Robinson, P . J . , P. Dunnill and M . D. Lilly: Biotechnol. Bioeng. [32] Kennedy, J . F., and J . E . Fox: Carbohyd. Res. 54 (1977), 13.

15 (1973), 603. [33] Kennedy, J. F., and J . E . Fox: in “Methods in Carbohydrate

Gel/f;G., and J . Boudrant: Biochim. Biophys. Acta 334 (1974), Chemistry”, ed. R. L. Whistler, Academic Press, New York, Vol.
476. VIII, (in press).
Van Leemputten, E., and M . Horisberger: Biotechnol. Bioeng. [34] Arrhenius, S.: Z. Physik. Chem. 4 (1889), 226.
16 (1974), 384. [35] Michaelis, L., and M . L. Menten: Biochem. Z. 49 (1913), 333.
Hasselbergrr, F . X . , B. Allen, E . K . Paruchuric, M . Charles and [36] Lineweaver, H., and D. Burke: J. Amer. Chem. SOC.56 (1934),
R. W. Coughlin: Biochem. Biophys. Res. Comm.57 (1974), 1054. 658.
Chapfin, M . F., and J . F . Kennedy: Carbohyd. Res. 50 (1976), [37] Hornby, W. E., M. D. Lilljf and E. M . Crook: Biochem. J. 107
267. (1968), 669.
Kennedy, J. F., S. A . Barker and C. A. White: Starke 29 (1977),
240.
Messing, R . A . : Process Biochem. 9 (1974), 26. Address of authors: Dr. 1.F . Kennedy, B. Sc., Ph. D., D. Sc., C.
Kennedy, J . F., and C. A . White: Starke 31 (1979), 93. Chem.,F.R.I.C.,andDr.C.A. Whire,B.Sc.,Ph.D.,C.Chem.,M.R.
Kennedy,J.F.,C. A . WhiteandC.L. Riddiford:Starke31(1979), I. C., Department of Chemistry, University of Birmingham, P. 0 .
235. Box 363, Birmingham B 15 2 T T (Great Britain). Present address for
Bernfeld, P.: Methods in Enzymol. 1 (1955), 149. C . A. W.: Department ongmistry, National Institute for Biological
Duwson, R. M . C., D. C. Elliott, W. H . Elliott and K . M . Jones Standards and Control, Hampstead, London NW3 6RB (Great
(eds.) : “Date for Biochemical Research”, Oxford University Britain)]
Press, London 1959, p. 192. (Received: April 6, 1979)

Rapid Determination of Dextrose Equivalent by

Cryoscopy*
By M. G. Fitton, Vilvorde (Belgien)

This paper describes the development of a novel method for Schnellbestirnrnung des Dextrose-Aquivalents durch Kryoskopie.
determination of Dextrose Equivalent of Malto-dextrins and glucose Diese Arbeit beschreibt die Entwicklung einer neuen Methode zur
syrups. The new method uses depression of freezing point as a Bestimmung des Dextrose- Aquivalents von Maltodextrinen und
measure ofthe number of molecules in solution ;Dextrose Equivalent Glucosesirupen. Die neue Methode benutzt die Gefrierpunktsernie-
is also a measure of the number of molecules in solution. The drigungals MaR fur die Anzahl der in Losung befindlichen Molekiile ;
advantages of the cryoscopic method are its inherent rapidity (2 min das Dextrose-Aquivalent ist ebenfalls ein Ma13 fur die Anzahl der in
per determination), its precision and the fact that previous expertise is Losung befindlichen Molekule. Die Vorteile der kryoskopischen
not required. Methodesinddie ihreigene Schnelligkeit (2 minje Bestimmung), ihre
Genauigkeit und die Tatsache, daR keine vorhergehenden Untersu-
chungen erforderlich sind.

1 lndroduction The disadvantages of the titration method are shown in Table

1. Probably the most important disadvantage of the titration
In this paper I am going to describe the development of a novel method as a control test for in-process use is that at best it
method for determining the Dextrose Equivalent (D. E.) of gives the value of D. E. at the time of taking the sample, which
Malto-dextrins and glucose syrups. The method is based on is not necessarily the D. E. of the batch at the time the result is
cryoscopy which is measurement of freezing point depres- available owing to the 15-30min time required for
sion: the time required for one determination of D. E. by determination.
cryoscopy is about 2min, no reagents or chemicals are
required, no previous training or expertise is needed and the Table 1.
results are equivalent to those given by the Lane-Eynon Disadvantages of D. E. Determination by Titration.
titration method. The precision of the cryoscopy method is
better than that of the manual titration approach. The 1. Time Consuming: 15 - 30 min for Determination
method is thus ideally suited to use as a rapid process control 2. Expertise Required to Produce Reliable Results
test. 3. Variation between Different Operators
Dextrose Equivalent is still the most fundamental index of 4. Standardised Reagents Required
acid or enzyme induced degradation of starch into glucose 5. Unsuitable as Process Control Test
syrups. It is determined by one of a number of alternative
titration techniques in which the reducing power of the syrup
sample is compared to the reducing power of the same weight
of D-glucose. D-glucose is arbitrarily assigned a D. E. value of 2 Theoretical Aspects
100 and the reducing power of the syrup is quoted as % of the
D-glucose figure. In order to accelerate the determination of Dextrose
Equivalent it is necessary to decide exactly what “Dextrose
* Lecture presented at the 30the Starch Convention of the Equivalent” means. “Dextrose Equivalent”, as shown in
Arbeitsgemeinschaft Getreideforschung at Detmold (FRG), April Table 2, is a comparison between the number of reducing end-
25-27, 1979. groups in a glucose syrup or related product compared with

Starch/Starke 31 (1979) Nr. 11, S. 381 -384 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979 381
0038-9058/79/1111-0381 %0?.50/0
Table 2.
Dextrose Equivalent, Schematic Definition.

Reducing Power of Syrup Solids

Dextrose Equivalent =
Reducing Power of D-Glucose
- Number of Reducing End-Groups in Syrup Solids
-
Number of Reducing Groups in D-Glucose
Number of Reducing Groups a (Molecular Weight)-’
Molecular Weight of D-Glucose
D . E. (X)= x 100
“Molecular Weight” of Syrup Solids

the number of reducing end-groups in the same weight (dry

basis) ofD-glucose (anhydrous). The result isexpressed as a %
figure.
As a syrup is a mixture of glucose polymers of different
individual sizes we cannot use the expression “Molecular
Weight” of syrup solids without further qualification.
Table 3 shows the different methods available for determining
the average molecular weight of polymer mixtures. It is the
column headed “Number Average Molecular Weight” which
is important. The determination of D. E. by titration is in
essence the determination of number average molecular
weight by the classical method of end-group analysis. The
expression of the result on a D. E. scale of zero to 100 is a
mathematical convenience.

Table 3.
Molecular Weight Averages of Polymers, Methods of Determina- Figure 1 . Automatic cryoscope.
tion.
3.1 Calibration of the Instrument
Number Weight Viscosity
Average Average Average The Lane-Eynon Dextrose Equivalent of glucose oligomers
M . W. M. W. M . W. deviates significantly from the theoretical D. E. of these
Ebulliometry Light Dilute materials. The general pattern of this deviation is shown in
Elevation of Boiling Point Scattering Solution Table 4. In orther words the determination ofnumber average
Viscosity molecular weight by the Lane-Eynon method of endgroup
End Group Analysis Ultracentrifuge analysis is inaccurate. A primary objective of this work was to
Depression of Freezing Point provide equivalence to D. E. obtained by the Lane-Eynon
Vapour Pressure method thus it is necessary to calibrate the instrument with
Decrease in Vapour Pressure syrups of known Lane-Eynon D. E. in the range of interest. In
Osmotic Pressure this way equivalence oflane-Eynon and Cryoscopic D. E.’s is
Osmotic Height Semi- assured.
permeable Membrane
Cryoscopy
Determination of Table 4.
Reactive End Groups Deviation between Lane-Eynon D . E. and Theorectical D. E.

Degree of Polymerisation Lane-Eynon D . E. Theoretical D . E.

One can suggest that alternative methods available for 1 (D-Glucose) 100 100
number average molecular weight may yield a D. E. We have (by Definition) (by Definition)
chosen cryoscopy because currently avaible equipment using 2 (Maltose) 58 52.6
thermistor temperature measurement is precise, rapid, and of 3 39 35.7
reasonable cost. 4 30 27.0
5 24.2 21.7
6 20.8 18.2
3 The Cryoscopic Method
Figure 2 shows calibration curves. Several syrups represen-
Figure 1 shows a commercially avaible instrument for ting a range of D. E. values were subjected to Lane-Eynon
determination of the number average molecular weight by D. E. determination by experienced personnel. These syrups
cryoscopy. Operation is extremely simple. The instrument were then prepared at dry substance levels between 10% and
reading is in milliosmols, i . e. molar concentration x lo3.The 20% using refractometric dry substance measurement on the
reading is thus directly dependent on the number average sucrose scale of the instrument. The sucrose scale does not
molecular weight of the sample and its concentration. At give accurate dry substance measurement with products of
constant dry substance the reading increases with increasing widely differing D. E., but this is not important because the
D. E. One cycle of operation takes 2 min. error is effectively the same for calibrants and samples.

382 StarchlStarke 31 (1979) Nr. 11, S. 381 -384

 97' I the only source of reducing power in the syrup - this
1100
assumption is generally valid. However the cryoscopic
approach is responding to the number of moles of material in
1000 solution, it is unaffected by the presence of high molecular
weight materials such as residual enzymes, proteins etc.. ;but
900 it can be affected by the presence of low molecular weight
800 inorganic salts, i. e. ash.
The presence of ash tends to give an increased cryoscope
700 reading and thus an artificially high final result. Thus it is
necressary to compensate for the effects of ash in order to
-
VI
0
600 produce reliable results.
5 Figure 4 shows the graph of cryoscope reading against
7 500
--- electrical conductivity for three different inorganic salts. The
= 400 graphs are linear and the gradients are very similar - a typical
value being 0.02. Thus cryoscope reading due to ash = 0.02 x
300 conductivity. By using conductivity as a measure of ash level
200 we do not materially affect the inherent rapidity of the
cryoscopic technique as an in-process control test.
100 Thus as shown in Table 5 :cryoscope reading due to glucose
L - I I I I I I I I I oligomers = Reading obtained - 0.02 x conductivity. This
10 11 12 13 14 15 16 17 18 19 expression is applied to all samples including calibrants
m Dry Substance by Refractometer I%J submitted to the cryoscope.
The validity of this ash compensation method is shown in
Figure 2. Calibration curves obtained with products of known Table 6 where extremely high ash levels were simulated by
Lane-Eynon D. E.
20.000-
The molar concentration reading of the instrument rises with
increased dry substance and with increasing D. E. of the
material in solution. Direct factorial conversion between the
molar concentration and freezing point depression is pos-
sible. Some commercially available instruments read milli- 15,000-
degrees directly.
Figure 3 shows this data in a more convenient form. -
Instrument reading of molar concentration is plotted against -
I
>
the Lane-Eynon Dextrose Equivalent previosly determined. L
-
c

Thus for a sample of unknown D. E. the method may be u

v
3

reduced to three steps : z

0
10,000-
1. Dilute sample to 15% dry substance sucrose scale on in
c,
a

refractometer. E
a,
2. Determine molar concentration or depression of freezing v)

point by cryoscopy.
3. Read D. E. of sample from calibration graph. 5,000-

20"

0 100 200
rn Miill - osmolality

Figure 4. Conductivity us. milli-osmolality for various concentra-

tions of CaCl,, KCI and NaCl in water.

Table 5.
Correction of Compensate for Ash.
Instrument Reading Due to Glucose Polymers =
5 10 15 20 25 30 35 40 15 50 55 60 65 70 75 80 85 90 95 100 Total Instrument Reading at Dry Substance X
Em Dextrose Equivalent I%l

Figure 3. Osmolality us. D. E. at 10, 15 and 20% dry substance.

-0.02 x [atConductivity
Dry Substance X 1
(pS)

Thus the Complete Method :

3.2 Effect of Low Molecular Weight Inorganic Salts 1. Dilute Sample of Syrup (etc.) to 15% Dry Substance (Sucrose
Scale Refractometer).
We have assumed so far that there are no sources of 2. Determine Electrical Conductivity in Micro Siemens.
interference in the comparison of D. E. by cryoscopy with 3 , Determine Molal Concentrations by Cryoscopy.
4. Instrument Reading - 0.02 x Conductivity = Corrected Re-
D . E. by Lane-Eynon. Lane-Eynon titration measures the ading.
reducing power of terminal groups on the glucose oligomers 5 . Obtain D. E. of Sample from Calibration Curve.
themselves and tacitly assumes that these terminal groups are

Starch/Starke 31 (1979) Nr. 11, S. 383 -384 383

Table 6. Table 7.
Validity of Ash Correction Method. Variation of D. E. Values (%n) Produced by Eight Untrained
Operators.
Lane- Ash Level on Conductivity Milli-Osmols Corrected
Eynon Dry Basis pS at 15% from Ash D . E. (YO) Operator Syrup 1 Syrup 2
D . E . (%) (Yn) D. S. (0.02
x Conduct.) 38.71 48.20
39.33 47.98
61.2 0.457 1080 21.6 60.45 39.22 47.80
0.557 1320 26.4 60.7 39.43 47.85
0.707 1680 33.6 61.3 39.43 47.85
0.957 2265 45.3 60.1 39.12 47.95
1.457 3450 69 .O 60.7 39.43 47.95
2.457 6990 140 61.2 39.22 47.80
5.457 12.870 257 60.22

adding inorganic salts to the syrup. The conductivity and Table 8.

cryoscope reading were determined for each sample and Side-by-Side Comparison of Lane-Eynon Dextrose Equivalent with
correction was applied using the method described. The D. E. by Cryoscopy.
corrected result is in close agreement with the value Lane-Eynon D. E.
determined by Lane-Eynon titration. D. E. (%n) by Cryoscopy (%)
So far I havedescribed the logic of themethod development in
some detail. This was necessary in order to establish the link 3.6 3.5
between two superficially different techniques : assessment of 12.8 12.4
reducing power by a titration method and measurement of 22.3 22.7
freezing point depression by cryoscopy. 29.5 29.7
37.0 36.8
3.3 Precision and Accuracy 41.5 41.6
48.5 47.5
Now I want demonstrate the precision of the cryoscopic 52.9 52.8
54.3 54.8
technique. A number of different operators who had not 61 .O 61.4
encountered cryoscopy previously were asked to determine 61.2 62.1
the D. E. of two glucose syrups. Calibration curves of syrups 61.6 61.2
in the same expected D. E. range as the samples were 62.3 62.4
provided. Table 7 shows the excellent precision of the result. 71.5 70.5
In discussing accuracy of the determination we must bear in 82.0 82.6
mind that Lane-Eynon D. E. which we are attempting to 97.1 96.7
100 100
match is in itself inaccurate. Thus accuracy in this context is
the closeness with which the cryoscopic D. E. approaches the
value given by the Lane-Eynon titration method. Table 8
shows a comparative table of D. E. values on a series of Table 9.
samples representing the full range of D. E. from maltodex- D. E. Determination by Cryoscopy Compared with Lane-Eynon
trin type products of low D. E. to values approaching that of Titration Method.
pure D-glucose ; this comparison is fully satisfactory.
In summary I wish to return to Table 1 in which we saw the Lane-Eynon Titration Cryoscopic
disadvantages of the titration approach. These can now be Method Method
compared with the advantages of the cryoscopie technique
1. Time required: About 2 min per
(Table 9). The new method is rapid, precise, does not require 15 - 30min determination
reagents or previous experience and is therefore ideally siid to 2. Expertise and prior no expertise needed
use as a control test where these qualities can be fully training needed
exploited. 3. Variation between max. Variation ,. 0.4 D. E
different operators between Operators
4. Standardised reagents no reagents required
Address of author: Michael G.Fitton, M. Sc.,CPC Europe Industrial required
R &D Center, Havenstraat 84, B-1800 Vilvoorde (Belgium). 5. Unsuitable for process Well suited to process
control control
(Received: May 23, 1979)

384 Starch/Starke 31 (1979) Nr. 1 I , S . 381 - 384