The spectral characteristics of solutions of eighteen representative monosaccharides in 79 per cent sulfuric acid have been determined for solutions maintained for 2 hours at 25° and 15 minutes at 100°. The use of these data for the identification of ketohexoses and 6-desoxyaldohexoses has been pointed out, as has their application to the quantitative determination of certain monosaccharides when present singly or in admixture with other monosaccharides, either as the monosaccharides or as components of oligosaccharides and polysaccharides

Ikawa, Miyoshi

Niemann, Carl

English

Caltech Authors - Main

A SPECTROPHOTOMETRIC STUDY OF THE BEHAVIOR OF 
CARBOHYDRATES IN SEVENTY-NINE PER 
CENT SULFURIC ACID* 
BY MIYOSHI IKAWA AND CARL NIEMANN 
(From the Gates and Crellin Laboratories of Chemistry,t California Institute of 
Technology, Pasadena) 
(Received for publication, May 10, 1949) 
Carbohydrates per se do not exhibit significant specific absorption in 
the 210 to 400 rnp region (1, 2), but, when exposed to the action of strong 
mineral acids, they are transformed into products which do absorb specifi- 
cally in this region and which will react with a variety of reagents to 
give colored addition products. This latter fact has been utilized in the 
development of calorimetric methods suitable for the identification and 
estimation of mono-, di-, and polysaccharides, as, for example, in the car- 
bazole-sulfuric acid method described by Dische (3, 4). Although a 
considerable amount of information is available with respect to the spectral 
characteristics of the various colored addition products (5-‘7), relatively 
little is known about the absorption spectra of solutions of carbohydrates 
in strong sulfuric acid, the medium used in most of these procedures. 
Bandow (8) determined the absorption spectra of solutions of a number 
of sugars in concentrated sulfuric acid after the solutions had been allowed 
to remain at room temperature for a day and noted that, while the spectra 
were not superimposable, the absorption curves for the hexoses, pentoses, 
and glucuronic acid were similar to that of furfural determined under 
the same conditions. In the course of a systematic study (6) of the vari- 
ables influencing the Dische carbazole method the spectra of solutions 
of glucose, galactose, mannose, fructose, and N-acetylglucosamine in 79 
per cent sulfuric acid were determined, and it was noted (6) that with 
fructose the strong extinction, observed with solutions that had stood at 
25” for 40 minutes, decreased when these solutions were subsequently 
heated at 100” for 15 minutes. In contrast to the above, solutions of the 
aldohexoses exhibited no significant specific absorption when allowed to 
stand at 25” for 40 minutes but did absorb strongly in the 315 rnp region 
after the solutions had been heated at 100” for 15 minutes. 
The above observations suggested the possibility of utilizing the ultra- 
* This investigation was supported in part by a research grant from the Division 
of Research Grants and Fellowships of the National Institut,es of Health, United 
States Public Health Service. 
t Contribution No. 1299. 
923 
924 SPISCTRA OF CARBOHYDRATES 
violet absorption spectra of strong sulfuric a.cid solutions of the saccharides 
for analytical purposes and we have now demonstrated that this pro- 
cedure is a practical one in a number of cases. The desirability of using 
79 per cent sulfuric acid as a medium was suggested by earlier studies 
in these laboratories (6), and it will be noted that sulfuric acid of this 
concentration is still an excellent solvent for practically all polyhydroxy 
compounds (9). 
I I I I I 
I I I ;L 
220 260 300 340 
Wave Length in Millimicrons 
FIG. 1. Spectra of heated solutions of the sldopentoses in 79 per cent sulfuric 
acid. Curve 1,100 y  of sylose; Curve 2, 100 y  of ribose; Curve 3,100 y  of arabinose; 
Curve 4,100 y  of lyxose. 
EXPERIMENTAL 
84 per cent (by weight) sulfuric acid was prepared from reagent grade 
acid. The carbohydrate solutions contained 100 y of anhydrous com- 
pound per ml., account being taken of water of crystallization whenever 
necessary. The glucosamine hydrochloride solution contained 100 y of 
free base per ml. The identity and purity of the individual compounds 
were checked by a determination of their optical rotation and their 
melting points. 
9 ml. portions of 84 per cent sulfuric acid contained in 15 X 150 mm. 
M. IKAWA AND C. NIEMANN 925 
test-tubes were thoroughly chilled in an ice bath. 1 ml. portions of the 
aqueous carbohydrate solutions were then added and the resultant solu- 
tions mixed by stirring with a glass rod, the tubes being immersed in the 
ice bath to avoid a rise in temperature. These solutions, which were 
uniformly 79 per cent in sulfuric acid, were placed in a bath kept at 25” 
for 2 hours or in a boiling water bath for 15 minutes. The spectra of 
these solutions were determined, at room temperature, after cooling in a 
water bath, with a Beckman model DU spectrophotometer equipped with 
Wave Length in Millimicronr 
FIG. 2. Spectra of heated solutions of two 6-desoxyaldohexoses in 79 per cent 
sulfuric acid. Curve 1,100 y  of rhamnose ; Curve 2,100 y  of fucose. 
1 cm. quartz cells. The values reported are averages of two or more 
separate determinations, with a maximum deviation of +5 per cent from 
the mean value. The curves were constructed from readings taken at 
10 rnp intervals except in regions of maxima and minima, where the in- 
terval was reduced to 5 and 2.5 mp. 
The absorption curves for solutions of glucose, galactose, mannose, 
fructose, and N-acetylglucosamine in 79 per cent sulfuric acid, after these 
solutions had been heated at 100” for 15 minutes, have been presented 
earlier (6). The absorption curves for comparable solutions of xylose, 
926 SPECTR-4 OF CARBOHYDRATES 
arabinose, ribose, lyxose, rhamnose, fucose, sorbose, glucosamine, glu- 
curone, and galacturonic acid are given in Figs. 1 to 3, inclusive. 
The salient spectral characteristics of 79 per cent sulfuric acid solutions 
of the carbohydrates investigated are given in Table I. Molecular ex- 
tinction coefficients were calculated from the relation B = l/cd log10 lo/l, 
where c = the final concentration in moles per liter of the solution and 
I I 
220 260 300 340 
Wave Length in Millimicrans 
FIG. 3. Spectra of heated solutions of various sugars in 79 per cent sulfuric acid 
Curve 1, 100 y  of glucurone; Curve 2, 100 y  of galacturonic acid; Curve 3, 100 y  oe 
sorbose; Curve 4,100 y  of glucosamine. 
d = the cell thickness in cm. In Table I values of A,,,. and emax. are 
given for solutions heated at 100’ for 15 minutes; values of ~31~ for solutions 
maintained at 100” for 15 minutes and at 25” for 2 hours, respectively, 
are recorded. Extinction coefficients were determined at 315 rnE.1, because 
practically all of the principal maxima lie in the region of 300 to 325 rnp 
and values obtained at 315 mp appear to be generally useful for compar- 
ative purposes. In addition, fructose solutions maintained at 25”, which 
were of particular interest because of the unusual behavior of the keto- 
hexoses, absorb maximally at this wave-length. 
The 2 hour period selected for observations at 25” was a compromise. 
TABLE I 
Spectral Characteristics of Solutions of Various Carbohydrates in 79 Per Cent Sulfuric 
Acid 
Carbohydrate - 
_ - 
Lax. 
- 
I 
-. 
D-Xylose 13.6 2.7 
n-Arabinose 7.7 0.46 
D-Ribose 8.5 0.82 
D-Lyxose 5.7 1.26 
D-Fucose 8.9 0.98 
n-Rhamnose 9.5 1.00 
n-Giucose 3.7 0.09 
D-Mannose 2.2 0.11 
D-Gala&se 3.6 0.13 
D-Fructose 3.0 11.5 
n-Sorbose 
fw 
309 
254 
307 
243 
307 
242 
299 
237 
327 
250 
327 
266 
308 
250 
305 
249 
300 
250 
313 
255 
299 
243 
294 
294 
14.3 
3.8 
8.3 
3.0 
9.2 
3.5 
6.6 
3.6 
11.8 
4.6 
12.6 
4.8 
4.0 
1.6 
2.3 
2.9 
4.9’ 
2.2 
3.1 
1.4 
5.0 
2.2 
9.3 
9.8 
3.9 10.6 
D-Giucurone 
D-Gaiacturonic acid 
N-Acetyl-D-glucosamine 
D-Giucosamine hydrochloride 245 1.4 
287 1.25 
325 1.75 
5.5 0.02 
5.6 0.17 
0.31 0.0 
1.5 0.04 
D-Mannitoi 0.09 0.0 
meso-Inositol 0.02 0.0 
Adonitol 0.0 0.0 
Sucrose 6.3 12.0 
Maltose 6.9 0.17 
Trehalose 6.9 0.03 
Lactose 7.1 0.24 
Melibiose 7.2 0.31 
RatEnose 9.8 11.6 
Melezitose 10.0 12.8 
Starch 3.2t 0.02t 
Glycogen 3.1t 0.06t 
Inulin 2.6t 9.6t 
I- 
- 
15 min. at 100’ / 2 hrs. at 25’ 
* This value is about 0.8 higher than that calculated from a previous determi- 
nation (6). 
t Calculated by taking the anhydrohexose unit (CJIroOa) as the molecular unit. 
927 
W8 SPECTRA OF CARBOHYDRATES 
A period longer than 2 hours at 25’ was not judged desirable, because 
solutions of the pentoses, notably xylose, began to absorb rather strongly 
and with a period of less than 2 hours the extinction coefficients of the 
solutions of sorbose and the fructose containing oligosaccharides were 
increasing too rapidly to permit the attainment of reproducible results. 
It was noted previously (6) that the extinction coefficients of the bands 
in the 300 to 325 rnp region decreased slowly when the solutions were 
maintained at 100” for prolonged periods. It appears that with a number 
of sugars hax. is attained in approximately 5 minutes heating at loo”, 
decreasing 10 to 15 per cent during the next few minutes of heating. 15 
minutes at 100” appeared to be a reasonable compromise, considering 
convenience, reproducibility, and general applicability. 
TABLE II 
Behavior of Classes of Monosaccharides in 79 Per Cent Sulfuric Acid 
At 25’,315 mp At loo’, 315 m,i 
ClaSS 
----I- -.-- 
e x 10-s LogI IO/P e x lo-~ Loglo IQ/P 
Aldopentoses and 6-Desoxyaldohexoses.. . . . >0.4 >0.025 >5.0 >0.35 
Aldohexuronic acids. . . . co.2 <O.OlO >5.0 >0.25 
Aldohexoses............................... co.2 <O.OlO c4.0 co 25 
Ketohexoses...............................i >lO.O >o .550 c4.0 I X0:25 
Polyols.. . I.. j co.1 ! co.005 co.1 j <0.005 
* Of solutions containing 10 y  of anhydrous compound per ml. of acid solution 
Results 
From the data contained in Table I certain generalizations, summarized 
in Table II, may be made with respect to the behavior of non-nitrogenous 
monosaccharides in 79 per cent sulfuric acid. It is evident that the infor- 
mation contained in Table II will permit one to assign a given non-nitrog- 
enous monosaccharide to a particular class and thus facilitate its ultimate 
characterization either by further spectrophotometric analysis, by means of 
the dat’a contained in Table I, or by paper chromatography (10). 
With binary mixtures of non-nitrogenous monosacchsricles it follows 
from the data given in Table I that those mixtures which contain a keto- 
hexose or a 6-desoxyaldohexose as one component are amenable to analysis 
by the above spectrophotometric procedure. Binary mixtures of two 
members of the same class (Table II) in most cases cannot be analyzed 
satisfactorily, nor can binary mixtures containing only aldopentoses, aldo- 
hexoses, or aldohexuronic acids, though in these cases a value for loglo 
lo/l greater than 0.25 for a solution containing 10 y of solute per ml., 
heated at 100” and observed at 315 rnl.c, will be indicative of the presence 
of an appreciable amount of aldopentose or aldohexuronic acid. 
M. IKAWA -4ND C. SIEMANN 929 
A number of color reactions suitable for distinguishing between aldo- 
ses and ketoses and based upon treatment of the sugar with sulfuric or 
hydrochloric acid and a second reagent have been described (11). In 
view of the fact that adequate spectrophotometers are now generally 
available, it would appear from the present investigation that direct 
spectrophotometric examination of the acid solution wit.hout the addition 
of a second reagent is the more satisfactory procedure, especially for quanti- 
tative observations. 
The applicability of the direct spectrophotometric method to oligosac- 
charides is illustrated by data contained in Table I. It will be noted 
that in general there is gdod agreement between the extinction coefficients 
TABLE III 
Analysis of Mixtures of Galactose and Fucose 
Mixture 
_ .-.--- .......... . -~--- .. 
Galactose ................................. 
Fucosc ................................... 
Galactose ................................. 
Fucose ................................... 
Galactose ................................. 
Fucose ................................... 
Galactose ................................. 
Fucose ................................... 
N-Acetylglucosaminet. ................... 
* Average of a duplicate analysis. 
- 
I 
-- Added 
Y 
54.9 
50.0 
82.4 
25.0 
27.4 
75.0 
54.9 
25.0 
33.7 
i 
-.- 
7 
58 
49 
84.5 
24 
27 
77 
57 
24 
Moles galactose -.-- 
Moles fucose 
Added Found 
-- -- 
1.00 1.08 
3.00 3.21 
0.33 0.32 
2.00 2.16 
__ - 
t The absorption at 300 and 327 mp was corrected for the slight amount due to 
t,he N-acetylglucosamine added. 
of solutions of the oligosaccharides and those expected on the basis of 
the component monosaccharides. The data clearly show that maltose, 
trehalose, lactose, and melibiose contain 2 aldohexose residues; sucrose, 
an aldohexose and a ketohexose residue; and raffinose and melezitose, 1 
ketohexose, and 2 aldohexose residues. 
Whereas the ketohexoses are distinguishable from the other classes of 
sugars listed in Table II because of the large extinction coefficients de- 
veloped in 79 per cent sulfuric acid solutions at 25”, the 6-desoxyaldo- 
hexoses are distinguishable from the other classes because of the position 
of the principal maxima developed in 79 per cent sulfuric acid solutions 
heated at 100”. It will be noted that the principal maxima of heated 
solutions of fucose and rhamnose are located at 327 mp; i.e., at a longer 
wave-length than the maxima of heated solutions of the other types of 
930 SPECTR.4 OF ChRBOHYURhTES 
monosaccharides. Fortunately the extinction coefficients of these maxima 
at 327 rnp are quite large, making it possible to resolve the spectra of 
solutions containing 6-desoxyaldohexose and other types of sugar into a 
“6-desoxyaldohexose peak” and a second peak characteristic of the other 
type of monosaccharide present in the mixture. 
Fucose and rhamnose are known to occur in a number of immunologically 
active polysaccharides (12), and in studies in these laboratories on prepa- 
rations of blood group A-specific substance from hog gastric mucosa it 
has been found that the amount of 6-desoxyaldohexose present in these 
preparations can be determined from the spectra of their solutions in 79 
per cent sulfuric acid after the latter have been heated at 100” for 15 
minutes. The values so obtained for the fucose and galactose content of 
preparations of blood group-A specific substance were in good agreement 
with values noted by other investigators.’ Several representative analyses 
of fucose-galactose and fucose-galactose-N-acetylglucosamine mixtures are 
given in Table III. These values were calculated from extinction values 
found at 300 rnp (galactose) and 327 rnp (fucose) for solutions of the mix- 
tures and of the pure monosaccharides. 
The direct spectrophotometric determination of carbohydrates by the 
above procedure should be used with caution in the investigation of complex 
natural products until interferences arising from non-carbohydrate com- 
ponents can be evaluated. Although it has been found that the method 
is applicable for the estimation of carbohydrates in the presence of signifi- 
cant quantities of representative simple proteins,’ no information is avail- 
able in regard to possible interference by other classes of organic com- 
pounds. 
SUMMARY 
The spectral characteristics of solutions of eighteen representative mono- 
saccharides in 79 per cent sulfuric acid have been determined for solutions 
maintained for 2 hours at 25” and 15 minutes at 100”. The use of these 
data for the identification of ketohexoses and 6-desoxyaldohexoses has 
been pointed out, as has their application to the quantitative determi- 
nation of certain monosaccharides when present singly or in admixture 
with other monosaccharides, either as the monosaccharides or as com- 
ponents of oligosaccharides and polysaccharides. 
BIBLIOGRAPHY 
1. Bednarczyk, W., andMarchlewski, L., Biochem. Z., 300, 42 (1938). 
2. Bosch, W. C., and Anderson, E. W., Phys. Rev., 61, 726 (1942). 
3. Dische, Z., Biochem. Z., 189, 77 (1927). 
1 Unpublished studies of the authors. 
M. IKAWA AND C. NIEMANN 931 
4. Dische, Z., Mikrochemie, 8, 4 (1930). 
5. Sorensen, M., and Haugaard, G., Biochem. Z., 260, 247 (1933); Corn@.-rend. Irav. 
Lab. Carlaberg, 19, No. 12 (1933). 
6. Holzman, G., MacAllister, R. V., and Niemann, C., J. Biol. Chem., 171,27 (1947). 
7. Dische, Z., and Shettles, L. B., J. Biol. Chem., 176, 595 (1948). 
8. Bandow, F., Biochem. Z., 294, 124 (1937). 
9. Hammett, I,. I’., Physical organic chemistry, New York, 266 (1940). 
10. Partridge, S. M., Biochem. J., 42,238 (1943). 
11. Tollens-Elsner, Kurzes Handbuch der Kohlenhydrate, Leipzig, 4th edition, 89- 
91 (1935). 
12. Stacey, M., Advances in Carbohydrate Chem., 2,164 (1946). 


A spectrophotometric study of the behavior of carbohydrates in seventy-nine per cent sulfuric acid

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