INDUSTRIAL
and
ENGINEERING
CHEMISTRY ANALYTICAL EDITIOS
+
Harrison E. Eowe, Editor
Analysis of Sugar Mixtures Containing Dextrose, Levulose, Maltose, and Lactose F. W. Z E R R A S A N D LOUIS SATTLER New l-ork Sugar Trade Laboratory, New York, N. 1-
D
Ii
URING the past few years methods for the determina-
tion of dextrose and levulose in sugar products have been studied in this laboratory. A procedure applicable to raw cane sugars has been published (17 , 18),and likev-ise one for cane molasses (6). Some food products also contain maltose and lactose, and the investigation has been extended to include these sugars as well. Sucrose can be estimated in the presence of the other four sugars by means of invertase, and for this reason only the four reducing sugars are considered in this article. With such complex sugar mixtures it is usually necessary t o resort to combined methods, as has been pointed out by Browne (8, S),because there are few reactions which permit the quantitative separation of one sugar from all the others. Selective fermentation with specific organisms has been employed successfully by a number of investigators, but the necessary pure cultures are not always readily available, they require painstaking technique in their propagation and application, and in the usual chemical laboratory used for sugar analysis i t is difficult to prevent contamination. Chemical methods have lately become available for the selective determination of levulose and of monosaccharides, and have been applied to the analysis of sugar mixtures. This suggested their possible use in the analysis of mixtures of the four sugars in question.
then
fr
= mg. of
dextrose (glucose)
of levulose (fructose) of maltose hydrate of lactose hydrate of apparent levulose by the method of Jackson and l l a t hews R: = nip. of dextrose plus levulose, expressed ~ b levulose, s by t h e copper acetate method of Steinhoff Ra = mg. of total reducing sugars, expressed as dextrose, by
F .If L H
= mg. = mg. = mg = mp
Fehling solution
L is determined separately R1 = 0.0806 G F Rz = aG F Ra = G bF dL
+ + + + + CAM
The factor 0.0806 in Equation 2 is the reducing ratio of dextrose to lerulose (12.4 mg. of dextrose have the sarnereducing power as 1 mg. of levulose). Factor a is the reducing ratio of dextrose to levulose in Steinhoff’s acetate method, and b, c, and d are the reducing ratios of levulose, maltose, and lactose, respectively, to dextrose for Fehling solution. The values of a, b, c, and d vary with the concentration, and are found from tables. By solving Equations 2 and 3 for G and F , we find
and
Principle of the hlethod
F = Rz
- aG
L , G, and F being knonn, Equation 4 gives
The determination of four sugars by combined methods requires four equations. In the absence of salts and organic impurities the total solids or the polarization may serve as criteria, but such cases are rare in practice, and it is generally necessary to depend on methods that are less affected by accompanying impurities. Lactose is the only sugar among the four that can be determined independently. It mag either be oxidized to mucic acid and weighed in this form, or else the other three sugars may be removed by fermentation v i t h yeast, and the residual lactose estimated by any suitable method. The dextrose and levulose may be determined by combining Jackson and Mathews’ modification of the X j n s method (8) for the selective determination of lewlose with the method of Steinhoff (14) for the selective determination of monosaccharides in the presence of disaccharides by means of a modified Barfoed reagent. The total reducing sugars are found with Fehling solution.
-If
=
Ra
-
(G
+ bF + dL) C
If the disaccharides had no reducing effect whatever on the reagents employed for the determination of the monosaccharides, as claimed by the original authors of the methods, the procedure and the calculation of the results would be simple. But i t has been found that both maltose and lactose have a slight reducing effect on the Jackson and Mathewe reagent, as well as on the Steinhoff copper acetate reagent This subject, and the procedure for applying the necessary corrections are discussed below.
Determination of Lactose The writers first tried the mucic acid method of Tollens. Kent, and Creydt (4, 9), as modified by van der Haar (6), The method lacks precision, as stated by van der Haar him669
670
INDUSTRIAL AND E N G I N E E R I l G CHElIISTItI
self, differencesof as much as 8 nig. of mucic acid being sometimes obtained in duplicate determinations. It is not, very accurate either and usiially gives low results i n tlie presence of other sugars or nonsugars. Tlie galactose, found from \-an 2 , should give lactose der Haar's Table I and multiplied ?q hydrate, Ixit experiments by the writers with known sugar mixtures have given an average factor of 2.2. K i t h low percentages of lactose in the mixt'ure the method gives fairly satisfactory results, sufficiently exact for practical purposes but it is not recommended. The method finally adopted is a modified form of the fermentation procedure of Hoffman, Schn-eitzer, and D d J y ( 7 ) .
\-Or>. 10, K O . 12
ayerage figures may be used t o correct. for the reducing effect of maltose and lactose. An average of 26.0 mg. of maltose hydrate and 25.6 nig. of lactose hydrate was found to be equivalent to 1 nig. of levulose.
Determination of Dextrose Plus Levulose The reduction method of St'einhoff has been retained with only slight changes, but the estimation of t,he reduced copper 11ad to be modified. Steinhoff acidifies the reaction mixture n-hile it is stili hot, and immediately adds standard iodine solution. -\ftcr cooling, the excess iodine is titrated back v i t h standard tliiosulfate. This procedure leads to uncertain results becaiise the iodine added to the hot solution is psrt,ly volatilized. Tlie m i t e r s have therefore adopted the iodometric nietliod of Sliaffer and Hartmann (19), modifying it to suit the particular coriditions. The folloving reagents are used:
The -ample is placed in a 500-nil. volumetric flaqk, and a thiii suspension of a mixture containing 35 grams of conil~res..ctl baker.+' yeact. 0.5 gram of ammonium sulfate, and 0.2 grain i i i sodium bisulfite in m t e r is added. The mixture in The fl further diluted n-ith n.ater t o a volume of about 400 ml. flask is closed ivith a stopper provided n-ith a delirery tube, tlic outer end of n-hich iq immerfied 1 em. belon the nurince of \v:iter in a beaker. The flaak is placed in a water bath or :lierriio..tnt LSntlirim :ire1 atc wlution, prepared b!. rlis.olrinp ZOO grams of kept at 30" C., and it iq shaken from time to time. c rry-t:xllixd ,-Lilt (CH3COOSa.3H20)in about 800 ml. of After standing a minimum of 4 hours. 15 ml. of a 20 I)er r e n t t \\Liter, roolinc, and niakinq up t o 1 liter. solution of neutral lead acetate are added, and t h e V(J1ullIc i, Sulfiii,ic arid, about 2 S,prepared by diluting 57 ml. of conmade up to tlie mark at 20" C. Sext, 1 gram of Filter-Ccl icentrated acid t o 1 litrr. added, and the contents of the flask are thoroughly s1i:iken and ?(it a-.ium iodidc-iodate solution, prepared tiy tli-iolring 3..1 filtered through a folded quantitative filter paper. The firht, 60 grams of poia--iuni iodide turbid portion of the filtrate is discarded. and exact1)lie solution is made alkaline by of t h e clew filtrate are collected in a drr lwlunietric fi roxide disqolved in a little water brnted for 200- and 220-ml. contents. To the 200-nil. 15 ml. of a pliosphate-oxalate solution are added, prcpnrctl liy ium oxalate, prepared by di..dissolying 7 grains of disodium phosphate (NacHP04,12H?O) -01~iiig165 pxms of the hydrated salt ( K ? C ~ O ~ . R : O in) 500 ml. and 3 prams of potassium oxalate (Ii2C204.H20) t o 100 1111. of l!ot v-ater, and cooling to room temperature. The flask is made u p to t h e 220-nil. mark at 20' C., 0.5 g r ~ i i i 0. I S tliio~i~lfate ;elution, exactly .-i andardized by i i ~ d o of Filter-Cel is added. and the contents are shaken r i g i ~ r o ~ ~ s l ~m(xt ~ . ric tletermin:it ion n-ith potassium dicliromate. Tlie solution is filtered through a quantitatiT7-e filter p:ipci~. :in11 Piixlilet copper -1.ilfate solution (Fellling I), prep:ired acro: (ling the clear filtrate is eolleclcd for tlie sugar determination, I>:ii.ii>>e ion. of tlie Association of Official Algricultural can be determined by any of the standard methods, siicli as the Munson and Kalker method used by the n-riters. +
The fermentation nietliod should be carried out i\-itli :I reliable lirantl of yeast, and its fermenting pcxvcr shoul~lbe checked by appropriate tests. Blank tests riin xitli a gootl yesst usually yield very lo^ reduction values, and the results are not t,rustv-orthy for corrections. It is a much better pract,ice to use pure lactose for the purpose. It is tllerefore recommended to run parallel deterniinat,ions with lactose alone, or with a knolvn sugar mixture approximating that of the sample. To piye an example, 1.056 grams of lartose n-ere treated in the 500-ni1. flask. After the fermentation, 200 ml. of the filtrate, containing 0.4224 gram of lactoqe, were made up t o 220 nil., and of the final filtrate 30 ml., containing 96 mg. of lactose, \ \ ? l e used for the sugar determination. Found, 0.1622 and O.lti01 gram of cupric oxide; average, 0.1612 gram of cupric oxide, corresponding to 128.8 mg. of copper and 97.5 nig. of lwtose hydrate. I n another experiment, a mixture of 1.036 pInn1s of lactose, 0.704 gram each of maltose and dextro,sc, nnd O.l?(j gram of levulose was similarly treated, and there was obtained 0.1626 and 0.1621, an average of 0.1624 gram of cupric oxitlr. corresponding to 98.2 mg. of lactose hydrate. The higher result i. due in part to the volume occupied by the yea-t a n d the lead precipitates in the flask$, and in tlie iecond ru-e pns-ibly t o 1111fermented reducing substancef
1
.j.O
3 2
2 3
7 0 3 0
5,s
11.1
10 12.9 14 9 10 8 1s 6
0 064
22.1 23.9 23.7 27.7
0.860 0.833
4 J
ii
T
8 'J
IO
13.3 1J,j 17.8 20.3 22.9
25.7
11
I? 13 14 13
,
20 3
29.4 31.4
16
17 18 10
"0 21 22
s
84 1
95 0
33 6 :75,8 38 4 40.9 43 8 16 0 ,IO 3
O.G40 0.829 0 922
0.9i0 0.901
0,944 0.916
0.886
0.808 0.782
O,i54 0.726 0.701 0.672 0.645 0 . 612 0 587 0.5.is
0,332
0; I : :
0 so3 0.952 1 000 0.980 0.953 0.914
0.897 0.8i0 0 845 0 . 819 0 793 0 . T67 0 747 0.715 0.688 0.666 0.640 0.590 0.583 0.555 0.530
0.82') I1 !I83 0.9;c, 0 081 0,955 0 930 0,905 0 880 0.855
0,830 0.805 0 .i 8 0 0.755 0.730 0.706 0.681 0.656 0.631 0.605 0,581 0.555
0.529
0.852 0.936 1.000 1.013 1.010 0.984 0 957
0.931
0 . 9C16
0,860 0,883 0,825 0.707 0.769 0 . 740 0.711 0 G81 0.6FA 0.621 0.093 0 5G4 0 536
Ten milliliter^ of the copper sulfate bolution and 20 ml of the sodiuiii acetate solution are pipetted into a 250-cc. widemouthed Eilenmeyer flask, and a nieawred amount of the sugar solution i p added. The quantity of siipar solution must he such that the thiosulfate corresponding to the copper reduced is 11ithin tlie limits of Table I, and that the thiosulfate coIresponding to the copper reduced from Fehling solution by the same quantity of sugar solution is nithin the limits of Table 11. A fexv preliminary experiments are usually necessary to ascertain the optimum amount for both determinations. l f t e r tlie sugar solution has been added, the volume is completed to a total of 50 ml. by the addition of water. After
..ISALl-TICAL EDITION
DECERlBER 15. 1938
thorough mixing, the Erlenmeyer is closed with a rubber stopper provided with a Bunsen valve, to prevent reoxidation of the reduced copper. The solution is placed in a briskly boiling T\-ater bath, the stop watch started, and the flask removed from the bath after exactly 20 minutes. It is then quickly cooled t o room temperature under a JTater tap. During thiz time the Bunsen valve must be vented from time to time t o prevent boiling caused by the vacuum. After cooling, 25 ml. of the iodide-iodate solution are carefully added from a pipet and mixed with the solution by gentle shaking. Then 40 ml. of t h r 2 S sulfuric acid are run in rapid1:- from a measuring cylinder, the flask being rotated to wash down the inside ivall. Thi. i- follon-ed by the addition of 20 nil. of the pota ium oxalate solution from a nieawring c:-linder. The content of the flack are \\-ell mixed until tlie precipitate is completely dissolved, and the excess iodine i.; titrated n-ith the standard thiosulfate. A blank i? run with water in-tead of sugar solution. Tlie difference between the thiosulfate titer of the blank and that of tlie sample is a direct measure of the cuprous oxide precipitated. TABLE 11. COPPERT a R r R a T E REIGEST (.\lilligranis of destrose corresponding t o varying volumes of thiosulfate solution, a n d reducing ratios of levulose, maltose, and lactose, n i t h respect t o dextrose) Reducing Ratios R3, Maltose Lactose Thiosulfate Destrose Levulose, b hydrate, c hydrate, d 7 -
.If 1. 1
2 3 4
1
6
;1
9 10 11 12 13 14
15 .-
16 17
18 19 20 21 22
23 24 25
-1fQ.
2.2 5.3
8.5 11 6 14.8 18.0 21.2 24.4 27 6 30.8 34.0 37.2 40.3 43.7 47 0 50.2 53,5 56.8 60.0
63.3 67.1 71.3 75.5 80.0 S5,l
0.648 0.815
0.868 0.893 0.907 0.914 0.918
0.921 0.922 0,922 0.921 0.920 0.91s 0.917 0.914 0.912
0.910 0.907 0.904 0.895 0,897 0,901 0,905 0,910 0 915
0.394 0.451 0.508
0.521
0.528 0,532 0.535 0 537 0.538 0.538 0.538 0.538
0.537 0.537 0.536 0.536 0.536 0.534 0.533 0.531
0.534 0.540 0.545 0.551 0.561
0 489 0.616
0.653 0,669 0.678 0.683 0 685 0 686 0.689 0.685
0.684 0.683 0.681 0.679 0.677 0.675 0.672 0.670 O.GG8 0.665 0.668
0.673 0.678 0.685 0 695
Results of duplicate determinations usually check within 0.1 to 0.2 ml. of thiosulfate. But larger discrepancies occur occasionally, due to the following causes: Differences in buret drainage, because of the relativelj- larger amounts of thiosulfate run out and the greater speed of their renioral in blank determinations, may cause an error amounting to 0.15 ml. difference of one drop in the quantity of the iodide-iodate solution pipetted out causes an error of as much as 0.15 nil. of thiosulfate, and for this reason the emptying of the pipet must be carefully standardized. The end point, determined viith starch solution. n-hile ,..harp, is affectedby light conditions. It is best to use a daylight lamp, and to prepare a blank solution of the copper reagent for compariwn. The precipitate must be completely dkiolved before the titration with thiosulfate. Without this precaution there may be fading of the end point, indicating incomplete solution. The boiling water in the bath causes a certain amount of agitation of the contents of the flasks, and variation:: in tlii affect the results. The 1%-atermust be boiling briikl, parts of the bath. It may be preferable t o u-e a comtanttemperature bath kept at 100” C. by the use of n higher boiling liquid, and to agitate the flasks mechanically. If the Erlenmeyer flasks are not thoroughly clean, the copper precipitate tends to creep u p on the n-all.. To prevent this, the flasks should be cleaned regular13 uith chromic acid mixture. The reducing effect of dextrose and lei-ulose, obtained from the National Bureau of Standards, and of mixtures of the two, on the copper acetate reagent was determined, and the results are shown in Table I. which gives the milligrams of
671
dextrose or levulose equivalent to the number of milliliters of thiosulfate found, and also the values of the factor a in Equation 3, for varying proportions between the two sugars.
Determination of Total Reducing Sugars I n this determination, 10 ml. of Soxhlet copper sulfate solution (Fehling I) and 10 nil. of alkaline tartrate solution (Fehling 11),both prepared according to tlie directions of the Association of Official Agricultural Chemists ( I ) . are mixed in a ~ ~ O - C C .n-ide-mouthed , Erleniiiey~r flask. The same quantity of sugar solution as was weti in the deteriiiination of dextrose plus l e d o s e is added, tlie 1-oluriie is made up to a total of 50 tnl., and the analysis is carried out exactly as described for that determination, except that only 25 ml. of tlie 2 S sulfuric acid are used instead of 40 nil. The reducing effect on the copper t’artrate reagent has been measured for dextrose, levulose, maltose, and lactose The results are shon-n in Table 11, which gives the milligrams of dextrose corresponding to varying milliliters of 0.1 S thiosulfate solution, and the ralues of the factors b, c , and d (reducing ratios of lerulose, maltose hydrate, and lactose hydrate with respect to dextrose) in Equation 4. These reducing ratios are very close to those for the Nunson and Walker method, as would be expected.
Reducing Power of Mixtures of Sugars
Two hypotheses have been used to account for t,he combined reducing effect of sugars present in mixtures. One of these assumes that the reducing effect is an additive property-for example, the copper reduced by a mixture of a mg. of dextrose and b mg. of levulose is expected to he the sum of the copper reduced by a nig. of dextrose and b ing. of levulose, each present alone. Schwartz (11) found, however, that the reducing power of mixtures is governed by a rule analogous to that observed by Voshurgh (16) for the specific rotation of sugar mixtures. This rule states that the specific rotations in a mixture of tv-o sugars are equal to the specific rotations lyliich each sugar would have if present a t the total sugar concentration. Similarly, the amount of copper reduced by a mg. of inrert sugar is the sum of one-half of the copper reduced by a mg. of dextrose and one-half of the copper reduced 11y a mg. of levulose. This “fractional proportionality” rule is illustrated by the following example from the reduction tables of Quisumbing and Thomas ( I O ) : Copper MQ.
200 mg. of i m e r t sugar alone 100 mg. of destrose alone reduce 100 mg. of levulose alone reduce 200 mg, of inrert sugar, according rule, XTould reduce 200 mg. of destrose reduce 200 me. of levulose reduce One-half of copper reduced b y 200 One-half of copper reduced b y 200 200 nip. of invert sugar, according portionality rule, would reduce
t o the simple additive
372.1 201.2 185.0
386.2 386.0 360.6
mg. of dextrose equals mg. of levulose equals t o the fractional pro-
193.0
180.3
__
373.3
The last figure checks nithin 1.1 mg. with the copper actually found. nhile the simple additivity rule gives 14.1 mg. of copper too i i i u c h . Xnalogou4y, the amount of copper reduced by a mixture of 50 mg. of dextrose and 150 mg. of levulose equals the sum of one-qua1 ter of the copper reduced by 200 mg. of dextrose and three-quarters of the copper reduced by 200 mg. of levulose. The simple additivity rule has been found t o give correct results, within the limits of error, in those cases where the reducing effect of one sugar is very small with respect to that of another, as, for instance, for mixtures of levulose and dextrose analyzed by the method of Jackson and M a t h e w . The frac-
INDUSTRIAL AND ENGINEERING CHEMISTRI-
672
tional proportionality rule is of more general application and gives results close to the truth when the reducing power of one sugar does not greatly differ from that of another sugar, as when a mixture of dextrose and levulose is analyzed with Fehling solution. But larger errors are produced when the reducing power of one sugar differs materially from that of another. This applies to the analysis of mixtures of dextrose and levulose by the Steinhoff acetate reagent, and of mixtures of a monosaccharide and a disaccharide by means of Fehling solution. This mag be seen from Table 111. TABLE 111. REDUCING POWEROF MIXTURES Thiosulfate Steinhoff Acetate Reagent Dextrose Levulose
Ma.
Mo.
50 37.5 12.5 37.5 12.5 Steinhoff Tartrate R eagent Dextrose Maltose 10 70 20 60 30 50 40 40 50 30 60 20 70 10 Mixture Levulose Maltose 70 10 60 20 50 30 40 40 30 50 20 60 10 70 50
Found
Calcd. by additivity rule
Calcd. by fractional proportionality rule
M1.
M1.
M1.
18.76 20.28 17.60
21,36 22.29
19.13 21.25
18.40
VOL. 10, NO. 12
sugars in varying proportions, and this has been done in establishing Table I, for the Steinhoff acetate method. Application of the same experimental procedure to the four sugars when determined by Fehling solution would entail an immense amount of work, and it has been necessary to depend on a fe\T check analyses of known mixtures, to test the use of Table 11,which gives the reducing ratios for the various sugars when present alone. Such check analyses are shown under the heading of “Method of Calculation.”
Reducing Effect of Maltose and Lactose on Steinhoff’s Copper Acetate Reagent Steinhoff stated in his paper that maltose has no reducing effect whatever on the copper acetate reagent, but the writers have not been able t o confirm this, and they have found that lactose likewise reduces this reagent.
18 02
TABLEIV. CORRECTIONS 15.12 16.46 17.84 19.24 20.51 21.70 22.73
15.58 17.10 18.53 19.93 21.28 22.59 23.51
15.05 16.33 17.61 18.89 20. 16 21.44 22.72
14.33 15.70 17.00 18.27 19,47 20.48 21.47
15.11 16.42
14.83
17.64 18.75
19.78 20.76 21.56
15.89 16.94 18.00 19.05 20.11 21.16
With the Steinhoff acetate reagent, both rules give high results, but those by the fractional proportionality rule check better with those found by experiment. The differences are smaller when the ratio of dextrose to levulose is high. With mixtures of dextrose or levulose and maltose, determined by means of Fehling solution, the results found are always lower than those calculated by the additivity rule. I n the case of the dextrose-maltose mixtures the differences increase with the ratio of monosaccharide to disaccharide; but in the case of the levulose-maltose mixtures the differences decrease with this ratio. The fractional proportionality rule gives smaller errors than the additivity rule, and mostly in the opposite direction. I n a few instances the found values check with the theoretical within the Limit of error. With a rise in the ratio of monosaccharide to disaccharide the differences first increase, and then decrease again. With a very ratio of levulose to maltose the experimental value is well below the theoretical. Since the reducing power of mixtures of two sugars is already a fairly complicated function of the reducing power of the components, it may be expected that the relations are much more complex when more than two sugars are present in mixtures. This entire subject requires further investigation. At present the best and most practical procedure for analyzing mixtures of sugars by combined reduction methods is that of Browne (S), who has shown that the formulas established by him, with the use of reducing ratios, give generally reliable results. He also called attention to the fact that in some cases, as with mixtures of monosaccharides and disaccharides, the reducing ratios are not constant. I t has since been found that the reducing ratios vary not only when determined for each sugar present alone, but also with the proportion between the sugars when present in mixtures. However, this circumstance can be readily taken care of by using reducing ratios found experimentally for mixtures of two
(To be applied t o milliliters of thiosulfate found, for varying quantitiea of maltose or lactose present in addition t o dextrose or levulose) Maltose Hydrate and Maltose Hydrate and Dextrose Levulose Dex- 200 mg. 100 mg. 50 mg. Levu- 200 mg. 100 mg. 50 mg. trose maltose maltose maltose lose maltose maltose maltose
Mg.
MQ.
Correction, Per Cent of 111. Thiosulfate 100 0 100.0 100.0 72.5 63.0 79.7 47.0 34 2 61.5 10 1 R 31.1 ~. 46.5 20.5
:
20 25 30 35 40 45 50 55 60 65
m ._
75
so
34.5 25.8 19.6 15 4 12.9 11.2 9.8 8.8 7.8 6.8 5.7 4.7 3 7
20.8 14 4 10.3 7.9 6.9 6.3 5.7 5.1 4.6 4 0 3.5 3.1 2 4
11.0 6.0
3.4
2 3 2.2 2.0 1.9 1.7 1.6 1 4 1 3 1 1 1 0
Lactose H j d r a t e and Dextrose 30 m g lactose lactose lactose 100 0 100 0 100 0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
200 mg. 100 mg.
0
71.5
10
17.3
15 20
29.5 19.0 13.4 9 4
25
30 35 40 45 50 55 60 65 70 73 80
i.2
6.4
6.1
5.7 5.4
5.0 4.7 4.4 4 0 3 7
62.6 30.5 16.0
7.6 4.3 2.8 2.5 2.4 2.2 2.1 1.9 1.7 1.5 1.4 1.2 1 0
54 5 22 6
9 7 3 4
1 5 1 4
1 3 1 2 1 1 1 0
1 1
0 9 0 8 0 7 0 6 0.5
n 5 10 15 20 25 30 35 40 45
PO 03
60 65 70 75 80
Correction, Per Cent of MI. Thiosulfate 100.0 100.0 100,o 75.3 41.3 60.4 24.1 39.0 56.5 41.2 25.0 13.7 28.6 17.8 8.8 20.4 12.0 5.9 13.8 8.3 3.5 9.2 6.5 5.0 4 5 4.1 3.7 3.3 2.9 2.5 2 1
5.2 3.2 2.4 2.2 2.1 1.9 1.8 1.6 1.5 1.3
2.4
1.6 1.5
1.4 1.3 1.2
1.1 1.0 0.9 0 8
Lactose Hydrate and Levulose LOOmg. 100mg. 50rng. lactose lactose lactose .oo 0 100.0 100.0 34.4 19.0 G0.7 19.3 11.7 39.8 1 1 . 2 6.6 26.4 6.6 2.1 15.8 3 . 8 1.0 10.3 0.9 5.6 2.0 2.4 0.9 0.8 1.0 0.8 0.8 0.8 0.7 0 9 0.7 0.6 0.8 0.6 0.5 0.7 0.5 0.5 0.6 0.4 0 6 0.4 0.4 0.3 0.5 0.3 0.2 0.4 0.3 0 2 0.1
The difficulties encountered in the preparation of pure maltose are well known. This sugar is generally made from starch by enzymatic degradation, and it must be separated from dextrose on the one hand and from higher saccharides on the other. There are no methods by which traces of these impurities may be determined. The writers have depended in their work on repeated crystallization of maltose and its effect on the reducing power toward the Steinhoff copper acetate reagent, and also on comparisons n-ith reputedly pure samples of maltose obtained from various investigators. It was not found possible t o prepare maltose with a reducing power less than that corresponding to 5 ml. of 0.1 N thiosulfate for 200 mg. of maltose. The reducing power of the same quantity of pure lactose was about one-half of that. Since lactose crystallizes much more readily than maltose, and yet exhibits reducing action toward the copper acetate reagent,
DECEMBER 1.5, 1938
ANALYTICAL EDITIOK
it appears probable t h a t the reducing effect of maltose is not caused by impurities. The reducing effect of maltose on the Steinhoff copper acetate reagent has been confirmed by Shapiro and Proferanzova (IS). Tauber and Kleiner (15) have also observed that their modified Barfoed reagent is reduced by 2 mg. of maltose or 3 mg. of lactose. Efforts have been made to discover a reagent which would be reduced by dextrose or levulose, but not by the authors' maltose or lactose. It was found that, if the Steinhoff reagent is diluted with water, or 0.5 gram of sodium salicylate is added to it, the reducing effect of the maltose or lactose becomes negligible, b u t the reducing effect of the modified reagent on dextrose is also lowered to such an extent that no practical advantage is gained. Even if later investigations should show t h a t maltose or lactose, purified by more efficient methods, have no reducing effect on the Steinhoff reagent, they are nevertheless a p t to affect the reducing power of dextrose or levulose in mixtures, as has been pointed out in the discussion of the reducing power of sugar mixtures. It would still be necessary to apply corrections for the maltose and lactose present, although the corrections would be different from those given in this paper. The amount of the corrections to be applied, assuming the maltose and lactose t o be pure, was evaluated by adding 50, 100, and 200 mg. of maltose hydrate, of lactose hydrate, and of equal parts of these sugars to varying quantities of dextrose, levulose, and invert sugar, respectively, and determining the reducing power of the mixtures on the copper acetate reagent. All the data were plotted and the figures given in Table IV were taken from the curves, for mixtures of maltose and dextrose, lactose and dextrose, maltose and levulose, and lactose and levulose. The tables shorn directly the per cent of the milliliters of 0.1 N thiosulfate to be deducted from those actually obtained, for 200, 100, and 50 mg. of either maltose or lactose in the presence of up to 80 mg. of either dextrose or levulose. The values for intermediate quantities of maltose or lactose and for intermediate quantities of dextrose and levulose are found by interpolation. This cross interpolation can be made more quickly by means of the curves, but the actual figures instead of the curves are reproduced in this paper, because the curves have to be drawn on a rather large scale t o be useful. The corrections to be applied for mixtures of maltose and lactose in the presence of mixtures of dextrose and levulose were found to accord with the fractional proportionality rule: The correction for maltose plus lactose is proportional to the partial concentration of either sugar, but on the basis of the total concentration of maltose plus lactose. Similarly, the correction to be applied for the effect of maltose or lactose on a mixture of dextrose and levulose is based on the total concentration of the last two sugars, and their proportion in the mixture. The use of the correction tables is explained in greater detail in the following example.
Method of Calculation The results of the analyses are calculated by a series of approximations which are continued until two successive calculations give practically identical results. A solution was prepared, containing in each 100 nil., 30 mg. of dextrose, 200 mg. levulose, 690 mg. maltose hydrate, and 90 mg. lactose hydrate. I n this first check analysis the mucic acid method was used for the determination of the lactose, 100-nil. portions of the solution being taken. This gave a n average of 14.6 mg. of mucic acid, corresponding, according to van der Haar's table, to 38.75 mg. of galactose, which multiplied by 2.2 is equivalent to 85.2 mg. of lactose in 100 ml., or 8.5 mg. in 10 ml.
673
Ten milliliters of solution gave 0.0728 gram of copper with the Jackson and Mathews reagent; this corresponds t o 23.3 mg. of apparent levulose (&). Ten milliliters of solution gave a titration value of 11.6 ml. of 0.1 A' thiosulfate with the Steinhoff copper acetate reagent, and of 20.4 ml. of equivalent to 25.0 mg. of levulose (R2): thiosulfate with the Steinhoff copper tartrate reagent, equivalent to 64.8 mg. of dextrose (&). Hence, L = 8.5 me RI = 23.3 Gg, RP = 25.0 mg.; a = 0.819 Rs = 64.8 mg.; b = 0.896, c = 0.532, d = 0,666 - . First approximation: 25.0 - 23.3 G = 0.819 - 0.081 2'3 mg' F = 25.0 - (2.3 X 0.819) = 23.1 mg. Percentage ratio of G to F is as 9 to 91; a = 0.813 25.0 - 23.3 = 2.3 mg. G = 0.813 - 0.081 F = 25.0 - (2.3 X 0.813) = 23.1 nig. Jf = 64.8 - [2.3 (23.1 X 0.896) 4- (8.5 X 0.666)l
+
=
67,9
0.532 Result of first approximation, for 10 mi. of solution: G = 2.3 mg. F = 23.1 mg. -If = 67.9 mg.
L = 8.5 mg. Second approximation, correction to R I . 67.9 mg. M equivalent to 67.9:26 = 2.61 mg. F 8.5 mg. L equivalent to 8.5:25.6 = 0.33 mg. F Total correction 2.94 mg. F Corrected R1 = 23.3 - 2.9 = 20.4 mg. Correction to Rz: Total dextrose plus levulose = 25.4 mg. Total maltose plus lactose = 76.4 mg.
Correction (Table IV)
% 25 F 25 F 25 G 25 G
j- 76
:M
9 2.5
+ L + 76 76 M
+ 76 L 25 F + 6 8 .If gives 9 X 6 8 : 7 6 25 F -+ 8 L gives 2.5 X 9 : 7 6 25 F + 68 ~ l ff 9 L 25 G + 68 M gives 10 X 68:76 25 G + 8 L gives 2.5 X 9 : 7 6 25 G + 78 M + 9 L 23 F + 68 , I I + 9 L gives 8.35 X 23:25 2 G f 68 .M + 9 L gives 9.25 X 2 : 2 5
23F+
10
2.5 8.05 0.30 -
8.35 8.95 0.30 9.25
7.68 0.14
8.42
2 G f 6 8 M f 9 L
Corrected thiosulfate titer, Steinhoff acetate reagent, is 11.6 (11.6 X 0.0842) = 10.6 ml. ~
-
Equivalent corrected K P = 23.2; a = 0.844
23.2 - 20.4 = 3.7 mg, 0.844 - 0.081 F = 23.2 - (3.7 X 0.844) = 20.1 mg. Percentage ratio of G to F is as 15.5 to 84.5; a = 0.835 23.2 - 20.4 = 3.7 mg. G = 0.835 - 0.081 F = 23.2 - (3.7 X 0.835) = 20.1 mg. 64.8 - [3.7 (20.1 X 0.896) (8.5 X 0.666)l = 70,3 mg. -If = 0.532 Result of second approximation, in 10 ml. of solution: G = 3.7 mg. F = 20.1 mg. Jf = 70.3 mg. L = 8.5 mg. Third approximation, correction to R1: 70.3 mg. M equivalent to 70.3:26 = 2.70 mg. F 8.5 mg. L equivalent to 8.5325.6 = 0.33 mq. F Total correction: 3.03 mg. F = 20.3 mg. Corrected R I = 23.3 - 3.0 Correction to RZ: Total dextrose plus levulose = 23.8 mg. Total maltose plus lactose = 78.8 mg. G =
+
+
INDUSTRIlL AIUD E?TGINEERI\G
674
Correction
+ +++ + + ++ + +
70
24 F 79 .V 24 F 79 L 24 G 79 41 24 G 79 L 24 F 4- 70!I. p r e s 10 5 X 70:79 24 F 9 Lgives 3.5 X 9 . 7 9 70 M 4- 9 L 24 F 24 G 70 -W gives 12.0 X 70:79 24 G 9 L gives 3.5 X 9:79 70 M 9 L 24 G 2 0 F 4-70 M 9 L giT-es 9 . 7 X 20:24 4 G 70 Jf 9 L gives 11.0 X 4 : 2 4 20F-t 4 G + 7 0 M + 9 L
10.5 3 5 12.0
3,s 9.3
0.4 9.7 10 6
0 4 -
+ ++
11 0 8 1 1 8 9.9
The corrected thiodfate titer, Sfeinhoff acetate reagent, i11.6 - (11.6 X 0.099) = 10.45 ml. Equivalent corrected I?? = 22.9; a = 0.845 22.9 - 20.3 = 3.4 mg. G = 0.848 - 0.081 F = 22.9 - (3.1 X 0.848) = 20.0 mg. Percentage ratio of G t o P is as 14.5 to 85.5; a = 0.839 22.9 - 20.3 G = 0.839 - 0.081 = 3.4 mg. F = 22.9 - (3.4 x 0.639) = 20.0 mg. 64.8 - [3.4 (20.0 X 0.896) + (8.5 X 0.666)l = 71,1 nlg. M = 0.532 Result of third approximation: G = 3.4 mg. F = 20.0 mg.
+
?l!f = 71.1 mg. L = 8.5 mg.
These values agree so closely with those obtained in the second approximation that further calculation is unnecessary. The final results are therefore: Taken .Ma.
Found MO. 3.4 20 0
3.0 20.0 69.0 9.0
Dextrose Levulose Maltose hydrate Lactose hydrate
71.1
8.5
Four other check analyses, in which the lactose was determined by the fermentation method, gave these results: ----3-
-2-
-4-
--3-----
CHEJIISTRI
J OL. 10, A.0. 12
The differences range from +0.53 to -0.31 ml. of thiosulfate, corresponding to about 2 to 3 mg. of maltose hydrate. They are in most cases somewhat larger than the experimental error of 0.1 to 0.2 i d . of thiosulfate, but the positive and negative errors nearly balance each other. It is prohable that the variation in tlie proportions of the individual sugars in the mixtures affects the reducing ratio of each sugar, as previously explained. The best check on the accuracy of the analysis of an u n k n o m sample is to prepare a mixture of the sugars in the proportions found, to analyze it, and to compare the results with those obtained in the analysis of tlie sample. Tlie errors in the results of t’he check analyses reported abol-e are not any larger than may be expected in the analysis of mixtures of four sugars by indirect methods, as has been pointed out by Broime (2). The limitations of such methods must always be kept in mind in the interpretation of the results obtained upon an unknown sample. The acruracy of the results, especially in the determination of maltose, ~vouldbe increased if at least one more sugar could be determined directly. Or t’he sum of two or three of the sugars might be ascertained by an independent method, as a check on bhe results. These possibilities will be inrestigated.
Summary Preyious studies on the determination of dextrose and lenlose have been extended to mixtures also containing maltose and lactose. I n the proposed method the total reducing sugars are determined by means of Fehling solution, the monosaccharides with Steinhoff’s modification of Barfoed’s reagent, and levulose by the method of Jackson and Xathews. Lactose is found by oxidation to mucic acid, or preferably by copper reduction after fermenting off the other sugars. Four equations are thus obtained from Tvhich the percentage of each sugar can be calculated. It has been found that both maltose and lactose have a slight reducing effect on Steinhoff’s reagent as Iyell as on Jackson and Mathews’ reagent, anti it is necessary to apply corresponding corrections. The quantities of dextrose, levulose, and lactose found in k n o m mixtures agree well with those taken, but the result for the Inaltoye is less reliable because it is obtained by difference.
Taken Found Taken Found Taken Found Taken Found Mg. M g . Mg. Jfo. Mg. Mg. Ms. -1fu. Dextrose Levulose 31alt ose hydrate Lactose hydrate
20 0 5 0
20.5 4.8
2.3 16.5
2.1 17.5
0.0 17.6
0.9 17.5
2F.4 5.4
27.5 5.6
20 0
17.5
28.5
26.4
35.0
38.7
20.7
1G.4
30.0
29.6
19.5
19.5
22.4
22.4
35.5
35.5
The quantities of dextrose, levulose, and lactose found check vel1 with those taken, but the maltose figures show larger discrepancies because this sugar is determined by difference, and the errors in all four determinations accumulate in this one result. I n two of the mixtures the maltose was found too high, in the others too lorn. The percentage error is generally smaller when the quantity of nialtose is high than when i t is
low. The principal criterion for the accuracy of the nialtoqc determination is the amount of the total sugars obtained with Fehling solution. Tlie milliliters of thiosulfate found upon eight different rriixtures of the four sugars compared as follows with those calculated by Formula 4 : Found
M1
I
20.39
4 5 6 7 8
22.35 21.31 17.20 16.63 14.44 16.76 20.40
Calculated
M1. 20.08 22.68 21.42 17.58 16.97 14.6i 16.23 20.71
L i t e r a t u r e Cited oc. Official Agr. Chem., Official a n d T e n t a t i v e M e t h o d s of Analysis, 4th ed., p. 477 (1935). ( 2 ) Browne, “ H a n d b o o k of Sugar Analysis,” pp. 472, 492, Xew I-ork, J o h n K i l e y 8: Sons, 1912. ( 3 ) BroTvne, J . d n z . Chem. Soc., 28, 439 (1906j; Intern. Sugar J . , 23, 35 (1921:. 14) Creydt a n d Tollens, Ann., 232, 205 (1885). ( 5 ) Erb a n d Zerban, IND.EXG.C H m r . , Anal. E d . , 10, 246 (1935). (6)H a a r , r a n d e r , A. W., Biochem. Z.,81, 263 (1017): “Xnleitung z u m S a c h w e i s , z u r T r e n n u n g u n d B e s t i m m u n g der hlonosaccharide und d i d e h y d s d u r e n , ” p. 123, Berlin, Gehriidcr Borntrseger, 1921. (7) Hoffman, Schweitzer, a n d D a l b y , ISD. EXG.CHEU., .inal. E d . , 8, 298 (1936). ;S) J a c k s o n and M a t h e m , Bur. Stu7~durdsJ . Reseurch, 8, 403 (1932). (9) K e n t a n d Tollens, Ann., 227, 222 (1885). (10) Quiaumhing a n d T h o m a s , J . Am. Chem. Soc., 43, 1503 (1921) (11) Schwarta, Riochem. Z., 224, 193 (1930). (12) Shaffer a n d H a r t m a n n , J. Biol. Chem., 45, 349 (1920). (13) S h a p i r o a n d Proferanaova, 2. W’irtachaflsgruppe Zuckerind., 85, 196 (1935). (14) Steinhoff, 2 . Spiritmind., 56, 64 (1933). (15) T a u b e r a n d Kleiner, J . B i d . Chem., 99, 249 (1932). (16) Vosburgh, J . A m . C h e m SOC.,43, 219 (1921). ESG. CHEJf., Anal. E d . , 8, 321 (1936). (17) Z e r b a n , IND. (18) Zerban a n d K i l e y , Ihid., 6, 354 (1934). RECEITEDSeptember 9 , 1938. Presented before the Division of Sugar Chemistry at the 93rd Meeting of the American Chemical Society, Chapel Hill, S . C., April 12 t o 15, 1937.
