1411
V O L U M E 24, NO. 9, S E P T E M B E R 1 9 5 2 Table 111. Dye Adsorption Indexes of Vitamin A Chromatographic Adsorption Mixtures (After exposure t o various atmospheric conditions.
x
10-8)
2641 Mixtures. Values
1 M g 0 : l Celite 3 M g 0 : l Celite:6 KazSO, 4-month Freshly 4-month Freshly ?ample prepared sample prepared i\ione 2.92 4.0 1.74 2.91 .. 0.325'' 20 hr. 0.oa COa Hi0 1.44 .. 20 hr. 2 3i Con 0.36 20 hr. 1 45 Ni Hi0 1:9i 2.ii 10 Inin. .. COS HzO 0 .37sn .. 0.oa .. con E80 45 niin. 45 min. .. 1.71 .. Cor HzO 1.28 2.3 .. Room atmos. 4 days ,. a Chromatographic effectiveness for iytamin 4 and carotene lost only in these samples. Treatment
+ ++ ++
.
-~~ _ _ _ _
~~
-
-
compared with those containing sodium sulfate. In both cases the mixtures that do not contain the sodium sulfate have the larger index, but actual chromatographic performance with carotene and vitamin A indicates that the sodium sulfate type is much stronger, as more compact bands are formed in this mixture. It appears that this kind of performance may possibly be accounted for by a mechanical increase of effective surface in the sodium sulfate adsorbent mixture. No doubt such a mechanical picture is oversimplified and could be elaborated upon from the standpoint of the electronic forces involved in such mixtures, but that is beyond the scope of this discussion. The data considered in Table I11 warn one that interpretive comparisons must be largely restricted to compositions of the
the same general type. The relative values given on the last line of Table I11 merely indicate the appreciable effect on adsorption index or activity by ordinary atmospheric exposure. The results of these studies indicate that it is possible to evaluate with precision the strength of a given adsorption mixture and that this evaluation may be correlated with chromatographic performance. Furthermore, it follows that such an evaluation of a magnesium oxide-Celite mixture determines the strength of the magnesium oxide itself from a practical viewpoint, since the Celite is inactive. In fact, a 1 to 1 mixture may be used advantageously for the evaluation of the magnesium oxide, as the distribution of dyestuff and the manipulative technique are thereby facilitated. Some time and material may be saved by adopting this procedure. as the mixture usually is the thing that has practical utility in the laboratory. I t is felt that a new and needed means is offered for more mccessful chromatography by the use of the adsorption index technique just presented in its relationship to the separation of vitamin .\ and carotene. LITERATURE CITED
(1) 11-ilkie, J. B., 1.Assoc. Ofic.Agr. Chemists, 32,455 (1949). (2) TTiikie, J. B., and DeWitt, J. B., Ibid., 28, 176 (1945). (3) Zettlemoyer, \. C., and Walker, W, C., Ind. Eng. Chent., 39, 69 (1947). RECEIVED for review April 15, 1952. Accepted July 3, 1952. Presented before the Division of Biological Chemistry a t the 121st Meeting of the AMERIC A K CHENICAL SOCIETY, Buffalo, N. Y.
Quantitative Paper Chromatography of D=Glucose and Its Oligosaccharides K. .J. DI-MLER, W. C. SCHAEFER, C. S. WISE, ANDC.E. RIST Y o r t h e r n Regional Research Laboratory, Peoria. Ill.
T
HE extension of paper chromatography to the qualitative
separation of mixtures containing di- and oligosaccharides ( I S ) has opened the way to solving the difficult problem of quantitative analysis of such mixtures. The procedure of quantitative paper chromatography presented here was established particularly for the analysis of mixtures of low molecular vieight polymers of D-glucose, such as those obtained in the enzymic hydrolysis of starch fractions ( 3 ) . I t is also directly applicable t o Dfructose and to oligosaccharides containing D-fructose alone or along with D-glucose. The present procedure combines the previously reported chromatographic techniques for oligosaccharides ( I S ) with elution of the resolved spots by an adaptation of the method of Dent ( 2 )and determination of the carbohydrate content by the colorimetric anthrone reaction (16,21). Quantitative paper chromatography of sugars has been described by several authors, usually in application to monosaccharides. Most of' their methods, however are not suitablr for the study of mixtures of glucose polymers of unproved structure and size. Thus the use of methods based on reducing power (6-7, I O , 18, E?),periodate oxidation ( I I ) , or comparison of spot intensity ( I > 8, 17) would be prevented by the lack of samples of the pure oligosaccharides for standardization and comparison. The ant,hrone method overcomes this difficulty, as it essentially determines the amount of monosaccharide which would be obtained on total hydrolysis. It offers advantages of speed and simplicity of operat,ion over other total carbohydrate methods, such as acid hydrolysis followed by determination of reducing power (6. 251, or distillation from phosphoric acid follolr-cd by
ultraviolet spectrophotometric measurement of hydroxymethyl furfural (24). On the basis of sensitivity the anthrone method is well suited to paper chromatography, as a determination ran be made in duplicate on 10 to 100 micrograms of carbohydrate. Since the studies described here were completed, a phenol-sulfuric acid procedure has been reported ( 4 ) for carbohydrate dctcrmination and applied in the quantitative paper chromatography of sugars in wheat flour ( I 4 ) , the known sugars being used for standardization. Evaluation of the merits and limitations of this method must await the publication of further drtails iPPIR4TLS $\I) REIGESTS
Paper chronmtography equipment, as described by Jeanes, Wise, and Dimler ( I S ) , or other suitable arrangements are satisfactory. In the present studies TJ-hatrnan No. 1 filter paper (45.5 X 57 cni. sheets) wab used. A stainless steel tray, 3 x 8 inches and 0.5 inch deep, holds six pairs of 2 X 2 inrh glass slide. in .z 10-inch diameter desiccator (see Figure 1). A spectrophotomet,er, mch as Coleman Universal spectrophotometer Model 11, is used. For the work reported here the cuvette for test, tubes was fitted with brass sleeves to accommodate selected 18 X 150 mm. borosilicate glass test tubes. The dinitrosalicylate spray reagent, contains 0.5 gram of 3,5dinitrosalicylic acid in 100 ml. of 1 S sodium hydroxide ( I S ) . -4mmoniacal silver nitrate spray reagent is prepared by dissolving 5 grams of silver nitrat'e in 95 ml. of water and adding 6 ml. (slight excess) of concentrated ammonium hydroxide ( l e ) . To avoid the danger of explosive silver residues, the solution should be freshly prepared and the unused portion discarded immediately in such a manner that no dr3- residue will form.
ANALYTICAL CHEMISTRY
1412
A method for qiiantitatively determining the diatrihution of oligosaccharides was required, for example, i n studies of the mechanism and kinetics of enzymio and acid hydrolysis of starch and related polysacoharides. A procedure is desorihed which is hased on using the anthrone reaction for the colorimetric measurement of the constituents resolved by paper chromatography and eluted from the paper. The analyses on known mixtures generally w-ere within 570 of theory. The procedure is of particnlar value heoause i t can he applied to all resolvahle polymers of glucose and fructose without need for samples of the polymers for standardization and without need for lcnowledge of the types of linkage or m o leoular size of the compounds. The method potentially is of even wider applicability, hecause other hexose derivatives and polymers ean be determined with the anthrone reagent.
Anthrone reagent is freshly prepared each day (Zf)hy dissolving 0.2 gram of anthrone in 100 ml. of concentrated sulfuric soid (assay 95-97%). PROCEDURE
Preparation of Chromatogram. Apply the carbohydrate solution in 1-MI. or smsller diquats on the starting line with a platinum loop, a micropipet, or an ultramicroburet. A pattern and spacing of spots which has been found convenient are shonn in Figure 2. I n the section to he analyzed (E?, Figure 2) sufficient applications of solution are made (adjacent to each other, superimposed, or a combination of the two) to give a t least 15 to 30 micrograms of the minor constituent after resolution (1 PI. of 1%solution of a sugar contains 10 micrograms). Each spot should he dried hefore another is superimposed on it or placed next to it. When the chromatogram is being spotted, apply the same amount of solution to a 4 X 6 om. piece of filter paper as in one group of spats on the chromatogram, This piece of filter paper, which is not developed, is referred to a6 the control and is used for the determination of the total amount of carbohydrate applied to the chromatogram. Develop the chromatogram with a suitable ~olventcombination such as l-but~nol-pyridine-water (6:4:3 parts hy volume) or fusel oil-pyridine-water (1:1:I), using single or multiple development as required (I$). Thefuseloil used had a distillation range of 121' to 129" C. Air-dry the developed chromatogram for about an hour a t room temperature. Removal of the last
Determination of Carbohydrate in Eluates. In 811 work involving the anthrone method for carhohydrate determination, u w special care to avoid the introduction of any trace of extreneour soluble or insoluble cmhohydrate m a t e r i d It is advisahle to reserve a special set of glassware for these determinations. Dilute each sample of eluate to 10 ml. or more, 80 that the carbohydrate content, if possible, will be in the range of 3 to 30 micrograms per ml. If lint is present, it must be avoided in pipetting or the solution filtered through B sintered-glass funnel. Place B 3-ml. aliquot of the diluted eluate in a lint-free clean 18 X 150 mm. horosilicate glass test tube selected for spectrophoiametric work. After cooling. the solution in a cold water hath (about 15'), layer 6 ml. of the anthrone reagent under the &queou6solution. With the tube still in the cold hath, mix the contents thoroughly with a clean stirring rod, which is then removed. Transfer the rack of cold tuhes to a boiling water hath. After 10 minutes (&ahout 15 seconds) of heating return the tuhes to a cold bath. Determine the optical denhties of the resultina solutions a t 620 mu aetainst a blank of water nlus reaeent. heated it the Same time. Calculate the concentration of u-gJucose 01. potential D-glucose in the eluate by the relation I
C = KD where C is the concentration in micrograms per ml., D the ohserved optical density and K a constant determined either from one or two standard gfucose solutions run a t the same time or, a t some possible sacrifice of accuracy, from a previously run set of glucose solutions of known concentrations. Correct the total weight of cwhohydrate recovered from each section of the chromatogram for the apparent glucose obtained on elution of the filter paver alone (in the present studies this amounted to about
control pieee"af paper or of the summation of the carbo&drate contents of the analped sertions of the chromatogram.
Cut off and sDrw the mide'strips with a suitahle reagent such
nitrate) to locate d e positions of thereducing sugars. Elution of Sugars for Analysis. From the unsprayed strip of the chromatogram cut off the sections corresponding to the desired carbohydrate spots a6 shown hy the guide stnps. Cut a shallow point on a short edge of each of the resulting pieces of filter paper. Insert the opposite edge (to ahout 0.5 em.) between
Figure 1. Assembly foor Elution of Carhohydrate from Sections of Paper Chromatogram Aenomblg p i u c d in humidified ohambsrdesiooalor containing water
..
For wider Dieces of Dan& the use of larger sizes of dass wduld he -
advisable.
I
Arrange the pairs of slides in the stainless steel tray as shown in Figure 1. With the tray supported in s dencctLtor containing a little water foor humidification. add water to the t r w . As the 1)aDers become wetted by capiilarv movement of the water up
the elution to'proceed foor 2 to 3 bours in the close3 desiccator, during which time ahout 8 to 12 drops of eluate containing all the carhohydrate will be collected from each section of paper (see also 16). The elution of oligosracchsridesof high molecular weight which have not moved from the starting line. sometimrs will re4uire longer (4 to 5 hours).
oligosaeoharide:
This eorre&on amountalto:
Corrected wt. = glucose found X (0.9
+
s)
where DP is the degree of polymerisation or number of glucose units per molecule. In most cases, however, i t is more Convenient to express all results as potential glucose (or fructose). Results. Typical results of the analysis of known mixtures of 0-glucose and maltose hydrate are given in Table I.
V O L U M E 24, N O ___
9, S E P T E M B E R 1 9 5 2 -
~~~
___
~
1413 ~
_
_
_
~
-
saccharides containing other monosaccharide units besidee glucose or fructose if the kind and ratio of units in each re('oiiiliohition 3listure Prepared, c; B a m l on Control Spottine" ~ _ _ Ratio of C o n l i i o n m t ~-~ qolved constituent were knon n, inalyzed 91 5 q1 6 91 4 30 that an appropriate facror !17. 1 8 5 8 4 could be calculated from the -~ 9 . 1 -- 8 6 106 2 100 0 100 0 100 0 anthrone values of the mono!I 3 8 4 8 6 8 9 saccharides or from known !lJ. 4 91 6 91 1 914 .~ samples of the oligosaccharide.. -1Oil-i 100 0 1 m 100 0 Such an extension of the pro50 8 cedure has been incorporated 49 2 100 0 in a study of the carbohydrate " Recovery of total carbohydrntc froiii control t n k r n as loo$:. constituents of soybean phoephatide fractions (90). In the chromatography step of the procedure, completeness For the chromatogrums from Tvhich thew data were obtained of resolution and uniformity of movement of the sugar spots are the sugar solution mis applied a i t h a Kirk micropipet (2.9) of particular importance. A number of factors influencing the of 1-pl. capacity, approximately 1 mg. of total carbohydrate being chromatography of sugars have been reported in the literature applied to the section to be analyzed and half that much to the control piece. The three results for each mixture (except 111) as well as being encountered in studies a t this laboratory. Some aye for three different rections of one sheet of paper, each with its of these are mentioned here only as a &wide rather than a~ a own control. The anthrone determinations were run in triplicate. complete discussion of factors involvcd in the paper chromatography of carbohydrates. The analysis of a mixture of mono-, di-, and trisaccharides is illustrated by the results in Table I1 on a known solution of Dglucose, maltose hydrate, and 3-a-isomaltosyl-D-glucose (panose, 26). The solution, containing 9% total carbohydrate, wm applied t'o the paper in the pattern of Figure 2, a 1-pl. quantity was placed at each position with a Gilmont ultraniicroburet ( 9 ) . The total weight of carbohydrate applied t o each of the tvio scctions to be analyzed was about 1 mg. of potential glucose. Over 90% of the carbohydrate in the measured volume applied t o the chromatogram and to the control was recovered. The application of the procedure to the mixture of oligoeaccharides in the 95% ethanol-soluble fraction from the partial hydrolysis of amylose with malt alpha-amylase (3)provided the results in Table 111. The components presumably are the members of the homologous series of 1,4-glucosidically linked oligosaccharides (1.9) but the names assigned in Table I11 are only tentative. I The results in the first colunin of Table I11 were obtained on a I _ . _ chromatogram spotted by a 1-pl. Kirk micropipet ( 2 3 ) and deFigure 2. Typical Pattern for Spotting Chromatograms veloped by a single descent with 1-butanol-pyridine-water Each spot may contain several superimposed applications of solution. Sections A and C are used as guide strips to locate the portions of B to ( 6 : 4 : 3 ) . The second column gives results on a chromatogram be cut off and analyzed for which the spotting was with a platinum loop (IS)and multiple development was used with three descending passages of the sanic solvent combination. Table I.
Kepresentatite Results of Analysis of Known Mixtures of D-Glucose and Maltose €I)drate Composition Yc - Ohyer\rd _ _ ~
~~~~~
~
~
~
~
~
-
DISCUSSION
The applicability of this procedure for quantitative paper chromatography of carbohydrates depends, of course, on the behavior of different sugars in the anthrone determination and on the ability to resolve the carbohydrates by paper chromatography. In the anthrone reaction, D-glucose and n-fructose and their polymers give the same intensity of color for equivalent weights of carbohydrate, as has been demonstrated by Morris (19) and others. The analysis of mixtures, therefore, does not require knowledge of the degree of polymerization or kind of glycosidic linkage in the resolved components as long as the constituent sugars are known to be limited to glucose and fructose. Sugars other than glucose and fructose give different degrees of color intensity in the anthrone reaction (16, 19). Representative results for some common sugars are shown in Table IV. It is apparent that the behavior of the monosaccharides is reflected in the di- and trisaccharides in Tyhich they occur, as noted also by Morris (19) for the color produced by lactose compared with glucose and galactose. Therefore, the present procedure for quantitative paper chromatography could be extended to oligo-
Table 11.
.inalysis of a Known Mixture of Mono-, Di-.
and Trisaccharide
Component
Composition as Prepared, %
D-Glucose hlaltose.Hz0 4-a-Isomaltosyl-Dglucose Total
29.2 34.6
316 .2 00,o
Observed Composition (as Potential Gluoose), 5% Based on Cont,rol Ratio of CompoSpottinga nents Analyzed 29.3 36.1
30.7 34.5
- _3 6_ . 8. _ _ _3 6. ._3 _ 101.5
102.2
28.6 35.4
30.3 34.0
36.0 100.0
35.7 100.0
Recovery of total carbohydrate froiii rontrol taken as 100%.
_____
~
~~
~~
~
.
.___
...
-
Table 111. Am)-lose Hydrolyzate, 95% Ethanol-Soluble Fraction Component D-GlUCOse Maltose.Hs0 "Maltotriose" "1Ialtotetraose" Higher saccharides Total
. a
Compo-ition (as Potential Glucose) Based on Control Spottingo, % 3.6 44.7 21.6 9.8 24.2 103.9
---
3.9 45.4 21.1 9.2 23.6 103.2
-
Recovery of total carboliydratr from control taken a s 100%.
1414
ANALYTICAL CHEMISTRY
Degree of resolution of two carbohydrates depends first of all on the differences in their distance of travel. This is controlled by the R/ values of the compounds and the distance of movement of the solvent. The choice of solvent combination and of method of development-e g., descending movement of solvent, multiple development, continuous developmentshould be made for the specific mixture being studied (IS). A second factor in the resolution of sugars for quantitative work is the size of the individual spots after development. Undesirably large spots may result from large diameter of initial spots, high R, value of the carbohydrate, or high levels of the component in the spot. Elongation or tailing of the spot can arise from too heavy local application of the mixture on the paper, too rapid movement of the solvent. and excessive amounts of salts in the mixture. In the present work the initial spot size was kept at 5 mm. or smaller diameter by using applications of 1 pl. or less To provide sufficient carbohydrate for analysis, spots were applied at adjacent position, as shown in Figure 2 Depending on the concentration of carbohydrate in the solution, superimposed applications also can be used on the adjacent positions. The pattern of spotting shown in Figure 2 has proved advantageous in minimizing the time of waiting for spots to dry. The section t o be sprayed should, of course, receive the same applications as one group of spots on the section to be analyzed, so that the guide strips will give a reliable indication of both the position and size of the spots. The accuracy and reproducibility of this quantitative paper chromatography procedure generally have been sufficient to give results n-ithin 5% of theory on known mixtures, as shown by Tables I and 11. Occasional variations approaching 10% were encountered, particularly for a minor constituent of a mixture. The anthrone method for carbohydrate determination. as used here, has given an average accuracy of about z i 2% in the optimum range of optical density (20 to 30 micrograms of n-glucose per ml. in this cme). Preservatives for sugar solutions being held for analysis may interfere and their effect should be tested on known solutions. Thus, results about 70% of normal were obtained when the h a 1 diluted sugar solutions were saturated with toluene Correct analyses were obtained after removal of the toluene by partial evaporation of the solution in an evaporating dish on the steam bath or by ether extraction. A thymol-saturated diluted carbohydrate solution gave none of the characteristic absorption at 620 mp after the anthrone reaction, the color being brown instead of greenish blue. No interference has been observed, however, from phenylmercuric acetate used a t a level of 1 p.p.m. The testing of chromatographic solvents a t low levels is also desirable t o avoid interfering effects that might arise from traces of such solvents as phenol, or of impurities in the solvents, left on the paper after development and drying. The use of control spots as a basis for calculating the total carbohydrate on the chromatogram was adopted to avoid uncertainties in the actual volume of delivery of the platinum loop or micropipet. Factors such as differences in viscosity and surface tension of the different carbohydrate solutions render the calibration of the loop or pipet with water or a solution of Dglucose more or less unreliable. With the control spots the only necessary precaution is that the operator be consistent in the manner of taking up the solution and applying it to the paper. Interspersing the control spotting with spotting of the chromatogram is beneficial in this respect. For the platinum loop, the volume of solution it carries is dependent on the rate of lifting the loop from the solution and its position relative to the surface With care, reproducibility of volume delivered has been within &lo% for individual loopfuls. When reducing oligosaccharides, made up of glucose, fructose, or both, are being studied, an approximate knowledge of the molecular size of the components should be obtainable from parallel
.
quantitative chromatograms by using the anthrone method to determine the carbohydrate weight and one of the reducing methods to determine the reducing end-group content of the spot.
Table IV. Comparison of Various Mono- and Oligosaccharides in the Anthrone Reaction
D-Glucose D-Fruc t ose D-Galnctoee D-Mannose L-Rhamnose L-Sorbose D-XVl O S P
Levoglucosan hlaltose.Hz0 Celloh;-. Treh; Sucrose Melezitose.2HzO Inulin Lactoee.Hz0 3Ielibiose.Z Hz0 Raffinose.5HzO
(Opticnl densities measured nt 620 mp) Recovery Baaed o n Relative Optical Monosaccharide ConstituDensity per Microgram, ents, % % of Theory 100 ...
98 57 55
49 76 7
10s 98 ind
107 102 111 74 72
75
...
...
98
98 99 100 102 102 102 102 105 104
As the molecular reducing power values of different oligosaccharide structures become established, such an approach will be increasingly ueeful. This extension of the quantitative paper chromatography of oligosaccharidee is under investigation. ACKNOWLEDGMENT
The authors are indebted t o S.C. Pan, E. R. Squibb and Sons, who mpplied a sample of 4-a-isomaltosyl-n-glucose (panose). LITERATURE CITED
(1) Brown, R.J., ANAL.C H m f . , 24,384 (1952). (2) Dent, C. E., Biochem. J . , 41, 240 (1947). (3) Dimler, R. J., Bachmann, R. C., and Davis, H. A , CmmZ Chem., 27, 488 (1950). (4) Dubois, M., Gilles, K., Hamilton, J. K., Reber, P. A., and Smith, F., Nature, 168,167 (1951). (5) Duff, R. B., and Eastwood, D. J.,Ibid.,165, 848 (1950). (6) Flood, A. E., Hirst, E. L., and Jones, J. K. N., J. Chem. SOC., 1948,1679. (7) Flood, A. E., Hirst, E. L., and Jones, J. K. N., Nature, 160, 86 (1947). (8) Gibbons, G. C., and Boissonnas, R. A., H e k . Chim. Acta, 33, 1477 (1950). (9) Gilmont, R., ASAL. CHEM.,20, 1109 (1948). (10) Hawthorne, J. R., Nature, 160, 714 (1947). (11) Hirst, E. L., and Jones, J. K. N., J . Chem. Sac., 1949, 1659. (12) Hough, L., Sature, 165, 400 (1950). (13) Jeanes, A., Wise, C. S., and Dimler, R. J., ~ A L CHEW, . 23, 415 (1951). (14) Koch, R. B., Geddes, W.F., and Smith, F.. Cereal C h a . , 28, 424 (1951). (15) Laidlaw, R. A., and Reid, 8.G., A-ature, 166,476 (1950). (16) McCready, R. hf., Guggolz, J., Silviera, V., and Owens, H. S., h A L . CHEM.,22, 1156 (1950). (17) hIcFarren, E. F., Brand, K., and Rutkowski, H. R., Ibid., 23, 1146 (1951). (18) Montreuil, J., BUZZ.sac. chim. biol., 31, 1639 (1949). (19) Morris, D. L., Science, 107, 254 (1948). (20) Scholfield, C. R., Dutton, H. J., and Dimler, R. J., J . Am. Oil Chemists Sac., 29, 293 (1952). (21) Seifter, S., Dayton, S.,Kovic, B., and ;\IuntwYler, E., Arch. Biochem., 25, 191 (1950). (22) Shu, P., Can. J . Research, 28B, 527 (1950). (23) Sisco, R. C., Cunningham, B., and Kirk, P. L., J . B b l . Chem., 139, 1 (1941). (24) Stone, J. E., and Blundell, 31. J., Can. J . Research, 28B, 676 (1950). (25) Williams, K. T., and Bevenue, A,, Cereal Chem., 28, 416 (1951). (26) Wolfrom, PI.L., Thompson, A , , and Galkowski, T. T., J . Am. Chem. Soc., 73,4093 (1951). RECEIVED for review April 18, 1952. Accepted July 11, 1952. Presented before t h e Division of Sugar Chemistry a t the 121st Meeting of the A M E R I CAN CHEMICAL SOCIETY, 3Iilwnukee, Wis.
