ACCELERATED GLUCOSE DISCOLORATION METHOD - A QICK …

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ACCELERATED GLUCOSE DISCOLORATION METHOD - A QUICK

TOOL FOR GLUCOSE STABILITY ASSESSMENT

Master Thesis in Analytical Chemistry, Lund University

Cristina Scret

Supervisor: Staffan Bergström, PhD, Specialist Analytical Chemistry,

Gambro Lundia AB

Abstract

The non-enzymatic browning of glucose was investigated by accelerating the glucose

degradation with or without heating glucose solutions for 1 h at 100°C with different reagents.

Evaluation of the glucose degradation was performed using two types of glucose, glucose A and

glucose B, in order to investigate the influence of the glucose manufacturing process on glucose

discoloration. The color formation was determined by measuring the UV-absorbance between

260-605 nm. The glucose degradation was also investigated by the formation of 5-

hydroxymethylfurfural and 2-furfural, which were determined by HPLC.

The accelerated glucose discoloration method was optimised in order to differentiate between

different glucose qualities with respect to color stability, i.e. the conditions selected were based

on maximizing the difference in glucose discoloration for glucose A and B. Direct measurement

of the absorbance difference at 350 nm, on 33 % (w/w) glucose/water solution kept at room

temperature for 1 h was found to be the optimum conditions for differentiating between glucose

A an B. Further investigation on Maillard reactions was made, and for the investigated

conditions, the reaction was found to be most accelerated for the glucose samples, 33% (w/w),

heated at 100°C for 1 hour in the presence of L-alanine.

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Table of Content 1. Introduction .............................................................................................................................. 3

1.1. Glucose in dialysis ........................................................................................................... 3

1.2. Glucose discoloration ....................................................................................................... 3

1.2.1. Caramelisation .......................................................................................................... 4

1.2.2. Maillard reaction ...................................................................................................... 4

1.2.3. Mechanisms of nonenzymatic browning .................................................................. 5

1.3. Scope of work ................................................................................................................... 5

2. Materials and Method ............................................................................................................... 6

2.1. Materials ........................................................................................................................... 6

2.2. Preparation of the glucose samples .................................................................................. 6

2.2.1. Preparation of Caramelisation Experiments ............................................................. 6

2.2.2. Preparation of Maillard Reaction Experiments ........................................................ 7

2.3. Ultraviolet (UV) Absorbance and Color Measurements .................................................. 8

2.4. High Pressure Liquid Chromatography (HPLC) determination of 5-HMF and 2-FA ..... 8

2.5. Method of Optimisation ................................................................................................. 10

2.6. Method of Robustness .................................................................................................... 10

3. Results and discussion ............................................................................................................ 11

3.1. Caramelisation ................................................................................................................ 11

3.1.1. Caramelisation of glucose in the presence of different acids ................................. 11

3.1.2. Caramelisation of glucose in the presence of water ............................................... 13

3.1.3. Caramelisation of glucose with metal chlorides as catalyst ................................... 14

3.1.4. Caramel colours ...................................................................................................... 15

3.2. HPLC determination of 5-HMF and 2-FA ..................................................................... 18

3.3. Maillard reaction ............................................................................................................ 19

3.4. Method of optimisation .................................................................................................. 22

3.4.1. Temperature optimisation ...................................................................................... 22

3.4.2. Concentration optimisation .................................................................................... 23

3.4.3. The pH optimisation ............................................................................................... 24

3.4.4. Time optimisation .................................................................................................. 26

3.4.5. Heating versus no heating ...................................................................................... 27

3.5. Method Robustness ........................................................................................................ 28

3.5.1. The robustness of the method ................................................................................. 28

3.5.2. Time ....................................................................................................................... 29

4. Conclusions ............................................................................................................................ 30

5. Acknowledgments .................................................................................................................. 30

6. References .............................................................................................................................. 30

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1. Introduction

1.1. Glucose in dialysis

D-glucose is a central substance for the human metabolism since it is a source of energy and a

metabolic intermediate. Most of this energy is delivered through aerobic or anaerobic respiration.

In human metabolism glucose is critical as a precursor for the production of proteins and in lipid

metabolism.

In humans the kidneys are used to purify the blood from various salts, metabolites and waste

products and when a person's own kidneys can no longer function adequately to maintain life,

dialysis is a procedure, which is primarily used to provide an artificial replacement for some of

the kidney's normal functions. Dialysis works on the principles of the diffusion of solutes and

ultrafiltration of fluids across a membrane in order to remove the harmful wastes, extra salts and

excess of water from the blood stream. There are two main types of dialysis: haemodialysis or

peritoneal dialysis.

Haemodialysis is a method, which removes wastes and water by circulating the blood outside

the body through an external membrane device, called a dialyzer. The dialyzer contains a semi-

permeable membrane, where the blood flows on one side of the membrane and dialysis fluid

flows on the other side of the membrane. The purification process is controlled by the diffusion

of the salts and waste products over the membrane, and the removal, or in some cases addition of

water, so called ultrafiltration, can be controlled by the pressure difference over the membrane.

Peritoneal dialysis is a method, which removes wastes and water from the blood, inside the

body using the peritoneal membrane of the peritoneum as a natural semi-permeable membrane.

The dialysis fluid is infused into the stomach cavity, and kept there for a defined time, while the

salts and waste products are equilibrated through diffusion into the fluid, which is then replaced

with given intervals. This type of treatment is normally only given to patients with some

remaining kidney function.

Glucose is by far the carbohydrate that is used most as osmotic agent in the peritoneal and

haemodialysis fluids today.

1.2. Glucose discoloration

The most common method of sterilisation for parental solutions of glucose is autoclaving, i.e.

heat treatment of the solution. During the sterilisation process some solutions could develop a

pale yellow color. The color formation increases with the time of heating, glucose concentration,

the presence of salts and by increased sterilisation temperature. The mechanism responsible for

this discoloration of the solution is the so called non-enzymatic browning, which is further

investigated in this work.

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Non-enzymatic browning of glucose could cause discoloration also during production and/or

storage of various glucose solutions. Caramelisation and Maillard reaction are known as two

main types of reaction pathways of non-enzymatic browning.

1.2.1. Caramelisation

Caramelisation involves heat treatment of monosaccharides in the absence of nitrogenous

compounds by removal of water at elevated temperature. The reaction starts the isomerisation of

the monosaccharides to low-molecular-weight (LMW) intermediates associated with aroma

compounds such as 5-hydroxymethylfurfural (5-HMF) and 2-furfural (2-FA), Figure 1, which

can undergo further condensation and polymerisation reactions to high-molecular-weight (HMW)

compounds associated with the color formation. These HMW compounds are commonly named

caramels.

Depending on processing, caramel colours can have positive or negative ionic charge.

Positively charged caramel colours are produced by reaction of ammonium with glucose and are

commonly found in soy sauces and beer, since they do not react further with proteins.

Negatively-charged caramel colours use sulphite as reactant, and are commonly found in acidic

soft drinks, (Schultz 2006).

Caramel colours have been used in a wide variety of food products and are produced by

controlled heating of carbohydrates with different reagents such as sodium hydroxide (NaOH),

sodium sulphite (Na2SO3) or ammonium chloride (NH4Cl).

There are 4 types of caramel colours:

Caramel Class I (also known as plain or spirit caramel, prepared with NaOH)

Caramel Class II (known as caustic sulphite caramel, prepared with sulphite containing

compounds such as Na2SO3 )

Caramel Class III (named ammonia or beer caramel, prepared with ammonia and used in

baker’s and confectioner’s caramel)

Caramel Class IV (known as sulphite-ammonia caramel, prepared with ammonia and

sulphite compounds and used as soft drink caramel or acid proof caramel) (Kamuf and

Nixon 2003)

Caramelisation generally requires temperatures over 200°C and a pH between 3 and 9, but the

presence of impurities in low concentration, e.g. iron ions and copper ions or other impurities,

could reduce the caramelisation temperature down to 40°C, (Coca et al. 2004).

1.2.2. Maillard reaction

The Maillard reaction involves a complex series of reactions between monosaccharides with

amino compounds such as amino acids or proteins. Reactive intermediates are formed that are

finally degraded to important flavour components such as 5-HMF and 2-FA, which react further

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to form brown pigments known as melanoidins. 5-HMF and 2-FA are considered to be precursors

of such polymer-compounds, (Tomasick et al. 1989).

1.2.3. Mechanisms of nonenzymatic browning

The glucose discoloration is still not a fully understood process and the mechanisms

responsible with glucose degradation are very complex, (Ramchander et al. 1975, Martins et al.

2003 and Coca et al. 2003).

Several studies have investigated various stages of the non-enzymatic browning reaction. These

studies have shown that the rate of the reaction is strongly dependent on the concentration of

glucose, ratio and chemical nature of reactants, temperature, time of heating, and pH. The

caramelisation process is dependent on heat and is catalyzed by acids and bases (Kroh 1994). At

a pH of 2.5 and lower a considerable amount of 5-HMF is produced (Tohji et al. 2007).

Glucose discoloration by the caramelisation process was shown to be accelerated by an increase

in temperature, concentration, pH and time of heating according to Buera and co-workers (1992).

Buera and co-workers (1987), Ajandouz and others (2001), Tanaka (2005) have studied the effect

of pH on glucose discoloration. According to their studies parameters such as temperature,

concentration, time of heating and pH have an acceleratory effect on glucose discoloration.

5-Hydroxymethylfurfural 2-Furfural

Figure 1. Structures of 5-HMF and 2-FA

Proteins or other carbohydrates such as maltose and isomaltose are present in various amounts

in the raw material used when manufacturing glucose. Thus, trace levels of these compounds

could be found also as impurities in the final product. Since other factors such as impurities and

the potential use of inhibitors such as sodium bisulphite, in the purification step, may cause

changes in color formation process in the glucose solutions, the glucose manufacture process may

very well have an influence on the amount of non-enzymatic browning of the glucose. The

presence of impurities in low quality products may favour the browning reactions (Coca et al.

2004).

1.3. Scope of work

The aim of this present work was to find an accelerated glucose discoloration method that could

be used as a quick tool for glucose stability assessment and to investigate the parameters

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influencing the non-enzymatic browning of glucose. The accelerated glucose discoloration

method thus evaluate the quality of glucose with respect to coloration stability of solutions and

could be used to find and exclude glucose that potentially will develop a yellow color in solutions

during the manufacturing process and/or during storage of final products containing the glucose.

The glucose discoloration was studied in glucose solutions prepared from two types of glucose,

herein named glucose A and glucose B. For these two types of glucose there had previously been

observed differences in color stability. Various parameters, as described below, were investigated

in order to find the conditions were the difference was maximized for the two types of glucose.

2. Materials and Method

2.1. Materials

Two types of D-glucose, glucose A and glucose B, produced with different processes were

chosen as test materials. Different reagents such as: amino acids (L-lysine, L-glycine, and L-

alanine), and chromium chloride were purchased from Sigma Aldrich (Steinheim, Germany),

hydrochloric acid (HCl), sulphuric acid (H2SO4), citric acid, phosphoric acid (H3PO4), sodium

sulphite, (Na2SO3), ammonium chloride,(NH4Cl) and sodium hydroxide (NaOH) were obtained

from Merck, (Darmstadt, Germany). All chemicals used were of pro analysis quality or equal.

2.2. Preparation of the glucose samples

In order to find a quick method for assessing the glucose stability with respect to discoloration,

several parameters, were investigated.

2.2.1. Preparation of Caramelisation Experiments

Caramelisation of glucose was tested by preparing the solutions according to the method CRA

E-20, (1987), Glucose Color Stability (Analytical Methods of the member Companies of the corn

Association, Inc), using the two types of D-glucose, glucose A and glucose B. The addition of

different reagents such as HCl, H2SO4, citric acid, H3PO4, chromium chloride, sodium sulphite,

ammonium chloride, and NaOH were investigated. The samples were heated in a Grant BT3,

block heater at various temperatures within the range of 20 to 140°C. The sample concentration,

pH and temperature were varied according to Table 1. The pH measurements were made with an

Orion pH Meter, model 290A. Where needed the pH of the solution was adjusted with HCl.

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Table 1. Levels of the variable of the glucose aqueous sample in caramelisation process

Reagents Factor Units Levels

Water Glucose concentration % (w/w) 33 33

pH un-adj.* un-adj.*

Temperature °C room 100

Time Hour 1 1

Citric acid Glucose concentration % (w/w) 20 20 20 20

Hydrochloric acid pH 1.5 1.5 1.5 1.5

Sulphuric acid Temperature °C 100 100 100 100

Phosphoric acid Time Hour 1 1 1 1

Chromium chloride Glucose concentration % (w/w) 33

pH

Temperature °C 100

Time Hour 1

CrCl2 concentration m Molar 20

Caramel classes Glucose concentration % (w/w) 33 33 33 33

at lower concentrations pH

of reagents Temperature °C 100 100 100 100

Time Hour 1 1 1 1

Caramel class 1 NaOH concentration (g/l) 0.5

Caramel class 2 Sodium sulphite (g/l) 0.5

Caramel class 3 Ammonium chloride (g/l) 0.5

Caramel class 4 Ammonium chloride+ (g/l) 0.5

Sodium sulphite (g/l) 0.5

Caramel classes Glucose concentration % (w/w) 33 33 33 33

at higher concentrations pH

of reagents Temperature °C 100 100 100 100

Time Hour 1 1 1 1

Caramel class 1 NaOH concentration (g/l) 5

Caramel class 2 Sodium sulphite (g/l) 5

Caramel class 3 Ammonium chloride (g/l) 5

Caramel class 4 Ammonium chloride+ (g/l) 5

Sodium sulphite (g/l) 5

*) Un-adjusted, i.e. pH 5-6

2.2.2. Preparation of Maillard Reaction Experiments

Since even low amounts of amino-compounds in glucose solutions can initiate Maillard

reaction and the fact that trace levels of protein can exist in the glucose as impurities from the

manufacturing process, Maillard reaction can also contribute to the yellow color formation in

glucose products besides caramelisation.

Aqueous model systems of Maillard reaction were prepared according to the same method

described above, using the two types of D-glucose and different amino acids: L-lysine, L-glycine,

and L-alanine with concentrations, pH, temperature and time of heating as presented in Table 2.

L-lysine, L-glycine, and L-alanine are the most commonly used amino acids in testing of

Maillard reaction. The pH of the model systems was adjusted with HCl and NaOH respectively.

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2.3. Ultraviolet (UV) Absorbance and Color Measurements

The absorbance of the liquid samples was analyzed after letting them to cool down to ambient

temperature. Absorbance was measured based on the method CRA E-20, (1987), in 1×4 cm

cuvette, using a Perkin Elmer Lambda 20 UV/VIS Spectrophotometer. The UV spectrum from

260-605 nm was recorded for each sample, and the glucose discoloration was calculated as the

absorbance difference between 350 and 600 nm, 450 and 600 nm and as absorbance difference

between λ max and 600 nm. The subtraction of the absorbance at 600 nm was made in order to

adjust the absorbance for any potential unspecific absorbance in the sample. All samples were

measured in triplicate. The calculations of the color, absorbance difference were made using the

equation:

Color, Absorbance difference ×100= (1)

2.4. High Pressure Liquid Chromatography (HPLC) determination of 5-HMF and 2-FA

5-HMF and 2-FA determination by HPLC were made with HPLC measurements performed on

Agilent 1100 equipment, equipped with a loop injector and a UV detector. An RP C18 column, 5

µm (150×4 mm), Supelco was used for separation. The mobile phase consisted of NaH2PO4×H2O

(0.05 M) at pH 5.5 and acetonitrile. The system was calibrated for 5-HMF in the range of 0.1-2.0

ppm and 0.02-0.5 ppm for 2-FA.

l

xAA nmx 100600,

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Table 2. Levels of the variables of the samples in the Maillard model system.


Parameter Factor Units Levels

pH 1.5 Glucose concentration

% (w/w) 33 33 33 33 33 33 33 33 33

L-Lysine concentration M 0.1 0.1 0.1

L-Alanine concentration M 0.1 0.1 0.1

L-Glycine concentration M 0.1 0.1 0.1

pH - 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

Temperature °C 100 100 100 100 100 100 100 100 100

Time Hour 1 1 1 1 1 1 1 1 1

pH 6 Glucose concentration

% (w/w) 33 33 33 33 33 33 33 33 33




pH - Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.*

Temperature °C 100 100 100 100 100 100 100 100 100

Time Hour 1 1 1 1 1 1 1 1 1

pH 9 Glucose concentration

% (w/w) 33 33 33 33 33 33 33 33 33




pH - 9 9 9 9 9 9 9 9 9

Temperature °C 100 100 100 100 100 100 100 100 100

Time Hour 1 1 1 1 1 1 1 1 1

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2.5. Method of Optimisation

The method was optimised by investigating the parameters found to affect the glucose

discoloration, such as temperature, pH, concentration and heating time. The investigated levels of

these parameters are presented in Table 3. The pH of the model systems was adjusted with HCl

and NaOH respectively when needed.

Table 3. Levels of parameters, which were used to optimise the glucose color stability method.


2.6. Method of Robustness

The robustness of the method was tested by investigating the repeatability and reproducibility

of the method. The repeatability of the method was tested by performing three different tests of

the method and the reproducibility of the method was tested by performing three different tests of

the method by another person, at a different day. The influence of mixing time was also

investigated as a robustness parameter.

Optimized parameter Factors Units Levels

Time temperature °C room 100 100 100 100 100 100 100 pH Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.*

glucose concentration % (w/w) 33 33 33 33 33 33 33 33 time hour 0 0.5 0.75 1 2 4 8 24

Temperature temperature °C 70 80 90 100 110 pH Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* glucose concentration % (w/w) 33 33 33 33 33 time hour 1 1 1 1 1

Concentration temperature °C 100 100 100 100 100 pH Un-adj.* Un-adj.* Un-adj.* Un-adj.* Un-adj.* glucose concentration % (w/w) 20 30 40 50 60 time hour 1 1 1 1 1

pH temperature °C 100 100 100 100 100 pH 1.5 3 5 7 9 glucose concentration % (w/w) 33 33 33 33 33 time hour 1 1 1 1 1

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3. Results and discussion

3.1. Caramelisation

3.1.1. Caramelisation of glucose in the presence of different acids

The acidic hydrothermal degradation of glucose was tested using different acids, HCl, H2SO4

and H3PO4 at pH 1.5. The acidified glucose samples (33%, w/w) were heated for 1 h at 100°C.

The results are presented in Figure 2.

Normalized difference

0%10%20%30%40%50%60%70%80%90%

100%

Glu

cose

A_c

itric

Glu

cose

A_h

ydro

chlo

ric

Glu

cose

A_p

hosphoric

Glu

cose

A_s

ulphuric

Glu

cose

B_c

itric

Glu

cose

B_h

ydro

chlo

ric

gluco

se B

_phosp

horic

Glu

cose

B_s

ulphuric

At 450nm-600

At 350nm-600

At λmax nm - 600

Figure 2. The effect of four different acids on glucose discoloration in glucose solutions from the two

types of glucose, at pH 1.5, heated for 1 h at 100°C.

At the absorbance difference, 350 nm, as can be seen in Figure 2, the glucose discoloration

increases, as expected, with the amount of added acid. The effect of the four acids was in

following order: sulphuric acid< hydrochloric acid <phosphoric acid <citric acid. The pH can be

related as a measure of the concentration of H+

present in the solutions and since the pH of the

used acids was adjusted to 1.5, higher amount of the weaker acids was needed in order to achieve

pH 1.5 in solutions. The same results showed that the acidic glucose degradation was more

accelerated for glucose B compared to glucose A, i.e. glucose A is shown to be more stable with

respect to discoloration.

Schrödter (1992) and Kroh (1994) suggested that the reaction of glucose degradation in acidic

medium proceeds principally via 1, 2- or 2, 3-enolisation of the sugar and ß-elimination of water

leading to the formation of 1-3- and 4-hexuloses. The reaction scheme is presented in Figure 3

and 4.

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Several studies on acidic thermal degradation of carbohydrates and color formation have been

performed and according to Kelly and Brown (1978) the anions of weak acids accelerate the

color formation, while the anions of strong acid had an inhibitory effect compared to the weaker

acids, which is confirmed by the obtained results. Girisuta (2006), Takeuchi et al. (2007),

reported that thermal acidic degradation of glucose leads to the formation of 5-HMF and this 5-

HMF formation is followed by re-hydration of 5-HMF to levulinic acid, Figure 5.

Figure 5. Glucose dehydration to 5-HMF and than 5-HMF re-hydration to levulinic acid.

At pH 1.5-2, the production of 5-HMF increases and then re-hydration of 5-HMF to levulinic

acid is accelerated by the use of strong acids since only a small amount of strong acid is needed

to catalyze this reaction, according to Takeuchi et al. (2007). This theory was confirmed by the

obtained results in this work, since the presence of strong acids such as sulphuric acid and

hydrochloric acid have a less accelerated effect on glucose discoloration compared to the weaker

acids. This effect can be explained by lower amount of 5-HMF, which is considered to be a

precursor to polymer formation. The lower amount of 5-HMF can be explained by further re-

hydration of 5-HMF to levulinic acid, which takes place in glucose solutions in the presence of

strong acids.

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3.1.2. Caramelisation of glucose in the presence of water

The results of glucose discoloration in pure glucose/water solutions heated at 100°C for 1 h

are presented in Table 4. In order investigate whether the glucose manufacture process has any

effect on the glucose discoloration, several batches from the two types of glucose were tested.

Table 4. Glucose discoloration in aqueous solutions for different batches of the two types of glucose, at

100°C for 1 h.

Glucose type At 450nm-600nm absorbance difference

At 350nm-600nm absorbance difference

At λmax-600nm absorbance difference

Glucose A Batch 1 0.128 0.658 6.868

Glucose A Batch 2 0.181 0.705 5.816

Glucose A Batch 3 0.174 0.724 6.084

Glucose A Batch 4 0.218 0.965 6.623

Glucose A Batch 5 0.067 0.382 4.636

Glucose B Batch 1 0.388 1.477 7.538

Glucose B Batch 2 0.382 1.524 8.653

Glucose B Batch 3 0.338 1.308 7.593

Glucose B Batch 4 0.268 1.303 8.168

Glucose B Batch 5 0.691 2.859 11.039

In aqueous solutions, glucose is found mainly in a cyclic structure, called hemiacetal and this

structure forms the two isomers α-D-glucose and β-D-glucose. Even if the reactivity of the

aldehyde group is blocked in the hemiacetal form, the small amount of D-glucose which remains

in open-chair form (0.8%) is enough for the glucose solution to show the typical reactivity of an

aldehyde group.

According to Watanabe (2005), glucose discoloration in aqueous glucose solutions occurs, by

isomerization of glucose to fructose, followed by dehydration of both glucose and fructose to 1,6-

anhydroglucose (AHG) and to 5-HMF and 2- FA respectively. The reaction pathway of this

glucose degradation in water is presented in Figure 6.

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Figure 6. Proposed reaction pathway of glucose in water, Watanabe (2005).

The results show that the color formation was more pronounced for glucose B compared to

glucose A for all the tested batches, thus an obvious difference in glucose quality with respect to

discoloration between the two types of glucose was verified.

3.1.3. Caramelisation of glucose with metal chlorides as catalyst

Metal chlorides have been reported to accelerate glucose degradation, and therefore the

glucose discoloration was investigated using CrCl2 as catalyst. The results are presented in Table

5. It was observed that CrCl2 gives a very strongly accelerated color formation for both types of

glucose, but no significant difference in glucose quality with respect to discoloration between the

two types of glucose could be observed. These results can be explained by when using CrCl2 as

reagent, the reaction rate is much more accelerated and glucose degradation to 5-HMF is more

complete.

Table 5. The effect of chromium chloride as catalyst on glucose discoloration, in glucose solutions,

which were heated for 1 h at 100°C. Measurements made on samples diluted 1:100, absorbance values re-

calculated by this factor.

Absorbance difference Glucose A

water/CrCl2

Glucose B

water/CrCl2

B/A Absorbance

factor

water/CrCl2

At 450nm-600nm 7.331 6.248 0.8

At 350nm-600nm 213.713 133.938 0.6

At λmax-600nm 330.259 326.595 0.9

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Previous work in this area, found in literature, showed that using CrCl2 as reagent will lead to a

high 5-HMF conversion yield from glucose and according Zao and co-workers (2007) this can

be explained by the effect of CrCl2 on glucose mutarotation of α-glucopyranose to ß-

glucopyranose and then isomerisation of ß-glucopyranose to fructose see Figure 7.

Figure 7. CrCl2 catalyses the mutarotation and then isomerisation to fructose, followed by dehydration to

5-HMF, Zao and co-workers (2007).

3.1.4. Caramel colours

The results of the investigation of glucose caramel classes by spiking glucose solutions with

NaOH (Caramel Class I), sodium sulphite, (Caramel Class II), ammonium chloride (Caramel

Class III) and with sodium sulphite and ammonium chloride (Caramel Class IV) and then heating

the samples at 100°C for 1 h, are presented in Figure 8.

16


0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Gluco

se A

+ 0,

05%

Na2

SO3

Gluco

se A

+0,5

% N

a2SO

3

Gluco

se B

+ 0,

05% N

a2SO

3

Gluco

se B

+ 0,

5% N

a2SO

3

Gluco

se A

+ 0,

05% N

H4Cl

Gluco

se A

+ 0,

5% N

H4C

l

Gluco

se B

+ 0,

05% N

H4Cl

Gluco

se B

+ 0,

5% N

H4C

l

Gluco

se A

+ 0,

05% N

H4Cl+

0,0

5%Na2

SO3

Gluco

se A

+ 0,

5% N

H4C

l+ 0

,5%

Na2

SO3

Gluco

se B

+ 0,

05% N

H4Cl+

0,0

5%Na2

SO3

Gluco

se B

+ 0,

5%µl N

H4C

l+ 0

,5%

Na2

SO3

Gluco

se A

+ 0,

05% N

aOH

Gluco

se A

+ 0,

5% N

aOH

Gluco

se B

+ 0,

05% N

aOH

Gluco

se B

+ 0,

5% N

aOH

At 450nm-

600nmAt 350nm-

600nm

Figure 8. Results of glucose discoloration for glucose caramel classes, at 100°C, for 1 h.

According to these results, the glucose discoloration was accelerated for Caramel Class I and

Caramel Class II, for both types of glucose.

3.1.4.1. Caramel Class I – Alkaline

The glucose discoloration was accelerated for Caramel Class I in alkaline conditions. No

difference with respect to discoloration could be observed for the two types of glucose since

glucose degradation is highly increased at high pH. The alkaline degradation of glucose gives

high molecular weight products, which are associated with color formation, according to Coca et

al. (2004). Monosaccharides in aqueous alkaline solutions undergo both reversible and

irreversible reactions according to De Bruijn, (1986).

3.1.4.2. Caramel Class II – Sodium sulphite

In our experiments the addition of higher amounts of sodium sulphite gave accelerated glucose

discoloration. This was not as expected, since sodium sulphite is known to have an inhibitory

effect on glucose discoloration. However, Hoffman and others (1999) and Nursten (2005),

reported that the presence of sodium sulphite has en inhibitory effect on glucose discoloration

only in earlier stages, likely by blocking the radical precursors, glyoxal and glucoaldehyde.

Glucose discoloration was found to be more accelerated at higher concentration of sodium

sulphite in the solutions, since after the induction period, sodium sulphite functions as catalyst in

5-HMF formation from 3-deoxyglucosone.

For Caramel Class II glucose discoloration was accelerated for both types of glucose and a

significant difference between them could no longer be observed. This observation can be

explained by the differences in the manufacture process between the two types of glucose. For

17

glucose type A the addition of substances with an inhibitory effect such as sodium bisulphite is

used in the purification step, while no additions are stated for glucose type B. The extra sulphite

addition in our experiments could possibly catalyse further formation of 5-HMF as a precursor to

color formation after the induction period for glucose type A. I.e. for glucose type A the

induction step is lower compared to type B for the same amount of sulphite addition.

In order to further investigate the effect of a low level of sodium bisulphite on glucose samples,

glucose type B, was spiked with different amounts of sodium bisulphite.

The results of spectroscopic determination of color formation and results of HPLC

determination of 2-FA formation show that the addition of sodium bisulphite at lowers levels

somewhat reduces both discoloration and 2-FA formation. The results of glucose discoloration by

spectroscopic determination are presented in Figure 9, in which samples of glucose type A were

plotted together with spiked glucose type B samples. The results of 2-FA formation are presented

in Figure 10.

As the results show, the addition of 30 ppm sodium bisulphite to a glucose B solution decreases

the discoloration to the same level of discoloration as a native glucose A solution.

The results obtained from HPLC analysis of 2-FA are presented in Figure 10, were the

concentration of 2-FA was plotted against the spiked amount of sodium bisulphite. The results

presented in Figures 9 and 10 allow the conclusion that the addition of lower levels of sodium

bisulphite to the glucose type B solutions has an inhibitory effect on the 2 –FA formation and

reduces the discoloration.

Sodium bisuphite spiking

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

Glucose A

native

Glucose B

native

Glucose B 5

ppm

Glucose B 10

ppm

Glucose B 20

ppm

Glucose B 30

ppm

Abs

350

nm

Figure 9. The effect of sodium bisulphite on 5-HMF formation from glucose type B, by spectroscopic

determination.

18

2-FA formation

5

5,5

6

6,5

7

7,5

8

8,5

0 5 10 15 20 25 30 35

Spiked amount Na-bisulphite (mg/l)

Co

ncen

trati

on

2-F

A (

mg

/l)

2-FA

(mg/l)

Figure 10. The effect of sodium bisulphite on 2-FA formation from glucose type B, by HPLC

determination.

3.1.4.3. Glucose Caramel Class III and Caramel Class IV

For Caramel Class III and Caramel Class IV, no or a lower glucose discoloration could be

detected.

3.2. HPLC determination of 5-HMF and 2-FA

The results of HPLC determination of formation of 5-HMF and 2-FA for caramelisation

process, using different reagents are presented in Table 6. It can be seen that when using CrCl2 as

reagent, the formation of 5-HMF and 2-FA was highest, for both types of glucose. When using

NaOH as reagent in the formation of Caramel Class I, 2-FA formation increases. This can be

explaining by the fact that in alkaline medium degradation of glucose occurs according to

Watanabe (2005), by isomerisation of glucose to fructose, followed by dehydration of both

glucose and fructose to 1, 6-anhydroglucose (AHG) and to 5-HMF and 2- FA respectively.

All the samples that have been subjected to heat did, however, produce some color, varying

from pale yellow to brown. The results of glucose discoloration in water, Caramel Class III and

Caramel Class IV, show that the heated samples, in which no visible yellow color could be

observed, can be explained by different reactivity of the used reagents.

When using strong reagents such as NaOH and CrCl2, the glucose discoloration was

accelerated so strongly for both types of glucose so that no significant difference in glucose

quality between the two types could be observed any more. This can be explained by when using

these reagents as catalysts the reaction of glucose degradation is more complete.

19

Table 6. HPLC evaluation of 5-HMF and 2-FA in the caramelisation process.

Glucose sample 5-HMF(ppm) 2-FA(ppm) Absorbance difference at 350nm

Supplier A/water <0.1 <0.02 0.382

Supplier B/water <0.1 <0.02 1.303

Supplier A Caramel Class I* 4.7 3.08 588

Supplier B Caramel Class I* 5.6 3.28 574

Supplier A Caramel Class II* <0.1 <0.02 615

Supplier B Caramel Class II* <0.1 <0.02 609

Supplier A Caramel Class III* 0.2 <0.02 0.454

Supplier B Caramel Class III* 0.2 <0.02 1.358

Supplier A Caramel Class IV* <0.1 <0.02 14.9

Supplier B Caramel Class IV* <0.1 <0.02 14.6

Supplier A/CrCl2** 186.6 0.27 214

Supplier B/CrCl2** 204.7 0.28 134

*) Samples of Caramel Classes were diluted 1:10.

**) Samples of glucose containing CrCl2 were diluted 1:100

3.3. Maillard reaction

The contribution of Maillard reaction to non-enzymatic browning of glucose was tested in

three different aqueous model systems, using two types of D-glucose and different amino acids:

L-lysine, L-glycine, and L-alanine. The effect of pH on Maillard reaction was also tested. The

effect of the amino acids on non-enzymatic browning in glucose solutions at pH 1.5 (HCl) and 9

(NaOH) and in the absence of amino acids is presented in Figures 11 and 12.

20

Normalized difference Maillard Reaction

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Glu

cose

A/L

ysin

e/NaO

H

Glu

cose

A/A

lanin

e/NaO

H

Glu

cose

A/G

lyci

ne/NaO

H

Glu

cose

B/L

ysin

e/NaO

H

Glu

cose

B/A

lanin

e/NaO

H

Glu

cose

B/G

lyci

ne/NaO

H

Glu

cose

A/L

ysin

e/HCl

Glu

cose

A/A

lanin

e/HCl

Glu

cose

A/G

lyci

ne/HCl

Glu

cose

B/L

ysin

e/HCl

Glu

cose

B/A

lanin

e/HCl

Glu

cose

B/G

lyci

ne/HCl

Glu

cose

A/N

aOH

Glu

cose

B/N

aOH

Glu

cose

A/H

Cl

Glu

cose

B/H

Cl

At 450nm-600nm

At 350nm-600nm

Figure 11. Effect of different amino acids on glucose discoloration, at pH 1.5 and 9 of glucose solutions

heated for 1 h at 100°C.

The glucose discoloration increased in the presence of amino acids. The acceleration effect of

amino acids on glucose discoloration was in following order: lysine > glycine >alanine. Glucose

discoloration increased, as expected with increased pH, shown in Figures 11 and 12. This is

consisted with the results reported by Weismann (1993).


0%

20%

40%

60%

80%

100%

Glucose

A/Lysine

Glucose

A/Alanine

Glucose

A/Glycine

Glucose

B/Lysine

Glucose

B/Alanine

Glucose

B/Glycine

Glucose

A/water

Glucose

B/water

At 450nm-600nm

At 350nm-600nm

Figure 12. Effect of different amino acids on glucose discoloration, at pH 6 of glucose solutions heated

for 1 h at 100°C.

21

The glucose discoloration was more accelerated for glucose type B compared to glucose type A

and therefore the same difference in glucose quality between the two types of glucose could be

observed in the Maillard reaction as well as for the caramelisation process.

The results of HPLC determination of 5-HMF and 2-FA formation in Maillard reaction are

presented in Table 7. The HPLC determination of 5-HMF and 2-FA confirmed the difference in

color formation for the two types of glucose obtained by spectrophotometric determination. The

same difference in stability of the glucose between the two types could be confirmed at pH 6 and

9, since 5-HMF was detected only in the glucose solutions containing glucose type B. For

glucose type A at the same conditions of reactions, no formation of 5-HMF and 2-FA could be

detected.

At pH 1.5, 5-HMF formation was detectable for both types of glucose, but more accelerated for

glucose type B. This difference in 5-HMF formation between the two types of glucose can only

be explained by the better quality of glucose A with respect to stability.

Table 7. HPLC evaluation of 5-HMF and 2-FA formation in Maillard reaction.

Glucose sample 5-HMF(ppm) 2-FA(ppm)

Supplier A Lysine + water <0.1 <0.02

Supplier A Alanine + water <0.1 <0.02

Supplier A Glycine + water <0.1 <0.02

Supplier B Lysine + water 0.2 <0.02

Supplier B Alanine + water 0.1 <0.02

Supplier B Glycine + water 0.1 <0.02

Supplier A Lysine + NaOH <0.1 <0.02

Supplier A Alanine + NaOH <0.1 <0.02

Supplier A Glycine + NaOH <0.1 <0.02

Supplier B Lysine + NaOH 0.1 <0.02

Supplier B Alanine + NaOH 0.1 <0.02

Supplier B Glycine + NaOH 0.1 <0.02

Supplier A Lysine + HCl 0.2 <0.02

Supplier A Alanine + HCl 0.3 <0.02

Supplier A Glycine + HCl 0.1 <0.02

Supplier B Lysine + HCl 0.9 <0.02

Supplier B Alanine + HCl 0.6 <0.02

Supplier B Glycine + HCl 0.8 <0.02

22

3.4. Method of optimisation

Optimisation of the accelerated glucose discoloration method was made by testing the

parameters, which affect non-enzymatic browning of glucose such as temperature, pH,

concentration and time and the results are presented in Figures 13, 15, 17, and 19. In order to find

the optimum parameters of accelerated glucose discoloration method, the difference in quality of

glucose with respect to discoloration between the two types of glucose was taken in to

consideration and results are presented in Figures 14, 16, 18 and, 20.

3.4.1. Temperature optimisation

The largest difference in glucose quality with respect to discoloration between the two types of

glucose was obtained at 90°C, Figures 13 and 14, which seems to be the optimum temperature in

the glucose discoloration assessment. The decrease in absorbance difference at 350 nm for 90-

100°C was confirmed by additional experiments, but remains unexplained. However, at the same

time the difference between the two types of glucose was increased, which is beneficial for

assessing the relative glucose quality with respect to discoloration.

Temperature optimization

0

0,2

0,4

0,6

0,8

1

1,2

75 80 85 90 95 100 105

Temperature ( °C)

Ab

so

rban

ce

Glucose A

Glucose B

Figure 13. Effect of temperature on glucose discoloration for 1 h heating of glucose samples (33%w/w) an absorbance difference of 350 nm.

23

Glucose B/Glucose A Absorbance Factor

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

60 70 80 90 100 110 120

Temperature (°C)

Abs

orba

nce

B/A Abs. Factor

Figure 14. Absorbance factor between the two types of glucose in temperature optimisation at an

absorbance difference of 350nm.

3.4.2. Concentration optimisation


glucose was obtained at 40 % (w/w) glucose concentration, Figures 15 and 16, which seems to be

the optimum concentration in glucose discoloration assessment. Even a higher glucose

concentration might have an inhibitory effect on glucose discoloration, according to Kjellstrand

et al. (2001).

Concentration optimization

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

10 20 30 40 50 60 70

Concentration (% w/w)

Ab

so

rban

ce

Glucose A

Glucose B

Figure 15. Effect of concentration on glucose discoloration, at an absorbance difference of 350 nm, at

100°C for 1 h.

24


0

1

2

3

4

5

6

10 20 30 40 50 60 70

Concentration % (w/w)

Ab

so

rban

ce

B/A Abs Factor

Figure 16. Absorbance factor between the two types of glucose in concentration optimisation at an

absorbance difference of 350 nm.

3.4.3. The pH optimisation


glucose was at pH 7, Figures 17 and 18, which seems to be the optimum in the assessment of

glucose discoloration.

pH optimization

0

5

10

15

20

25

30

35

40

45

50

0 2 4 6 8 10

pH

Ab

so

rban

ce

Glucose A

Glucose B

25

pH optimization

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 2 4 6 8 10

pH

Ab

so

rban

ce

Glucose A

Glucose B

Figure 17. Effect of pH on glucose discoloration at an absorbance difference of 350 nm, by heating of

glucose samples (33% w/w) at 100°C for 1 h. The lower graph shows pH 1.5-7 in a larger scale


0

0,5

1

1,5

2

2,5

3

0 2 4 6 8 10

pH

Ab

so

rban

ce f

acto

r

B/A Abs. Factor

Figure 18. Absorbance factor between the two types of glucose in pH optimisation at an absorbance

difference of 350 nm.

26

3.4.4. Time optimisation

The largest difference in glucose quality with respect to discoloration between the two types

of glucose was obtained when heating the glucose sample at 90°C for 1 h and without heating of

glucose samples at time of 0 h, Figures 19 and 20, which seem to be the optimum times in

accelerated glucose discoloration method.

Different times at ambient temperature were further investigated, and showed that results had a

larger variation when the samples were measured directly after apparent dissolution of the

glucose i.e. time 0. The repeatability was significantly improved when allowing the samples to

dissolve thoroughly for 1 h prior to the absorbance determination at ambient temperature.

Time optimization

0

2

4

6

8

10

12

0 5 10 15 20 25 30

Time (hours)

Abs

orba

nce

Glucose A

Glucose B

Figure 19. Effect of time of heating on glucose discoloration at an absorbance difference of 350nm by

heating of glucose sampes (33% w/w) at 100°C.

27


0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0 5 10 15 20 25

Time (hours)

Ab

so

rban

ce

B/A Abs. Factor

Figure 20. Absorbance factor between the two types of glucose in time optimisation at an absorbance

difference of 350 nm.

3.4.5. Heating versus no heating

Since a larger difference in quality of glucose with respect to discoloration between the two

types of glucose was obtained without heating of the glucose samples, more tests were performed

in order to find the optimum time and temperature. The results are presented in Figures 21 and

22.

Glucose discoloration before heat

-0,03

0,02

0,07

0,12

0,17

0,22

260 310 360 410 460 510 560 610

Wavelength (nm)

Ab

sorb

ance

A water Day1

B water Day 2

A water Day 2

B water Day 2

A water Day 3

B water Day 3

Figure 21. The effect of water on glucose discoloration before heating of glucose sample (33%, w/w), in

three different days.

28

Glucose discoloration after heat

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0,5

260 310 360 410 460 510 560 610

Wavelength (nm)

Ab

sorb

ance

A water Day 1

B water Day 1

A water Day 2

B water Day 2

A water Day 3

B water Day 3

Figure 22. The effect of water on glucose discoloration after heating of glucose sample (33%, w/w) for 1

h, in three different days.

According to the obtained results in the experiments with or without heating of the glucose

samples, the color formation was more accelerated for glucose type B compared to glucose type

A and therefore an obvious difference in glucose quality with respect to discoloration between

the two types of glucose could be observed. Since a larger difference in discoloration between

the two types of glucose could be observed when mixing glucose with water, without heating of

the samples, (Figure 21), and the optimum temperature for the glucose discoloration method was

found to be at ambient temperature.

According to the obtained results, based on the difference in quality between the two types of

glucose, the optimum conditions for accelerated glucose discoloration method as a tool for

glucose stability assessment were found to be in aqueous glucose samples 33% (w/w), kept at

room temperature for 1 h.

3.5. Method Robustness

3.5.1. The robustness of the method

The robustness of the method was tested by investigating the repeatability and reproducibility of

the chosen method and the results are presented in Table 8.

29

Table 8. Robustness test of the accelerated glucose discoloration method in two different days, each day

represent an average of three tests.

Tests Absorbance difference Glucose Type A

Type A RSD% Glucose Type B

Type B RSD %

Day 1 At 450nm-600nm 0.072 3.5 0.244 2.1

At 350nm-600nm 0.244 6.1 1.106 1.0

At λmaxnm-600nm 1.575 1.4 4.788 1.1

Day 2 At 450nm-600nm 0.064 13.6 0.248 2.3

At 350nm-600nm 0.241 6.3 1.106 0.3

At λmaxnm-600nm 1.571 3.0 4.651 0.4

Day 1-Day 2 At 450nm-600nm 0.068 10.5 0.246 2.2

At 350nm-600nm 0.242 5.6 1.106 0.7

At λmaxnm-600nm 1.573 2.1 4.720 1.8

According to above the results, the tested method is repeatable and reproducible.

3.5.2. Time

The influence of time on the glucose discoloration at ambient temperature was tested and the

results are presented in Table 9. No influence of time on color formation in glucose sample could

be observed at ambient temperature. The method is robust at ambient temperature with respect to

time.

Table 9. Effect of time in glucose solutions at room temperature.

Time Glucose A Glucose B B/A Absorbance factor

0,75 0.203 1.009 5.0

1 0.237 1.068 4.5

2 0.236 1.149 4.9

4 0.223 1.104 4.9

6 0.225 1.077 4.8

24 0.198 1.078 5.5

48 0.233 0.974 4.2

30

4. Conclusions

The optimum conditions for assessing the glucose stability with respect to discoloration was

found to be when mixing glucose with water, 33% (w/w), keep the samples at ambient

temperature for 1 h before determination of the color, absorbance difference at 350 nm by

UV/Vis spectrophotometer in a 4 cm cuvette.

For Maillard reaction, based on the difference between the two types of glucose, the, optimum

conditions were found to be the glucose-L-alanine model system, when heating the samples (33%

glucose, w/w) at 100°C for 1 h.

The stability of glucose with respect to discoloration and 5-HMF formation was proven to be

better for glucose type A compared to glucose type B.

The results showed that glucose manufacturing process plays an important roll in non-enzymatic

browning of glucose.

The proposed glucose discoloration method could be used as a quick tool to separate glucose that

potentially will develop yellow color in the manufacturing process and/or during storage of final

products containing glucose.

5. Acknowledgments

The author is grateful to Gambro Lundia AB Sweden for the financial support for this project and

would like to specially thank specialist Staffan Bergström for all his support and work in the

present study as well as to the personal at the Analytical Chemistry Group at Gambro Lundia AB.

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