1037
V O L U M E 2 8 , NO. 6, J U N E 1 9 5 6 Table 111. Results of Analyses of Five Rat Liver Homogenates Calciuni 0 0 0 0 0
(Xicroinole per mg. nitrogen) Magnesium 0 300 0 330 0 378 0.296 0 323
046‘3 0349
0387 0414 OFYR
and gives a poor end point, a small amount of standard magnesium solution (0.025 ml.) was added t o each calcium sample. T h e titration was then carried out as for magnesium, and from this titer (expressed in moles) n a s subtracted the moles of magnesium added. This procedure is successful because the chelating agent reacts first Jl-ith calcium, and only then n i t h magnesium. T h u s the end-point change is due t o the chelation of magnesium, n-hich provides a good color change. RESULTS
Trials of t h e above separation and titration procedure on solutions of known concentration indicated recoveries of about 100%
for calcium and 97% for magnesium, with deviations of less than 5%. X typical set of d a t a demonstrating this is shown in Table 11, which gives the micromoles found after ~ e p a r a t i o non , each of four columns, of identical known mixtures of calcium and magnesium. T h e results of analyses done on some samples of normal rat liver homogenates are given in Table 111. Before homogenization, t,hese livers were perfused free of blood with 0.25.1f sucrose solution. l l o r e detailed results on rat liver honiogenates and cell fractions will be presented elsevihere. LITERATURE CITED
(1) Biedermann, W., Schwaraenbach, G , Chimia (Sicztz ) 2 , 56 (1948). ( 2 ) Diehl, H., Goets, C. .1., Hach, C. C . , J . Am. Tl’afer Works Assoc. 42, 40 (1950). RECEIVEDfor review December 16, 1955. Accepted 3Iarcli 12, 19%. Supported b y funds under C o n t r a c t Konr-222((31) between t h e Office of Kava1 Research a n d t h e University of California. R . L. Grisnold was r,9 . Public Health Service postdoctoral research f e l l o w 1952-33.
Detection of Carbohydrates on Paper Chromatograms M. L. WOLFROM and J. B. MILLER D e p a r t m e n t of Chemistry, The O h i o State University, Columbus 70, O h i o
Carbohydrates and related substances are well indicated on developed paper chromatograms by successively applying aqueous sodium metaperiodate, aqueous potassium permanganate, water, and an acid solution of benzidine.
A
S A means of detecting material on paper chromatograms, the joint use of sodium metaperiodate and potassium permanganate is particularly a t t r a d i v e because of its a i d e applicability. Thus, such treatment should detect not only compounds t h a t are easily oxidized by permanganate b u t also compounds whose periodate cleavage products are so oxidized. T h e permangmate-periodate system as a n oxidizing medium for olefins has been investigated in detail by Lemieux and coworkers ( d ) , who have also reported its use in the detection of carbohydrates on paper chromatograms (S). A similar procedure, which has been in use in this laboratory for some time, is somewhat faster and less sensitive t o an arid atmosphere, and t e m of reagents Jl-hich results in a dark blue spot. T h e chromiitograms are permanent for some months and the sensitivity somexvhat exceeds t h a t of t h e Lemieux and Bauer method. EXPEXIAIENTAL
The production of blue spots results from the oxidation of benzidine t o “henzidine blue” by manganese dioxide. T h e benzidine reagent has the i‘olloil-ing composition ( 1 ) : 1 gram of benzidine, 8 grams of trichloroacetic acid, 20 ml. of anhydrous acetic acid, 12 nil. of xvater, and 160 nil. of absolute ethanol. T h e air-dried chromatogram is first sprayed v-ith a 17caqueous solution of sodium netaperiodate. ;ifter 3 t o 4 minutes, t h e chroniatogram is sprayed with a freshly prepared 1%; aqueous solution of potassium permanganate. I n 5 minutes, sites of t h e largest amounts of material viill show as green, yellow, or bro\l-n spote. T h e paper is then washed free of permanganate color with distilled water. This treatment will frequentl?. reveal faint broxvn spot? previouely covered bT- the excess of permanganate solution. h f t e r air-drying, the chromatogram is sprayed ivith the benzidine reagent, Xl-hich instantly converts the bron-n spots t o dark blue ones and frequently reveals spots not previously
seen. T h e background is initially light blue, changing t o n h i t e on drying and then t o b r o a n after about 1 a e e k . This method will detect 0.5 y of mannitol, 2.4 y of glucose, and 7 . 8 y of sucrose, as 5 X 6 m m . oval spot., xl-hen these substances are placed directly on K h a t m a n S o . 1 paper. Distilled water on R h a t m a n KO. 1 paper m a y give a faint blue ring (not a spot). DISCUSSION
The sensitivity, wide applicability, and speed of this method make it attractive. T h e benzidine reagent (I) may be used alone as a supplementary test. As with any aqueous spray reagent, too heavy application will cause the spots to dipperse, thus decreasing the sensitivity. Failure to obtain a white background, indicating the presence of manganese dioxide, n-ill lessen the contrast and hence the sensitivity. T h e source of such manganese dioxide may be: permanganate oxidation of the periodate cleavage products of the paper ( 2 , 5); colloidal manganese dioxide in the permanganate reagent; failure to n-azh the paper thoroughly; or failure t o cover the paper completely Tvitli the periodate spray, thus leaving those uncovered portions open t o rapid permanganate oxidation. Exposing the paper, particularly when wet, to a reducing atmosphere such as one containing hydrogen sulfide, d l destroy the manganese dioxide spot.. ACKNOWLEDGMENT
One of the authors (J. B. 11.)is pleased t o :tcknon-ledge the support of t h e Procter & Ganihle Co. fellomhip held during 1955-1966. LITERhTL-RE CITED
(1) Bacon, J. S. D., Edelnian, J., Biochem. J . ( L o ~ Z o n )48, 114
(1951). Cifonelli, J. d.,Smith, F., ;\XAL. CHEST. 26, 1132 (1964). Lemieux, R . U., Bauer, H. F., I b i d . . 26, 920 (1954). (4) Lemieux, R. U., Rudloff, E. \-on, Can. J . Chem. 33, 1701 (1955). (5) IIetzenberg, R . L., IIitchell. 13. K., J . .4m. C h e m . SOC.76, 4187 (1954). (2) (3)
RECEIVED for revieiv December 1 3 , 1955, Accepted \ l a r c h 8, 1956.
