in different

concentrations of fructose in the solution. The 0% of fructose concentration is represented by

solubility of CO2 in pUTO Water.

6.0

5.8

5.6 -

5.4


af 5.0



0 4.6






4.0

3.8

3.6 E Pressure 7.58 MPa
0 5 10 Pressure 10.34 MPa
Concentration (g of fructose/100g of distilled water)

Figure 4-1. Solubility of CO2 VeTSus concentration of fructose solutions, at 7.58 and 10.34 MPa,
and at a constant temperature: 40 oC. Vertical bars denote 0.95 confidence intervals.

Figure 4-1 shows the solubility of CO2 at 7.58 MPa (blue line), and at 10.34 MPa (no

continue red line). At 7.58 MPa, the mean solubility of CO2 in pUTO Water (0% of fructose) did










not decrease significantly at 5% of fructose; nevertheless, at 10% the difference is noticeable. At

10.34 MPa, the mean solubility of CO2 in pUTO Water decreased significantly while concentration

of fructose in the solution increased to 5% and to 10%. Tables 4-6, 4-7 and 4-8 display the

analysis of variance (ANOVA), the LS mean analysis and the LSD test, respectively.

Table 4-6. Analysis of variance (ANOVA) of solubility of CO2 in fructose solutions of different
concentrations.
Sum of Mean F Mlil utpe Ajse
Source DF PrF > util Futil 2 2st
squares square value R R2 R
Model 5 2.5671 0.5134 24.05 0.0007 0.9759 0.9524 0.9128
Error 6 0.1281 0.0213
Corrected Total 11 2.6952

Table 4-7. Least Square (LS) Mean analysis of solubility of CO2 in fructose solutions of
different concentrations.
Solubility Solubility
Pressure Fructose Solubility Solubility Standard Coefficient
mean standard
(M Pa) (%) --1-95.00%/ +95.00% deviation of variation
(g/100g') error
7.58 5 4.77 0.1158 4.15 4.80 0.11 0.02
10.34 5 5.47 0.1158 5.15 5.80 0. 14 0.03
7.58 10 4.47 0.1158 4.14 4.79 0.07 0.02
10.34 10 4.68 0.1158 4.36 5.00 0. 17 0.04
'Grams of CO2 per 100 g of fructose solution.

Table 4-8. P-values from the least Signifieant Difference (LSD) test of solubility of CO2 in
fructose solutions of different concentrations.
7.58 MPa 10.34 MPa
Treatments
0% 5% 10% 0% 5% 10%
0 % -0.1732 0. 0042 0. 0023 0.9476 0. 0071
7.58
5 % 0.1732 -0. 0258 0. 0006 0.1577 0. 0495
MPa
10 % 0. 0042 0. 0258 -0. 0001 0. 0039 0.6426
0 % 0. 0023 0. 0006 0. 0001 -0. 0025 0. 0001
10.34
5 % 0.9476 0.1577 0. 0039 0. 0025 -0. 0066
MPa
10 % 0. 0071 0. 0495 0.6426 0. 0001 0. 0066
Note: Values in italic font are less than a = 0.05; thus, the correspondent groups are significantly
different.

In the ANOVA (Table 4-6), the p-value denotes (0.0007) that at least one of the treatments

was significant difference between one or more treatments (0, 5 and 10 % of fructose

concentration). In the LS mean analysis (Table 4-7), the maximum coefficient of variation

(0.04) reveals that the experimental procedure has an acceptable error, below 5%. The LSD test










showed the mean solubilities that were statistically different. According to Table 4-8, the

fructose concentration of 5% (4.8 oBrix) decreased significantly the solubility of CO2 at pressure

of 10.34 MPa; however, at 7.58 MPa solubility did not change considerably. The 10% (9.7

oBrix) concentration of fructose reduced considerably the solubility of CO2 in the solution, at

both pressures (7.58 and 10.34 MPa).

4.3 Solubility of Carbon Dioxide in Malic Acid Solutions

Solubility of CO2 in malic acid solutions was measured at a constant temperature of 40 oC

and at pressures of 7.58 and 10.34 MPa. Figure 4-2 shows the effect of increasing the malic acid

concentration of a solution on the solubility of CO2-

6.0

5.8



S5.65. i



o o



4.8 -

S4.6

Vm 4.4

4.2


4.0 E Pressure at 7.58 MPa
0 1 2 3 4 5 -1- Pressure at 10.34 MPa
Concentration (g of malic acid/100~g of distilled water)

Figure 4-2. Graph of solubility of carbon dioxide versus concentration of malic acid solutions, at
two different pressures: 7.58 and 10.34 MPa, and at a constant temperature: 40 oC.
Vertical bars denote 0.95 confidence intervals.























































Table 4-11. Least Square (LS) Mean analysis of solubility of carbon dioxide in malic acid
solutions of different concentrations.


7.58 0.5 4.47 0.0387 4.36 4.58 0.03 0.01
10.34 0.5 4.90 0.0387 4.79 5.00 0.08 0.02
7.58 5 4.47 0.0387 4.35 4.57 0.02 0.00
10.34 5 4.68 0.0387 4.68 4.90 0.07 0.01
'Grams of CO2 per 100 g of malic acid solution.


Table 4.-9 shows the experimental data. The results of the ANOVA and LS mean analysis,

and LSD test are shown in Tables 4-10, 4-11 and 4-12. According to Table 4-10, at least one of

the treatments (0, 0.5 and 5 % malic acid concentration, at both pressures) is significantly

different because the p-value (Pr>F = 0.0007) is lower than at = 0.05. In addition, the maximum

coefficient of variation was 0.02 that shows a good experimental performance (Table 4-1 1).

Table 4-9. Experimental data of CO2 Solubility in solutions with different concentration of malic
acid.
Pressure Malic acid CO2 CO2 Solubility Mean St DW4 Difference5
(MPa) solution (g) (slt') (g) (g/100g2) (g/100g2) Dev3 (g/100g2) (g/100g2)
0.5% Malic Acid Concentration


7.58 172
7.58 172
10.34 169
10.34 169


3.933 7.73
3.902 7.66


4.49
4.46


4.47


0.03 4.77


0.08 5.51


0.30


0.02


4.191 8.23 4.84
4.315 8.48 4.96 4.90
5% Malic Acid Concentration


7.58 172 3.900 7.66 4.45
7.58 172 3.902 7.70 4.48 4.47 0.02 4.77 0.31
10.34 171 4.129 8.11 4.69
10.34 172 4.237 8.32 4.84 4.79 0.07 5.51 0.77
'Standard liters. 2Grams of CO2 per 100 grams of solution. 3Standard Deviation in grams of CO2 per 100
grams of solution. 4Mean Solubility of CO2 in distilled water. 5Differences between DW and Mean.


Table 4-10. Analysis of variance (ANOVA) of solubility of carbon dioxide in malic acid
solutions of different concentrations.
Sum of Mean F Multiple Multiple Adjus
Source DF Pr > F22
squares Square Value R R2 R
Model 5 1.4727 0.2945 24.06 0.0007 0.9760 0.9525 0.912
Error 6 0.0734 0.0122
Corrected Total 11 1.5461


;ted

9


Solubility
Mean
(g/100g')


Solubility
Standard
Error


Pressure Mahic
(MPa) Acid (%)


Solubility Solubility Standard Coefficient
-95.00% +95.00% deviation of variation










Table 4-12. P-values from the least Signifieant Difference (LSD) test of solubility of carbon
dioxide in malic acid solutions of different concentrations.
7.58 MPa 10.34 MPa
Treatments
0 % 0.5 % 5 % 0 % 0.5 % 5 %
0 % -0. 0346 0. 0312 0. 0005 0. 3028 0.8900
7.58
0.5 % 0. 0346 -0.9408 0. 0001 0. 0085 0. 0286
MPa
5 % 0. 0312 0.9408 -0. 0001 0. 00 78 0. 0259
0 % 0. 0005 0. 0001 0. 0001 -0. 0014 0. 0006
10.34
0.5 % 0.3028 0. 0085 0. 00 78 0. 0014 -0.3638
MPa
5 % 0.8900 0. 0286 0. 0259 0. 0006 0.3638
Note: Values in italic font are less than a = 0.05: thus, the correspondent groups are significantly
different.

Table 4-12 shows the difference between treatments. At pressure of 7.58 MPa, there was

significant difference between solubility in water (0% malic acid concentration) and solubility in

5 % of malic acid concentration; however there was no significant difference between 0.5% and

5% of malic acid concentration. At pressure of 10.34 MPa, solubility in water was significantly

different than 0.5% and 5% of malic acid concentrations; however there was no significant

difference between 0.5% and 5% of malic acid concentrations, as in 7.58 MPa of pressure.

According to Meyssami and others (1991), the presence of acids (e.g. ascorbic acid, citric

acid, etc.) contributes to the reduction of solubility of CO2 in the solution. They reported a

decrease in pH at pressures of 0 and 5.5 MPa and temperature of 32, 37 and 42 oC, in

comparison with pH of pure water. At those conditions of pressure and temperature, the pH

dropped in the binary water- CO2 System from about 5.68 to 3.19 (difference was approximately

2.5 units), while at the ternary system (water-acid- CO2), the pH dropped from about 3.74 to 3.13

(difference was around 0.61 units).

Due to lack of experimental studies in the area of measurement of CO2 Solubility in model

and in real liquid foods, no more references are cited to compare with current experimental

results.









CHAPTER 5
SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR FUTURE STUDY

5.1 Summary

The use of CO2 in Dense Phase Carbon Dioxide (DPCD) technology is not optimized;

excess of CO2 is used to reach saturation, due to unknown solubility of CO2 in the liquid foods.

The high pressure CO2 Solubility apparatus was designed and built to measure CO2 Solubility in

liquid foods at high pressures and different temperatures. The apparatus was tested at a constant

temperature of 40 oC, and at pressures of 7.58 and 10.34 MPa. Solubility values were reported in

grams of CO2 per 100 grams of liquid. The average experimental error (coefficient of variation)

was 0.02%.

Adding fructose to a liquid solubility of CO2 decreased significantly. At pressure of 7.58

MPa of, solubility of CO2 in water was 4.77 10. 15 g/100 g of distilled water. When the

concentrations of fructose in the solution were 5 and 10%, solubilities of CO2 in the solutions

were 4.5510. 11 and 4. 1210.07, respectively. At pressure of 10.34 MPa, the CO2 in Solution with

water was 5.5110.20 g/100 g of distilled water, and when the amount of fructose in the solution

increase to 5 and 10 g/100 g of solution, solubilities of CO2 were 4.7810. 14 and 4. 1910. 17,

respectively.

Adding malic acid to a solution also decreased solubility of CO2. The reference used to

compare was the experimental solubility of CO2 in water. At 7.58 MPa of pressure, the amount

of CO2 dissolved in the solutions of 0.5 and 5% of malic acid concentration was 4.4710.03 and

4.4710.02 g/100 g of solution. At 10.34 MPa of pressure, solubilities of CO2 in Solution with 0.5

and 5% of malic acid concentration were 4.9010.08 and 4.7910.07 g/100 g of solution.









5.2 Conclusions

It is possible to conclude that the apparatus is a functional, accurate and precise instrument

to measure solubility of CO2 in mOdel liquid foods. The significance of this study is that the

experimental apparatus could offer an advantage to the food industry, primarily to soft drink

industry, and to the DPCD users, by eliminating the need to use excess CO2 and therefore

reducing the production cost.

This apparatus could be used for the measurement of CO2 in real liquid foods. Versatility

of the apparatus allows carbonation of various samples, at conditions above critical temperature

and pressure, and below density of 0.9 g-mli. In addition, the side connection between Vessel 1

and Vessel 2 ensures a correct liquid circulation and an appropriate measurement of solubility.

5.3 Suggestions for Future Study

Future works in the area of DPCD technology should focus on first experimentally

determining and then thermodynamically predicting the "theoretical" level of CO2 to use, at any

given pressure and temperature, and for any liquid food of known composition. More studies on

the effect of temperature on the solubility of CO2 in Solutions with different concentrations of

fructose and malic acid should be performed.

This experimental apparatus is a valuable instrument to measure CO2 Solubility in liquid

food to reduce the need to use excess CO2 to reach saturation, at the conditions tested.

Moreover, experiments under pressures higher than 10.34 MPa could be performed to determine

the effect on density changes of CO2 On the accuracy of the apparatus.









APPENDIX A
DESIGN OF THE HIGH PRESSURE CARBON DIOXIDE SOLUBILITY APPARATUS


Figure A-1. Pictures of the high pressure carbon dioxide solubility apparatus.
























Figure A-2. Picture of the mobile part of the apparatus.


Figure A-3. Picture of the teflon tape used in every male connection.


Figure A-4. Picture of the CO2 tank connections. The carbon dioxide tank, the three-way valve
1 (TV1), and the pressure gauge 1 (PGl).

























Figure A-5. Pictures of the high pressure pump (HHP). A) From the front, micrometers. B)
From the back, cooling j acket.


Figure A-6. Picture of the heat exchanger (HE).










1.4-20 UNC
SCarW R rFr BRNor
2 REQUIRED;


PISTON
SENSORW


Figure A-7. Back Pressure Regulator (BPR) diagram (Tescom Industrial Controls 2006).


A B
Figure A-8. Pictures of the back pressure regulator (BPR) used in the machine. A) From the
side. B) From the top.

























Figure A-9. Picture of the heating tape (around the BPR) and the variable autotransformer.


Figure A-10. Picture of the Hart Scient
peristaltic pump.


; high precision water bath and the Masterfle









//


Figure A-11i. Picture of the thermocouple used inside the vessel 1.











Figure A-12. Picture of the metering valve (MV).














Figure A-13. Picture of the gas meter (GM).
















































Figure A-15. Picture of the two way valve (V).


Figure A-16. Picture of the quick connect stem (female NPT) and body (male NPT).


Figure A-14. Picture and diagram of the cross angles connector.



























Figure A-17. Vessel i. A) Picture of vessel i. B) Diagram of vessel 1 (Milton Poy 1986).






~NPT j 8

~t ~
T st
~ I~


Figure A-18. Diagram of vessel 2 (Milton Roy 1986).























Figure A-19. Picture of vessel 2 with the hole at the bottom.


Figure A-20. Picture of the forged stainless steel gate valve.



























A B
Figure A-21. Picture of the bottle and the stopper. A) Picture of the 5 gallons bottle. B) Picture
of the stopper in the opening of the bottle.









APPENDIX B
THE HIGH PRESSURE CARBON DIOXIDE SOLUBILITY APPARATUS PROCEDURES


Figure B-2. Pictures showing the sample being pumped.


Figure B-1. Picture of the mobile part of the equipment, before immersion into the cooler.




































Figure B-3. Picture of disconnecting QC6.


Figure B-4. Picture showing when V4 was opened to collect the liquid sample.




































Figure B-5. Picture showing the measurement of the liquid sample collected.









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BIOGRAPHICAL SKETCH

Thelma Francisca Calix Lara was born on 1984, in Tegucigalpa, Honduras, Central

America. After graduating from high school in December 2001, she entered the Escuela

Agricola Panamericana (University of Zamorano), earning her Bachelor of Science in

Agroindustry in December 2005. During spring 2005, she performed an internship at the Food

Technology of Fruits and Vegetables Department at the Center of Investigation in Food and

Development (CIAD) in Mexico. During fall of the same year, she was offered an assistantship

to pursue her graduate studies, advised by Dr. Murat O. Balaban. In December 2007, she earned

a Master of Science in Food Science and will continue her studies towards a doctoral degree.





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1 DESIGN AND TESTING OF AN APPARATU S TO MEASURE CARBON DIOXIDE SOLUBILITY IN LIQUID FOODS By THELMA FRANCISCA CALIX LARA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Thelma Francisca Calix Lara

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3 To those that have guided and inspired me

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4 ACKNOWLEDGMENTS I wish to express my special gratitude to my major advisor, Dr. Murat O. Balaban, for his valuable support, guidance and for being an exampl e of motivation and hard work to us. I also like to thank my advising committee, Dr. Charles A. Sims and Dr. Allen F. Wysocki, for their guidance, assistance and time for my research. I am very grateful for my lab partners and friends, Luis, Jose, Max, Milena, Alberto, Zareena, Mutlu, Diana, Wendy, Yavuz, Maria and Giovanna for their assistance and for making the work in the lab such an enjoyable experience. I thank infinitely to my parents, Winston and Sagrario, my sister and my brother, Lourdes and Winston, for their unconditional love and sup port during my entire life. They have been my primary inspiration to pursue my goal. Finally, my dearest thanks to Jorge, for all the support and happiness he brought to my life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION..................................................................................................................14 2 LITERATURE REVIEW.......................................................................................................17 2.1 Carbonated Soft Drinks....................................................................................................17 2.2 Carbon Dioxide............................................................................................................. ....19 2.2.1 General Information...............................................................................................19 2.2.2 Applications............................................................................................................22 2.3 Carbonation................................................................................................................ .......23 2.3.1 Definition............................................................................................................... .23 2.3.2 Methods and Apparatus for Carbonation...............................................................29 2.4 Supercritical Carbon Dioxide...........................................................................................31 2.4.1 Definition............................................................................................................... .31 2.4.2 Dense Phase Carbon Dioxide.................................................................................33 2.4.3 Supercritical Flui d Extraction (SFE)......................................................................34 2.6 Constituents that Affect Carbon Di oxide Solubility in Beverages...................................35 2.6.1 Carbonated Soft Drinks..........................................................................................35 2.6.1.1 Carbohydrate sweetener...............................................................................36 2.6.1.2 Acidulants.....................................................................................................38 2.6.2 Milk..................................................................................................................... ...39 3 MATERIALS AND METHODS...........................................................................................40 3.1 Design of the High Pressure Carb on Dioxide Solubility Apparatus................................40 3.2 Preparation of the Samples...............................................................................................46 3.2.1 Distilled Water........................................................................................................46 3.2.2 Fructose................................................................................................................. .46 3.2.3 Malic Acid..............................................................................................................46 3.3 Experimental Procedure....................................................................................................47 3.3.1 Preliminary Procedure............................................................................................47 3.3.2 Pumping the Liquid Sample into the System.........................................................48 3.3.3 Carbonating the Sample.........................................................................................48 3.3.4 Measuring Solubility..............................................................................................49 3.3.5 Cleaning the Apparatus..........................................................................................50

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6 3.4 Experimental Design........................................................................................................50 3.5 Data Analysis.............................................................................................................. ......51 4 RESULTS AND DISCUSSION.............................................................................................53 4.1 Solubility of Carbon Dioxi de in Distilled Water..............................................................53 4.2 Solubility of Carbon Dioxide in Fructose Solutions.........................................................55 4.3 Solubility of Carbon Dioxide in Malic Acid Solutions....................................................58 5 SUMMARY, CONCLUSIONS AND SUGG ESTIONS FOR FUTURE STUDY................61 5.1 Summary.................................................................................................................... .......61 5.2 Conclusions................................................................................................................ .......62 5.3 Suggestions for Future Study............................................................................................62 APPENDIX A DESIGN OF THE HIGH PRESSURE CARBON DIOXIDE SOLUBILITY APPARATUS...................................................................................................................... ...63 B THE HIGH PRESSURE CARBON DI OXIDE SOLUBILITY APPARATUS PROCEDURES..................................................................................................................... .73 LIST OF REFERENCES............................................................................................................. ..76 BIOGRAPHICAL SKETCH.........................................................................................................81

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7 LIST OF TABLES Table page 2-1 Physical properties associat ed with the phase diagram of CO2.........................................20 2-2 Quality standard of carbon dioxide....................................................................................21 2-3 Soft drink components a nd their typical use level.............................................................36 2-4 Properties of bulk sweeteners............................................................................................37 2-5 Acidulants used in beverage formulation..........................................................................38 3-1 Different levels of each variable in the two phases of the experiment..............................51 4-1 Experimental results of solubility of CO2 in pure water....................................................53 4-2 Summary of the analysis of vari ance (ANOVA) of the solubility of CO2 in pure water.......................................................................................................................... .........54 4-3 Summary of the least square mean (LS Mean) analysis of th e solubility of CO2 in pure water..................................................................................................................... ......54 4-4 P-values from the least significant differe nce (LSD) test between experimental mean data and literature data of solubility of CO2 in pure water................................................54 4-5 Experimental results of CO2 solubility in solutions with different concentration of fructose....................................................................................................................... ........55 4-6 Analysis of variance (A NOVA) of solubility of CO2 in fructose solutions of different concentrations................................................................................................................. ...57 4-7 Least Square (LS) Mean an alysis of solubility of CO2 in fructose solutions of different concentrations.....................................................................................................57 4-8 P-values from the leas t Significant Difference (LSD) test of solubility of CO2 in fructose solutions of different concentrations....................................................................57 4-9 Experimental data of CO2 solubility in solutions with different concentration of malic acid..................................................................................................................... ......58 4-10 Analysis of variance (ANOVA) of so lubility of carbon dioxide in malic acid solutions of different concentrations..................................................................................59 4-11 Least Square (LS) Mean analysis of solubility of carbon dioxide in malic acid solutions of different concentrations..................................................................................59

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8 4-12 P-values from the least Significant Difference (LSD) te st of solubility of carbon dioxide in malic acid solutions of different concentrations...............................................60

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9 LIST OF FIGURES Figure page 2-1 Beverage sectors and segments..........................................................................................17 2-2 Global beverage consumption by category, 1998-2003....................................................18 2-3 Beverage consumption by countries (1998-2003).............................................................18 2-4 Carbon dioxide phase diagram...........................................................................................19 2-5 Density-pressure isot herms for carbon dioxide.................................................................20 2-6 Carbon dioxide delivery process........................................................................................22 2-7 Carbonation: CO2 chart in solution with pure water..........................................................24 2-8 Solubility of carbon dioxide in water as a function of pressure and temperature..............25 2-9. Carbonation methods....................................................................................................... ..29 2-10 Measurement of carbonation..............................................................................................30 2-11 Phase equilibrium in the H2O and CO2 system..................................................................32 2-12 Variation of physico-chemical properties of CO2 at near-critical conditions....................34 2-13 Diagram of supercritical fluid extraction equipment.........................................................35 3-1 Schematics of the high pressure CO2 solubility apparatus................................................41 4-1 Solubility of CO2 versus concentration of fructose solutions, at 7.58 and 10.34 MPa, and at a constant temperatur e: 40 C. Vertical bars de note 0.95 confidence intervals.....56 4-2 Graph of solubility of carbon dioxide vers us concentration of malic acid solutions, at two different pressures: 7.58 and 10.34 MPa, a nd at a constant temperature: 40 C. Vertical bars denote 0.95 confidence intervals..................................................................58 A-1 Pictures of the high pressure ca rbon dioxide solub ility apparatus.....................................63 A-2 Picture of the mobile part of the apparatus........................................................................64 A-3 Picture of the teflon tape us ed in every male connection..................................................64 A-4 Picture of the CO2 tank connections. The carbon dioxi de tank, the three-way valve 1 (TV1), and the pressure gauge 1 (PG1).............................................................................64 A-5 Pictures of the high pressure pump (HHP)........................................................................65

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10 A-6. Picture of the heat exchanger (HE)....................................................................................65 A-7 Back Pressure Regulator (BPR) diagram...........................................................................66 A-8 Pictures of the back pressure re gulator (BPR) used in the machine..................................66 A-9 Picture of the heating tape (around the BPR) and the variable autotransformer...............67 A-10 Picture of the Hart Scientific high precisi on water bath and the Ma sterflex peristaltic pump........................................................................................................................... .......67 A-11 Picture of the thermocouple used inside the vessel 1........................................................68 A-12 Picture of the metering valve (MV)...................................................................................68 A-13 Picture of the gas meter (GM)...........................................................................................68 A-14 Picture and diagram of the cross angles connector............................................................69 A-15 Picture of the two way valve (V).......................................................................................69 A-16 Picture of the quick connect stem (female NPT) and body (male NPT)...........................69 A-17 Vessel 1.................................................................................................................. ............70 A-18 Diagram of vessel 2....................................................................................................... ....70 A-19 Picture of vessel 2 with the hole at the bottom..................................................................71 A-20 Picture of the forged stainless steel gate valve..................................................................71 A-21 Picture of the bottle and the stopper..................................................................................72 B-1 Picture of the mobile part of the equi pment, before immersion into the cooler................73 B-2 Pictures showing th e sample being pumped......................................................................73 B-3 Picture of disconnecting QC6............................................................................................74 B-4 Picture showing when V4 was ope ned to collect the liquid sample..................................74 B-5 Picture showing the measuremen t of the liquid sample collected.....................................75

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DESIGN AND TESTING OF AN APPARATU S TO MEASURE CARBON DIOXIDE SOLUBILITY IN LIQUID FOODS By Thelma Francisca Calix Lara May 2008 Chair: Murat O. Balaban Major: Food Science and Human Nutrition Carbon dioxide (CO2) is a non-toxic, inexpensive, chem ically inert, non-flammable fluid and a safe solvent. It has multiple applications in the beverage industr y, such as carbonation and dense phase carbon dioxide (DPCD) tec hnology, a cold pasteurization process. The solubility of CO2 in water depends on pressure a nd temperature (Dodds and others 1956). Increasing pressure in creases solubility of CO2 and increasing temperature decreases it. In addition, the presence of other dissolved substances could have a positive or negative effect in CO2 solubility (Meyssami and others 1991). DPCD is a continuous cold pasteurization method that affects microorganisms and enzymes under pressures below 50 MPa (7,250 psi) without the use of high temperature treatment s. Currently, the use of CO2 in DPCD treatments is not optimized, since solubility of CO2 in liquid foods at different temperatures, pressures, and compositions is not known. Therefore, excess CO2 is used to assure saturation. If CO2 solubility could be predicted at any temperature, pre ssure and composition for a liquid food, then the amount of CO2 used would be slightly above this theo retical value, and the process would be more economical. The first step towards this type of prediction is the experimental measurement of CO2 solubility in model liquid food systems and its correlation with pressure, temperature and composition. Therefore, the objectives of this study were: 1) to desi gn, build and test a high-

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12 pressure carbon dioxide solubility appara tus, 2) to compare the volume of CO2 that dissolves in pure water with the current literature; and fina lly 3) to determine the maximum amount of CO2 in solution with model liquid foods of known compos ition (binary solutions of fructose and malic acid). An experimental system to measure the CO2 solubility was designed and tested. The pressure and temperature were controlled in th e system, and the design assured an accurate measurement of solubility. Experimental results of CO2 in solution in pure water were compared with the literature, a nd there was no significant difference at pressures of 7.56 MPa (1100 psi) and 10.3 MPa (1500 psi), at a constant temperat ure of 40C. At pr essures of 7.58 and 10.34 MPa, solubilities of CO2 in water were 4.77 .15 and 5.51 0.20 g/100 g of distilled water, respectively. By increasing the concentration of malic acid or fructose, solubility of CO2 decreased significantly. At 7.58 MPa of pressure, the amount of CO2 dissolved in the solutions of 0.5 and 5% of malic acid c oncentration was 4.47.03 and 4.47.02 g/ 100 g of solution. When the concentrations of fructose in the solu tion were 5 and 10%, solubilities of CO2 in the solutions were 4.55.11 and 4.12.07, respectively (at 7.58 MPa). At 10.34 MPa of pressure, solubilities of CO2 in solution with 0.5 and 5% of malic acid concentration were 4.90.08 and 4.79.07 g/100 g of solution. When the amount of fr uctose in the solution increased to 5 and 10 g/100 g of solution, solubilities of CO2 at 10.34 MPa were 4.78.14 and 4.19.17, respectively. This equipment design and method can experimentally measure CO2 solubility in real liquid foods, and therefore is a significant contribution to the DPCD field. This is the first step in the generation of experimental data for the thermodynamic modeling of CO2 solubility in liquid

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13 foods. The experimental apparatus could offer an advantage to the food industry, primarily to soft drink industry, and to the DPCD users, by eliminating the need to use excess CO2 and therefore reducing the production cost.

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14 CHAPTER 1 INTRODUCTION Carbon dioxide (CO2) has numerous food applications and many others are being developed, providing simple and economic process alternatives to the beverage industry. CO2 gas is used in the carbonation of soft dri nks, beers and wines because many studies have demonstrated that CO2 prevents fungal and bacterial growth (Eastoe and others 1996; Del PozoInsfran and others 2006b; Gunes and others 2005). In addition, CO2 enhances the physical properties and sensory attribut es of carbonated beverages (Ka ppes and others 2007; Del PozoInsfran and others 2006a). In 1998 and 2003, the global beverage consum ption volume reached an estimated 1.2 trillion liters and 1.4 trill ion liters, respectively. Carbonated so ft drinks represented 14% of the total in both years, increasing by 3.0 million liter s during that period (Roethenbaugh 2005). In 2003, the United States was the biggest consumer, with 30% of the total volume of carbonated soft drinks, followed by Mexico with 9%. However, the overall compound annual growth forecasts a growing concern for health and welln ess. That forecast will drive the growth of bottled water and still drinks c onsumption in the coming years; sa ve for soft drinks, which have increased in importance in recent years agains t other beverage categories (Roethenbaugh 2005). Carbon dioxide is a non-toxic, chemically inert, non-flammable flui d and a safe solvent for process applications (Eastoe and others 1996). Carbon dioxide has a specific gravity of 1.53 (based on air), and its molecular weight is 44.01 grams per mol and/or 22. 4 liters per mol (Steen 2005; Subramaniam and Busch 2002). In food applications, CO2 can be present as a gas, liquid or solid, depending on temperat ure and pressure. Accordi ng to the phase diagram, CO2 has a triple point (-56.6 C at 0.52 MPa=5.11 atm), a sublimation range and a critical point (temperature 31 C and pressure 7.4 MPa=73 atm). The tr iple point is the condition that CO2

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15 simultaneously exist as a solid, liquid and gas. The sublimation range is when CO2 gas can become solid CO2 (dry ice), without passi ng through the liquid phase (and vice-versa). The critical point is the temperature above which it is impossible to liquefy the gas (Steen 2005). Most of the studies about supercritical CO2 have been mainly focused on supercritical extractions. However, the purpose of this project was focused on the area of dense phase carbon dioxide (DPCD) pasteurization, to design and build an experimental apparatus to measure solubility of CO2 in liquid foods. DPCD is a cold pasteurization method that kills microorganisms, inactivates certain enzymes a nd viruses at pressures below 50 MPa without applying high temperature treatments (Damar and Balaban 2006). The microbial inactivation effect of DPCD is partly base d on gas dissolution under pressure in a microbial cell that, when decompressed to atmospheric pressure, causes fatal damage (Balaban and others 1991; Park and others 2002). Furthermore, there is the theory that high pressure car bon dioxide dissolves in water to produce carbonic acid, thus the pH will be temporarily lowered and therefore inactivates enzymes (Damar and Balaban 2006). Kincal and others (2005) te sted a continuous high-pressu re carbon dioxide system for microbial reduction in orange ju ice, and Arreola and others (1 991) studied the effects of CO2 on some quality attributes. Results showed that the treatment was able to cause a 5-log reduction of the natural flora in spoiled juice and coul d achieve a 5-log reduction of pathogenic Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes Balaban and others (1991) stated that pectinesterase wa s inactivated under supercritical conditions, suggesting that its inactivation was due to the combination effect s of pressure, temperature, pH reduction and process time. Dagan and Balaban (2006) measur ed the effect of DPCD pasteurization on beer quality using a continuous system, and the pro cess was able to produce a clear, consistently

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16 fresh-tasting beer with extended shelf life. In DPCD, the use of carbon dioxide is not op timized, since solubility is not known, and not predicted. Thus, excess carbon dioxide is used to assure saturation. In addition, Ji and others (2007) mentioned that the reduction of greenhous e gases emissions has become an issue of worldwide concern (CO2 represents about 64% of the e nhanced greenhouse effect). If CO2 solubility could be measured at any temperature, pressure and composition of food, the amount of CO2 used would be slightly above this theoretical value, an d the process would be more economical. Therefore, the objectives of this study were: To design and build a high-pre ssure apparatus to measure CO2 solubility in liquid foods. To test the accuracy and pr ecision of the high-pressure CO2 solubility apparatus, and compare with the literature the amount (v/v) of CO2 dissolved in pure water. To determine the maximum amount (v/v) of CO2 in solution with a model liquid food of known composition (solution of sugar or acid).

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17 CHAPTER 2 LITERATURE REVIEW 2.1 Carbonated Soft Drinks According to Roethenbaugh (2005), soft drinks are sweetened water-based beverages, usually balanced by acidity, and their primary func tion is hydration. Soft dr inks are divided into two large groups, the ready-to-dri nk (RTD), and the concentrated or diluted-to-taste products. RTD products are subdivided into carbonated pro ducts. Figure 2.1 shows the beverage sectors and segments. Figure 2-1. Beverage sectors a nd segments (Roethenbaugh 2005). The dominant world players in the carbonate d RTD soft drink industry are Coca Cola, Pepsi and Cadbury-Schweppes. Together with thei r associated brand names, they constitute over half the worlds consumption of such products In 2003, carbonated soft drinks became the worlds second most popular beverage after tea. In 1998 and 2003, the global beverage consumption volume reached an estimated 1.2 trilli on liters and 1.4 trillion liters, respectively, and approximately fourteen percent (14%) of the total were carbonated soft drinks (Figure 2-2) (Roethenbaugh 2005).

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18 Figure 2-2. Global beverage consump tion by category, 1998-2003 (Roethenbaugh 2005). Over the period 1998-2003, the global consum ption of beverages was dominated by the United States (15%), followed by India (10%) and Ch ina (9%) (Figure 2-3). In carbonated soft drinks the United States repres ented thirty percent (30%) of global consumption, followed by Mexico (9%). The fastest growing consum ing countries between 1998-2003 were BosniaHerzegovina (160% increase), Se rbia (86%) and China (86%). Figure 2-3. Beverage consum ption by countries (1998-2003).

PAGE 19

19 2.2 Carbon Dioxide 2.2.1 General Information Carbon dioxide (CO2) is a non-toxic, inexpensive (appr oximately 4.85 cents/kg in 2007), chemically inert, non-flammable fluid and a safe solvent for process applications (Eastoe and others 1996, Steen 2005). It has a specific gravity of 1.53 (based on air) under conditions of room temperature and atmospheric pressure, a nd its molecular weight or molar mass is 44.0098 0.0016 grams and/or 22.4 liters per mol (Steen 2005; Subramaniam and Busch 2002; Span and Wagner 1996). The molecule of CO2 is linear with a double bond between the carbon and oxygen atoms (O=C=O) (Song 2002). The effects of temperature and pressure on the state of CO2 (physical properties) are shown in Figure 2-4. The triple point (-56.6 C at 0.52 MPa=5.11 atm) is the condition in which CO2 can be present as a solid, liquid or gas (the thre e phases coexist). The sublimation range is when CO2 solid (dry ice) directly becomes gas (and vice-versa), for example at -78.8 C and 1 atm (Song 2002; Steen 2005, Levelt 2000). Figure 2-4. Carbon dioxide phase diagram (Steen 2005).

PAGE 20

20 Above the critical point (temperature 31 C and pressure 7.4 MPa=73 atm) it is impossible to liquefy the gas, and CO2 exists as a fluid or dense gas (S ong 2002). At atmospheric pressure and room temperature (25 C), CO2 cannot be present as liquid. To liquefy CO2 it is necessary to compress and cool between the triple point and th e critical point conditio ns (Steen 2005). The density, viscosity and diffusion coefficient associated with different phases of CO2 are shown in Table 2-1. Table 2-1. Physical prop erties associated with the phase diagram of CO2 (Koutchma and Murakami 2005; Buhler and others 1998). States Density (g/ cm3) Viscosity (cP) Diffusion coefficient (cm2/s) Gas 0.002 0.014 0.01 Super Critical 0.467 0.02-0.12 0.0001 Liquid 1.0 1.0 0.00001 In CO2, the density ( ) increases rapidly around th e critical pressure (pc). When the temperature decreases, the change in is more remarkable and it does not move to higher pressures as fast as at high temperatures; thus, becomes difficult to c ontrol around the critical temperature (Clifford and Williams 2000). Figure 2-5 shows four density-pressure isotherms. Figure 2-5. Density-pressure isotherms for carbon dioxide (Clifford and Williams 2000).

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21 According to Steen (2005), the European Industrial Gases Association, the Compressed Gases Association of America a nd the International A ssociation of Beverage Technologist have described the standard quality of CO2 for use in foods and beverages. Table 2-2 shows the specifications for commercial liquid CO2. Table 2-2. Quality standard of carbon dioxide (Steen 2005). Component Specification Assay 99.9% v/v min. Moisture 50 ppm v/v max. (20 ppm w/w max) Acidity To pass JECFA* test Ammonia 2.5 ppm v/v max. Oxygen 30 ppm v/v max. Oxides of nitrogen (NO/NO2) 2/5 ppm v/v max. each Non-volatile residue (particulates) 10 ppm w/w max. Non-volatiles organic residue (oil and grease) 5 ppm w/w max. Phosphene 0.3 ppm v/v Total volatile hydrocarbons (calculated as methane) 50 ppm v/v max. of which 20 ppm v/v max. non-methane hydrocarbons Acetaldehyde 0.2 ppm v/v max. Benzene 0.02 ppm v/v max. Carbon monoxide 10 ppm v/v max. Methanol 10 ppm v/v max. Hydrogen cyanide <0.5 ppm v/v Total sulphur 0.1 ppm v/v max Taste and odor in water No foreign taste or odor JECFA: Joint FAO/WHO Expert Committee on Food Additives. In commercial use, there are several methods of CO2 production. Fermentation and direct combustion are two typical processes. Ferm entation consists of mixing a carbohydrate-based solution with yeast and oxygen in a fermenter; CO2 and alcohol are obtained. The CO2 is passed through a separator to eliminate the foam; then CO2 is compressed. Finally, CO2 is washed with water to remove water-soluble impurities. Direct combustion consists of burning a hydrocarbon fuel, such as light oil or natural gas, to produce a gas containing less than 0.5% oxygen by volume. The gas is cooled, scrubbed (to remove impurities) and passed through an absorbent tower that gets in contact with a CO2 absorbing solution. This solution is then pumped to a striping tower, used to release the CO2 in vapor form (Steen 2005).

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22 Carbon dioxide is delivered as a liquid to the customer, often by road tanker, and then it is transferred to pressurized vessels (20.5 bars) of 5-50 tons capac ity at -17C; temperature is controlled by a small refrigeration unit. To change CO2 liquid to CO2 gas is necessary to use a carbon dioxide vaporizer. Every ba tch supplied must have a certifi cate of conformance to ensure that there are no dange rous residues in CO2, such as benzene, and to assure the safety of the user. A hazard analysis and critical control points (HACCP ) survey of the installed system is required (Steen 2005). Figure 2-6 shows how CO2 is delivered: A liquid and gas phases are connected in both tanks (Steen 2005). Figure 2-6. Carbon dioxide de livery process (Steen 2005). 2.2.2 Applications Carbon dioxide has numerous applications in the food and beverage industries. CO2 liquid or solid is used for quick freezing, surface freezing, chilling and refrigeration in the transportation of foods. Cold sterilization can be obtaine d with a mixture of 90% CO2 and 10% ethylene oxide; CO2 has a stabilizing effect on the ethylen e oxide and diminishes the hazard of explosion. CO2 gas is used in the processing of car bonated soft drinks, beers and wines. CO2

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23 provides sensory characteristics. For consumers, CO2 can be a pleasurable and sought-after sensation; while, for some people, CO2 can be an irritating or even painful sensation (Dessirier and others 2000). CO2 bubbles are responsible for several se nsations because they stimulate the mechanoreceptors on the tong and because of chem ogenic origin by formation of carbonic acid (Descoins and others 2004). In addition, dense phase CO2 is used in beverages due to its effective antimicrobial effects (Eastoe and others1996). During the last 20 to 30 years, considerable interest has centered on supercritical fluid extraction (SFE) using CO2 (Bamberger and others 1999; Harris and Yung 1995). Isolation of flavors, decaffeination of coffee beans, remova l of hexane from soybean oil, extraction of triglycerides, cedarwood oil, cedarleaf oil, carotenoids, and an thocyanins (a type of flavonoid or a group of phenolic compounds (Netzel and ot hers (2006)) are some examples of SFE applications (Bamberger and others 1999). Identification of a cetylated soybean lecithin components was also performed to test th e surfactant potential of supercritical CO2 (Nasir and others 2007). 2.3 Carbonation 2.3.1 Definition Carbonation is the impregna tion of a liquid with CO2 gas (Steen 2005) and its effectiveness depends on the quantity of gas in solution with the soft drink (Mitchell 1978). Solubility of gases in liquids has been studied by many investigators sinc e the early nineteenth century (Battino and Clever 1965; King and others 1992) Solubility of a solute (solid, liquid or gas) is the analytical compositi on of a saturated solution, expresse d in terms of the proportion of the designated solute in a designated solvent (K arukstis and Van Hecke 2000). Dodds and others (1956) state that solubility of CO2 in water depends on its pr essure and temperature in equilibrium with the solution. Pressure has a direct effect on the solubility of CO2 (pressure

PAGE 24

24 increases the solubility of CO2). On the other hand, with increas ing temperature, solubility of CO2 decreases. The presence of other substances could have a positive or negative effect in solubility (Descoins and others 2006; Meyssami and others 1991). In addition, solubility of CO2 has an opposite behavior than solubility of soli ds (e.g. sugar or salt) in water. Most solids dissolve more readily in hot water than in col d. Figure 2-7 shows how the relationship between pressure and temperature affect the volume of CO2 in solution with pure water at relatively low pressures. Figure 2-8 shows the effect of te mperature and pressure on the solubility of CO2 in pure water. Figure 2-7. Carbonation: CO2 chart in solution with pure water (Steen 2005).

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25 Figure 2-8. Solubility of carbon dioxide in wate r as a function of pressure and temperature (Dodds and others 1956).

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26 When CO2 is dissolved in water, it forms carboni c acid (Equation 2-1). Carbonic acid, in combination with the product, produces the acid ic and biting taste in carbonated beverages, making the beverage more palatable and visually attractive (Steen 2005; Descoins and others 2006). CO2 (gas, liquid) CO2 (aq) (2-1) CO2 (aq) + H2O H2CO3 K = 1.6 x 10-3 H2CO3 (aq) + H2O HCO3 (aq) + H3O+ (aq) pKa = 6.35 HCO3 (aq) + H2O CO3 -2 + H3O+ pKa = 10.33 According to Steen (2005), Henrys law postulated by William Henry (1774-1836) states that the amount of gas dissolved in a given volume of solvent is proportional to the pressure of the gas with which the solvent is in equ ilibrium. The universal ideal gas law is: pV = mRT (2-2) p is the absolute pressure in atmospheres (atm), V is the volume of the gas (m3), m is the number of moles of gas, R is the specific gas constant (J kg mol-1 K-1) and T is absolute temperature (in Kelvins). CO2 molar mass ( M ) is 44.0098 0.0016 g mol-1 and its molar gas constant ( Rm) is 8.314510 0.000210 J kg mol-1K-1. The specific gas constant for CO2 ( R = Rm/ M ) is 0.1889241 0.0000116 kJ kg-1 K-1(Span and Wagner 1996). Diamond and Akinfiev (2003) st ate that solubility of CO2 in terms of the Henrys law can be expressed as follows: ) ( ) ( 0 ) ( 22 2) (aq CO T P H y T P COk y f aq xCO (2-3) CO2(aq) and y are the mole fractions of carbon dioxide in the aqueous and non-aqueous phase respectively, kH(P,T) in MPa is the Henrys constant of carbon dioxide in pure water on a mole fraction basis at specified pressure and temperature conditions, 0CO2 (P,T) is the fugacity of pure

PAGE 27

27 carbon dioxide (in MPa) at specified pressure and temperature conditions, CO2(aq) is the asymmetric activity coefficient ca rbon dioxide in Henrys law ( CO2(aq) 1 as CO2 (aq) 0) and y is symmetric (Raoults law) activity coe fficient of carbon dioxide in the non-aqueous phase ( y 1 as y 1). To calculate solubilities using Eq. (3 ), it is necessary to assume that CO2 (aq) and y are equal to unity and 0CO2 (P,T) for the CO2-rich vapor and for the CO2-rich liquid are calculated from the fundamental equation of state, expre ssed in form of the Helmholtz energy (Span and Wagner 1996): ,rRT T A (2-4) where A is the specific Helmholtz energy, is mass density, T is temperature, R is the gas constant, is the dimensionless Helmholtz energy ( = A/RT ), is the reduced density ( = / c) and is the inverse reduced temperature ( = Tc/T). The dimensionless Helmholtz energy ( ) is split into a part depending on the ideal gas behavior ( ) and a part which takes into account the residual fluid behavior ( r). The critical density of carbon dioxide ( c) is 467.6 kg/m3 and critical temperature (Tc) is 304.1282 K (Span and Wagner 1996). The vapor pressure ( ps) and the densities of saturated li quid () and saturated vapor () can be determined from an equation explicit in by simultaneous solution of the equations: 1 'r sRT p (2-5) 1 "r sRT p ln 1 1r r sRT p

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28 which correspond to the equality of pressure, temperature and specific Gibbs energy in the coexisting phases (Span and Wagner 1996). The Gibbs energy (GE) is used to describe the nonideality of the H2O-rich phase in the equation of stat e (Costa-Gomez 2007; Jdecke and others 2007; Krichevsky and Kasarnosky 1935). The Helm holtz energy of the ideal gas is given by: T Ts RT T h T A (2-6) where the enthalpy h of the ideal gas is a function of the temperature only, and the entropy s of the ideal gas depends on temperatur e and density (Span and Wagner 1996). Also, y (mole fraction of CO2 in the non-aqueous phase) is estimated according to the Lewis-Randall rule (at low pressures): total watersatP P y 1, (2-7) where Pwatersat is the pressure of liquid-vapor equilibrium of pure H2O (in MPa) and Ptotal is the total pressure (in MPa), at a specifie d temperature. At high pressures, y is determined from the following empirical equation: y = 1 (0.1256 t 0.0212) x 10-3 P (0.065 t + 1.121) x 10-5, (2-8) where t is temperature in C and P in MPa. Henrys constant ( kH) is calculated from the equation of state (Akinfiev and Diamond 2003): 5 0 0 0 01000 2 ln ln 1 ln2 2 2T b a M RT f kO H O H W O H H (2-9) where 0H2O is the fugacity (in MPa) and 0H2O is the density (in g cm-3) of pure water at determined P and T (in Kelvins), R is the gas constant (8.31441 cm3 MPa K-1 mol -1), MW is the molar mass of H2O (18.0153 g mol-1), and (dimensionless), a (in cm3 g-1) and b (in cm3 K0.5g1) are empirical parameters (Diamond and Akinfiev 2003; Akinfiev and Diamond 2003).

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29 Many of the parameters and thermodynamic coef ficients require experimental data for accurate calculation. Such CO2 solubility data for real liqui d foods is rare or non-existent. 2.3.2 Methods and Apparatus for Carbonation The complexity and cost of the equipments us ed to carbonate and to measure solubility of CO2 in liquids are extended from mass spectro meter to the simple manometric van Slyke apparatus, used to measure gas quantities released from blood (B attino and Clever 1965). Figure 2-9 shows different schemes of carbonation methods. The most common method is the bubbler that consists of sparging CO2 into the liquid under pressure to form small bubbles of gas which can be easily absorbed by the liquid (Steen 2005). The waterfall method represents the early carbonators which used to operate at around 4C. Figure 2-9. Carbonation methods (Steen 2005). According to Steen (2005), the rate of flow and the pressure of CO2 are critical to ensure the correct carbonation level. When the pressure increases, the gas bubbles formed at the sparger will be smaller and the gas bubbles surface area will faci litate the gas absorption by the liquid.

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30 In addition, the greater surface area of liquid exposed to CO2 will increase the rate of absorption of CO2 by the liquid. Figure 2-10 shows an example of a typical apparatus to measure carbonation which consists of a jig to restrain the container, a nd a piercer connected to a pressure gauge. The container is shaken before the pressure is m easured and the release valve is opened until the pressure gauge reads zero. For a second time, the container is shaken and the pressure is retaken and the temperature measured. The volume of carbonation is then obtained from a carbonation chart by finding the point that relates the conditions of temperature and pressure previously measured (Steen 2005). Figure 2-10. Measurement of carbonation (Steen 2005). The purpose of releasing the pressure for the fi rst time is to exclude other gases from the head space of the can (container) or from the beverage. Air has approximately one fiftieth the solubility of CO2 in a liquid. Air constituents are appr oximately 79% nitrogen and 21% oxygen. Due to the differing solubility and proportion of oxygen and nitrogen, the dissolved air actually

PAGE 31

31 contains 35% oxygen and 65% nitrogen (low solubi lity). The presence of oxygen could create spoilage problems (Steen 2005). This method is not practical or safe unde r pressures used by the DPCD method. 2.4 Supercritical Carbon Dioxide 2.4.1 Definition According to Subramaniam and others (2002) supercritical phase reactors are gaining acceptance in industry. DuPont has announced a US$ 40 million demonstration plant for supercritical polymerization. Tu rner (2006) states that supercri tical gas or liquid occurs when the temperature increases over th e critical point. In the CO2 phase diagram (Figure 2-4), the curves represent coexistence between the phases. When the curve representing the liquid + gas phase ends at the critical point the distinction between vapor and liquid disappears (Levelt 2000) and the densities of the two phases become identical (Clifford and Williams 2000). In supercritical fluid mixtures, it is important to predict the phase equilibria to successfully develop and operate the physical separation processes and solubility models (Stratmann and Schwiger 2004). The fundamental equation of phase equilibria establis hes that, at a given temperature and pressure, the fugacity of a component i in the gas phase, g ifmust be equal to the fugacity of the same component in the solid phase s if (Valderrama and others 2006). Binary fluid mixture phase diagrams are usually repr esented as two-dimensi onal projection of P-T (pressure-temperature) (Levelt 2000). Phase equ ilibria diagram between th e binary mixture of water and CO2 system is represented in Figure 2-11.

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32 Figure 2-11. Phase equilibrium in the H2O and CO2 system (Diamond and Akinfiev 2003). LCEP denotes the lower critical end-poi nt of the system. Quadruple point Q1 indicates the equilibrium between H2O-ice, CO2-clathrate-hydrate, CO2-bearing water and CO2-vapor (IceCla-Laq-V). The quadruple point Q2 shows the equilibrium between CO2-clathrate-hydrate, CO2bearing water, CO2-liquid and CO2-vapor (Cla-Laq-LCO2-V) (Diamond and Akinfiev 2003; Longhi 2004). At LCEP, liquid and gas phases critic ally merge to form a single fluid phase in the presence of a non-critical so lid phase (McHugh and Krukonis 1994). Carbon dioxide clathrate hydrate is formed at temperatures below approximately 10 C (Someya and others 2005; Anderson 2002). The clathrate hydrate is a non-stoichiometric crystalline compound, suggested as a natural ga s storage medium, formed by a modification of the crystal structure of ice (Someya and others 2005; Ferdows and Ota 2005; Anderson 2002).

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33 The CO2 hydrate is a structure -I hydrat e. Structure-I hydrate consists of 46 water molecules and 8 guest molecules at most per unit cell and fo rms two smaller cages of pentagonal dodecahedron (512) and six larger cages of tetrakaidodecahedron (512612). CO2 hydrates consist of three parts: interaction between water-water mol ecules, interaction between water-CO2 molecules and interaction between CO2-CO2 molecules. According to A nderson (2002), once carbon dioxide clathrate begins to form, it becomes nearly impossible to assess the partitioning of CO2 between the liquid and solid phase. Regarding solubility of CO2, several studies state that solubility with the hydrate would decrease with decreasing te mperature and pressure (Ferdows and Ota 2005). 2.4.2 Dense Phase Carbon Dioxide Dense phase carbon dioxide (DPCD) processing, which includes liquid and supercritical CO2, is a continuous cold pasteurization method that affects microorganisms and enzymes under pressures below 50 MPa (7,252 psi) without appl ying high temperature treatments, based on the hypothesis that high pressure CO2 dissolves in water to produce car bonic acid, thus the pH value will be temporarily lowered and able to inactivate enzymes (Damar and Balaban 2006; Del PozoInsfran and others 2006b; Ishikawa and others 1995; Shimoda and others 2001). Moreover, DPCD system removes dissolved oxygen, preven ting degradation of several compounds, such as antioxidant phytochemicals; thus DPCD process decreases phytoc hemical and antioxidant losses that occur during storage of juices (Del Po zo-Insfran 2006a; Subramaniam and others 2001). Fraser (1951) was the first to prove that DPCD technology can disrupt ba cterial cells and some years later, Taniguchi and others (1987) showed the e ffect that supercritical CO2 (20 MPa, 35 C, 2 hr) has on the enzyme activity. Furthermore, supercritical CO2 has been recognized for sterilizing and inactivating viruse s. Usually, supercritical reacti on media have the potential to increase reaction rates, selectivity of chemical reactions, and facilitate separation of reactants, products and catalysts after reaction. In addition, principles for gas or liquid phase reactions may

PAGE 34

34 be applied to supercritical phase reactions (Figure 2-12) (Subramaniam and Busch 2002; Subramaniam and others 2002). Figure 2-12. Variation of phys ico-chemical properties of CO2 at near-criti cal conditions (Subramaniam and others 2002). 2.4.3 Supercritical Fluid Extraction (SFE) Extraction is an important method to isol ate natural odor/flavor components and other natural products such as essential oils. The pr oduct obtained from disti llation/extraction, vacuum distillation, extraction with li quid solvents and adsorption ha s solvent residues (Bamberger and others 1999). However, supercritical CO2 extraction results in a pr oduct without solvent residues and with a better taste/aroma, because it is a non-thermal process (Marteau and others 1995; Palazo lu and Balaban 1998). According to Subramaniam and Busch (2002), CO2 has limited reactivity; thus, supercritical CO2 has many chemical applicati ons to replace conventional organic solvents. To perform supercritical fluid extractions it is necessary to pump CO2 through

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35 a vessel previously filled with sample. After extraction, the supercritical fluid is depressurized for collection of extracted compounds (Schneider and others 2000). According to Clifford (1999), the factors that control su percritical fluid extrac tion are: the solute must be sufficiently soluble in the supercritical fluid to be removed by solution in the fluid flow; the solute must be transported sufficiently rapidly from the interior of the matrix in which it is contained and; the solute must be released by the matrix. A superc ritical fluid instrument includes a high-pressure pump (one or two) to carry the supercritical fluid, a high pressure vessel to contain the sample, a polar cosolvent (ethanol) if necessary, a restrict or and a collection vial (Turner 2006). Figure 213 shows a schematic of SFE equipment. Figure 2-13. Diagram of s upercritical fluid extracti on equipment (Turner 2006). 2.6 Constituents that Affect Carbon Dioxide Solubility in Beverages 2.6.1 Carbonated Soft Drinks Carbon dioxide solubility can be affected posit ively or negatively by different constituents present in the formulation of different soft dri nks. The term soft drink applies to beverages containing flavors and/or fruit juices together with other c onstituents of technological or nutritional value designed to enhance the appearan ce and stability of the product and to ensure that its sensory properties remain intact du ring a reasonable shelf life (Taylor 2005). Some categories of ingredients are discussed in more detail in the following sections, in terms of CO2

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36 solubility. The functional constituents of soft drinks and their typical usage levels are listed in Table 2-3. Table 2-3. Soft drink components and their typical use level (Taylor 2005). Component Typical use level Water Up to 98% v/v when high-intensity sweeteners in use Sugars 7-12% m/v when sole source of sweetener Fruit juice Widely variable usage High-intensity sweeteners Use based upon sucro se equivalent (e.g. aspartame might be employed at 0.40-6% m/v as sole sweetener) Carbon dioxide 0.30-6% m/v Acids (e.g. citric) 0.05-0.03% m/v Flavours Nature-identical and artif icial: 0.10%-28% m/m. Natural: up to 0.5% m/m Emulsifier 0.1% m/v Colours (naturals or synthetic) 0-70 ppm Preservatives Statutory limits apply (e.g sorbic acid up to 250 ppm in EU) Antioxidants (e.g. BHA) Less than 100 ppm, subject to user-country legislation Quillaia extract (saponins) Up to 200 mg/l (EU), up to 95 mg/l (USA) Hydrocolloids (mucilaginous gums) 0.1-0.2% per GMP, minimum amount required to create desired effect Vitamins/Minerals ADIa applies aADI: acceptable daily intake. 2.6.1.1 Carbohydrate sweetener According to Bessley (1990), the first asso ciation between sugars and carbonated soft drinks occurred in the seventeen th century when lemon juices containing natural sugars were added to spring water. Since Captain Cook, th e use of carbohydrate sweeteners in juices and drinks has increased, in order to preserve th e juice for long periods (ODonnell 2005; Bessley 1990). In the past 10-15 years, the profile of bulk and intense sw eetener use in soft drinks has changed (technical improvements in the manu facture of bulk sweetener with different carbohydrate profiles that deliver equivalent sweetness and mout hfeel properties to sugar at lower cost). In addition, due to an increasing consumer awareness of healthy eating and concern about the growing incidence of obesity and Type 2 diabetes, interest in low calorie carbonated soft drinks has increased considerably (OD onnell 2005). An extensive number of carbohydrate sweeteners are used in soft dri nk industry, providing sweetness, m outhfeel, stability and color (in

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37 some cases). Table 2-4 illust rates the properties of some car bohydrate sweeteners used in soft drinks. Table 2-4. Properties of bul k sweeteners (O Donnell 2005). Approval for use in soft drinks Sweetener/ carbohydrate Relative sweetness (sucrose=1) Solubility (g/100g water at 20C) Insulindependent metabolism Caloric value (kcal/g) EU USA Sucrose 1 200 Yes 4 N/A N/A Glucose syrup 0.6 Higha Yes 4 N/A N/A HFGS 0.9-1.0 Higha Yes 4 N/A N/A Fructose 1.2-1.8 374.83 No 4 N/A N/A FOSb 0.3-0.6 75 No 2 N/A N/A Inulin 0 10 No 1 N/A N/A Polydextrose 0 80 No 1 Yes Yes Trehalose 0.45 40.8 Yesc 4 Yes Yes Tagatose 0.92 62.0 No 1.5 No Yes a Solubility good, but varies with pr oducts of different specifications. bFructo-oligosaccharides. cGlucose and insulin responses lower than glucose. In general, sugars diminish the average level of CO2 solubility, at pressures from 1 to 6 bars and pH set at 3.1(Descoins and others 2006). In addition, sugars contribute sweetness and body to the drink, act as synergis t and give balance to flavor (S teen 2005). Carbohydrate sugars used in carbonated soft drinks can be divided into those in a dry, granular form (e.g. granulated sucrose) and those in a liquid or syrup form (e.g liquid sugar (sucrose in solution with water and glucose-type syrup produced from maize or wheat, e.g. glucose syrup or high-fructose glucose syrup)). Granulated sugar is a dry, crystallized disaccharide extracted from sugar beet and sugar cane called sucrose and liquid sugar consists of an aqueous solution of sucrose at a saturated concentration of 67 Brix at 20 C (Bessley 1990). In United States, high-fructose corn syrups dominate the carbohydrate-sweetened soft drinks sector; however, in Europe the use of high-fructose glucos e syrup (HFGS) is restricted by production quotas. Fructose (levulos e) is a sugar in crys talline or syrup form. It is produced by hydrolyzing sucrose to a glucose-fructose mixtur e; the resulting monosaccharides (glucose and fructose) are separated using chromatography and the fructose is crystallized. Its sweetener

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38 capacity is higher than sucrose (Table 2-4); thus it can be used at lower concentrations to decrease the level of energy content in the form ulations. The absorption and metabolic process of fructose by the body is very slow and does not depend of insulin production, being appropriate for diabetics (ODonnell 2005). 2.6.1.2 Acidulants The use of acidulants is an essent ial part of beverage formulati on. Acidulants contribute to sharpness, sourness, background to flavor and increase thirst-quenc hing effects in soft drinks (Steen 2005). In addition, acidul ants can indirectly prevent disc oloration and rancidity, by acting as a synergist to antioxidants (Taylor 2005). Table 2-5 shows the list of the most common acidulants used in soft drinks. Table 2-5. Acidulants used in beverage formulation (Taylor 2005). Acidulants Molecular weight Melting point (C) Citric acid : 2-hydroxy-1,2,3-propane tricarboxylic acid 192.1 152-154 Tartaric acid (D-tartaric): 2,3-dibydroxy butanedioic acid 150.1 171-174 Phosphoric acid: orthophosphoric acid 98.0 42.35 Lactic acid (DL-lactic): 2-hydroxy propanoic acid 90.1 18 Malic acid (D-malic): 2-hydroxy butanedioic acid 134.1 98-102 Fumaric acid: trans -butenedioic acid 116.1 299-300 Acetic acid: ethanoic acid 60.0 16-18 Citric acid is the most widely used acid in fruit-flavored beverages. It used to be commercially produced from lemons, limes or be rgamots by concentrating and precipitating the juice. Recently, it is produced using enzymes st arting from glucose and ot her sugars. Tartaric acid is naturally present in grapes, currants, blac kberries and cranberries. Tartaric acid has a sharper flavor than citric acid; t hus, it is used in lower concentra tion to provide equivalent palate acidity. However, tartaric acid has lower solubility than citric acid. Phosphoric acid is used in soft drinks for its astringency to complement the dry and balsamic characteristic of cola drinks. It has a drier and sharper flavor th an citric and tartaric acid. Mali c acid is closely associated with

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39 apples and it is a prefe rred acidulants in low-calorie drinks. It is also used to mask the off-taste of some sugar subst itutes (Taylor 2005). 2.6.2 Milk Skim milk contains about 9% total solid, casein (2.5%), whey proteins (0.5%) and lactose. Solubility of CO2 in milk has had the same behavior as in water, at diffe rent conditions of pressure and temperature. However at 25C, solubility of CO2 in milk was less. At 5.5 MPa and at 38 and 50 C, solubility of CO2 in milk was very similar to the solubility of water because casein was completely precipitated. This proved that casein was competing with CO2 to get in solution (Tomasula and Boswell 1998).

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40 CHAPTER 3 MATERIALS AND METHODS 3.1 Design of the High Pressure Carbon Dioxide Solubility Apparatus The concept of the solubility of a gas in a liquid at high pressures is simple. After the gas is saturated in the liquid, isolate a known amount of liquid at high pr essure, reduce its pressure and measure the amount of gas that comes out of the li quid. The ratio of the mass of gas to that of liquid gives solubility. However, there are many design-related and practical challenges. Designing, building and tes ting the high pressure CO2 solubility apparatus took one year and a half. The idea started from previous attempts to build an apparatus to measure CO2 solubility, under high pressures, without success. The princi ple of the machine was to assure that the CO2 measured was previously in solution in a liquid of known mass. Several versions of the machine were tested with water. The main dilemma was compressed gas (CO2) trapped in the system, during processing under high pre ssures, giving the impression that solubility of CO2 was three or even four times higher than literature values. The solution was to generate a system able to recirculate the liquid during pr ocessing; based on the hypothe sis that the density of CO2 is lower than the density of water (or liqui d food), at experimental conditions. The experimental apparatus was designed to accurately measure the amount (v/v) of CO2 in solution with water or any other liquid. Ther e are two vessels: vessel 2 is above vessel 1. Vessel 1 has a capacity of approximately 200 ml a nd can be operated at temperatures between 0 to 45 C and pressure up to 24 MPa. Figure 3-1 show s the schematic drawing of the apparatus. Figure 3-1 shows the CO2 tank, high pressure pump (HPP), h eat exchanger (HE), pressure gauge (PG), back pressure regulator (BPR), thermoc ouple (T), metering valve (MV), gas meter (GM), water bath, bottle, stopper, fume hood (FH), two way valves (V), three way valves (TV), vessels and quick connects (QC). Figure A-1 shows some pictures of the equipment. From the CO2

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41 tank until QC3, the apparatus is fixed, and be tween QC3 and BPR the apparatus is mobile (Figure A-2). Figure 3-1. Schematics of the high pressure CO2 solubility apparatus. To connect various pieces of the apparatus toge ther, it was necessary to use Teflon tape in the threads. The Teflon tape avoids leaks at hi gh pressures. The Teflon tape has to be applied clockwise to every male connecti on (Figure A-3). Leaks were detected using a soap solution, from ACE Hardware Corp (Oak Brook, IL). The CO2 tank was ordered from Airgas South, Inc (in Gainesville, FL). It had a siphon tube, and contained 34 kg of liquid CO2 (99.91% of purity). The tank valve was labeled as V1 in Figure 3-1. It was connected to TV1, a three-way valve to purg e the line, followed by a pressure gauge PG1 (Figure A-4), to control and regulate the flow of CO2 from the tank. TV1, TV2 and

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42 TV3 were ordered from Swagelok Company (Jac ksonville, FL, model SS-83XKF4). They were made of stainless steel, and can hold 41.2 MPa of pressure at temperature of 38 C. TV2 was used to depressurize the line from the tank, in case of emergency. TV3 was also used to depressurize the line from the tank, during processing. The high pressure pump (HPP) is from Milton Roy Company in Ivyland, PA (model X-10, serial N M229150-1). It wa s used to pump liquid CO2 through the system, to increase the pressure up to 27.5 MPa (Figure A-5). To liquefy CO2, it was necessary to use a heat exchanger (HE), containing a solution of water (50%) and antif reeze (50%), to reach very low temperatures (approximately -15 C). The heat exchanger (H E) was from Fisher Scientific, model 900 and serial N 870075 (Figure A-6). The back pressure regulator (BPR) was used to regulate the pressure in the system. It was purchased from TESCOM Industrial Controls, Elk River (part nu mber is 26-1762-24). Its body and trim are made of stainless steel, the back-up ring is made of Teflon, the O-rings are made of buna-N, the seal is made of CTFE and remaining pa rts are made of stainle ss steel. Its controlled pressure range is from 0.34 to 41.37 MPa. Its inlet and outlet port type is NPT and the size is Its weight is 5 lbs (2.2 kg) and the BPR am bient operating and fluid media temperature is 40 C to 73.8 C. Figure A-7 shows the schematics of the high pressure/back pressure regulator and Figure A-8 shows the BPR used in the machine. To control the pressure, the back pressure regulator is turned cl ockwise (to increase pressure) or counter-clockwise (t o decrease it). When the syst em is depressurized using the BPR, the exiting CO2 expands, cools and may turn to solid (dry ice), and the BPR may be obstructed. To prevent this, it was necessary to use an electri cal heating tape around the BPR, and a variable autotransformer (Staco Energy Products, Dayton, OH) used to control the voltage

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43 supplied to the belt. The percent used for the ex periment varied between thirty and forty percent (30-40%) of 110 V. This setup is shown in Figure A-9. The water bath was a cooler wi th a capacity of about 60 L wate r. The water in the cooler was recirculating through plastic tubes to a sm all high precision water bath (Hart Scientific, Utah; model 6035) that was set at 46-50 C to increase and/or main tain a constant temperature in the system. To recirculate the water, it was nece ssary to use a Masterflex peristaltic pump (Cole Parmer, Chicago; model 7553-50), set around 3.5 in the speed control dial (Figure A-10). To control the temperature in the cooler a nd inside the system, it was necessary to use thermocouples. The thermocouple used in the co oler was ordered from Omega Engineering, part # JMQSS-125G-12. The thermocouple was ma de of stainless steel, 12 of length and 0.125 of diameter. The thermocouple inside the system was also ordered from Omega Engineering (part # TQSS-116G-12 and QC# PL040052). The thermocouple was made of stainless steel, 1/16 of diameter and 12 of length. It was desi gned to withstand vibration, high temperature and high pressure (Figure A-11). To connect this thermocouple to the system, a Swagelok tube fitting (part # SS-100-1-4) was used. It is a male connector 1/16 and male NPT 1/4. The metering valve (MV) was ordered from Swagelok Company (Jacksonville, FL, model # SS-31RF4). It is a medium-flo w high pressure (34.47 MPa) mete ring valve. It was used to control the flow of the CO2 coming out of the system (Figure A-12), to be measured using the gas meter (GM). The GM was obtained from Ali cat Scientific (Tucson, AZ), model number M1SLPM-D (CO2). It is calibrated to measure CO2 specifically, at a maximu m flow of 1 standard liter per minute (Figure A-13). The GM is based on the accurate measurement of volumetric flow at a standard temperatur e and pressure (0.1 MPa and 25 C). The volumetric flow rate is

PAGE 44

44 determined by creating a pressure drop across a unique internal re striction, name as Laminar Flow Element (LFE), and measuring the difference on pressure across it. Its operating principle is known as the Poiseuille Equation: L r P P Q 8 ) (4 2 1 (3-1) where Q is the volumetric flow rate, P1 is the static pressure at the inlet, P2 is the static pressure at the outlet, r is the radius of the restriction, is the (eta) absolute viscosity of the fluid, and L is the length of the restriction. Since r (radius) and L (length) ar e constant; Equation 3-1 is rewritten as: P K Q, (3-2) where K is a constant factor calcul ated from the restriction geometry, P is the differential pressure and the absolute viscosity. Viscosity of carbon dioxide at 0.1 MPa and 25 C is 149.33 gcm-1sec-1. The volumetric flow rate units ar e SLPM (standard liters per minute). The cross-angled connector between the MV, V4, T and Ve ssel 1 was designed and made of stainless steel to withsta nd high pressures. Based on the buoyancy principle, the angle connections allow any possible bubble in the system to move to the top of the equipment. This reduces / eliminates the error of excess CO2 reading in the gas meter (GM) when the system is depressurized (Figure A-14). The two way valves (V), and quick connects (QC) were ordered from Swagelok Company (Jacksonville, FL), and are made of stainless steel. The two way valves (straight ball valves) are model SS-83KF4, 1/4 end connections size an d female NPT end connection type. Their pressure rating is 41.37 MPa at 38 C (Figure A-15). The QC are also made of stainless steel, their pressure rating is 41.37 MPa at 21.11 C. The quick connect stems are model SS-QF4-S-

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45 4PF (female NPT) and SS-QF4-S-4PM (male NP T). The quick connects bodies are model SSQF4-B-4PF (female NPT) and SS-QF4-B4PM (male NPT) (Figure A-16). Vessel 1 has a maximum allowable working pres sure of 34.4 MPa at 121 C. It was made of stainless steel, its capacity is 150 ml, and its end size connectio ns are 1/4 NPT (Figure A-17). Vessel 2 has a maximum allowable working pressu re of 68.95 MPa at 22 C. Its body and cover were made of stainless steel; the o-ring of Buna N; and the cap of alloy steel. Its capacity is 55 ml, and its end size connections are 1/4 NP T (Figure A-18 and Figure A-19). The main difficulty was that the gas that remained in vessel 1 at the end of the saturation period had to bubble up to vessel 2 through valve V4, and at th e same time some liquid from vessel 2 had to flow down to vessel 1 by gravity. Because of the small inner diameter of the valve V4, this did not happen, and some gas was left trapped in vessel 1. Since this gas was under pressure, upon de-pressurization its volume e xpanded greatly, and gave false r eadings about solubility. Valve V4 was replaced with a larger diameter gate valv e (a forged stainless st eel gate valve, Figure A20, class 1500, 0.38 of opening diameter, NPT connection, model number 15GT-6L-U-G, from NEWCO/OIC Valves, Jacksonville, FL) with the expectation of easier CO2-liquid exchange between vessels 1 and 2. However, th is valve leaked. Another solution had to be found. To recirculate the liquid from the top vess el 2 to the bottom vessel 1, it was necessary to drill an opening (1/4 NPT) in the bottom of ve ssel 2 (Figure A-19) and connect it to the bottom of vessel 1. This assured that the liquid pressu re at the top would force the liquid to flow from vessel 2 to vessel 1 by gravity by the side arm, a nd therefore push any gas remaining in vessel 1 to bubble up to vessel 2 though valve V4. Therefore, it was assured that after the flow of CO2 from the bottom of vessel 1 (to reach saturation) was stopped, a ll gas remaining in vessel 1 would bubble up to vessel 2. The accuracy of the experimental solubility measurement depended

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46 on no un-dissolved gas remaining in vessel 1. Please note that at pressures where the density of CO2 becomes higher than that of the liqui d, this system will not work properly. The plastic bottle has a capacity of 5 gallons and it was made of high density polyethylene (HDPE). The opening of the bottl e was restricted by a stopper ( number 11) with two holes. One opening was for the incoming CO2 (with possible entrained liqui d), and the other was for the CO2 to go out to the gas meter. The tube attach ed in the outgoing opening was shorter, to allow just the CO2 to exit, and be measured at the GM (Figure A-21) 3.2 Preparation of the Samples 3.2.1 Distilled Water To verify that the high-pressure CO2 apparatus worked properly, it was necessary to compare its results with the l iterature values (v/v) of CO2 dissolved in pure water. The distilled water used was obtained from the building source (triple filtration). Th e volume of distilled water used was approximately 500 ml per run. 3.2.2 Fructose The D-Fructose (reagent grade) was ordered from Fisher Scientific (Fair Lawn, New Jersey; lot number 068019). The sample with 10% fructose and 90% water (200 grams of fructose in a total solu tion of 2000 ml with distilled water) corresponds to 9. 7 Brix, and the sample with 5% fructose and 95% water (100 gram s of fructose in a to tal solution of 2000 ml with distilled water) corresponds to 4.8 Brix. The fructose was measured using an analytical balance (model PG603-S), Mettle r Toledo. The Brix data wa s measured with a portable refractometer, model 13-946-20, from Fisher. 3.2.3 Malic Acid The malic acid (food grade, C4H2O5) in powder was ordered from Presque Isle Wine Cellars (Philadelphia, PA). The sample with 0.5% malic acid and 99.5% water (10 grams of

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47 malic acid in a total solution of 2000 ml with distilled water) had a pH of 2.35. The sample with 5% malic acid and 95% water (100 grams of ma lic acid in a total so lution of 2000 ml with distilled water) had a pH of 1.79. The pH va lues were measured using an expandable ion analyzer (EA 920, model Q027A), from Orion Research Incorporated (Cambridge, MA). 3.3 Experimental Procedure 3.3.1 Preliminary Procedure First, the HE was turned on for about 20 minutes before starting the HPP (HE had to reach approximately -15 C). During th at period, the cooling jacket of the HPP was getting covered with a thin layer of ice. At the same time, th e high precision water bath was turned on, to start warming up the water inside of the cooler. Sin ce the warming process could take about 1.5 to 2 hours, batches with water (approxim ately 2 liters per batch) were heated in the microwave and added to the cooler, to speed up the process. In the stationary part of the apparatus, it was important to ve rify that V1 (that corresponds to the tank) and V2 were closed, TV1 was turned to the right and TV2 was turned to the left. The previous procedure wa s performed to prevent CO2 to escape out of the apparatus, at the time to open V1; therefore, to avoid gas inhalation. Then, before su bmerging the mobile part in the cooler, it was important to confirm that V5 a nd MV were closed, and that V3 andV4 were opened. TV3 should be turned to the left or facing up and the BPR should be slightly open (clockwise), to hold a back-pressure of approxi mately 2.76 MPa. Also, QC4, QC5, QC6 and QC7 should be already connected (Figure B-1). When the complete apparatus was connected (s tationary and mobile parts), and the plastic tubes coming from TV3, BPR and GM were pl aced inside of the fume hood (FH), it was necessary to ensure that between QC6 and GM there was only CO2. For that reason, V1 was slightly opened until approximately 2.76 MPa wa s reached (pressure was confirmed in PG2).

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48 Next, V2 was slowly opened and the BPR was regul ated to increase the pressure up to 1.38 MPa (pressure was confirmed in PG3); then, V2 was closed. The GM was zeroed and cleared (Alicat Scientific 2007), and the stopper wa s verified to be in place. Then, the MV was opened slowly and the CO2 coming out of the system was measured unt il read about 0.250 st andard liters /min, MV was closed. The system was depressurized, a nd when the pressure was 0 psig (pressure read at PG3), QC3 was disconnected. 3.3.2 Pumping the Liquid Sample into the System Before starting, QC8 should be disconnected and a plastic tube s hould be connected to QC8 stem. The sample was held in a small cont ainer of approximately 2 liters. By using the peristaltic pump, the sample was pumped through a plastic tube to the stem connection of QC3. The liquid was pumped until both vessel 1 and 2 were filled. When the system was full, water started coming from the plastic tube connected to the QC8 stem (Figure B-2). The peristaltic pump was turned off. The plastic tube in th e QC3 stem was removed, and the QC3 stem was quickly connected to its QC3 body. The plastic tube in the QC8 stem was also removed, and QC8 body was connected to its corresponding stem. 3.3.3 Carbonating the Sample Before starting the carbonation process, it was necessary to verify that the temperature inside of the system was the desired temp erature. Then, V1 was opened until reaching approximately 2.76 MPa (pressure was confirmed in PG2); then, it was closed. Next, V2 was opened very slowly and the BPR was turned to incr ease the pressure, at the same time. The flow rate of the CO2 with water coming from the BPR should be very slow. The previous step was performed to ensure that the system was filled with CO2, and that there were no other gases. The V2 was closed, the BPR was regulated to hold approximately 6.89 MPa and the HPP was set to about 77% of flow rate using the micrometers (Figure A-5).

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49 V1 was opened again and the HPP was turned on. The variable autotransformer was also turned on. V2 was opened very slowly and, if some gas with water was coming out of the system, the BPR was opened to increase th e pressure, until around 1.03-1.38 MPa above the desired pressure was reache d. At that point, the CO2 flow rate of the HPP was reduced to about 37%, by turning clockwise the micrometers. The HPP remained on during 1 hour and 10 minutes to reach saturation of CO2 in the liquid. During this time, CO2 continuously bubbled through the liquid (entering from the bottom of vessel 1, and exiting off the top of vessel 2 through BPR), and saturated the liquid with CO2. 3.3.4 Measuring Solubility After the saturation time, with CO2 passing through the system, V2 was closed. Immediately after this, the HPP was turned off; and V1 was closed. Next, TV3 was very slowly turned to the right (clockwise); and V2 was opened very slowly, while it was warmed up with some hot water from the cooler. The variable tr ansformer was turned off. When the pressure was 0 MPa (pressure was confirmed in PG2), QC3 and QC4 were disconnected. Then, the mobile part of the equipment was shaken from si de to side, while still immersed in water (around 1 minute per side) and up/down (20 times). The purpose of this was to loosen any CO2 bubbles attached to the inner surfaces of vessel 1, and allow them to bubble up to vessel 2. This procedure took around 3-5 minutes. If the pressure (re ad in PG3) was just at the desired level, first V3 and then V4 were closed. However, if the pressure was still higher than desired, it was necessary to adjust it to the desired level. The variable transformer was turned on a nd the BPR was turned very slowly, to depressurize the top part of V4. The GM was again zeroed and cleared. At this point, it was certain that there was only liquid phase in vesse l 1, and the dissolved CO2 in that liquid. The MV was slightly opened to start depressurizati on, and this continued

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50 until all the dissolved gas came out of the system and the pressure read in PG3 was 0 MPa. The maximum flow rate allowed in the GM was 0.350 slpm (standard liters per minute), to prevent that the system cooled by expansion of CO2. To expand and read all the CO2 that was in solution took around 20-25 minutes. When th e volumetric flow rate in the GM was 0 slpm, QC8 was disconnected and the rest of the mobile part was removed from the ice cooler. As soon as the equipment was removed, QC5 was disconnected under a small container of 2 liters of volume, to allow the liquid coming out of vessel 2 to be collected. Then QC7 was disconnected. All the end connections were dr ied. QC6 was disconnected and all the liquid in the plastic tube and in the bottle was collected in a containe r of 5 liters (Figure B-3). V4 was opened inside of the same container of 5 liters (Figure B-4), subsequently TV3 was turned to the left and all the liquid from vessel 1 was collected. At the e nd, the liquid collected was measured using a graduated cylinder (Figure B-5). This amount wa s the basis of the solu bility calculations. To convert the volumetric flow (sta ndard liters) to mass flow of CO2, it was necessary to divided it by 22.4 sltmol-1, and then, multiply it by 44.01 g mol-1. Solubility was calculated as the rate of grams of CO2 in 100 grams of liquid. 3.3.5 Cleaning the Apparatus The mobile part of the apparatus was sepa rated by disconnecting th e QCs. The various parts were washed in a solution of water with so ap. Then, the parts were rinsed with abundant water, and consequently, they were rinsed with distilled water. 3.4 Experimental Design The carbonation process variables were pressu re, temperature and composition. The pH and Brix of the model liquid samples were al so measured. Experimental conditions were decided with the goal of meas uring the solubility of CO2 above its critical point (temperature 31 C and pressure 7.4 MPa) and below high CO2 density levels at high pressures. The first

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51 phase of the experiment consiste d of testing the high pressure CO2 solubility apparatus, and comparing the experimental solubility values of CO2 in pure water with literature data. Two levels of pressure were used: 7.58 MPa (74.85 atm) and 10.34 MPa (102.07 atm) at 40 C. Carbon dioxide density at those pressures and te mperature was approximately 0.2 g/ml and 0.65 g/ml, respectively (Clifford and Williams 2000). The second phase of the experiment consis ted in determining the solubility of CO2 in a model liquid food of known composition (different c oncentrations of fructose and malic acid). Two levels of fructose concentration (5% and 10% w/v), and two levels of malic acid concentration (0.5% and 5% w/v) were used. The two concentrati ons of fructose and malic acid were selected according to the typical usage leve ls of functional constituents in soft drinks (Taylor 2005). Every solubility measurement wa s performed in duplicate. Table 3-1 shows the different levels of each carbonation variable. Table 3-1. Different levels of each vari able in the two phases of the experiment. First Phase Treatment Temperature Pressure Model Liquid 1 40 C 7.58 MPa Distilled water 2 40 C 10.34 MPa Distilled water Second Phase Temperature Pressure Model Liquid 3 40 C 7.58 MPa Fructose 5 % m/v 4 40 C 7.58 MPa Fructose 10 %m/v 5 40 C 10.34 MPa Fructose 5 % m/v 6 40 C 10.34 MPa Fructose 10 % m/v 7 40 C 7.58 MPa Malic Acid 0.5 % m/v 8 40 C 7.58 MPa Malic Acid 5 % m/v 9 40 C 10.34 MPa Malic Acid 0.5 % m/v 10 40 C 10.34 MPa Malic Acid 5 % m/v 3.5 Data Analysis Statistical analysis consisted of performing an analysis of variance (ANOVA) and mean separation to evaluate the differe nce between solubilities at the di fferent conditions of pressure and composition, at 40 C. The analysis was di vided into two phases. In the first phase

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52 significant differences between the experimental a nd literature values were evaluated. The null hypothesis (Ho) was: At least one of the experimental mean results is different from literature results. The alternative hypothesis (H1) was: Means of the experimental results are equal to literature results. The second phase consisted in determining if th e reduction in the solubility of CO2 in solution with pure water was significant, by in creasing the solute (fructose or malic acid) concentration in the liqui d. The null hypothesis (H0) was: Means of the solute experimental results are equal to the expe rimental mean reference. The alternative hypothesis (H1) was: There was significant difference between solute experi mental results and the mean of experimental reference. The statistical data analysis wa s conducted by performing a Least Square Mean (LSMean) analysis and the Least Significant Differen ces (LSD) test, with P< 0.05. The software programs used were STATISTICA 97 and SAS 9.1.

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53 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Solubility of Carbon Dioxide in Distilled Water To demonstrate the accuracy and precision of the high-pressure CO2 solubility apparatus, it was necessary to compare the experimental results of CO2 solubility in pure water with the literature values. The CO2 solubility chart developed by Dodds and others (1956) was used as reference. Also the chemical/ther modynamic simulation program Aspen PlusTM 12.1 (Schweiss 1997) was used as a simulation reference. The CO2 solubility chart (F igure 2-8) shows the solubility of CO2 in pure water (kg/100 kg of water), at pressures from 1 to 70.93 MPa, and at temperatures from 0 to 120 C. Accord ing to this chart, solubility of CO2 at 110.34 MPa and 40 C is 5.68 kg/100 kg of pure water. At condi tions of 7.58 MPa and 40 C, solubility of CO2 is 5.00 kg/100 kg of water. The Aspen PlusTM 12.1 was designed to pr edict the behavior of chemical systems (Schweiss 1997). According to th is program, at a constant temperature of 40 C, solubilities of CO2 in pure water at 7.58 MPa and 10.34 MPa of pressures are 4.6 and 5.7 kg/100 kg of liquid, respectively. The experimental results of solubility of CO2 in pure water obtained from the apparatus, at a constant temperature of 40 C, are displayed in Table 4-1. Table 4-1. Experimental re sults of solubility of CO2 in pure water. Pressure (MPa) Distilled water (g) CO2 (slt1) CO2 (g) Solubility (g/100g2) Mean (g/100g2) St. Dev. Literature (g/100g2) Difference (g/100g2) 7.58 171 4.063 7.98 4.67 7.58 168 4.176 8.20 4.88 4.77 0.15 5.00 0.23 10.34 169 4.625 9.08 5.38 10.34 169 4.863 9.55 5.65 5.51 0.20 5.68 0.17 1Standard liters. 2Grams of CO2 per 100 g of pure water. 3Standard Deviation in g of CO2 per 100 g of pure water. According to Table 4-1, the mean solubility of CO2 in water at pressure of 7.58 MPa is 0.23 g/100 g lower than the literature. Also, at pr essure of 10.34 MPa, the mean of solubility of

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54 CO2 is 0.17 g/100g below the reference. Those diffe rences could be attributed to the time of saturation with CO2. If the time of passing CO2 through the system increases, solubility of CO2 in liquids would increase until it reaches saturation. This has been demonstrated in preliminary experiments (data not shown). According to stat istical analysis using Statistica 97 and SAS 9.1 programs, those differences were not significantly differe nt. Tables 4-2, 43 and 4-4 show the summary of the analysis of variance (ANOVA) summary of least square mean (LS Mean) analysis and the least significant diff erence (LSD) tests, respectively. Table 4-2. Summary of the analysis of variance (ANOVA) of the solubility of CO2 in pure water. Source DF Sum of squares Mean square F value Pr > F Multiple R Multiple R2 Adjusted R2 Model 1 0.5462 0.5462 17.77 0.0519 0.9480 0.8988 0.8482 Error 2 0.0614 0.0307 Corrected Total 3 0.6076 Table 4-3. Summary of the least square mean (LS Mean) analysis of the solubility of CO2 in pure water. Pressure (MPa) Solubility Mean (g/100g*) Solubility Standard Error Solubility -95.00% Solubility +95.00% Standard deviation Coefficient of variation 7.58 4.77 0.1239 4.24 5.30 0.15 0.03 10.34 5.51 0.1239 4.98 6.05 0.20 0.03 *Grams of CO2 per 100 g of pure water. Table 4-4. P-values from the least significant difference (LSD) test between experimental mean data and literature data of solubility of CO2 in pure water. Experimental data Literature data Treatments 7.58 MPa 10.34 MPa 7.58 MPa 10.34 MPa 7.58 MPa 0.0519 0.4045 0.0499 Experimental data 10.34 MPa 0.0519 0.1390 0.4776 7.58 MPa 0.4045 0.1390 0.1059 Literature data 10.34 MPa 0.0499 0.4776 0.1059 Note: P-values in italic font are lower than = 0.05; thus, the correspondent groups are significantly different. Table 4-2 shows that the null hypothesis (Ho) was not rejected, due to a P-value higher than = 0.05 (Pr>F = 0.0519); therefore, at least on the experimental mean results is different from the literature. Table 4-4 shows significant difference between the pressure of 7.58 MPa,

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55 from experimental mean data, and the pressure of 10.34 MPa, from literature results. However, within the same conditions of pressure, th ere was no significant difference between the experimental mean results and the literature data; differential values were higher than = 0.05, and the average coefficient of variation level was 0.03. In addition, the confidence interval for pressures of 7.58 and 10.34 MPa were 5.51.76 and 4.77.37 g of CO2/100g of liquid, respectively. The corresponding literature values for each pressure fits within the confidence interval. Therefore, th e experimental results, obtained fr om the high-pressure carbon dioxide solubility apparatus are statisti cally acceptable and the apparatus is a functional instrument to measure CO2 solubility in liquids at th e conditions of pressure and temperature tested in this study. 4.2 Solubility of Carbon Dioxide in Fructose Solutions Solubility of CO2 in fructose solutions was measured at a constant temperature of 40 C and at two levels of pressures (7.58 and 10.34 MP a). Table 4-5 shows the experimental results of CO2 solubility in the different fructose so lution concentrations (5% and 10%). Table 4-5. Experime ntal results of CO2 solubility in solutions with different concentration of fructose. Pressure (MPa) Fructose solution (g) CO2 (slt1) CO2 (g) Solubility (g/100g2) Mean (g/100g2) St. dev3 DW4 (g/100g2) Difference5 (g/100g2) 5% Fructose Concentration 7.58 169 3.983 7.82 4.63 7.58 172 3.913 7.69 4.47 4.55 0.11 4.77 0.23 10.34 172 4.102 8.06 4.68 10.34 170 4.228 8.31 4.89 4.78 0.14 5.51 0.73 10 % Fructose Concentration 7.58 170 3.608 7.09 4.17 7.58 172 3.563 7.00 4.07 4.12 0.07 4.77 0.66 10.34 170 3.523 6.92 4.07 10.34 172 3.774 7.41 4.31 4.19 0.17 5.51 1.32 1Standard liters. 2Grams of CO2 per 100 grams of solution. 3Standard Deviation in grams of CO2 per 100 grams of solution. 4Mean solubility of CO2 in distilled water. 5Differences between DW and Mean.

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56 According to Table 4-5, by increasing the fructo se concentration in a liquid, solubility of carbon dioxide decreases. Descoins and others (2004) stated that at conditions of 6 atm of pressure and at pH of 3.1 an increment of the concentration of sugar (0 10 and 40 g/1 of sugar) yields a decrease of the av erage level of dissolved CO2 due to different orientation of the water molecules around the solute particles; therefore, water is less free to interact with other compounds, in this case CO2. Figure 4-1 shows the change in solubility of CO2 in different concentrations of fructose in the solution. Th e 0% of fructose concentration is represented by solubility of CO2 in pure water. Pressure 7.58 MPa Pressure 10.34 MPa 0510Concentration (g of fructose/100g of distilled water) 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Solubility (g of CO2/100g of solution) Figure 4-1. Solubility of CO2 versus concentration of fructose solutions, at 7.58 and 10.34 MPa, and at a constant temperature: 40 C. Ver tical bars denote 0.95 c onfidence intervals. Figure 4-1 shows the solubility of CO2 at 7.58 MPa (blue line), and at 10.34 MPa (no continue red line). At 7.58 MPa, the mean solubility of CO2 in pure water (0% of fructose) did

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57 not decrease significantly at 5% of fructose; nevert heless, at 10% the difference is noticeable. At 10.34 MPa, the mean solubility of CO2 in pure water decreased significantly while concentration of fructose in the solution increased to 5% a nd to 10%. Tables 4-6, 4-7 and 4-8 display the analysis of variance (ANOVA), the LS mean an alysis and the LSD te st, respectively. Table 4-6. Analysis of varian ce (ANOVA) of solubility of CO2 in fructose solutions of different concentrations. Source DF Sum of squares Mean square F value Pr > F Multiple R Multiple R2 Adjusted R2 Model 5 2.5671 0.5134 24.05 0.0007 0.9759 0.9524 0.9128 Error 6 0.1281 0.0213 Corrected Total 11 2.6952 Table 4-7. Least Square (LS) M ean analysis of solubility of CO2 in fructose solutions of different concentrations. Pressure (MPa) Fructose (%) Solubility mean (g/100g1) Solubility standard error Solubility -95.00% Solubility +95.00% Standard deviation Coefficient of variation 7.58 5 4.77 0.1158 4.15 4.80 0.11 0.02 10.34 5 5.47 0.1158 5.15 5.80 0.14 0.03 7.58 10 4.47 0.1158 4.14 4.79 0.07 0.02 10.34 10 4.68 0.1158 4.36 5.00 0.17 0.04 1Grams of CO2 per 100 g of fructose solution. Table 4-8. P-values from the least Significan t Difference (LSD) test of solubility of CO2 in fructose solutions of different concentrations. 7.58 MPa 10.34 MPa Treatments 0 % 5 % 10 % 0 % 5 % 10 % 0 % 0.1732 0.0042 0.0023 0.9476 0.0071 5 % 0.1732 0.0258 0.0006 0.1577 0.0495 7.58 MPa 10 % 0.0042 0.0258 0.0001 0.0039 0.6426 0 % 0.0023 0.0006 0.0001 0.0025 0.0001 5 % 0.9476 0.1577 0.0039 0.0025 0.0066 10.34 MPa 10 % 0.0071 0.0495 0.6426 0.0001 0.0066 Note: Values in italic font are less than = 0.05; thus, the correspondent groups are significantly different. In the ANOVA (Table 4-6), the p-value denotes (0 .0007) that at least one of the treatments was significant difference between one or mo re treatments (0, 5 and 10 % of fructose concentration). In the LS mean analysis (Tab le 4-7), the maximum coefficient of variation (0.04) reveals that the experimental procedure has an acceptable error, below 5%. The LSD test

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58 showed the mean solubilities that were statisti cally different. According to Table 4-8, the fructose concentration of 5% (4.8 Brix) decreased significantly the solubility of CO2 at pressure of 10.34 MPa; however, at 7.58 MP a solubility did not change considerably. The 10% (9.7 Brix) concentration of fructose reduced considerably the solubility of CO2 in the solution, at both pressures (7.58 and 10.34 MPa). 4.3 Solubility of Carbon Dioxide in Malic Acid Solutions Solubility of CO2 in malic acid solutions was measured at a constant temperature of 40 C and at pressures of 7.58 and 10.34 MPa. Figure 42 shows the effect of increasing the malic acid concentration of a soluti on on the solubility of CO2. Pressure at 7.58 MPa Pressure at 10.34 MPa 012345Concentration (g of malic acid/100g of distilled water) 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Solubility (g of CO2/100g of solution) Figure 4-2. Graph of solubility of carbon dioxide versus concentr ation of malic acid solutions, at two different pressures: 7.58 and 10.34 MPa, and at a constant temperature: 40 C. Vertical bars denote 0.95 confidence intervals.

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59 Table 4.-9 shows the experimental data. The results of the ANOVA and LS mean analysis, and LSD test are shown in Tables 4-10, 4-11 and 412. According to Table 4-10, at least one of the treatments (0, 0.5 and 5 % malic acid concentr ation, at both pressu res) is significantly different because the p-value (Pr>F = 0.0007) is lower than = 0.05. In addition, the maximum coefficient of variation was 0.02 that shows a good experimental performance (Table 4-11). Table 4-9. Experimental data of CO2 solubility in solutions with different concentration of malic acid. Pressure (MPa) Malic acid solution (g) CO2 (slt1) CO2 (g) Solubility (g/100g2) Mean (g/100g2) St Dev3 DW4 (g/100g2) Difference5 (g/100g2) 0.5% Malic Acid Concentration 7.58 172 3.933 7.73 4.49 7.58 172 3.902 7.66 4.46 4.47 0.03 4.77 0.30 10.34 169 4.191 8.23 4.84 10.34 169 4.315 8.48 4.96 4.90 0.08 5.51 0.02 5% Malic Acid Concentration 7.58 172 3.900 7.66 4.45 7.58 172 3.902 7.70 4.48 4.47 0.02 4.77 0.31 10.34 171 4.129 8.11 4.69 10.34 172 4.237 8.32 4.84 4.79 0.07 5.51 0.77 1Standard liters. 2Grams of CO2 per 100 grams of solution. 3Standard Deviation in grams of CO2 per 100 grams of solution. 4Mean solubility of CO2 in distilled water. 5Differences between DW and Mean. Table 4-10. Analysis of variance (ANOVA) of solubility of carbon dioxide in malic acid solutions of different concentrations. Source DF Sum of squares Mean Square F Value Pr > F Multiple R Multiple R2 Adjusted R2 Model 5 1.4727 0.2945 24.06 0.0007 0.9760 0.9525 0.9129 Error 6 0.0734 0.0122 Corrected Total 11 1.5461 Table 4-11. Least Square (LS) Mean analysis of solubility of car bon dioxide in malic acid solutions of different concentrations. Pressure (MPa) Malic Acid (%) Solubility Mean (g/100g1) Solubility Standard Error Solubility -95.00% Solubility +95.00% Standard deviation Coefficient of variation 7.58 0.5 4.47 0.0387 4.36 4.58 0.03 0.01 10.34 0.5 4.90 0.0387 4.79 5.00 0.08 0.02 7.58 5 4.47 0.0387 4.35 4.57 0.02 0.00 10.34 5 4.68 0.0387 4.68 4.90 0.07 0.01 1Grams of CO2 per 100 g of malic acid solution.

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60 Table 4-12. P-values from th e least Significant Difference (LSD ) test of solubility of carbon dioxide in malic acid solutions of different concentrations. 7.58 MPa 10.34 MPa Treatments 0 % 0.5 % 5 % 0 % 0.5 % 5 % 0 % 0.0346 0.0312 0.0005 0.3028 0.8900 0.5 % 0.0346 0.9408 0.0001 0.0085 0.0286 7.58 MPa 5 % 0.0312 0.9408 0.0001 0.0078 0.0259 0 % 0.0005 0.0001 0.0001 0.0014 0.0006 0.5 % 0.3028 0.0085 0.0078 0.0014 0.3638 10.34 MPa 5 % 0.8900 0.0286 0.0259 0.0006 0.3638 Note: Values in italic font are less than = 0.05; thus, the correspondent groups are significantly different. Table 4-12 shows the difference between treatmen ts. At pressure of 7.58 MPa, there was significant difference between solubility in water (0 % malic acid concentration) and solubility in 5 % of malic acid concentration; however ther e was no significant difference between 0.5% and 5% of malic acid concentr ation. At pressure of 10.34 MPa, solubility in water was significantly different than 0.5% and 5% of malic acid c oncentrations; ho wever there was no significant difference between 0.5% and 5% of malic acid c oncentrations, as in 7.58 MPa of pressure. According to Meyssami and others (1991), the pr esence of acids (e.g. ascorbic acid, citric acid, etc.) contributes to the reduction of solubility of CO2 in the solution. They reported a decrease in pH at pressures of 0 and 5.5 MPa and temperature of 32, 37 and 42 oC, in comparison with pH of pure water. At those conditions of pressure and temperature, the pH dropped in the binary waterCO2 system from about 5.68 to 3.19 (difference was approximately 2.5 units), while at the ternary system (water-acidCO2), the pH dropped from about 3.74 to 3.13 (difference was around 0.61 units). Due to lack of experimental studies in the area of measurement of CO2 solubility in model and in real liquid foods, no more references ar e cited to compare with current experimental results.

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61 CHAPTER 5 SUMMARY, CONCLUSIONS AND SUGG ESTIONS FOR FUTURE STUDY 5.1 Summary The use of CO2 in Dense Phase Carbon Dioxide (DPCD) technology is not optimized; excess of CO2 is used to reach saturation, due to unknown solubility of CO2 in the liquid foods. The high pressure CO2 solubility apparatus was designed and built to measure CO2 solubility in liquid foods at high pressures and different temperatures. The appa ratus was tested at a constant temperature of 40 oC, and at pressures of 7.58 and 10.34 MPa. Solubility values were reported in grams of CO2 per 100 grams of liquid. The average expe rimental error (coeffi cient of variation) was 0.02%. Adding fructose to a liquid solubility of CO2 decreased significantly. At pressure of 7.58 MPa of, solubility of CO2 in water was 4.77 .15 g/100 g of distilled water. When the concentrations of fructose in the solu tion were 5 and 10%, solubilities of CO2 in the solutions were 4.55.11 and 4.12.07, respectively. At pressure of 10.34 MPa, the CO2 in solution with water was 5.51.20 g/100 g of distilled water, and when the amount of fructose in the solution increase to 5 and 10 g/100 g of solution, solubilities of CO2 were 4.78.14 and 4.19.17, respectively. Adding malic acid to a solution al so decreased solubility of CO2. The reference used to compare was the experimental solubility of CO2 in water. At 7.58 MPa of pressure, the amount of CO2 dissolved in the solutions of 0.5 and 5% of malic acid concentration was 4.47.03 and 4.47.02 g/100 g of solution. At 10. 34 MPa of pressure, solubilities of CO2 in solution with 0.5 and 5% of malic acid concentration were 4.90.08 and 4.79.07 g/100 g of solution.

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62 5.2 Conclusions It is possible to conclude that the apparatus is a functional, accurate and precise instrument to measure solubility of CO2 in model liquid foods. The signif icance of this study is that the experimental apparatus could offer an advantage to the food industry, primarily to soft drink industry, and to the DPCD users, by eliminating the need to use excess CO2 and therefore reducing the production cost. This apparatus could be used for the measurement of CO2 in real liquid foods. Versatility of the apparatus allows carbonation of various samples, at conditi ons above critical temperature and pressure, and below density of 0.9 gml-1. In addition, the side connection between Vessel 1 and Vessel 2 ensures a correct liquid circulation and an appropriate measurement of solubility. 5.3 Suggestions for Future Study Future works in the area of DPCD technology should focus on first experimentally determining and then thermodynamically pr edicting the theoretical level of CO2 to use, at any given pressure and temperature, and for any li quid food of known compos ition. More studies on the effect of temperatur e on the solubility of CO2 in solutions with diffe rent concentrations of fructose and malic acid should be performed. This experimental apparatus is a valuable instrument to measure CO2 solubility in liquid food to reduce the need to use excess CO2 to reach saturation, at the conditions tested. Moreover, experiments under pres sures higher than 10.34 MPa coul d be performed to determine the effect on density changes of CO2 on the accuracy of the apparatus.

PAGE 63

63 APPENDIX A DESIGN OF THE HIGH PRESSURE CARB ON DIOXIDE SOLUBILITY APPARATUS Figure A-1. Pictures of the high pressu re carbon dioxide solu bility apparatus.

PAGE 64

64 Figure A-2. Picture of the m obile part of the apparatus. Figure A-3. Picture of the teflon ta pe used in every male connection. Figure A-4. Picture of the CO2 tank connections. The carbon di oxide tank, the three-way valve 1 (TV1), and the pressure gauge 1 (PG1).

PAGE 65

65 A B Figure A-5. Pictures of the high pressure pump ( HHP). A) From the front, micrometers. B) From the back, cooling jacket. Figure A-6. Picture of the heat exchanger (HE).

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66 Figure A-7. Back Pressure Regulator (BPR) diagram (Tesco m Industrial Controls 2006). A B Figure A-8. Pictures of the back pressure regu lator (BPR) used in the machine. A) From the side. B) From the top.

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67 Figure A-9. Picture of the heating tape (ar ound the BPR) and the variable autotransformer. Figure A-10. Picture of the Hart Scientific high precision water bath and the Masterflex peristaltic pump.

PAGE 68

68 Figure A-11. Picture of the thermo couple used inside the vessel 1. Figure A-12. Picture of the metering valve (MV). Figure A-13. Picture of the gas meter (GM).

PAGE 69

69 Figure A-14. Picture and diagram of the cross angles connector. Figure A-15. Picture of the two way valve (V). Figure A-16. Picture of th e quick connect stem (female NPT) and body (male NPT).

PAGE 70

70 A B Figure A-17. Vessel 1. A) Pi cture of vessel 1. B) Diag ram of vessel 1 (Milton Roy 1986). Figure A-18. Diagram of vessel 2 (Milton Roy 1986).

PAGE 71

71 Figure A-19. Picture of vessel 2 with the hole at the bottom. Figure A-20. Picture of the forg ed stainless steel gate valve.

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72 A B Figure A-21. Picture of the bottle and the stopper. A) Picture of th e 5 gallons bottle. B) Picture of the stopper in the opening of the bottle.

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73 APPENDIX B THE HIGH PRESSURE CARBON DIOXIDE SOLUBILITY APPARATUS PROCEDURES Figure B-1. Picture of the mobile part of th e equipment, before immersion into the cooler. Figure B-2. Pictures showi ng the sample being pumped.

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74 Figure B-3. Picture of disconnecting QC6. Figure B-4. Picture showing when V4 was opened to collect the liquid sample.

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75 Figure B-5. Picture showing the measur ement of the liquid sample collected.

PAGE 76

76 LIST OF REFERENCES Alicat Scientific. 2007. Opera ting manual: 16 series mass and volumetric precision gas flow meter. Tucson, AZ: Alicat Scientific. Available from: http://www.alicatscientif ic.com/documents/manuals/Gas_Flow_Meter_Manual.pdf Accessed Apr 2006. Anderson GK. 2002. Solubility of carbon dioxide in water under incipient clathrate formation conditions. J Chem Eng 47: 219-22. Arreola AG, Balaban MO, Marshall MR, Peplow AJ, Wei CI, Cornell JA. 1991. Supercritical carbon dioxide effects on some quality attributes of single strength orange juice. J Food Sci 56: 1030-33. Akinfiev NM, Diamond LW. 2003. Thermodynamic description of aqueous nonelectrolytes at infinite dilution over a wide range of state parameters. Geochim Cosmochim Acta 4 (67): 613-29. Balaban MO, Arreola AG, Marshall MR, Peplow AJ, Wei CI, Cornell JA. 1991. Inactivation of pectinesterase in orange juice by supercritical carbon di oxide. J Food Sci 56: 743-46. Bamberger A, Sieder G, Maurer G. 2000. High-pressure (vapor + liquid) equilibrium in binary mixtures of (carbon dioxide + water or acetic acid) at temp eratures from 313 to 353 K. J Supercrit Fluids 17: 97-110. Battino R, Clever HL. 1965. The solubility of gases in liquids. Chem Rev 66: 395-463. Bessley PM. 1990. Carbohydrate sugars. In: Mitche ll AJ, editor. Formulation and production of carbonated soft drinks. Glasgow and L ondon: Blackie and Son Ltd. p 37-55. Buhler E, Dobrynin AV, DeSimone JM, Rubins tein M. 1998. Light-scattering study of diblock copolymers in supercritical carbon dioxide: CO2 density-induced micellization transition. J. of Macromol 31: 7347-55. Clifford AA. 1999. Supercritical fluid extr action. In: Letcher TM, editor. Chemical thermodynamics. Paris, France: Bl ackwell Science Ltd. p 95-104. Clifford AA, Williams JR. 2000. Introduction to supe rcritical fluids and their applications. In: Clifford AA, Williams JR, editors. Supercriti cal Fluid: Methods and Protocols. Totowa, NJ: Humana Press Inc. p 1-16. Costa-Gomez MF. 2007. Low-pressure solubility and thermodynamics of solvation of carbon dioxide, ethane, and hydrogen in 1-hexil-3methylimidazolium bis (trifluoromethylsulfonyl) amide between temperatures o 283 K and 343 K. J Cehm Eng 52: 472-5. Dagan GF, Balaban MO. 2006. Pasteurization of beer by a continuous dense-phase CO2 system J Food Sci 3(71): E164-9.

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77 Damar S, Balaban MO. 2006. Review of dense phase carbon dioxide t echnology: microbial and enzyme inactivation, and effects on food quality. J Food Sc 71: R1-R11. Del Pozo-Insfran D, Balaban MO, Talcott ST. 2006a. Enhancing the retention of phytochemicals and organoleptic attributes in muscadine grape juice through a combined approach between dense phase CO2 processing and copigmentation. J Agric Food Chem 54: 670512. Del Pozo-Insfran D, Balaban MO, Talcott ST 2006b. Microbial stability, phytochemical retention, and organoleptic at tributes of dense phase CO2 processed muscadine grape juice. J Agric Food Chem 54: 5468-73. Descoins C, Mathlouthi M, Moual M, Hennequi n J. 2006. Carbonation monitoring of beverage in a laboratory scale unit with online measurement of dissolved CO2. J Food Chem 95: 541-53. Dessirier JM, Simons CT, Carstens M, OMa hony M, Carstens E. 2000. Psychophysical and neurobiological evidence that the oral sens ation elicited by carbonated water is of chemogenic origin. J Chem Senses 25: 277-284. Diamond LW, Akinfiev NN. 2003. Solubility of CO2 in water from 1.5 to 100 C and from 0.1 to 100 MPa: evaluation of lite rature data and thermodyna mic modeling. Fluid Phase Equilibria 208: 265 Dodds WS, Stutzman LF, Sollami BJ. 1956. Carbon di oxide solubility in wa ter. Ind Eng Chem 1 (1): 92-95. Eastoe J, Bayazit Z, Martel S, Steyler D, H eenan R. 1996. Droplet structure in a water-in-CO2 microemulsion. Langmuir. 12 (6): 1423-24. Ferdows M, Ota M. 2005. Molecular simulation study for CO2 clathrate hydrate. J Chem Eng Technol 28 (2): 168-73. Fraser D. 1951. Bursting bacteria by re lease of gas pressu re. Nature 167: 33-4 Gunes G, Blum LK, Hotchkiss JH. 2005. Inactivation of yeasts in grape juice using a continuous dense phase carbon dioxide processing system. J Sci Food Agric 85: 2362-68. Harris JG, Yung KH. 1995. Carbon dioxides liqu id-vapor coexistence curve and critical properties as predicted by a simple mo lecular model. J Phys Chem 99: 12021-24. Ishikawa H, Shimoda M, Kawano T, Osajima Y. 1995. Inactivation of enzymes in an aqueous solution by micro-bubbles of supercritical ca rbon dioxide. J Biosci Biotech Biochem 59 (4): 628-31. Ji Y, Ji X, Feng X, Liu C, Lu L, Lu X. 2007. Progress in the study on the phase equilibria of the CO2-H2O and CO2-H2O-NaCl systems. J Ch em Eng 15 (3): 439-48.

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78 Jdecke M, Prez-Salado A, Maurer G. 2007. Expe rimental investigation of the solubility of CO2 in (acetone + water). J Chem Eng 52: 1003-9. Kappes SM, Schmidt SJ, Lee SY. 2007. Relations hip between physical properties and sensory attributes of carbonated bevera ges. J Food Sci 72: S1-S11. Karukstis KK, Van Hecke GR. 2000. Chemistry c onnections: the chemical basis of everyday phenomena. San Diego, CA. Academic Press. p 11-28. Kincal D, Hill WS, Balaban MO, Portier KM, Wei CI, Marshall MP. 2005. A continuous high pressure carbon dioxide system for microbial reduction in orange juice. J Food Sci 5(70): M249-54. King MB, Mubarak A, Kim JD, Bott TR. 1992. The mutual solubilities of water with supercritical and liquid carbon dioxide. J Supercrit Fluids 5: 296-302. Koutchma T, Murakami E. 2005. Effect of carbo n dioxide and pressure processing on microbial and enzyme inactivation in food and beverages. Chicago, IL: National Center Food Safety and Technology. Available from: http://www.foodtechinternational.com/papers /effectofcarbondioxide.htm Accessed Aug 2007. Krichevsky IR, Kasarnovsky JS. 1935. Thermodynami cal calculation of solubilities of nitrogen and hydrogen in water at high pressure s. J American Chem Soc 57: 2168-71. Levelt JMH. 2000. Supercritical fluids: their properties and applications. In: Kiran E, Debenedetti PG, Peters C, editors. Supercri tical fluids: fundament als and applications. Dordrecht, The Netherlands: Kluwer Academic Publishers. p 1-29. Longhi J. 2004. Phase equilibria in the system CO2-H2O I: new equilibrium relations at low temperatures. Geochem Cosmochem 69 (3): 529-39. Marteau, Obriot J, Tufeu R. 1995. Experimental determination of vapor liquid equilibria of CO2 + limonene and CO2 + Citral mixtures. J Supercrit Fluids 8: 20-24. McHugh MA, Krukonis VJ. 1994. Superc ritical fluid extraction: prin ciples and practice. Newton, MA: Butterworth-Heinemann. p 27-84. Meyssami B, Balaban MO, Teixeira AA. 1991. Pr ediction of pH in model systems pressurized with carbon dioxide. Biot ech Program 8: 149-54. Milton Roy. 1986. Standard system options. In : Milton Roy Company, editor. Instruction manual: supercritical extraction laboratory methods development system. Iviland, PA. p 14-16. Mitchell AJ. 1978. Soft drinks constituents and combination principles. In: Green LF, editor. Developments in soft drinks technology. L ondon, U.K: Applied Science Publishers Ltd. p 102-142.

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79 Nasir MI, Bernards MA, Charpentier PA. 2007. Acetylation of s oybean lecithin and identification of components for solubility in supercriti cal carbon dioxide. J Agric Food Chem (55): 1961-9. Netzel M, Netzel G, Tian Q, Schwartz S, Kon czak I. 2006. Sources of an tioxidant activity in australian native fruits: iden tification and quantification of anthocyanins. J Agric Food Chem 54: 9820-26. ODonnell K. 2005. Carbohydrate and intense sweet eners. In: Ashurst, editor. Chemistry and technology of soft drinks and fruit juices. Oxford, U.K: Bl ackwell Publishing Ltd. p68-89. Palazo lu TK, Balaban MO. 1998. Supercritical CO2 extraction of lipids from roasted pistachio nuts. Transactions ASAE 41 (3): 679-84. Park SJ, Lee JI, Park J. 2002. Effect of a comb ined process of high-pressure carbon dioxide and high hydrostatic pressure on the quality of carrot juice. J Food Sci 5(67): 1827-34. Roethenbaugh G. 2005. Trends in beverage ma rkets. In: Ashurst, editor. Chemistry and technology of soft drinks and fruit juices. Oxford, U.K: Bl ackwell Publishing Ltd. p15-34. Schneider GM, Kautz CB, Tuma D. 2000. In: Ki ran E, Debenedetti P, Peters C, editors. Supercritical fluids: fundamentals and applic ations. Dordrecht, The Netherlands: Kluwer Academic Publishers. p 31-68 Schweiss DA. 1997. Aspen PlusTM web site. Cambridge, MA: University of Michigan Available from: http://www.engin.umich.edu/~CRE/hel p/software/html/aspen/credits.htm Accessed Oct 2007. Shimoda M, Cocunubo-Castellanos J, Kago H, Mi yake M, Osajima Y, Hayakawa I. 2001. The influence of dissolved CO2 concentration on the death kinetics of Saccharomyces cerevisiae. J Applied Micro 91: 306-11. Someya S, Bando S, Chen B, Song Y, Nishio M. 2005. Measurement of CO2 solubility in pure water and the pressure effect on it in the presence of clathr ate hydrate. J Heat and Mass Transfer 48: 2503-7. Song C. 2002. CO2 conversion and utilization: an overv iew. In: Song, Gaffney, Fujimoto, editors. CO2 conversion and utilization. ACS Symposium 809. Washington, DC: ACS Publication Division. p 364-86. Span R, Wagner W. 1996. A new equation of st ate for carbon dioxide co vering the fluid region from the triple point temperature 10 1100 K at pressure up to 800 MPa. J Phys and Chem Ref Data 6 (25): 1509-96. Steen D. 2005. Carbonated beverages. In: Ashur st, editor. Chemistry and technology of soft drinks and fruit juices. Oxford, U. K: Blackwell Publishing Ltd. p150-81.

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80 Stratmann A, Schweiger G. 2004. Fluid phase equ ilibria of binary mixtures with supercritical solvents with in-situ concentration measurements by Raman spectroscopy. In: Brunner G, editor. Supercritical fluids as solvents a nd reaction media. Amsterdam, The Netherlands: Elsevier BV. p 85-120. Subramaniam B, Busch DH. 2002. Use of densephase carbon dioxide in catalysis. In: Song, Gaffney, Fujimoto editors. CO2 conversion and utilization. ACS Symposium 809. Washington, DC: ACS Publica tion Division. p 364-86. Subramaniam B, Lyon CJ, Arunajatesan V. 2002. Environmentally benign multiphase catalysis with dense phase carbon dioxide. Applied Catal B Env 37: 279-92. Taniguchi M, Kamihira M, Kobayashi T. 1987. Effect of treatment with supercritical CO2 on enzyme activity. Agric Biol Chem 51(2): 593-4 Taylor B. 2005. Other beverage ingredients. In: Ashurst, edito r. Chemistry and technology of soft drinks and fruit juices. Oxford, U.K: Blackwell Publishing Ltd. p 90-128. Tescom. 2006. High pressure/back pressure catal og. Elk River, MN: Tesc om Industrial Controls. Available from: http://www.tescom.com/pdf_files/05432617.pdf Accessed Apr 2006. Tomasula PM, Boswell RT. 1998. Measurement of th e solubility of carbon dioxide in milk at high pressures. J Supe rcrit Fluid 16: 21-6 Turner C. 2006. Overview of modern extraction t echniques for food and ag ricultural samples. In: Turner, editor. Modern extraction techni ques. ACS Symposium 926. Washington, DC: ACS Publication Division. p 3-19. Valderrama JO, Robles PA, De la Fuente JC. 2006. Determining the sublimation pressure of capsaicin using high-pressure sol ubility data of capsaicin + CO2 mixtures. J Chem Eng 51: 17837.

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81 BIOGRAPHICAL SKETCH Thelma Francisca Calix Lara was born on 1984, in Tegucigalpa, Honduras, Central America. After graduating from high school in December 2001, she entered the Escuela Agricola Panamericana (University of Zamo rano), earning her Bachelor of Science in Agroindustry in December 2005. During spring 2005, she performed an internship at the Food Technology of Fruits and Vegetabl es Department at the Center of Investigation in Food and Development (CIAD) in Mexico. During fall of the same year, she was offered an assistantship to pursue her graduate studies, advised by Dr. Mu rat O. Balaban. In December 2007, she earned a Master of Science in Food Science and will co ntinue her studies towards a doctoral degree.


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