present in urine interfered slightly. This interference was removed by the addition of cyanide as described in the experimental procedure. Comparison with Other Methods.
Calcium was determined in seven different samples of urine by t h e present method, by t h e complexometric method of Wlson (9),and by the oxalate precipitation method of Shohl and Pedley (8). I n Table IV, the vaIues for calcium obtained by the present procedure compare favorably with the other methods, as do the values for calcium plus magnesium with the Wilson procedure (9). The method of Sholil and Pedley requires approxiniately 24 hours for completion and the procedure of Kilson require 2 t o 3 hours. Precision of Method. To calculate the standard deviation of these procedures, calciuni and calcium plus magnesium were determined on 20
individual aliquots of the same urine. The magnesium content of the urine was obtained by difference between the calcium and the calcium plus magnesium determination. The results of this experiment were (in millimoles per liter): Ca = 8.41 =t0.032; Ca hIg = 14.44 =t0.033; AIg = 6.03 =k
+
0.177. Twenty triplicate titrations of urinary calcium where the amount of calcium varied from 2.23 to 8.94 millimoles per liter agreed within i 2.9%. ACKNOWLEDGMENT
The authors n-ish to acknowledge the technical assistance of Thomas Austin. They also express their appreciation to Geigy Industrial Chemicals for supplying saniples of EGTA used in this experiment.
LITERATURE CITED
(1) Berger, E., Clin. Chem. 1 , 249 (1955). (2) Brunisholz, G., Genton, M., Plattner. E., Helv. Chim. $eta 36, 782 (1953), (3) Fiske, C. H., Logan, M. A , , J . B i d . Chem. 93, 211 (1931). (4) Golby, R. L., Hildebrand, G. P.,
Reilley, C. N., J . Lab. Clin. M e d . 50. 498 11957). (5) Hildebrand, G.' P., Reilley, C. S . , A h A L . CHEK 29, 258 (1957). (6) Horner, I\-.H., J . Lab. Clin.M e d . 45, 4.51 14.5.5) 951 i (1955).
((7) 7 ) Schmid, sei;; R. W., Reilley, C. K., AXAL. CHEU.29, 264 (1957). (8) Shohl, A. T., Pt Pedley, F. G., J . Bio2. Chem. 50. ,537 t(1922). L H L L I. (9) W&on, A,' A , , ' J . Comp. Pathol. Therap. 63, 294 (1953).
RECEIVED for review July 29, 1957. Accepted November 26, 1957. Supported in part by research grant (PHS8-248) from the National Institute of ilrthritis and Metabolic Diseases, Sational Institutes of Health, Public Health Service.
Infrared identification of Disaccharides JONATHAN W. WHITE, Jr., C.
R.
EDDY, JEANNE PETTY, and NANCY HOBAN
Eastern Regional Research laboratory, Philadelphia, Pa.
b The value of infrared spectra for the identification of amorphous disaccharides and their acetates, by comparison with spectra of known disaccharides and their acetates, i s demonstrated. Infrared spectra of ten amorphous disaccharides of Dglucose, of D-glucose and D-fructose, and of their 0-octaacetates are presented over the range of 650 to 1500 cm.J Potassium bromide disks were used. All spectra differ in sufficient detail to allow differentiation among closely related disaccharides.
A
chromatographic analysis has been of inestimable value in separation of mixtures of sugars, their identification by chromatographic evidence alone is not conclusive. Relative rates of migration on paper or column, together with color reactions v i t h spray reagents, only contribute to identification. Physical properties of isolated samples or derivatives are also required. Heretofore, crystallization of the sugar or derivative has been considered imperative before an identification is unequivocal. Sugar samples isolated by chroniatographic means may be so small that i t is practically impossible to crystallize LTHOUGH
506
ANALYTICAL CHEMISTRY
them. Hence, melting point, x-ray diffraction, and crystallographic study are eliminated as tools for identification. Determination of optical rotation does not require crystalline samples, but values may become only approxiniations as sample weights decline into the lower milligram range. The infrared absorption spectrum of a compound is increasingly used for its identification and analysis. As a unique physical property that is not primarily dependent on crystallization, it offers a most useful criterion for identification of disaccharides. Infrared spectra of all types of carbohydrates have been published. I n the first of a series of papers on this subject, Barker and his colleagues (5) stated that determinations of infrared spectra offer poll-erful means for the comparison of supposedly identical samples of carbohydrates. They examined the spectra of a variety of carbohydrates and their derivatives, but have not published them in sufficient detail for comparison purposes. They noted that spectra in the 730- to 960cm.-' range allow assignment to the CYor p-series of D-glucopyranoses, Khistler and House (20) have also reported that certain regions of absorption are characteristic of the configuration of the anomeric carbon.
Kuhn (13) has published the spectra of 79 carbohydrates and derivatives over the range 8.0 to 15.0 microns. Of these, only t n o are of sugars included here. Kuhn's spectra were determined with an amount of snniple in the beam too small to permit maximum utility of the curres for comparison purposes. Infrared spectra of a large number of sugar acetatrs and related compounds have been determined a t the National Bureau of Standards (12). Crystalline materials were used but the spectra were all determined in solution. The pressed-disk technique is useful when water-soluble materials are examined. It also prrmits the use, where necessary, of less-than-milligram samples ( 1 ) . Some difficulties have been experienced in comparing disk spectra with those obtained from mulls (d, 3, 8). Barker et al. (6) have ascribed the changes they previously (3)noted during aging of disks containing certain carbohydrates to the relatively high moisture content of potassium bromide disks. They did not find any such changes in any of the three disaccharides included in their study. The m l u e of infrared spectra in identification of larger molecules such as trisaccharides was cited by Whiffen (19), n h o noted that identification by melting point and rotation
becomes rather uncertain a t this level of complexity. With potassium bromide disks and a standardized procedure for converting the sugar to the acetate, infrared fpectra may be obtained of quality sufficient to allon- identification with 10 mg. of amorphous sugar, ~ i t h o u tthe use of supplementary condensing systems in the spectrophotometer. I n these spectra of the free sugar and the acetate, as many as 20 to 30 items of information are availalile from one sugar for comparison n ith an ~mkiionn, contrasted n i t h one for a melting point. Degree of crystallinity has :i c o n d e r ahle influence on solid-state infrared spectra. The spectra reported here are those of noncrystalline sligars arid acetates; the procedure adopted is intended to avoid introducing crystallinity as a variable. The spectra of these amorphous materials do not shon- as much detailed structure as those of crystalline substnncrs; the number and intensitie. of bands are cvnsiderably reduced. This numerical reduction of bands is compensated for bv including the spectruni of the acetate in the comparison. APPARATUS A N D REAGENTS
Spectra were obtained with a Perkinlllmer Model 21 sliectrophotonieter. The resolutioii dial n-as set a t 930, IIO reference was used in the rear beam, :itid the optical balaiice was turned niaxinium clockn-ise. (Preliminary espwinients indicated that although disks of very good optical quality could he pressed with freeze-dried sugars in potassium bromide, disks containing the sugar concentration required could not be removed from t,he tlie without cwcking.) A die Ohat could be inserted into the speetrophotonieter without rcmoval of the disk v a s made by niodifyiiig commercial punches and dies designed for punching sheet niet,al. The punch was 0.500 inch in diameter with :I straight shank. The dies were 1.00 inrh in outside dianietcr and 1.187 iiic~hcs long. The intcrnal diameter, originally 0.497 inch, was enlarged to O.5OOd inch. The vacuuni chamber \vas similar to that of the press distribu t i d by Jarrcl-Ash Co. (Catalog KO. H920) with the addition of s c r e w for applj-ing forcc to remove the punch fmni (lie aftw pressing. Thme dies fit' the microrcll adapter of the spectrophotometer, except that the t x o shelves for positioning the microcell interfere. A new adaptrr n-as made having remorable shelves. K t ' h this arrangenicnt, t'he die rontaining t,he potassium Immide disk could be inserted directly into the microcell adapter for obtaining the spectrum. Leniieux et al. (14) and Grendon and Love11 ( I O ) have also designed dirs iyhich could be inserted directly into a Model 21 spectrophotometer. Disaccharide
Samples.
Sucrose,
maltose, turanose, cellobiose, a n d gentiobiose were obtained commercially. Isomaltose was obtained froni enzymatic hydrolysis of K R R L B-512 dextran. Leucrose. sample X R R L 3674-50-E. Maltulose was prepared b y isomerization of maltose by limev-ater a t 35' C. for 3 days under toluene. After excess maltose was removed b y digestion with honey invertase (ZW), the maltulose was separated by charcoal column and paper chromatography. From 1 gram of maltose was obtained 103 mg. of amorphous maltulose, [a]'," = f 5 2 " [+,52.8" (18), +56.2" (7')l. The reducing power of the sugar was 41.7% of that of glucose (maltose was 43.974) n-ith Shaffer-Somogyi reagent 50. The sugar consumed 91% less iodine in the hypoiodite oxidation than did maltose, shoning the reducing group to be ketose. I t s osazone showed a n identical w a y ponder diffraction pattern to that of maltosazone. Inulobiose. A disaccharide fraction n-as prepared by tlie partial hydrolysis of inulin as directed by Pazur and Gordon ( 1 7 ) . Kigerose. Five preparations of this sugar were as follons: 1. By acid reversion from u-glucose a t The University of Kebraska. 2 . By acid reversion essentially as described by Pazur and Budovich (!6), [ a ] : = fSG"; Pazur and Budovich rcportcd f87". 3. From sakiibiose octaacetate. N a t w d a et al. s h o w d sakbbiose to be identical n-ith nigerose ( 2 5 ) . X sample of crystalline sakbbiose acetate (30 mg.) was treated in methanol (2 nil.) with 0.1 nil. of 0.4Sbarium methylate (11) a t 5" C. for 24 hours. The suspension was treated with 0.4 ml. of 0.1.1- sulfuric acid and centrifuged, and the supernatant was evaporated to dryness, dissolved in m t e r , and deionized b y ion exchange. 1. By hydrolysis of nigeran. Kigeran was prepared from Aspergillus niger mycelia as described by Yuill ( 2 4 . It was subjected to partial acid hydrolysis followed by destruction of nialtose by baker's yeast. d disaccharide was obtained by charcoal colunin cliromatographj- corresponding to that obtained by Barker et al. ( 4 ) from nigeran hydrolysis. Yields n-ere too Ion- to permit reliablr optical rotation determination. 5. By isolation from honey. d reducing disaccharide n as isolatrd from honey by carbon column and paper chromatographic procedures (21). It was hydrolyzed t o D-g~UCoseonly and it migrated on paper slightly faster than maltose. PROCEDURE
Sugars. T o a solution of t h e sugar (3 mg. in 10 i d . of n.ater) is added 500 mg. of potassium bromide (Harshaw infrared quality). A second solution (10 ml.) is prepared a t 0.25 of t h e concentration of t h e first and 500 mg. of potassium bromide is added. Both solutions are freezedried and t h e d r y products are transfcrred t o a mullite mortar and lightly
ground. ,4400-mg. sample is weighed, dried for 3 hours in vacuo at 8.5" C., and stored (as are disks) in a desiccator. The spectra of the disks are determined immediately after pressing. The disks (diameter, 1.26 em.; thickness, approximately 0.1 em.) are pressed for 10 minutes a t a total force of 10 tons (50 tons per square inch). Sugar Acetates. T h e acetates are prepared by t h e procedure described by French (Q), which has been found satisfactory for routine preparation of milligram quantities of completel>, a w t ylated disaccharides for infrared spectra. The sugar (5 to 10 mg.) is mixed with a n equal weight of fused sodium acetate and 0.05 to 0.10 ml. of acctic anhydride in a 10 X 75 mm. tc,st tubc. The suspension is boiled by touching the test' tube to a hot platt until solution occurs (about 1.5 minutos). Tlic sample is spread over thc lon-rr half of the inside of the test tube by rotating it' as it cools and solidifies. A stream of n-arm air is directed into t h r trst tube until the residual acetic anhydride is evaporated (about' 4 to G hours). The residue is extracted scyeral tinics n-ith a few millilibers of benzene whirh is filtered through sintercd glass arid evaporated dry on tlic stram bath. Yields are 70 to 80% of theory. The octaacetates are deposited froni acetone solution onto pon-dcretl (200meshj pobassium bromidc. T o 410 mg. of potassium broniitlc in a iiiullitc~ mortar is added the sugar acetate ( 3 . 2 mg. in 0.10 to 0.15 nil. of acet,one). The entire volume should be takrn u p by tlie dry powder. I t is mixcd in the mort,ar hy gtintle ruhbing until after the solvent evxporates. A d i l u t d sample is made up similarly using 0.5 mg. of acetate and 410 nig. of potassium bromide. An alternative procedure for preparation of the dilute samples is to poi\-drr the original disks after use and mix a portion with the required amount of potassium bromide. Reduction of disk thickness by pressing one fourth of the maberial at' the original concentration is not satisfactory for routine n-ork, as the strengt,h of the disks is too Ion a t the reduced thickness. RESULTS A N D DISCUSSION
The infrared spectra of these disaccliarides and their octaacetates are shon n in Figures 1 and 2 . Any one of these carbohydrates may be distinguished from the remainder by comparison of spectra. The range included is from 650 to 1500 em.-', as the spectra a t higher frequencies are of no value for this purpose. Concentrations are: sugars, 1.5 and 6.0 mg. per gram of potassium bromide; acetates, 1.5 nig. and 8.0 to 9.0 nig. per gram of potassium bromide. Effect of Crystallinity. I n Figure 3 are shown two spectra of sucrose. T h e upper curve is t h a t of crystalline sucrose, mixed u5th 200-mesh potassium bromide and dried a t 100' C. in vacuo for 1 hour before pressing. VOL. 30,
NO. 4,
APRIL 1958
507
WAVE
7
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9
8
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_ _ I _ _ _
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U
c ACETATE
100
I 1 I
1
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f
CELLOBIOSE
0
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~~~~
O I500
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ACETATE
I
L
_I300
1100 FREQUENCY,
900 CM'
700
1500
1300
I100 FQEQUENCY,
?PO
700
CH'
Figure 1. Infrared spectra of various disaccharides and their octaacetates in potassium bromide disks, 1.1 to 1.2 mm. thick
508
ANALYTICAL CHEMISTRY
LENGTH,
WAVE
&
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7 100 I
p
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7
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INULOBIOSE
-
-0 1500
1300
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900 CM-'
700
I 500
I300
900
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FREOUENCY.
700
CM"
Figure 2. Infrared spectra of various disaccharides and their octaacetates in potassium bromide disks, 1 . 1 to 1.2 mm. thick
VOL. 30, NO. 4, APRIL 1958
509
WAVE
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Figure 3.
?,OO CM
U K I-
Infrared spectra of sucrose
Considerably greater detail is shown than in the lorver curve, which is t h a t of a freeze-dried sucrose-potassium bromide solution prepared as described above, b u t not heated after drying. The upper curve s h o w detail which is the sanie or better than that of a spectrum of crystalline sucrose from a mineral oil mull. The curves have been vertically displaced t o facilitate comparison. T o investigate the possibility that the differences in spectra are due to formation of Ion--temperature sucrose hydrates in the freeze drying, samples of sucrose-potassium bromide mixtures dried in this way were heated in a vacuum oven. Young and Jones (23) have reported that two sucrose hydrates formed a t freezing temperatures: C12H22011, 2.5 H20, nielt'ing point 45.7"C.; arid C1?H,2011. 3.5 H20,melting point 27.8" C. Both decomposed to anhydrous sucrose a t their melting points. Heating of the dry sucrosepotassium bromide mixture for 4 hours a t 110' C. in a vacuum of 0.7 nini. of mercury produced no change in the spectrum, except a slight' lowering of the water band a t 1630 em.-' Reproducibility. Figure 4 s h o w the spectra of the five preparations of 3-O-cr-~-glucopyranosyl-~-glucose. The curves have been vertically displaced t o facilitate comparison. From top t o bottom the curves shoiv preparation froni sak6boise: nigerose by acid reversion (this laboratory), nigerose by acid reversion (University of Sebraska), nigerose from nigeran, and nigerose from honey. All maxima fall within + 5 cni.-l of the mean value for each band. Four replicate preparations viere made of sucrose octaacetate by the procedure outlined above. Their spectra were superimposahle. Four of the sugar acetate spectra shown here were also published by Isbell and cov,-orkers ( 1 % ) . Their spectra were determined on solutions of recrystallized materials. The spectra resemble those shown in this paper closely, except that minor peaks are
510
700
ANALYTICAL CHEMISTRY
FREOUENCY,
Figure 4.
CM-I
Infrared spectra of five preparations of
3-O-cu-D-glUCOpyranOSyl-D-glUCOSe
niore difficult to compare a t the lower concentrations used by Isbell -e.g., the spectrum of gentiobiose octaacetate shown here differs in showing more structure in the 650- to 1000-cm.-1 region. Thus the infrared spectra of amorphous acetates deposited on potassium bromide resenible solution spectra rather than solid-phase spectra of crystalline materials. The use of the procedure outlined here for the identification of the disaccharides of honey will he described in a forthcoming publication. ACKNOWLEDGMENT
The authors gratefully acknowledge gifts of sakkbiose octaacetate from Kiyoshi Aso, Tohoku University, Sendai, Japan; of nigerose from J. H. Pasur, University of Sebraska; and of isomaltose and leucrose from F. H. Stodola, Sorthern Vtilization Research and Development Division. U.S. Department of Agriculture. The assistance of R . C. Carden, C. S. Fenske, and C. T. Leander in operation of the spectrophotomcter and the niodification of the potassium bromide disk die by R. R. Calhoun is also acknonledgcd. LITERATURE CITED
(1) .2nderson, D. H., Koodall, S . B., A4?;a~. CHEW 25, 1906 (1953).
(2) Bak, E., Christensen, D., S e t a Chena. Scand. 10, 692 (1956). (3) Barker, S. A,, Bourne, E. J.. Neeley, K. B., Whiffen, D. H., Chem. & Ind. (London) 1954, 1418. (4) Barker, S. A., Bourne, E. J , Stacey, XI., Ibad., 1952,756. (5) Barker, S. .4,, Bourne, E. J., Stacey, M., Whiffen. D. H., J . Ch,ern. Soc. 1954,171. ' (6) Barker, S. +., Bourne, E. J., Weigel, H.. Whiffen. D. H.. Chem. & Ind. (Londonj 1956, 318.
(7) Corbett, W.II.,Kenner, J., J . Chenz. Soc. 1954, 1789. (8) Farmer, F'. C., Chem. B Ind. (Lond o n ) 1955.586. French, D.,'J. Am. Chenz. Soc. 77, 1024 (1955). Grendon, H. T., Lovell, H. T., Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 2, 1956. Isbell, H. S., Bur. Standards J . Research 5 , 1185 (1930). Isbell, H . S., Smith, F. .4., Creitz, C., Frush, H. L., IIoyer, J. D., Stewart, J. E., J . Research S a t / . Bur. Standards 59, 41 (1957). Kuhn. L. P.. ANAL.CHEX.22. 276 (1960).
'
(14) Lemieux, R. C., Epp, -A.., Bauer, H. F., Division of Carbohvdrate Chemistry, 128th ?\Ieet,ing,"ACS, IIinneapolis, IIinn., September 1955.
(15) Ilatsuda, K., Hiroshima, G., Shibasaki, K., Aso, K., Tohoku J . A q r . Research 5 , 239 (1954). (16) Pazur, J. H., Budovich, T., J . Am. Chem. Soc. 78, 1885 (1956). (17) Pazur, J. H., Gordon, A. L., Ibid., 75, 3458 (1953). (18) Peat, S., Robert, P. J. P Whelan, IT..J.. Biochem. J . 51. x& 119523. (19) Khiffen: D. H., Chkn. B Ind. (London)1957, 129. (20) Khistler, R. L., House, L. R., .IYAL. CHEM.2 5 , 1463 (1953). (21) . , Khite, J. K . , Jr., Hoban, Ti., unpublished data. (22) JThite, 3. W.,Jr., Ilaher, J., Arch. Biochem. Biophys. 42, 360 (1953). (23) 1-oung, F. E., Jones, F. T., J . Phys. B CoIloid Chenz. 53, 1334 (1949). (24) Tuill, J. L., Chem. B I n d . (London) 1952, 755. '
RECEIVEDfor review July 12, 1957. Accepted Sovember 7 , 1957. The Eastern Regional Research Laboratory is a laboratory of the Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. Mention of trade names does not imply endorsement of the Department of A4griculture over products of a similar nature not named.
