KINETIC STUDIES ON CARAMELISATION OF REDUCING SUGARS

N. A. Ramaiah and M. B. Kumar

National Sugar Institute, Kan$ur, India

I. INTRODUCTION

Forination of caramel by alkaline destruction of reducing sugars is a reaction of inarlied importance to the sugar industry and has attracted the attention of nunier- ous workers from the beginning of this century. Maillard13 was apparently the first to observe the formation of colouring matter when the reducing sugars were heated. Spengler et al.21 noticed that prolonged heating of sugars at pH g and above gradually destroyed the sugar, with the formation of colouring matter, known as caramel. Lobry de Bruyn and Alberda van Ekensteinlo observed that in alkaline solutions, both glucose and fructose undergo a transformation resulting in an equilibrium inix- ture of glucose, fructose and mannose. Upon prolonged heating in alkaline solutions of the hexoses, formation of various acids, such as lactic, formic, dihydroxybutyric, succinic acids, etc. was recordedg,I4,15. Ramaiall and Agarwall7 examined chromato- graphically the coinposition of the carainel produced by the destruction of reducing sugars and found that five compounds of very high molecular weights were produced. Binkley3 determined the molecular weights of some of the carainel products in molasses; they were found to have inolecular weights as high as 23000 f 400.

Elucidation of the mechanism of the caramelisation of reducing sugars has been the subject of recent interest. Bainford and Collins1 studied kinectically the re- arrangenient of glucose and fructose in alkaline solutions. Apart from this study, as far as it is ltnown to the present authors, no systematic kinetic studies seein to have been made in caramelisation of reducing sugars from which it is possible to forinulate

I N . A. RAMAIAH et al. I a mechanism for the reaction. The kinetic studies of carainelisation or destruction of reducing sugars is therefore of considerable interest. The present paper reports a detailed investigation on the destruction of a few hexoses such as glucose, fructose, mannose etc. and also suggests a mechanism for the same.

I 2. E X P E R I M E N T A L

The following sugars were used (i) Glucose, (ii) Fructose, (iii) Mannose, (iv) Sucrose. These were Analar B.D.H. samples. Their purity was examined polarimetrically. (a) Sodium hydroxide, (b) potassium hydroxide and (c) calcium hydroxide were used as the alkalies. Of these (a) and (b) were pure coinmercial sainples while the calcium hydroxide was obtained by calcination of lime stone and has a purity of 95%.

Carbonates, sulphates, chlorides, etc. of sodium, potassium and calcium, inagnesiunl were used as the salt constituents. Phosphates of sodium and potassium were also used. The effect of sodium tetraphenylborate on the alkaline destruction of reducing sugar was also studied. Excepting the last mentioned compound, the selection of all the salts was based on their reported presence in cane juice. While all the salts were B.D.H. pure chemicals, tetraphenylboron was prepared in the authors' laboratories as follows, by employing Heyl'ss method. This involves the addition of boron trifluoride ethanate to phenyl inagnesiuin bromide and saturating the clear solu- tion with sodium chloride, when the compound precipitates. I t was purified by chloroforin and vacuum drying.

The following chemicals were used for analytical estimation of reducing sugars by Vargollci'szz and Felllings methods: potassium ferricyanide, copper sulphate, inethylene blue, sodium potassium tartrate. They were also pure Analar sainples.

For chromatographic studies, organic solvents, such as ethanol, butanol, chloroform and acetone were purified by distillation and used.

!

1 3. EQUIPMENT

The colour measurements were made on a Beckinan DU spectrophotometer. Beck- man Hz type pH meter was used with glass and calomel electrodes for measuring the hydrogen ion concentration. With solutions of pH more than 11, Acco pH meter was used. Conductivity studies were made on surface conductivity bridge, employing a cell with a corresponding constant of 0.91. Chromatographic analyses were made with the help of an indigenous wooden chamber fitted with glass panes. I t was provided with means to hold the solvents and chromatographic paper.

4. E X P E R I M E N T A L PROCEDURE FOR KINETIC STUDIES

Earlier studies on the kinetics of reducing sugars in alkaline media made by Bamford oLbiMEand eLl+ refer to the heating of a known concentration of glucose in an alkali of

kpown strength, and estimation of the ainount of lactic and saccharanic acids pro- duced at different intervals of time. In the present studies, the kinetic studies were

I77O PROCESSING

made primarily by following the loss of reducing sugar at different intervals of time. The accompanying changes, such as the formation of colouring matter, organic acids, other reducing sugars, etc. were also investigated. For this purpose the reactants, viz. solutions of alkali and reducing sugar were initially kept separately in Erlenmeyer flasks iininersed in a thermostat, which was previously maintained at a desired teinperarure. When the solutions attained the constant temperature, aliquots of these were talcen out by calibrated pipettes and transferred simultaneously to another flask imnlersed in the same bath. When the pipettes'were half empty, tlie reaction was considered to have started. At regular intervals of time, known volumes of the reaction mixtures were pipetted out and run into measuring" flasks containing ice cold distilled water to chill the reaction. The volumes of the flasks were inade up and these solutions were used for quantitative determination of reducing sugar, pH, colour and organic acids. Of these, the estimation of reducing sugar, pH and colour involved conventional methods. For the determination of the total organic acid content of the system, the conductometric method developed by Ramaiali and d l + and describ,ed elsewhere in detail, was employed. N RT&%J+~W

5 . RESULTS

Only typical results obtained from a series of experiments were reported here. The inain object of the presentation of the results has been to put forward essential and important experimental findings which have a bearing on the development of a mechanism of the caramelisation process. The other results which are of minor consequence or of repetitive nature are eliminated.

5.I Kinet ic studies o n loss of veducing sugavs

Fig. I gives a typical set of results on the changes occurring in the concentration of reducing sugar with length of tlie reaction time. Curves I, 2 and 3 refer to fructose, glucose and mannose, respectively. Kinetic analyses of these data showed that the reaction was first order with reference to the loss of sugar. Second and third order equations were found to be inapplicable to these results. Table I gives the data on the first order constants K for destruction of different sugars. For glucose at 60" C, K , was 3.35 x 10-4 sec-I. From these data the therinodynamic quantities associated with the reaction were calculated and these were given in Table 2. The energy of activation of the destruction of the hexoses corresponded to 20.0 f 0.5 lccals. Relnarlcably enough, the energy of activation and other thermodyna~nic quantities such as entropy change, etc. are rougl~ly the saine for reactions with glucose, fructose, niannose etc. These experiments were repeated several times and the results were found to be nonspurious and reproducible.

Effect of different salts on destruction of reducing sugars was studied in detail. These investigations were initially carried out as follows. To known quantities of reducing sugar solution, salts were separately added such that the total ionic strength was the sanze and heated at a fixed temperature. The progress of the reaction was found to be too slow to investigate the influence of the salts per se. For this reason, the influence of different salts on Kl was carried out at a fixed pH or at a known

. - 0 4 0 80 120

Time in minutes . Fig. I . Destruction of reducing sugars a t 70' C. NaOH, = 0.625 M. Curve I, Fructose, 0.555 M. Curve 2, Glucose, 0.555 M. Curve 3, Mannose, 0.555 M.

TABLE I

FIRST ORDER RATE CONSTANTS FOR DESTRUCTION O F GLUCOSE, FRUCTOSE AND MANNOSE

Temp. 60" C, Redncing sugar 0.555, NaOH 0.625 Time lnin Glucose- Fructose Mannose

X 10-4sec-1 X I O - ~ S ~ C - ' x I O - ~ S I ~ C - '

TABLE 2

DETERMINATION O F THERMODYNAMIC PROPERTIES FOR THE DESTRUCTION O F REDUCING SUGARS

' Thermodynamic Glucose Fructose Mannose property

K 3.352 x 10-4 sec-I 4.212 x 10-4sec-I 3.25 X 10-4 sec-I AH 19.69 kcals 19.40 ltcals 19.74 lrcals dl? 24.84 licals 24.62 kcals 24.78 ltcals AE 20.35 licals 20.06 lrcals 20.40 lrcals d s - 15.4 cals/"/inole - 15.67 cals/"/mole - 15.12 cals/"/mole +lb/Kt 2.58 2.54 2.6

TABLE 3

INFLUENCE O F VARIOUS SALTS ON TIlE VELOCITY CONSTANT K

Salt Concentration I<

Glucose alone - 3.352 X 10-4 sec-I

Sodium carbonate 0. I 0.25 0.35

Potassium carbonate 0.1

0.25 0.35

Calcium carbonate 0.10

0.25 0.35

Sodium phosphate 0.10 3,144 x 10-4

0.25 3.01 x 10-4

Sodium sulphite 0.1 3.200 x 10-4

0.25 3,080 x 10-4

0.35 3.000 x 10-4

Sodiuln chloride

Sodium tetraphenyl borate 0.10 3.352 x 10-4

0.20 3.352 X 10-4

0.30 3.352 X 10-4

TABLE 4

Bj CONSTANTS O F ANIONS FOR THE DESTRUCTION O F REDUCING SUGARS

Carbonate (C0,Z-) +0.25 Phosphate (PO4,-) -0.22

Sulphite (S0,Z-) -0.24 Chloride (CI-) -0.10

Sulphate (So42-) -0.18 Hydroxyl ion (OH-) +0.55 Tetraphenyl borate 0.00

PROCESSING

concentration of alkali. The change in the magnitude of K over the blank containing only the alkali, gave the influence of the salts. The data obtained are given in Table 3. I t is of interest that from these results, the cations like K+, Na+, Li+, CaZ+, etc. have no influence on K, on the other hand the anions such as C032-, SO,Z-, S032-, PO,3- etc. exhibited remarkable effect on the magnitude of K. Suggestively enough, C02, is catalytic, while SO,Z-, PO,3-, etc. have inhibitory effect on the destruction of reducing sugars. These results on the influence of salts on the velocity constant of destruction of reducing sugars were analysed by Bronstead's equations for the primary salt effects on kinetics of cheinical reactions. Table 4 gives the Bj constants for the role of different salts. These values also point out the significant role of different anions on the reaction. I t is of interest to note that sodium tetraphenylborate, however, has no influence on the velocity constant. This is of marked significance and will be referred to later again.

I

N. A. RAMAIAH et al. I773

5.2 Colour fohnat ion- Induc t ion period

I t is known that during the destruction of reducing sugars in alkaline media, brown colour (caramel) is produced. This has an absorption maximum at 415 inp in the visible region (see Fig. 2). A few attempts have been made earliei-19~6 to employ the absorption of caramel at this wavelength to follow the kinetics of the reactions. For

I reasons elucidated and discussed in detail elsewhere, this procedure has not been used by us for study of the kinetics of the reaction and the evaluation of therino- dynamics thereof. Nevertheless, the data in Fig. 2 indicate an important factor

I,--- 400 440 480 520

Wave length (my)

Fig, 2. Absorption spectrum of caramel.

Fig. 3 . Development of colour during the destruction of glucose. Curves I and 3 = blank, 2 and 4 = with calcium lactate.

relating to the formation of caramel during destruction of reducing sugar. Reference may be made to Fig. 3, which indicates the time development of colour during the heating of glucose in 0.625 M NaOH (curves I, 2, 3). The formation of the colour is very slow at the beginning of the reaction indicating the existence of an illduction period in the process of colour developinent. This, however, does not appear in the curve representing the loss of sugar with time (see curve I, Fig. I). Further, the induction period is reduced or eliminated by the external addition of organic acids component, such as lactate, etc. (see curves 2, 4, Fig. 3). This observation suggests that the induction period is associated with the organic acid content of the reactions system.

5.3 Quantitative investigation of organic acid content produced during cavanzelisation

I t is known that when alkaline solutions of reducing sugars are heated, the concentra- tion of alkali present in the system is reduced and the pH of the solution falls. Cukes11 and othersIZ,5,II,7 detected chroinatographically the forination of organic

PROCESSING

I acids, of which lactic acid was found to be predominant. This accounts for the

I observation that despite its absence in raw cane juice, lactic acid is found in large

I quantities in n~olasses due to destruction of reducing sugars in factory operations.

I Quantitative assessment of the exact ainount of organic acid produced when reducing sugars are heated for different intervals of time, could not be made on account of the cuinbersoine procedures involved in quantitative chromatography. A simple elegant conductoinetric illethod developed in the authors' laboratories13 made this investigation possible. Table 5 gives the data on the total organic acid produced

IABLB 5

I PRODUCTION O F ORGANIC ACIDS DURING THE DESTRUCTION OF GLUCOSE AT 60' C

Concentration of glucose 0.556 M Concentration of NaOH 0.625 M

Amount of reducing sugar de- stroyed (g/ moles/l)

Percentage of reducing sugar destroyed

Amount of reducing sugar con- tributed to acid procluc- tion (g/moles/l)

0.0089 0.0179 -

0.0366 0.0527 0.0677 0.0733 0,0777

Destructed sugar convert- ed to acid, %

I at different intervals of time, when 0.55 mole of glucose was heated at 60" C. Column

I 2 shows the amount of reducing sugar utilised for the formation of lactic acid. I t is of interest to note from those results that the entire quantity, 100% of the reducing sugar destroyed in the initial stage of the reaction is totally utilised for the production of organic acids. With the progress of the reaction, this proportion decreases indi- cating that production of organic acids is one of the preliilliilary steps in the caramel- isation process. Siinple calculations showed that the ainount of alliali consumed was equivalent to the organic acid produced. The role of alliali in the caramelisation process seeins only to neutralise the acids formed.

5.4 Chromatog~a$hic s t ~ d i e s on the com$osition of cayamel

These studies relate primarily to the investigation of the nature of reducing sugars undergoing change or those formed during the heating of glucose, fructose, etc. in alkaline solutions. The method employed was due to Patridge16 and described by us elsewhere in detailI7. The composition of the reaction mixture was chromato- graphed before the start and at different intervals of time during the progress of the reaction. Figs. 4, 5 and 6 give the results obtained with glucose, fructose and nlannose respectively. In these experiments zero relates to the system before the'

I start of the reaction. The data indicate obviously the presence of one reducing sugar

I taken; chron~atogram 2 shows the composition of the reaction mixture after 2-3

I min. The presence of three reducing sugars: glucose, fructose and mannose was

N. A. RAMAIAH et al. I775 - T~me ~n rnlnutes Tlme ~n minutes

0 3 5 7 10 30 0 2 4 5 10 30

Fig. 4. Kinetics of caramelisation of glucose at 70" C.

Fig. 5. Kinetics of caramelisation of fructose a t 70' C.

Fig. 6. Kinetics of caralnelisation of mannose a t 70" C.

noticed in all the reaction systems. Whatever reducing sugar: glucose, fructose or mannose, was taken as the reacting species, the forination of a niixture of glucose, fructose and mannose has been noticed, in accord with the Lobry de Bruyn-Alberda van Ekenstein transformation. Further, chromatograms 3-6 show the forillation of higher illolecular weight compounds after 3-4 min of reaction time, when the colour formation in the system begins to occur. These high molecular weight conipounds could not, however, be detected a t early stages (see chromatograms 2, Figs. 4 and

. 5). The RF values 'of the final products are given below: Spot No. R ~ { X roo) 1. 0

2. 2.0 3. 4.4 4. 6.6 5. 9.5 6. 13.0 7. 14.9

1 7 7 ~ PROCESSING

I t is of marked interest to note that the composition of the final products is independent of the hexose taken as the reactant. This observation and the same identical values of energy of activation and thermodynamic properties of the reaction (see above) suggest that whatever may be the hexose talien for heating in alltaline medium, one of the componeilts of the Lobry de Bruyn-van Eltenstein equilibrium mixture or intermediates thereof forms the basic species entering the rate-controlling reaction, which yields the same final products.

The following is a brief rCsurnC on the important findings relating to the c'aramelisa- tion process occurring when the reducing sugars, such as hexoses, are heated in alltaline solutions.

(a) When hexoses are heated, Lobry de Bruyn-Alberda van Eltenstein trans- formation occurs with the formation of glucose, fructose and mannose.

(b) Loss of reducing sugar content obeys a first order law. The energy of activation, entropy changes, etc. are roughly the same. The chailge in the entropy is negative.

(c) Anions of the salts exhibit a inarlted role on the liiiletics of the reaction. (d) Formation of organic acids is one of the preliminary steps for the develop-

ment of coloured caramel. (e) The end products which are the same for different reactants, are compounds

of high molecular weights. Lobry de Bruyn-Alberda van Eltenstein transformation has been well investi-

gated by many worlters20~~j. The transIorination appears to proceed through epimeri- sation and isomerisation.

i H-C=O HO-C-H CH20H H-C-01-1 H-C=O II

H-7 -OH n I ?-OH $=O II HO-5 I

HO-f-H I I I I I

HO-C-H -HO-C-H-HO-C-H _ HO-C-H HO-C-H I - I - I - I = = H-c-OH H-c-OH H-c-OH H-c-OH H-&--OH I I I

I H-C-OH H-C-OH H-C-OH H-C-OH H -C -OH

I I I I I

G-Glucose Eneaiol D-Fructose Enediol D-Mannose ftra?sl (cis)

(1) (11) (Ill) (IV) (V)

I t is also reported that the above transformation occurs through the formation of intermediate enediol formation (I1 and IV).

The formation of the equilibrium mixture does not materially affect the reducing nature of the reacting system. In other words the kinetic studies by the authors do not relate to the above epimerisation and isomerisation processes.

The loss of reducing power of the system during the progress of reaction is apparently due to loss of one of the Lobry de Bruyn-Alberda van Ekenstein equilib- rium products or the intermediary species. No data exist to indicate the specific

I N. A. RAMAIAH et al. I777

one of these products entering further steps of the reaction of caramelisation. In what follows it is assullled that the actual species undergoing the rate-controlled reaction is enediol (11) or (IV) molecule. The possibility of the reacting hexoses, viz. glucose, fructose, mannose etc. entering the rate-controlled reaction was not con- sidered because of the similar energy of activation and other thermodynamic properties and on account of the same end products of the caramelisation reaction. A coinlnon intermediate enediol (E) inolecule seems to be the species taking part in the rate- controlling reaction.

Since the experimental data indicate that the reaction is a first order one and the ions such as OH-, CO;z-, etc. play an important role in the reaction, thefollo\ving scheme may be considered.

I E + O H - 2 E . . . . OH- - (A)

E . . .OH-+A+ B + OH- (B)

In this, E is the enediol molecule and E....OH- is the intermediate conlplex with negative charge formed by the combination of glucose and hydroxyl ion. Reaction (B) refers to the unimolecular breakdown of the E.. . .OH- into reaction intermediates and OH-. I t further suggests that the role of OH- is but catalytic in accord with the data presented earlier. Mechanisms of chemical reactions with the postulate of the formation of an intermediate charged complex are not uncommon. To quote a familiar case, the inversion of sucrose in acid solutions involves the formation of an intermediate complex with positive cllarge45.

This was found helpful to elucidate the catalytic nature of the H+ ions and inore so, the influence on the reaction kinetics of the anions C1-, Br-, I-, SO:-, etc. on the basis of Guggenheim-Bronstead theory4. The anions affect the kinetics of the positively charged complex in (C). Similar considerations do not hold good for the elucidation of the influence of salts on the kinetics of caramelisation of reducing sugars from the standpoint of the mechanism suggested in the above steps (A) and (B). This scheme which refers to the formation of a negatively charged complex E-OH- predicts that the cations and not the anions of salts have a pronounced effect on the alltaline decompositioil of reducing sugars.

The role of other anions in the caramelisation process seem therefore to be different from what one finds in the case of the inversion of sucrose. I t is postulated that all the anions combine through covalent linkage to one of the hydrogen atoms of the enediol molecule to form the intermediate complex (E-X). Thus in case of the influence of SO,Z- on the destruction of a hexose, the mechanism suggests

HO-C-H HO-C-H I C-OH

II C-OH

I HO-C-H

I HO-C-H - - - - - so2- I C +so;- - I

H-C-OH H-C-OH I

H-C-OH I

H-C-OH I

CH20H I CH20H

Enedlol lntermedlate complex

177~ ' PROCESSING

,In this, the combination of S0,z- is shown with C, as an example. According to this, reaction B refers to

HO-C-H HO-C-H II C-OH

II C-OH

I HO-C-H ..--. I

HO-C-H

I - + + so:-

H- C-OH H-C-OH I I

I H-C-OH

I H-C-OH

I CH20H CH20H

No data exist to identify the exact enediol, cis or tvans (I1 or IV) that is entering the above inechanisn~. One can expect differences in kinetic data with cis and tvans enediol.

The above ilzechanisin suggests that all anions influencing the caramelisation process combine with one of the hydrogen atoms of the enediol molecule by covalent linkage. From this one would expect that those salts which cannot conibine with any of the hydrogen atoms of the enediol inolecule through covalent bondage, should not have any influence on the destruction of reducing sugars. In this connection, it is of interest to consider the data on the effect of tetraphenylboron on the carame-

C I 010-

a, X

P - +

Temp 60°C

$ 005- A_x-o-~-.-x-o-~-

a, x/"-

8 - A/ Glucose 0.222M G i ~ '

0 2 - NaOH 0.055M

S /-*-O-X-~- m n

4 O l - 2

I /

. / '

px , 0 80 160 2 4 0

Time in mlnutes

Fig. 7. Influence of Na(CGH,),B on colour developmeilt and on destruction of glucose. NaHO 0,055 M. Glucose 0.222 M. 0-0 = blank; x-x = with 0.1 M Na(C6H,),B; A-A = with 0.2 M Na(C&),B.

lisation process (see Fig. 7). This ion cannot combine with any other atom by covalent linkage. The results in Fig. 7 show, suggestively enough that tetraphenylboron does neither enhance or inhibit the caramelisation process.

N. A. RAMAIAH et al. I779

orm mat ion of organic acids during the heating of the alkaline solutioils of hexoses is a subject which attracted the attention of many workers. The following mechanism was suggested for the formation of lactic acid from hexoses:

H-C=O CHOH CHOI-I I II II H\ /OH

H-C-OH C-OH - C-OH I - I

?OH

HO-C-H - HO-C-H I - -

HO -C-H2 I

H-C-OH I

H-C-OH T

I I LH3 7-0 l

H -C-OH H-C-OH C HO I

COOH

CH20H I I

H2COH Glycerol - CHOH I

CHOH dehyde I

CH20H I CH3

Lactlc acld

Production of high inolecular weight caramel products occurs through condensation of the aldoses orland hexoses with acids produced during the reaction.

7. CONCLUSION

The following mechanism has been considered possible for caramelisation of reducing sugar.

Hexoses Z Enediol (E) , . . . (1)

E -f A- Fast E . . .A- . . . . (11)

E . ..A- 4 Organic acids f Slow

aldehydes + A- . . . .. (iii)

Aldehydes or hexoses + organic acids 4 Caramel products . . (IV)

Here A-refers to an anion such as OH-, COi-, S0,Z-, etc. which influences the destruc- tion of reducing sugar. The kinetic data obtained on the loss of reducing sugar presented in this communication refers oilly to the reaction (111) above, which indicates the forination of the intermediate organic acids, the presence of which has been detected and analysed. The last reaction indicates a process of formation of high ilzolecular weight caramel compounds. Kinetics of this reaction (IV) are of interest. I t follows from above that till an adequate quantity of organic acids has not been produced to enter reaction (IV), no colour development during the process of heating of reducing sugars in alkaline solutions occurs, in accordance with the observation that there exists an induction period during the process of colour forination in the reaction of caralnelisation of reducing sugars (see Fig. 3). Sug- gestively enough, when acids, such as lactic acid, are added initially to the reaction mixture, this induction period is eliminated.

REFERENCES

I BAMFORD, C. A. AND J. R. COLLINS (1955) I<inetlc studies of carbohydrates in allralllle con- ditions. PYOC. Roy. SOC. (London), A228 100.

2, BAMFORD, G, A., D. BAMPORD AND J. R, COLLINS ( ~ 9 5 0 ) Kinetlcs of rearrangement of glucose and frnctose in alkalinc solutions. Pyoc, ROY. SOC. (London), A204. 85.

1780 PROCESSING

3 BINIZLEY, W. W. (1960) (i) An estimation of the molecular weight of the dialysed brow~lillg products from cane final molasses. Intern. Sugar J., 6 2 : 36. (ii) The isolation of membrane diffusion of certain products from cane'final molasses. Intern. Sugar J. , 6 2 : 176.

4 BROXSTEAD, J. N. AND E. A. GUGGENHEIM (1927) Theory of acid base catalysis. J . A m . CAem. Soc., 4 9 : 255. (a) E. A. GUGGENHEIM, and L. A. WI'SSMAN (1950) Kinetic salt effects on inversioil of sucrose, Proc. Roy . Soc. (London) A 2 0 3 : 17.

5 BUCH, M. L., R. MONTGOMERY AND W. L. PORTER (1952) Identification of organic acids on paper chromatograms. Anal . Clzem., 2 4 : 289.

6 Doss, K. S. G. AND S. K. GHOSH (1948) Icinetics of developmellt of colour in sugar solutions. Proc. S . T . A . ( I n d i a ) , 17: 51.

7 EVANS, W. L. (1947) Some less familiar aspects of carbohydrate chemistry. Chem. Revs., 31: 537.

8 HEYL, W. (1954) Coinplex metal tetraphenyl boron salts. Brit . Pdtegt 705, 719, Mar. 17. '

9 ISBELL, H. S. (1942) Preparation of lower aldonic acids by oxidation of sugars in alkaline medium. B u r . Standards, J. Res. ( W a s h . ) , 2 9 : 227.

10 LOBRY DE BRUYN AND ALBERDA VAN EKENSTEIN (1899) Action of alkalies on the sugars. Rec. Trav. Chim. , 2 6 : 81.

11 LUGG, J. H. W. AND B. T. OVERELL (1948) One and two dimensions partition chromatographic separation of organic acids. Aus t . J. Sci . Res., A I : 98.

12 LUIZESH, H. (1956) Degradation products of caramelised sugars. Naturwiss., 4 3 : 108. . 13 MAILLARD, L. C. (1961) Synthesis of humus lilre substances by the interaction of amino acids

ancl reducing sugars. A n n . Chim. , 5 : 258. 14 MONTGOMERY, R. (1953) Observations on allraline degradation of sugars. I n d . Eng . Chem., 4 5 :

1744. 15 NEB, J. U. (1914) Dissociatioll reactions in the sugar group 111. A n n . , 403: 2014. 16 PATRIDGE, S. M. (1946) Application of the paper partition chromatograms to the qaantitative

analysis of reducing sugars. Nature, 158: 270, 17 RAMAIAH, N. A,, S. K. D. AGARWAL AND J. I<. P. AGARWAL, ( 1 9 ~ 3 (i) Chromatographic studies.

on the kinetics of the production of caramel. Proc. I n d . Acad. Sci., 4 5 : 97. (ii) Composition of caramel prepared by heating sugars. Current Sci . ( I n d i a ) , 2 6 : 81.

18 RAMAIAH, N. A,, R. NATRAJAN AND G. D. CHANSARIZAR (1961) A method for estimatioll of organic acid component in technical sugar solutions. Sharkara, 4 : 13.

19 RAMAIAII, N. A. AND S. K. D. AGARWAL (1960) 011 the kinetics of caramelisation of sugars. Proc. S . T . A . ( I n d i a ) , 2 8 : 101.

20 SOWDEN, J. C. AND R. SCHAFFER (1952) The reaction of D-glucose, D-maimose and D-fructose in 0.035 N NaOH a t 35" C. J. A m . Chem. Soc., 7 4 : 499.

21 SPENGLER, O., S. BOTTGER AND B. SEELIGER (1936) The chemistry of darlce~ling of sugar juices. 2. Wirtschaftsgruppe Zuckerind., 86 : 193.

22 VARGOLLCI, V. AND A. IONNESCUE (1920) New method for the volumetric determination of reducing sugars. Bull . Soc. Chim. ( R o u m a n i a ) , 2 : 381.

23 WOLFROM, M. L. AND W. L. LEWIS (1920) Reactivity of methylated sugar 11. Action of dilute alltali on tetramethyl glucose. J. A m . Chem. Soc., 5 0 : 837.