Color Test for Identification of Glucose - Analytical Chemistry (ACS

Anal. Chem. , 1947, 19 (12), pp 1032–1035. DOI: 10.1021/ac60012a028. Publication Date: December 1947. ACS Legacy Archive. Note: In lieu of an abstra...
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V O L U M E 19, NO. 1 2

1032 I1 received a potassium hydroxide treatment during removal of hemicelluloses-1, a sodium chlorite treatment during delignification of the wood residue, and another potassium hydroxide treatment for the extraction of hemicelluloses-2. Materials removed by the last of these treatments were 12.57& but only 3.5% were recovered. This indicates that some hemicelluloses and considerable a-cellulose Tvere degraded to the extent of being unrecoverable from the dilute potassium hydroxide solution by the procedure employed. O'Dwyer's method (?) is similar to the one proposed in this article in that a single extraction with alkali was used. Her method was applied t o the maple wood used in the present research and a yield of 5 . 9 5 of oven-dry, lignin-free, ash-free hemicelluloses was obtained in comparison \Tith 14.6% by the mcthod proposed in this paper. T h e method described in this paper appears suitable for the extraction and precipitation of hemicelluloses equivalervt to approximately 14.5c7, of maple wood. These yields are higher than those obtained directly from wood by other single-eutraction methods commonly used. The procedure can be carried out in a single )Torking day, which is less time than that required for the methods commonly used directly on wood. SUMMARY

A method employing treatments with concentrated and dilute potassium hydroxide solutions is described for the preparation of heniicelluloses directly from hardwoods.

Yields based on the ash-free, lignin-free, oven-dry hemicellu-

loses obtained are approximately 14.570 of the weight of ovendry sugar maple Tvood. These yields are higher than those obtained directly from wood by previously published singleextraction methods. The time required for isolating hemicelluloses directly from wood has been shortened, LITERATURE CITED (1) Anderson, Ernest, J . B i d . Chem., 112,531 (1935-6). (2) Anderson, Ernest, Seeley, Millard, Stewart, W.T., Redd, J. C . , and Westerbeke, Don, Ibid, 135,189 (1940). (3) Anderson, Ernest, Seigle, L. W.,Kranarich, P. TV., Richards, L., and Martiny, W. TV., Ihid., 121,167 (1937). (4) Clark, E. P., J . Assoc. Oficial Agr. Chem., 15,136 (1932). ( 5 ) Forest Products Laboratory, Madison, Wis., Rept. R-19 (revised 1939). (6) Kurth, E . F . , and Ritter, G. J., J . Am. Chem. Soc., 56, 2720 (1934). (7) O'Dwyer, If.H., Biochcm. J . , 17, 501 (1923). (8) Ihid., 20, 656 (1926). (9) Preece, I. A . , Ihid..3 4 , 2 5 1 (1940). (10) Sands, Lila, and Nutter, Pauline, J . Biol. Chem., 110,17 (1935). (11) Thomas, B . B . , P a p e r l n d . a n d p a p e r W o r l d , 27,374 (1945). (12) Wheeler, H . , and Tollens, B., Ann., 254, 304 (1889); Ber., 22, 1046 (18S9). (13) Whistler, R. L., Martin, 8.R., and Harris. XI., J . Research Natl. Bur. Standards, 24,13 (1940). (14) Wise, L. E., Murphy, M.,and D'Addieco, A . A , Paper Trade J., 122 (2),35 (1946).

RECEIVED February 10, 1917. Presented before the Division of Cellulose SOCIETY, Chemistry at the 111th Meeting of the i i J I E R I c . 4 S CHEJIICAL Atlantic City, S . J .

Color Test for Identification of Glucose SZILOIIO HESTRIN' AND JACOB IIAGER Hormone Research Laboratory and Department of Hygiene and Bacteriology, Hebrew Cnirersity, Jerusalem

4 color test is described whereby glucose can be detected in a small sample to the exclusion of many other sugars, among them the aldohexoses, galactose and mannose. The test is based on the fact that gIucose, unlike the other sugars examined, forms no color w-hen heated only with phosphoric acid under the conditions described but forms a characteristic lilac color when pyrocatechol is subsequently added to the reaction system. The test

I

T H.16 been demonstrated repeatedly that although given

sugars may differ only in configuration they can still be distinguished, in suitable instances, by color teats which are similar in principle to the LIolisch reaction (1, 2, 3, 6 ) . .ittention has been directed to the finding that some aldohexoses (glucose, galactose, and mannose'i form different colors \Then heated in phosphoric acid n i i h pyrocatechol (4). Phosphoric acid as a medium of carbohydrate color reactions has some noteworthy merits: it is more convenient t o handle than sulfuric acid; it does not produce appreciable heat on admixture with the test sample even if some water is present; it does not char or otherwise discolor amino acids even at 100' C. I n view of these merits, further study of the color reactions which carbohydrates undergo in the presence of phosphoric acid seems of intercst. The present communication reports a color test based on the use of a pyrocatechol-phosphoric acid reagent whereby the sugar unit of a carbohydrate can be recognized as glucose to the exclusion of some other sugars. Under the conditions of the test, glucose (free or bound) remains colorless in phosphoric acid 1 Present address, Department of Biological Chemistry, School of Medicine, Washington University, St. Louis, Mo.

is inapplicable if other sugars are present. Nitrites and tryptophane interfere. The test is \alia in the presence of other aniino acids as well as certain proteins (gelatin and edestin). Purines and polyhydric alcohols do not interfere. 4 numerical method is proposed for distinguishing between the colors produced with p?rocatechol b? glucose, galactose, and mannose. Examples are gi\en of application to oligosaccharides and polyaldohexosides. wlution. but reacts with pyrocatechol to form a characteristic lilac color. EXPERIMEKTAL

Materials. Sugars, pure crystalline (British Drug Houses); amino acids, pilre crystallinc preparations (various sources); orthophosphoric arid, c . P . , specific gravit,y 1.75 (Hopkins and Killiams); polysaccharides and proteins. from various sources, indicated \There known; mannan, prepared from carob bean gum by partial hydrolysis ( 5 ) ; pyrocatechol, crystalline resublimed preparation (Merck). Procedure. Powder the sample and add an amount which may be expected to contain 0.2 to 1.0 mg. of carbohydrate t o a dry test tube, preferably one equipped with a ground-glass stopper. Add 1 ml. of 85% orthophosphoric acid. (If sample is availablc om!y as an aqueous solution, the test can be carried out by adding 0.05 ml. of sugar solution to 1.0 ml. of 90% phosphoric acid.) Heat in a boiling water bath for 15 minutes, shaking after 1 minute t o hasten solut'ion. Observe whether color has formed. If none has formed, heat for another 15 minutes and again examine. If no color has formed, add t o t.he warmed solution 4 ml. of a freshly prepared solution of 0.2% pyrocatechol in 85% hosphoric acid. Heat the mixt'ure for bserve the color and compare with that another 15 minutes. of a control run simultaneously on glucose.

8

D E C E M B E R 1947 Table I.

Color Reaction of Aldohexoses with Pyrocatechol

Carbohydrate None Glurose Jlaltose Trehalose Starch Cellulose b Galactose Mannose Mannan Glucosamine Glucose Galactose Glucose 1- galactose, 1 : l Lactose.

Expt. a

h

1033

Carbohydrate fsaoa Added, Alp. (1.0 Cm.) ... 0.00 1.0 0.70 1.0 0.54 1.0 0.47 1.0 0.67 1.0 0.56 0.8 0.49 0.41 0.5 0.5 0.40 3.0 0.02 1 2 1 08 1.2 1.28 1.2 1.2

fsda/‘sro

...

0; 50 0.50 0.47 0.49 0.52 0.82 1,09 1.10

...

0.46 0 . 76

0.94 1 00

0.60 0.69 0.49 2.; 1.82 c Glucose 1.70 0.46 2.7 Cellulose h 1 74 0.43 2.7 Glycogenc 0.77 2.5 2.04 Galactose a Values should be regarded as approximation in x-ien. of weighinaerror ( e 0 . 3 mg.). b Cellophane. C Prepared f r o m rabbit heart.

.

Color Colorless Lilac Lilac Lilac Lilac Lilac Red-bron-n Brown Brown Colorless Lilac Red-brown Red-lilac Red-tiiac Lilac Lilac Lilac Red-brown considerable

DISCUSSIOR

Color Formation by Various Carbohydrates on Heating in Phosphoric Acid. The tests were made in accordance with the method which has been detailed. The findings map be summarized as follons: Glucose, glucosamine, diglucoses (maltose, trehalose 1, polyglucoses (starch, glycogen, cellulose), and glucuroziic acid: no color formation on heating for 15 or 30 minutes in phosphoric acid. b. Mannose, galactose, pentoses (xylose, arabinose, lyxose, ribose), methyl pentose (rhamnose), ketoses (sorbose, fructose;, oligosaccharides containing a nonglucose sugar unit (sucrose, lactose, melibiose, raffinose I , and polysaccharides containing a nonglucose unit (inulin, mannan 1 : greenish-yellon to amber brown color on heating for 15 minutes in phosphoric acid. (Galactose, lactose, and melibiose give a relatively weak color. If galactose is present in low amounts the weak amber color reaction produced in phosphoric acid alone map be difficult t o discern. H o m v e r . t,he red color formed with pyrocatechol distinguishes galactose from glucosr.) a.

Table 11.

Color Reaction of Sugars with Pyrocatechola

(Direct method) Concentration of Sugar. %o Espt. Sugar JIg.,’lIl. 1.26 0 7 a Glucose 0.92 0.8 Cellulose 1.02 0.8 Glycogen 0.99 0.8 Starch 1.44 0 3 Mannose 1.05 0.4 Mannan b Glucose 0 7 1.69 u-Phenyl gluooside 1.2 1.63 ~ J I e t h y glucoside l 0.c 1.26 n-Glucose-1-phoaphate (dipotaisium salt) 1.5 1.54 Trehalose 0.8 0.77 0.8 1.65 Cellobiose Maltose 0.8 1.57 0.22 c Glucose lJ20 0.20 Galactose 0.14 0.50 Lactose 0 36 0.39 Melibiose 0.36 0.17 1.06 d Ribose 0.17 1.85 d - X y 1ose 0.2 1.01 d-ilrabinose 0.93 1-Arabinose 0 2 Lyxose 0.2 ... 0 2 0 25 e Gllicuronic acid 0.2 1.15 Rhamnose f Sorbose 0.03 0.58 Fructose 0.03 0.58 a See footnotes t o Table I.

.

1.o 09

n.8

07 0.6

G

The absence of color in phosphoric acid shows that none of the sugars in group b is present. Dehn, Jackson, and Ballard ( I ) have reported on the behavior of carbohydrates on heating in various mineral acids. Under the conditions of their xvork they found formation of color with certain diglucoses and polyglucoses in hot phosphoric acid and no formation of color with others. Undnr the conditions used by the present authors the behavior of the different di- and polyglucoses was uniform. Dehn et al. do not state the carbohydrate concentration m-hich n-as employed by them for their tests. It can be shoirn that if the amount of carbohydrate added is sufficiently large (more than 2 mg. per ml.) the different glucoses (glucose, maltose, glycogen, starch, and cellulose) form a visible pinkish color on heating in phosphoric acid for 30 minutes. I n Figure 1 relative amounts of color formed in phosphoric acid by the three common aldohexoses are shoJvn as a function of the sugar concentration. There is a particularly large difference in color-generating activity betneen glucose and mannose. The extinction-concentration curves are all straight lines. I n a mixture of glucose and mannose in a proportion of 1 to 1 the amount of color produced is n-ithin a limit of 3% the amount of color formed hy the mannose constituent alone. Further study of this reaction n-ith a view to its application to the determination of mannose in pr(’serice of glucose seems therefore worthn-hile.

%Iz/%o

0.56 0.59 0.57 0.57 1.17 1.20 0.53 0.57 0.52 0 51 0.55 0.51 0.50 0.5.i

0.05

0.74 0.77 0.80 0.69 0.76 0.81

.

,

,

0.92 0.75 0.55 0.67

Color Lilac Lilac Lilac Lilac Brown Brown Lilac Lilac Lilac Lilac Lilac Lilac Lilac Lilac Red-brown Lilac-red Lilac-red Red Lilac-red Red Red Red Red Red-brown Lilac Lilac-red

0.5 0.4

/

/

/



03 0.2 01 2

Y

0

5

Figure 1.

10

15 20 25 30 35 hlg. of Sugar per 5 111. of .4cid

3

40

4

45

Production of Color by Aldohevoses in Phosphoric Acid Heating time, 15 minutea ’3. Mannone X . Galactose 0 . Glucose

50

Color Formation by Reaction of Aldehexoses with Pyrocatechol* Attention has been directed to the usefulness of the pyrocatechol color ieaction in the differentiatlon of glucose, galactose, and mannose ( 4 ) . The nature of t h r colors produced by this group has been studied in greater detail. Transmission was measured n i t h the help of a Pulfrich stufenphotometer (?wept where otheiwise specified) on a solution layer of 1.0 mi. I n the experiments summarized by Figures 2 and 3 and Table TI the sugars were heated Tvith a 0.2% solution of pvrocdtechol in phosphoric acid directly-i.e., without pretreatment in the same acid alone. I n the experiments shown in Table I the standard two-step procedure was applied. The data bring out clearlv the different transmission properties of the products formed by the different sldohexoses. A characteristic feature of the glucose product is its marked ab500 mp (filter S50). T h e sorption peak in the region of ratio e s r s / e s l o is sufficiently different in the cases of the three

V O L U M E 19, NO. 1 2

1034

The method here proposed is valid as a means of identifying glucose only if no other colorInterferance 1n forming substance is present in Amount of Formation of Color Reaction of Added I n Hap01 Glucose the test material. Common Substance, InHnPO4 and with PyroDescription of .4dded Added Substance Substance Mg. alone glucose catechol pentoses (d- and I-arabinose, ribose, xylose, lyxose, and rhamiGelatin Eastman, purified 50 n n n Albumin Armour, bovine plasma, nose) when heated in the pyrocrystalline 50 Pink Pink i n n catechol reagent produce colors Edestin 3 times recrystallized 50 !' a-Lactodobulin Electrouhoretically Dure 50 n Brown which are intermediate in tone Mixture used by (5)-with SUPAmino i c i d plement of methionine, no between the red-brown given n n n tryptophane 50 Yellow i(red) Tryptophane ...... .......... 0 5 g by galactose and the lilac given n n n Glucosamine Recrystallized hydrochloride 3 by glucose (see Table 11). Purine Uracil, adenine uric acid, xanthine, hbpoxanthine, Moreover, two ketoses (fructose n n guanine 2 n Polyhydric alcohol Sorbitol, mannitol, adonitol, and sorbose) and one pentose n n n inositol 1 (xylose) yield colors which are n n n F a t t y acid Palmitic stearic 2 n n n Salt NaCl. a k m o n i u m sulfate 10 not certainly distinguishable in Brown Brown Sitrite NaNOt 1 1 terms of e843/cs10 from the color n = no formation of color, or does not interfere in color terrt. i = interferes. given by glucose (cf. Figure 2 g = green fluorescence. and Table 11). It is particularly fortunate that the presence of nonglucose sugars (hexoses and pentoses) is revealed by their color in the first step of the standard conimon aldohexoses (glucose, galactose, and mannose) to afford procedur'e-Le., on heating in phosphoric acid alone. Specific a clear-cut differentiation between them. The ratio, while sensitesting for presence of these sugar interfcrants is therefore untive t o differences of configuration within the sugar unit, is indenecessary. Glucuronic acid on heating with pyrocatechol forms pendent of the nature of the interglucosidic linkage, the polya red-brown color differcnt from that given by glucose. Like the merization degree of the sugar unit, and the carbohydrate conlatter, however, it does not form color on heating with phosphoric centration. While it was difficult to obtain good reproduction of acid alone. I t is desirable therefore to demonstrate absence of the absolute amount of colot formed by a given sugar, the ratio glucuronic acid from the test material by an appropriate independesr3/cssoremainedconstant within *7y0 (see Tables I and 11) with ent method. any given procedure. An identification of glucose on the basis of behavior in the 1.30 pyrocatechol-phosphoric acid test is necessarily of a presumptive nature. I t is always possible that units which are different from 1.20 glucose but behave' in the test in a manner similar t o glucose can be present in a biological material. Influence of Noncarbohydrate Substances on the Color Reac1.10 tion. Observations concerning the effect of certain groups of noncarbohydrates on the color reaction have been assembled in 1.00 Table 111. It is noteworthy that both tryptophane and nitrite interfere with the test. The absence of interfering color forma0.90 tion in the presence of any amino acid, n i t h the exception of

Table 111. Effect of Noncarbohydrate Substances on Color Reaction

0.80 1.2

0.70

1.1 1.0

0.60 .9

0.50

8

3VI .7

.

0.40

W

0.30

.G .5 .4

0.20

.3 .2

0.10

.I 380

400

Figure 2.

420

440

460 A , mp

480

500

520

540

560

Extinction values meaaured with Beckman spectrophotometer Heating time, 15 minutes Aldohexose concentration, 0.4 mg. per ml. 0 . Mannose Ketohexose concentration 0.04 me. Der ml.

%:

fi:

0

Colors Formed by Reaction of Hexoses with Pyrocatechol

~~~~~~

1

MG. SUGAR

Figure 3.

PER

ML.

Reactions of Aldohexoses with Pyrocatechol Heating time, 15 minutes 0 . Mannose X. Galactose 0 . Glucose

D E C E M B E R 1947

1035

tryptophane, is an important merit of the present method. It can be expected, therefore, that a tryptophane-free protein such as gelatin will not be a n interferant, and this prediction is in fact confirmed. I t is evident, furthermore, t h a t the reaction can also be applied safely in the presence of certain proteins which contain tryptophane-e.g., edestin. Yet clearly caution is indicated where the test is being applied on a n unknown protein material in which tryptophane can be demonstrated. As may be seen from Table 111, both plasma albumin and p-lactoglobulin, interfere with the glucose test.

LITERATURE CITED

Dehn, W., Jackson, K., and Ballard, D., 1x0. EXG.CHEII., SSAL. ED.,4, 414 (1932). Dische, Z., Milcrochemie, 8 , 4 (1930); 7, 33 (1929). Gurin, S., and Hood, D., J.Bid. Chem., 1 3 1 , 2 1 1 (1939). Hestrin, S., and Mager, J., Sature, 158, 95 (1946). Len-, B., and Gortner, R., Arch. Biochem., 1 , 3 2 5 (1943). Sorensen, M.,and Haugaard, G., Biochem. Z.,260, 247 (1933). RECEIVED October 1,1846.

Systematic Qualitative Tests for Certain Acidic Elements in Organic Compounds EDWARD L. BENNETT, CLARK W. GOULD, J R . ~ ,ERNEST H. SWIFT, AND CARL NIERIANB Gates and Crellin Laboratories of Chemistry, California Institute of'Technology, Pasadena, Calif. A system for the detection of nitrogen, chlorine, bromine, iodine, arsenic, sulfur, and phosphorus in a single 1-mg. sample of an organic compound and for carbon and fluorine in separate 1-mg. samples is described. The procedures are applicable to compounds whose boiling points are greater than approximatel>60" C. and any of the above elements can be detected w-hen present to the extent of 1 to 5 % of the sample weight.

0

S E of the most sensitive and reliable qualitative tests for

.

the presence of nitrogen in organic compounds is the modified Emich test ( 1 , 4, 5 ) described by Johns (3). I n this test the sample is pyrolyzed in the presence of calcium oside and zinc and the liberated ammonia is detected with the aid of litmus. Because of the general applicability of this test it appeared deQirableto exploit the pyrolytic technique, so that a single ignition could be used not only for the detection of nitrogen but albo for the systematic qualitative identification of certain other acidic elements. Early in 1944 a system was developed in these laboratories which provided for the qualitative detection of nitrogen, chlorine, bromine, iodine, sulfur, phosphorus, and arsenic; a n outline of this system is shown in Table I. SYSTEMATIC TESTS

A. Combustion of Sample and Test for Nitrogen. A combustion tube is prepared from a freshly cleaned 12-em. length of Pyrex tubing 3 mm. in outside diameter by constricting a 2- to 3-mm. portion of the tube to an inside diameter of 0.5 to 0.7 mm. a t a distance of 3 cm. from one end of the tube. Acid-washed and freshly ignited asbestos is introduced into the long arm of the combustion tube and is pressed firmly into the near side of the constriction with the aid of a clean glass rod until a plug 2 to 3 mm. in length is obtained. 1

Present address, General Aniline & Film Corp., Easton, P a .

Table I.

A misture of equal parts of calcium oside, ordinarily prepared by the ignition of calcium oxalate, and SO-mesh zinc powder (sulfur- and arsenic-free) is introduced in small portions, and with tapping, into the long arm of the combustion tube until a 20-mm. column of the misture is in position immediately adjacent to the asbestos plug. The liquid or solid sample contained in a 2- to 3-mm. segment of a 0.5-mm. inside diameter thin-walled capillary tube 10 to 15 mm. in lsngth is placed on top of the calcium oside-zinc column and the end of the long arm of the combustion tube is sealed with the aid of a forceps. The open end of the combustion tube is inserted into a 4-cni. length of 5-mm. outside diameter Pyres t,ubing for a distance of approsimately 1 cm. and in the other end of the sleeve is placed a strip of red litmus paper approsimately 3 mm. in width. Since ordinary red litmus paper map not be sufficiently sensitive, strips of ordinary blue or neutral litmus paper are suspended in distilled water and just enough 1 1: perchloric acid is added t o change the color of the paper to pink. The papers are then washed with distilled water until a treated paper when pressed against a piece of neutral litmus paper will not turn the latter red. The prepared papers are pressed between soft filter papers and then stored in a moist condition in a sealed cont,ainer. The combustion tube assembly prepared as described above is placed on a wire gauze bearing a 10 X 30 mm. hole, in such a way that, the portion of the tube bearing the calcium oxide-zinc mixture is over the hole. The asbestos plug in the combustion tube is first heated to a dull red glow with a small sharp flame from a Bunsen burner, and the burner is moved toward t,he sealed end of the tube so as nest to heat the calcium oxide-zinc zone to glowing and, finally, the space bearing the sample. This heating operation need not consume more than 2 minutes. The presenre

Systematic Detection of Nitrogen, Halogen, .Arsenic, Sulfur, and Phosphorus in Organic Compounds

A. Fuse 1-mg. sample with Zn and CaO. Test gas with litmus Gas: N H I (blue litmus color: nitrogen present) Residue: CaIz, C,aBn, CaCIz, CastPo&, CaO, ZnIz, ZnBrz, ZnC11, ZnS, Zn~Asr,Zna(P03r, Zn B. Leach residue with water Solution: C a + +, I -, Br-, Cl-. Treat separate portions as indicated in B-1, B-2, and B-3 R s i d u e : Car(P04)1, CaO, ZnO, ZnS, ZniAst. Zna(PO,):,

zn

.4dd A g S O HSOa

B-1.

and

AgI, AgBr, AgCl (halogen present)

Ppt.:

8 - 2 . Add chloramineT, fluorescein. and acetic acid

B-3. Add starch, HCzH302, and KaXOz

P i n k color: eosin, tetraiodofluorescein (bromine and/or iodine present)

Starch-iodine color (iodine present)

C. Add HCIO, Gases: HIS, AsHa C-1 Collect gases separately in HgCh and P b (CzH302)~ Ppt.: P b S (black. sulfur present), HgAsHxCl (yellow, arsenic present)

S o h tion : HaPO4. Z n + + , C a + + , HCIO, D. Add portion to (NH4)11LloO4 and HNOa on test paper. Add benzidine and NaCzHaOz Blue color: phosphorua present