Paper Chromatography of Sugar Alcohols and Their Glycosides J. CERBULIS Stephen
F,W h i t m a n & Son,
Inc., Philadelphia 5, Pa.
p-iinisidine is found to be a very valuable reagent for the detection of sugar alcohols and their glycosides on paper chromatography. The filter paper is sprayed with the reagent, and after being heated at 100' C. for 10 to 15 minutes, turns light brown. Sugar alcohols and their glycosides leave white spots. These white spots can be seen after several months and can be distinguished from yellow or brown sugar spots. The heat and reagent do not affect the consistency of filter paper which can be handled and stored without any difficulties. p-Anisidine is a useful reagent of greater sensitivity than ammoniacal silver nitrate for detection of sugar alcohols and their glycosides.
M
.
ANY reagents have been used for the detection of sugars by paper chromatography ($), but only a few reagents have been used for the detection of sugar alcohols. Ammoniacal silver nitrate in different variations has been the most widely used reagent for polyols ( 1 4 , 16). During the last several years boric acid-pheiiolphthaleiii, boric. acid-phenol red ( 6 ) , boric acid-methyl red ( 1 3 ) ) vanillin-perchloric acid ( 5 ) , lead tetraacetat,e ( 4 ) , and alkaline periodatepermanganate ( 7 ) have also heen recommended as reagents for t,he detection of polyols. The author has found p-nnisidine phosphoric acid ( 1 2 ) to be R very valuable reagent for the detection of polyols on paper strips. This reagent', used for 2 years in research on carbohydrates in the cacao bean, has been proved to be somewhat superior to ammoniacal silver nitrate ( 1 7 ) and the procedure is much iimpler. With p-anisidine detection is possible for sugar alcohols, glycosides of sugar alcohols, and reducing and nonreducing sugars in the same run. EXPERIMENTAL
Apparatus and Materials. Chromatographic apparatus consisted of a cylindrical glass jar, 25 em. in diameter and 45 em. in height, Kith a tight-fitting ground-glass cover, which served as the chamber. Filter paper, Whatman S o . I . H. Reeve Angel & Co., Inc., Yew York. Reagent. pAnisidine reagent was prepared by dissolving 0.5 gram of panisidine in 3 ml. of phosphoric acid (sirupy, 85 to 88%). Small amounts of precipitate, if any, were not filt,ered off. The solution was diluted with 100 ml. of 80 volume Yc aqueous methanol, Procedure., The solutions of polyols (about 8yoconcentration) were applied in triplicate as spots ( 5 mm. in diameter) about 2 c-m. apart on a starting line 4 cm. above the edge of the paper sheet. The chromatograms were developed by the ascending method for 24 hours, After the solvent had advanced to about 32 cm., the sheet was dried in air for 1 hour, immersed in a shallow pan containing the p-anisidine reagent, removed, and gently blotted to remove excess reagent. Then the sheet was heated a t 100" C. for approximately 10 to 15 minutes until the paper turned light brown. The sugar alcohols appeared as white spots on a light brown background. Floridoside and umbilicin gave brown spots (Table I). A marked contrast in the color was noted between the spots and the background. Replicate R/ determinations were found to agree within &2%. As a check, sugar alcohols were also determined by ammoniacal silver nitrat,e ( 1 7 ) and vanillin-perchloric acid ( 5). RESULTS AND DISCUSSION
The p-anisidine reagent is recommended for the detection of sugar alcohols and their glycosides on paper chromatography. 1
Present address, 2848 N . Park .ive., Philadelphia 32, Pa
The analytical results are \!WN I I in Table I. The Rj values n ere determined using Whatman S o . 1 filter paper and a mixture of 1-propanol-ethyl acetate-water as the irrigant. The spots 01 almost rill pol~-ols 11ere clearly distinguishable. ~-Volemitol, 1-D-mannitol-monoacetate, and dulcitol gave somewhat weaker spots, which were recognizable. Only n-perseitol gave a very weak spot test, or none a t all, with the 8% concentration used. Paper strips on which p-anisidine reagent was used could be stored very easily and the spots could be distinguished after several months. The sensitivity of ammoniacal silver nitrate reagent W V Ysimilar ~ to the p-anisidine reagent. Umbilicin and 1-D-mannitol monoacetate treated with ammoniacal silver nitrate gave very weak spots. Chromatograms were handled without any difficulties. Vanillin-perchloric acid reagent proved to be as satisfactor! as the other reagents, but the procedure was more complex. According to the recommendations of Godin ( 5 ) ,the strips were heated for a longer period a t 85' C. The disadvantages of this reagent were: The spots faded rapidly and required marking immediately upon removing the strips from heat; and on heating the paper strips became very brittle.
Table 1.
Rj Values of Polyols
Color of Spot with Lit. Average ~ A n i s i d i n e Polyols Cited Rfa Reagent 1-v-hlannitol iiionoacetate (8) 0.63 Whit e Glycerol 0.58 White Erythritol 0.49 White L-Arabitol 0.43 White .Idonit01 (ribitol) 0.40 White Pinitol 0.36 White Floridoaide (a-~-galactopyranosyl-2-glycerol) ( 1 6 ) 0.36 Brown Gmbilicin (3-~-arabitol-~-D-galactofuranoside) ( 2 1 ) 0,35 Brown D-Mannitol 0.34 White White D-Sorbitol (D-glucitol) 0.31 Dulcitol ,(galactitol) 0.31 White o-Volemitol 0.28 White D-Perseitol 0.25 N o spot 3-Floridoside-a-v-mannoside !9) 0 . 1 8 White I-D-Rlannitol-P-D-glucopyranoside f8) 0.15 White White Inositol 0.11 1- and 7-D-l'olemitol-P-D-glucoppranoside'~ ,:lo) 0.11 White 1,F-D-Mannitol-di- (8-D-glucopyranoside) 0.05 White Oalactinol(0-a-salactopyranosyl-myo-inositolj 0.03 White ' Values represent average of triplicate determinations on Whatman No. 1 paper by ascending method run in solvent system of I-propanol-ethyl acetate-water (7:1:2, vol. %) ( 1 ) a t 20-21' C . h Mixture of both substances substances. -
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\CKNOW LEDGMEYT
The author wishes to express his appreciation to C. Clay for .t~mulating comments and suggestions and to T. H. Sharp for his interest in this Fork. He also wishes to thank the following for generous supply of various compounds: Pinitol o-Perseitol Galart in01 Floridosidr, Umbilicin o-Voiemitol I-D-llannitol monoacetate '3-Floridoside-a-uniannoside l-D-hIannitol-@-Dglucopyranoside 1- and 7-D-VolemitolP-D-glycopyranosidr 1,B-D-Mannitol-di(8-D-alucopyranoside)
1400
.i.B. Anderson. Forest Products Laboratory, Uni-
versity of California, Berkeley, Calif. .A. A. Benson. Radiation Laboratory, University of California, Berkeley 4, Calif. R . J. Brown and R. F. Serro, The Great Western Sugar Co., Denver, Colo. 13. Lindberp. The Royal Institute of Technology, Division of Organic Chemistry. Stockholm 70. qveden
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L U M E 27, NO. 9, S E P T E M B E R 1 9 5 5 LITERATURE CITED
dlbon, N., and Gross, D., A n u l y s t , 77, 410 (1952). Block, R. J., Le Strange, R., and Zweig, G., "Paper Chroni:rtography," pp. 78-91, Academic. Kew York. 1952. 13rown, R. J., and Serro. R . F.,.T. Am. Chern. Soc.. 75. 1040 (1953). Buchanan, J. G., Dekker. C. A,. and Long, A. G.. .J. L'hein. Soc.. 1950, p. 3162. Godin, P., 4ature. 174, 184 (1954). Hockenhull, D. J. D., Ibid.. 171, 952 (1953). Lemieux, R. U., and Rauer. H. F., ANAL. CHEM..26, 920 (1954). Lindberg, B.. Acta C'hem. Scnird.. 7, 1119 (1953). Ibid., 8, 869 (1954).
Lindberg. B., and Paju, J.. I b i d . , 8 , 517 (1954). Lindberg, B., lfisiorny, A.. and Wachtmeister, C. A., Ibid., 7, 591 (1953). hlukherjee, S., an2 Srivastal-a, H. C., S a t u r e , 169,330 (1952). Parikh. 3 . S . , Parikh, J. AI., and Godbole. d. PI:.. Current Sci. (India),23, 53 (1954). Partridge, 3. 31.,Biocheni. J . , 42, 238 (1948). Putman, E. W., and Hmsirl. T T . Z.. J . -47% Chern. Soc., 76, 2221 11954). Trevelyan, W.E., Procter, D. P., and Harrison, J. S., Nature. 166, 444 (1950). Williams. K. T., and Hevenue. -4.. .J. Sssoc. Ofic.Agr. Cheinists, 36, 969 (1953). K E C E I V F Dfor review February 4. 1955.
Accepted M a y 3, 1955
Colorimetric Determination of l o w Concentrations of Dissolved Oxygen in Water L. S. BUCHOFF', N. M. INGBERZ, end J. H. BRADYI Chemical Engineering Laboratory,
u. s. N. Engineering Experiment
Station, Annapolis,
Md.
The existing need for simplicity aud sensitivity to lo\r conr,entration suggested a colorimetric method. This paper rei-ounts a successful search for, and development of, such a method. h search of the literature showed that several previous illvestigators have explored this field. Procedures based on coloI developed Jvith the iodine or chlorine released in a modified Rinkler procedure were tried hut abandoned, because of the manipulations involved (6). Records of several reagents which react directly with dissolved oxygen to yield colors were found. and four of these colors were surveyed. A4midole ( 2 ) ) ndurol (9), pyrogallol (8), and indigo carmine (4)produced colors whirti :&rerelated to dissolved-oxygen roncent'rat'ion. Trail confirmed the findings of previous investigators that all of these reagent,,, mere unstable, the developed colors were inconsistent, or the test v a s insensitive. Despite t8his negative information, furt,hrr investigation was devoted to indigo carmine, because of th(*wid(, EED water for high-pressure boilers must be fret, w s ( ~ i i - range of colors which it develops. Upon oxidat,ion t,he bright yellon--green color of reduced indigo carmine change8 to orange. tially of dissolved oxygen, ivhich causes pitting of the steel to red. to purple, to blue, and finally to blue-green in the conisurfaces. Mechanical deaeration can reduce the dissolvedpletely oxidized form. Three prohlems needed to be solved t,o oxygen content of feed miter to the range of approximately 0.01 provide a usable indigo carmine method: development of :m p.p.m. (10 p.p.b.). Chemical reducing agents, notably sodium air-etahle, reduced, indigo carmine reagent: development of it sulfite, are employed for .scavenging the last traces of dissolvrd procedure by which a measured amount of reagent could bt. oxygen. ils insurance t,hat these mechanical and chemicd pro?ewes are effective, boiler operators ncled a method for a~ccunit,t~ added to a representative, segregated water sample; and calibration of developed colors arid provision of' color standards for ?omdetermination of dissolved-oxygen conwntration of 20 p.p.11.or parator determinations. less. Methods currently employed ior the detctmiination of dissolved DEVELOPMENT O F INDIGO C i R M I N E REAGENT oxygen are based upon t,hat of Winkler. The so-called "straight Efimoff ( 4 ) employed :i 0.1% solution of indigo carmine in Winkler" is insensitive to dissolved oxygen in conwntratiow water to \\-hich was added 1%of glucose as reducing agent and 1% below 20 p,p,b., and is suhjrct t o interference by eithei, oxidizing of potassium carbonat,e as pH controller. This was stored under or reducing materials which m:~y tw present i n thr s:tmplr. mineral oil to prevent atmospheric oxidation. This harrier is Modifications developed by Yrhwartz arid Gurntly ( f i ) , :inti t)). Adams, Rarnett, and Keller ( 1 ) . offclr p i ' ion and :tccui'ac.y ~ i ' not completely effective, and pipetting the reagent with a protcetive slug of oil above and b ~ l o wit in the pipet is too coinplithe order of 2 p.p.b. Both have 1)t:en used widely for ieferer \vorli. mted for boiler-plant operators. Potassium hydroxide was SUI)that of Adams et al. having hcen adopted as the referee mc.thoct of the -4merican Society for Testing lluterials. Ho~vcvci, 110th of stituted for potassium carbonate in the reducing solution. In :an attempt to sharpen the colors. :i one to one mixture of glycerol these methods are cumberxmr :tnd dcmanding. They requircx and m t e r was substituted for n--stc:I,as solvent for the reagent. duplex sampling in Fpecial glassware, skilled manipulation i i i This not on13 brightened the colors, but provided also for morv sampling and titration. and the use of a comparatively expensivct rapid initial reduction of indigo r::trmine and a more air-stxhle c.1ectrometric titrator. Neit,her is i d i d for frequent cwntioi iwtgent solution. malyeis by povrer-plant operators. ' r .
I here is a need for a less coniplex and more accurate method for determining dissolved oxygen concentrations in the 0 to 50 parts per billion range. 4 colorimetric method has been developed employing reduced indigo carmine. The oxygen is estimated either hl comparing by eye with artificial color standards, or with a spectrophotometer. Oxygen in concentrations of 0 to 25 p.p.b. can be determined by eye with an average deviation of 2 p.p.b. The procedure adopted is free of the difficulties of air entrapment. reagent blank, and complicated sampling. I t can be used b> inexperienced boiler plant personnel to detwminr dissolved oxygen accurately in boiler feed water. o r in precise laboratory determination.
F
1 2
Present address, Westinghouse Electric Gorp., Pittsburgh, Pn Present address, Army Chemical Center. F:deewnod, I I d . Present address. United States r \ r n i y .
STABILITY OF SOLUTIONS
The air stability of the stock indigo carmine solution was evaluated by storage tests. Typical stock solutions were preparrd
