AIDS FOR THE ANALYST Inexpensive Glass Chamber for One- and TwoDimensional Ascending Paper Chromatography Thomas Miwa' and Harry Zeitlin, Chemistry Departmeni, The university of Hawaii, Honolulu, Hawaii.
glass chaiiiliers for ascending kinper chroI-matographyplate may be constructed from niaterials readi1.v availVEXPESSII-E
able in the laborator!-. T h e total time required for the coinpletion of six chaiiihei,s was several days; the last chamber was completed in leas than an hour. Each chamber coat S2.80, about oue tenth the cost o f a coiniiiercial chamber purchased previouslj,. T h e homemade chanibers, the dimensions of which are 12 X 123Is X 24 inches, h a r e stood up n-ell in a year and i~ half of use and have several advantages over the relatively eY]ien4\-e commercial cylintler~. .-I$;JE.\IBLY OF CH.41IBER
The following materials are required for a single chamber. Five single-weight nindow panes (12 x 24 X 3/]6 inch), l M 3 , ' * inches of 1-inch white adhesive tape, '2 ounce of Seal All fastdrying plastic cement (-4lIen Products Corp., Detroit 34, Nich,), and 481/4 inches of 111 State ruhber sponge weather stripping 3 ib X inch). ,t
permitted t o overh:ing I ,'& inch on all sides. --in>-exposeti cement is covered n i t h fast-drJ-ing plastic cement to jirovitte to1eranr.e in the fitting. T h e solvent niistures n-ere placed i n 10-inch borosilicate glase pie plates which were set on t h e bottom o!' the chnmbers. T h e ,y the use of the chambers hai-e k e n excellent and reproducible. Korkers n-ho had access tCJ l o t h t lie commercial and the homemade t >-pe$ex h ihi t ed a c1e.i ded preference for the latter.
Figure 1
Construction. All glass surfaces are cleaned with acetonc prior t o cementing and weather stripping. One pane of window glass is c u t into two, using a glass-cutting tool, t o make t h e top and bottom sections of a chamber (Figure 1). T h e other four v-indow panes are left intact, t o form the four sides of the chamber. One side is attached by cement, followed immediately by adhesive tape, t o the 12-inch length of the bottom. T h e next side pane is attached in similar fashion t o t h e 11S/8-inch base, flush against t h e previously attached side and a t right angles t o it. This side pane will protrude inch beyond t h e 1ljj8-inchbase, tlie thiclcness of t h e glass, T h e third side pane ,is attached qiniilarly along the 12-inch base, flush n i t h t h e 3,lI6-inch protrusion. T h e fourth pane is attached similarly along the 115/8inch base and flush over t h e edges of t h e two sides a t right angles T O them. If cementing and application of t h e adhesive are done jjroperly, a square chtimher is obtained with over-all dimensions of 12 X 128/, X 24 inches. ITeather stripping is applied t o the edges of t h e top pane, which fit flush with t h e chamber, and is 1 Present address, Biochemistry D e p a r t m e n t , College of hgriculture, I - n i w r P i t y of Tyisconcin. LIadisun. 1%.
Desalting Amirjo Acid Solutions by Ion Exchange Gerald C. Mueller, Grace Bowman,and Ailene Herranen, McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wis. HE presence of inorganic salts in estracts of biological mateT r i a l s constitutes a serious obstacle in t,he separation of amino acids b y paper chromatography. A number of procedures have been reported for the removal of salts from 3iich solutions (i, 3, 5 , 6); however, the need for a simple routine procedure still esisted. T h e folloving method has been developed in which Loth amino acids and cations (sodium, potaspiurn, magnesium, and calciumj are first, adsorbed on D o s e s 50 (hydrogen fornij and the majority of the amino acids are subsequently eluted free of the inorganic saltP with 0.8-Y hydrochloric acid in 559/, ethyl alcohol. Fifteen amino acids are recovered quantitatively in this eluate; four additional compounds are r e c o v e d from the column with 6 S hydrochloric acid (acpeous) after the selective elution of EOdium chloride n-ith 11-hydrochloric acid (aqiieous'i. JI-hile thr. p r v c ~ d u r ehas been utilized pr,iniai ilj. for the Eepa-
1357
ANALYTICAL CHEMISTRY
1358 ration of small quantities of radioactive amino acids formed by tissue preparations in physiological salt solutions in vitro, it also has been applied successfully t o perchloric acid extracts of rat tissues. EXPERIMENTAL
Dowex 50 resin (200- to 400-meshJ 12% cross-linked) was prepared in the hydrogen form according t o the directions of Hirs, Moore, and Stein ( 2 ) . Before use batches of this resin were washed with 55% ethyl alcohol until the eluates were no longer turbid. Resin columns, 0.8 em. in diameter and 5 cm. in height, were prepared from aqueous suspensions of this resin. T h e appropriate glass tubing sealed onto the bottom of 50- or 100-ml. Erlenmeyer flasks provided convenient columns for this operation. Samples of amino acids or tissue extracts containing 25 mg. of sodium chloride or potassium chloride, with or without smaller amounts of magnesium or calcium ions, dissolved in 10 t o 20 ml. of water or 0.02-V perchloric acid ere placed on the columns. T h e columns were successively eluted with 5 nil. of distilled water, 40 ml. of 0.8-V hydrochloric acid in 557, ethyl alcohol, 10 ml. of 1N hydrochloric acid in water, a n d 10 ml. of 6 N hydrochloric acid in water. T h e elutions were conducted at a flow rate of approximately 1 ml. per minute with the aid of slight air pressure. With a manifold as many as 12 columns have been operated simultaneously with ease. T h e eluates were customarily evaporated t o dryness under a stream of air in a water bath a t 55" C. T h e samples were neutralized t o approximately p H 6.3 prior t o preparation of the paper chromatograms. T h e chromatographic svstems of PIlcFarren ( 4 )were employed to separate the amino acids in t h e hydrochloric acid-ethyl alcohol fraction.
Table I .
original solution, no special efforts were made to improve this fraction for the present work. The recovery on all amino acids was considered quantitative within the limits of analytical methods employed. A column of the above dimensions will permit the isolation of amino acids from 3.0 ml. of an isotonic salt solution. If more salt is present in the extract, the volume of the column should be increased proportionately by expanding the diameter of the column; the volume of the eluates should also be increased proportionately. As the percentage of ethyl alcohol in the eluting hydrochloric acid solution was increased, the affinity of the resin for the majority of amino acids was decreased relative t o the affinity for sodium and potassium ions. In comparison calcium and magnesium were retained even more tenaciously. The possible utility of aqueous alcohol solutions in other chromatographic procedures using ion exchange resins is suggested again by these results. Hirs, Moore, and Stein have reported the value of ethyl alcoholbuffer mixtures in the separation of several amino acids with similar chromatographic characteristics ( 2 ) . The application of this procedure to 4% perchloric acid extracts of whole blood, kidney, and liver was tested. -4fter approximately 80% neutralization of the perchloric acid with potassium hydroxide and removal of the precipitated potassium perchlorate, the samples were desalted as described. Excellent chromatographic resolution of the amino acids in the ethyl alcohol-hydrochloric acid eluate derived from 0.5 ml. of blood or 100 mg. of fresh liver and kidney was obtained using the p H 12 buffered-phenol solvent svstem of PIIcFarren (4).
Recovcry of Amino .4cids after Desalting with
Dowex 50 (Hydrogen Form)
LITERATURE CITED
%
Amino .4cid Recovery 103 Aspartic acid 103 Glutamic acid 101 Serine 95 Threpnine 100 Glycine 101 Alanine 102 Methionine 98 Phenylalanine 91 Leucine 9.5 Isoleucine 94 Valine 97 Tyrosine 106 Prolinea 112 Hydroxyproline" 107 Tryptophana 109 Argininea*5 106 Histidine% 6 RR Lysinea, 5 102 Cystine", b Amino acid recoveries determined gravimetrically on 10-nig. samples. All other amino acid recoveries determined colorimetrically on l-ir mole samples. b Eluted with 10 ml. of 6 5 hydrochloric acid as in experimental work.
T h e recovery of known quantities of the individual amino acids was determined either gravimetrically or colorimetrically with ninhydrin on ammonia-free samples a c d r d i n g to the procedure of Troll a n d Cannan (?). One-micromole samples of each amino acid in the presence of salt were used when the colorimetric method was employed. I n the gravimetric trials 10 mg. of each amino acid was similarly employed without overloading the column. RESULTS
After a 5.0-ml. water wash t o remove anions and neutral compounds, the following amino acids were eluted quantitatively with 40 ml. of 0.8S hydrochloric acid in 557, ethyl alcohol: aspartic acid, glutamic acid, serine, threonine, glycine, alanine, methionine, phenj-lalanine, isoleucine, valine, tj-rosine, proline, hydroxyproline, and tryptophan (Table I ) . A 10-ml. aliquot of lAVhydrochloric acid in water next eluted the sodium completely as the chloride salt. Four additional amino acids (arginine, histidine, lysine, and cystine) \yere recovered in a final 10 ml. of 6 S hydrochloric acid in water. The latter group 1w.s contaminated with pot,assium, magnesium, and calcium ions when present in the
(1) Consden, R., Gordon, A. H., and Martin, 4.J. P., Biochem. J . 41, 590 (1947). (2) Hirs, C. H. W , Iloore, Stanford, and Stein, W. H., J . Diol. C'hrm., 195, 669 (1952). (3) Kit, Saul, and Xwapara, Jorge, Cancer Research, 13, 694 (1953). (4) JIcFarren, E. F., A s . 4 ~ CHEW, . 23, 168 (1951). ( 5 ) IIcFarren, E. F., and Mills, J. A , , I h i d . , 24, 650 (1950). (6) Piee, K. -4.,Tooper, E. B.. and Fosdick, L. S., J . B i d . Chem., 194, 69 (1952).
(7) Troll, TValter, and Cannan, R. K., Ibid., 200, 803 (19S3). WORKsupported by a grant from the Alexander and Margaret Stewart Trust F u n d , grant C-1897 (C) from the U. S. Public Health Service and a n institutional grant from the American Cancer Society.
Improved Purification of Tetramethylammonium Chloride for Polarographic Studies P. L. Pickard and W. E. Neptune, Department of Chemistry, University of Oklahoma, Norman, Okla. HE
high negative discharge potential of tetramethylani-
Tmonium chloride makes it useful as a supporting electrolyte
for polarographic studies. I n addition, its solubility in alcohol enhances its value in work on many water-insoluble organic compounds. The technical grade quaternary salt is contaminated and frequently shows several reduction waves before the discharge potential of the tetramethylammonium ion. T h e usual purification method is recrystallization from ethyl alcoholas many as four recrystallizations frequently being necessaryand is accompanied by high loss of the product. A superior recrystallization medium has been found t o be methanol in acetone. About 10 grams of the salt is dissolved in 100 ml. of hot 25% methanol in acetone. T h e hot solution is filtered, 100 t o 110 ml. of acetone is added, and the solution is allowed t o cool. The crystalline material is filtered and dried in a vacuum desiccator. The yield of salt of sufficient purity for use as a supporting electrolyte in aqueous or alcoholic media is 60 t o 6570.
