Analysis of caramels by capillaryelectrophoresis and ultrafiltrationLouise Royle* and Catherine M RadcliffeDepartment of Food Science and Technology, The University of Reading, Whiteknights, PO Box 226, Reading, RG6 6AP, UK

Abstract: Class I, III and IV caramels are distinguished from each other by capillary electrophoresis at

pH 2.5 and 9.5. The majority of the colour is shown to be in the high molecular weight (MW) fraction of

all the caramels. The high MW Class I and Class IV caramel peaks migrate with a negative charge at

both pHs but the Class IV caramel also has several sharp peaks from low MW components compared to

only a small neutral peak from the Class I caramels. The Class III caramels have a high MW peak which

is positively charged at pH 2.5. The migration time, and hence charge, of the high MW peak of Class III

caramels is shown to be related to the caramel's nitrogen content.

# Crown copyright 1999

Keywords: caramel; Class I caramel; Class III caramel; Class IV caramel; capillary electrophoresis

INTRODUCTIONThe International Technical Caramel Association

divides caramels into four different categories accord-

ing to the reactants added to the sugar during

caramelisation.1 Class I caramels are produced by

heating sugar at high temperature without the addition

of nitrogenous `catalysts'; they are used for colouring

spirits such as whisky. Class II caramels have small

amounts of sulphite added as reactant, and are used in

aperitifs. Class III caramels have ammonia as reactant,

producing highly coloured caramels which are widely

used in beers and foodstuffs. Ammonium sulphites are

used as reactants for production of Class IV caramels;

these highly coloured, negatively charged caramels are

used in cola type beverages. By varying the exact

processing conditions, a wide range of products within

each of these classes can be obtained. The relative

consumption of caramels in the UK and world-wide

differs considerably, being respectively: 5% and 2% for

Class I; <1% and 1% for Class II; 70% and 28% for

Class III and; 25% and 70% for Class IV.1,2

Spectrophotometric measurement of colour is cur-

rently used for the determination of caramel concen-

tration. The different classes are distinguished by the

addition of positively charged DEAE cellulose or

negatively charged phosphoryl cellulose to an acidi®ed

solution of the caramel, which causes precipitation of

Class IV or Class III caramels, respectively.3 These

methods are fairly crude and cannot be used reliably

with foodstuffs where other food components can

interfere with both the colour measurement and the

precipitation reactions. Therefore, more speci®c ana-

lytical methods for caramels are needed. Coffey et al4

used ion-pair HPLC of a low-molecular-weight

marker compound for Class III caramel to estimate

the amounts of this caramel in various foods. Coffey etal5 also used capillary electrophoresis (CE) to analyse

Class III caramels and identi®ed some peaks which

were present only in this class of caramel. We have

found CE more successful for the analysis of high MW

melanoidins which are similar to caramels, than

HPLC, as these compounds adhere to the HPLC

columns.6 Also, we7 have shown that CE can be used

to both identify and quantify Class IV caramels in soft

drinks.

This study aims to identify, and distinguish

between, the major caramel classes (I, III and IV)

using CE.

MATERIALS AND METHODSMaterialsA range of Class I, III and IV caramels was obtained

from British manufacturers. Caramel solutions were

made up at 5mgmlÿ1 in high purity water which was

prepared in-house with a Purite Labwater RO50 unit

(Purite Ltd, High Wycombe, UK) and used through-

out. Combustion elemental analysis for nitrogen and

sulphur content was performed by either Medac Ltd

(Egham, UK) or The University of Kent (Canterbury,

UK) on samples dried at 105°C.

Sodium hydroxide solutions for CE (1.0 and 0.1M)

were obtained from Fluka (Gillingham, UK). Phos-

phate buffer was prepared from 30mM sodium

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1709±1714 (1999)

* Correspondence to: Louise Royle, Department of Food Science and Technology, The University of Reading, Whiteknights, PO Box 226,Reading RG6 6AP, UKContract/grant sponsor: Ministry of Agriculture, Fisheries and Food, UK(Received 27 February 1998; revised version received 22 March 1999; accepted 13 May 1999)Neither Her Majesty’s Stationery Office nor the Department of the Ministry of Agriculture, Fisheries and Food, UK accept any responsibility forthe accuracy of any recipe, formula or instruction published in this Article or in the Journal.

# Crown copyright 1999. Reproduced with the permission of the Controller of Her Majesty's Stationery Of®ce.J Sci Food Agric 0022±5142/99/$17.50 1709

dihydrogen orthophosphate monohydrate (AnalaR

grade, Aldrich, Gillingham, UK) adjusted to pH 2.5

with hydrochloric acid (AnalaR grade, Aldrich).

Carbonate buffer was made by mixing 50mM solu-

tions of disodium carbonate and sodium hydrogen

carbonate (GPR grade, BDH) to give pH 9.5. All

solutions were ®ltered before use with a 0.2-mm PVDF

®lter (Whatman, Maidstone, UK).

Uncoated fused-silica capillaries were obtained

from Hewlett Packard (Bracknell, UK).

MethodsCapillary electrophoresis

A Hewlett Packard HP3D CE with diode-array

detection and an HP3D CE ChemStation for instru-

ment control, data acquisition and data analysis was

used throughout.

At the beginning of each day, the capillary was

preconditioned by ¯ushing successively with 1M

NaOH for 10min, 0.1M NaOH for 5min and water

for 5min. CE running conditions were: buffer: pH 9.5,

50mM carbonate buffer (changed every four runs) or

pH 2.5, 30mM phosphate buffer (changed every run);

capillary: 40cm to detector, 48.5cm total length,

50mm ID, X3 bubble; voltage: 20kV with pH 9.5

buffer, 30kV with pH 2.5 buffer, ramped up over

0.3min; temperature: 25°C; injection: 50mbar, 5s;

preconditioning: 3min 0.1M NaOH ¯ush, 3min

buffer ¯ush; detection: 200, 280, 360 and 460nm

with a band width of 10nm, plus optional full spectra

collection, 195±600nm.

Ultra®ltration

Caramel solutions were prepared at 5mgmlÿ1 in

water. This `total' caramel solution was separated into

high and low MW fractions using Microcon centrifu-

gal ®ltration devices with low-protein binding, regen-

erated cellulose ultra®ltration membranes of 5000

Dalton nominal molecular weight (NMW) cut-off

(Millipore, Watford, UK). A 200ml sample of the

caramel solution was ultra®ltered by centrifugation

and the LMW fraction collected. The retained HMW

fraction was then washed with 2�200ml of water,

reconstituted to 200ml, then 0.22mm ®ltered using an

Amicon micropure separator (Millipore, Watford,

UK).

RESULTS AND DISCUSSIONThe 11 Class III caramels analysed had nitrogen

contents of 9.08, 8.53, 8.26, 7.65, 6.63, 6.38, 5.83,

5.76, 4.84, 4.29 and 4.00%. The four Class IV

caramels analysed had nitrogen contents of 5.35,

0.86, 0.52 and 1.02% and sulphur contents of 8.90,

2.59, 2.07 and 1.79%, respectively. The ®ve Class I

caramels were assumed to have little or no nitrogen or

sulphur content, as neither ammonium nor sulphite is

added during their manufacture.

During the caramelisation reaction, some low MW

byproducts are also produced4 and ultra®ltration

membranes with a nominal MW cutoff of 5000

daltons were used as a means of separating the high

MW caramels from these components. The MW range

of caramels produced is expected to be wide, so,

although the high MW fraction should only contain

caramel, some lower MW caramels may be present in

the low MW fractions.

All the electopherograms (e-grams) shown are taken

at 200nm, because this is the most sensitive wave-

length for detection of all the components in the

caramel mixtures. The high MW caramels absorb over

the full wavelength range of the instrument (195±

600nm) with very similar absorbance spectra (Fig 1).

As there are no distinguishing differences between the

spectra from the different caramels, this peak is

referred to as the `coloured' caramel peak throughout.

The other, sharp peaks detected at 200nm show

Figure 1. Absorption spectrum of the coloured caramel peak from a ClassIV caramel.

Figure 2. E-grams obtained in carbonate buffer at pH 9.5, detection at200nm of Class I caramel as (a) total caramel; and ultrafiltration fractions(b) >5000 NMW and (c) <5000 NMW.

1710 J Sci Food Agric 79:1709±1714 (1999)

L Royle, CM Radcliffe

absorbance only in the UV and therefore are not

coloured.

Analysis in carbonate buffer at pH 9.5At pH 9.5, the electro-osmotic ¯ow (EOF) is fast

(2.9min), as all the silanol groups on the capillary are

ionised. This ¯ow is fast enough to carry negatively

charged compounds past the detector, which is located

near the cathodic end of the capillary, even though

they are migrating in the opposite direction. At pH 9.5,

all the caramel classes would be expected to be

negatively charged.

All the Class I caramels ran as a single, broad,

coloured peak without any signi®cant spikes from low

molecular weight (MW) compounds, except for a

small neutral peak migrating with the EOF. Figure 2a

shows an e-gram typical of this class of caramel. When

the ultra®ltration fractions are examined (Fig 2b above

and Fig 2c below 5000 NMW), it is clear that most of

the broad coloured peak is of high MW, although there

are some components within this peak which are of a

lower MW.

Figure 3 shows e-grams of a Class III caramel with

5.83% nitrogen content. At pH 9.5, all the Class III

caramels analysed gave similar e-grams with a large

neutral peak, and a coloured peak with a mean

migration time of between 3.4 and 4.0min. The broad

coloured peak consists of high MW caramels (Fig 3b).

The majority of the low MW compounds migrated as

though neutral, with only a small number of positively

and negatively charged peaks detectable (Fig 3c).

Figure 4 shows e-grams of a high-nitrogen high-

sulphur Class IV caramel. Previous work7 has shown

that the migration time of Class IV caramels in

carbonate buffer at pH 9.5 is related to the caramel's

sulphur content; the higher the sulphur content, the

longer the migration time of the caramel peak.

Ultra®ltration gives a clear separation between the

high MW coloured caramel peak (Fig 4b) and the low

MW components (Fig 4c). The Class IV caramels

contain the highest number of peaks from low MW

components of all the caramels analysed at this pH.

Analysis in phosphate buffer at pH 2.5At pH 2.5, the EOF is very slow (about 25min), as

most of the silanol groups on the capillary are not

charged. Compounds which are negatively charged at

this pH are not carried past the detector, as they are

migrating in the opposite direction faster than the

EOF.

The e-grams of the Class I caramel (Fig 5) at this pH

show only one peak, which is neutral and runs with the

EOF. This peak is from low MW components (Fig

5c), but its total area is greater than that seen from

neutral components at pH 9.5. This could be due to

compounds which are negatively charged at pH 9.5

becoming neutral at pH 2.5 and/or absorbance

changes due to different buffer interactions. However,

no peak is seen for the high MW coloured caramels,

Figure 3. E-grams obtained in carbonate buffer at pH 9.5, detection at200nm of Class III (5.83% nitrogen) caramel as (a) total caramel; andultrafiltration fractions (b) >5000 NMW and (c) <5000 NMW.

Figure 4. E-grams obtained in carbonate buffer at pH 9.5, detection at200nm of Class IV (5.35% nitrogen, 8.90% sulphur) caramel as (a) totalcaramel; and ultrafiltration fractions (b) >5000 NMW and (c) <5000 NMW.

J Sci Food Agric 79:1709±1714 (1999) 1711

Analysis of caramels

indicating that the Class I caramels are still negatively

charged and thus have isoelectric points of less than

pH 2.5.

Figure 6 shows e-grams typical of the Class III

caramels. This is the only class of caramel where the

high MW coloured caramel component is positively

charged at pH 2.5. Thus the Class III caramels have

isoelectric points between pH 2.5 and 9.5. Figure 6b

shows that the majority of the broad peak is made up of

high MW coloured caramel. Figure 6c shows that

there are low MW compounds present, some posi-

tively charged and some neutral. It also shows that

some of the coloured peak is of a NMW lower than

5000. Figure 6a is similar to the e-gram obtained by

Coffey et al5 of Class III caramel analysed in 30mM

phosphate buffer at pH 1.9, suggesting that the large

peaks picked up by their HPLC analysis were low MW

neutral compounds.

In Fig 7, the e-grams of the Class IV caramel show

sharp peaks for the low MW compounds, some of

which are positively charged and some neutral at this

low pH (Fig 7c). The coloured, high MW caramel

peak is not seen (Fig 7b), indicating that, like the Class

I caramels, the Class IV caramels are still negatively

charged and thus also have isoelectric points of less

than pH 2.5. All the different Class IV caramels gave

e-grams similar to Fig 7, but with progressively lessFigure 5. E-grams obtained in phosphate buffer at pH 2.5, detection at200nm of Class I caramel as (a) total caramel; and ultrafiltration fractions(b) >5000 NMW and (c) <5000 NMW.

Figure 6. E-grams obtained in phosphate buffer at pH 2.5, detection at200nm of Class III (5.83% nitrogen) caramel as (a) total caramel; andultrafiltration fractions (b) >5000 NMW and (c) <5000 NMW.

Figure 7. E-grams obtained in phosphate buffer at pH 2.5, detection at200nm of Class IV (5.35% nitrogen, 8.90% sulphur) caramel as (a) totalcaramel; and ultrafiltration fractions (b) >5000 NMW and (c) <5000 NMW.

1712 J Sci Food Agric 79:1709±1714 (1999)

L Royle, CM Radcliffe

marked low MW peaks as the caramel sulphur content

decreased.

Figure 8 compares four of the Class III caramels in

the early region of the e-gram where the positive,

largely high MW, components appear. When the

migration times of this broad peak (taken at the apex

of the parent peak) from all 11 Class III caramels are

examined, a plot of mean migration time (from three

runs) vs % nitrogen gives a moderately good ®t to a

straight line (Fig 9, r2=0.858), whereas a plot of mean

peak width at half height (from three runs) vs %

nitrogen gives a somewhat lower ®t (Fig 10,

r2=0.706). This shows that at pH 2.5 the mean

migration time and peak width at half height are

related to the nitrogen content of the Class III caramel

in a more or less linear fashion. This relationship can

be explained by the nitrogen-containing chemical

groups carrying a positive charge at this pH; therefore,

the higher the nitrogen content, the higher the positive

charge on the caramel and the shorter its migration

time. The decrease in peak width with increasing

nitrogen content could be due to a narrowing of the

range of size-to-charge ratio of caramel, as the

likelihood of an even nitrogen distribution increases,

and/or there could be less band broadening with

decreasing migration time due to capillary-wall inter-

actions.

CONCLUSIONSThe high MW component of Class I caramels migrates

as a broad coloured peak at pH 9.5, but this peak is not

detected at pH 2.5, suggesting an isoelectric point of

less than 2.5. There is only one small neutral peak of

low MW detected at both pH 9.5 and 2.5, presumably

due to the sugar solution undergoing a less complex

caramelisation reaction in the absence of added

chemicals than in their presence, as with Class III

and IV caramels.

Class III caramels all have similar e-grams at pH 9.5,

a broad coloured peak of negatively charged high MW

caramel and a large sharp peak from neutral low

MW components. At pH 2.5 the coloured high MW

caramel migrates as a positively charged broad band.

The mean migration time and width at half height of

this peak are shown to be related to the caramel's

nitrogen content. There are also several positively

charged and neutral low MW components detectable

at pH 2.5. The Class III caramels have isoelectric

points between 2.5 and 9.5.

For the Class IV caramels, the high MW coloured

caramel migrates as a broad band at pH 9.5, the mean

migration time being related to the caramel's sulphur

content.7 This peak is not detected at pH 2.5,

suggesting isoelectric points of less than 2.5. There

are also many low MW components detected at both

pH 9.5 and 2.5.

The major caramel classes can now be distinguished

by CE in simple aqueous solutions. Class III and Class

Figure 8. E-grams obtained in phosphate buffer at pH 2.5, detection at200nm of Class III caramels with nitrogen contents of (a) 9.08%, (b) 7.65%,(c) 5.83% and (d) 4.00%.

Figure 9. Plot of migration time at 200nm (mean of three runs, relativestandard deviations <1%) vs % nitrogen, for CE analysis of Class IIIcaramels in phosphate buffer at pH 2.5.

Figure 10. Plot of peak width at half height at 200nm (standard deviationsshown) vs % nitrogen, for CE analysis of Class III caramels in phosphatebuffer at pH 2.5.

J Sci Food Agric 79:1709±1714 (1999) 1713

Analysis of caramels

IV caramels can be further categorised according to

their nitrogen or sulphur content, respectively. Since

the type of caramel present can thus be identi®ed, the

quanti®cation of caramels in food has become a

realistic possibility. Future work will investigate the

degree to which other food components affect these

analyses.

ACKNOWLEDGEMENTSThe authors thank Professor HE Nursten and Dr JM

Ames (The University of Reading, Department of

Food Science and Technology) and Dr L Castle (CSL

Food Science Laboratory, Norwich) for their helpful

comments and discussions, the caramel producers

who kindly donated samples for analysis, and MAFF

for funding the work.

REFERENCES1 International Technical Caramel Association, Guide to the Speci-

®cations of Caramel Colour (unpublished). Atlanta, Ga, USA

(1984).

2 MAFF (Ministry of Agriculture, Fisheries and Food), Dietary

intake of food-additives in the UK: initial surveillance. Food

Surveillance Paper No 37, HMSO, London (1993).

3 Licht BH, Shaw K, Smith C, Mendoza M, Orr J and Myers DV,

Development of speci®cations for caramel colours. Food &

Chemical Toxicology 30:383±387 (1992).

4 Coffey JS, Nursten HE, Ames JM and Castle L, A liquid

chromatographic method for the estimation of Class III caramel

added to foods. Food Chem 58:259±267 (1997).

5 Coffey JS and Castle L, Analysis of caramel colour (Class III).

Food Chem 51:413±416 (1994).

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Identi®cation and quanti®cation of Class IV caramels using

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1714 J Sci Food Agric 79:1709±1714 (1999)

L Royle, CM Radcliffe