Two-Dimensional Chromatography of Amino Acids on Buffered

ANALYTICAL CHEMISTRY

396 Solvent removal by steam distillation ( 4 ) minimized “ghost” spots and thus made pretreatment of the paper XTith oxalic acid unnecessary (6). LITERATURE CITED

Brown, F., .l‘uture, 167, 411 (1951). (2) Brown, F., and Hall, L. P., Ibid., 166, 66 (1950).

(1)

(3) Buch, &I. L., Montgomery, R., and Porter, IT. L . , A s a ~CHEM., . 2 4 , 4 8 9 (1952). ( 4 ) Denison, F. IT., J r . , and Phares, E. F., Ihid., 24, 1628 (1952). (5) Hiscox, E. R., and Berridge. K.J., .Vatwe, 166, 522 (1950). (6) Kennedy, E. P., and Barker. H. .I., - 4 x . i ~CHEX, . 23, 1033 (1951). (7) Stark, J. B., Goodban, .1.E., and Owens. H. S.,I b i d . , 23, 413 (1951). RECEIVED for reriem September 19, I%?.

.iccepted December 1.5, 1932.

Two-Dimensional Chromatography of Amino Acids on Buffered Papers A. L. LEVY

AND

DkVID CHUNG

Zforrnone Research Laboratory, Department of Biochemistry, Z-nirersity of California, Berkelel., Calif.

Previous systems for two-dimensional chromatography of amino acids-e.g., collidine-phenol-0.3% ammonia-were not entirely satisfactory owing inter alia, to irregularly shaped spots, lack of reproducibility, incomplete separation, discoloration of the paper, and unpleasant odor. The solvent system described (4:1:5 butanol-acetic acid-water-1 :1 rn-cresol-phenol, pH 9.3 borate buffer, Figure 3), overcomes these difficulties, allowing completion of the chromatogram in 40 hours. As analysis of peptide and protein hydrolyzates is established routine in many laboratories, iniprovements in technique are to be welcomed.

S

INCE the classical paper by Consden, Gordon, and Martin ( 2 ) in 1944 on the analysis of protein hydrolyxates by tn-odimensional paper chromatography using the system collidinephenol-O.3% ammonia (coal gas), a variety of other solvent combinations have been employed for this purpose ( I ) . I n this communication, the authors wish to record a new two-dimensional system for the qualitative analysis of amino acid mixtures, which, in this laboratory, has proved more satisfactory than any others so far tried. The authors’ experience with the sepnrate solvents in one-dimensional runs, and with procedures for protein hydrolysis, is also briefly described. Butanol-Acetic Acid. The butanol-acetic acid-water (4:1:5) mixture of Partridge (9) has proved the most generally satisfactory solvent for one-dimensional papers, affording a reproduciblr pattern of compact spots, allowing detection of minimal quantiticls of amino acids, and being not unpleasant t o handle. The reproducibility of this system is doubtless related t o the buffering action of the acetic acid, and to its relative insensitivity to temperature change. For control purposes it was found convenient to divide the natural amino acids into two individually resolvable groups of nine (mixtures A and B, Figure l), and to run them on either side of the unknovn. From the chromatogram it will be seen that the pairs threonine-glutamic acid, methionine-valine, isoleucine-phenylalanine, and to a lesser extent glycine-serine, are inseparable in butanol-acetic acid. Nixture .4 was prepared by dissolving 200 micromoles each of lysine, aspartic acid, glJ-rine, thre?nine, proline, valine, tryptophan, phenylalanine, and leucine in 2 ml. of 1 S hydrochloric acid, and making up to 10 nil. with 0.1 S hydrochloric acid: mixture B , by dissolving 200 micromoles each of cystine, histidine, arginine, serine, glutamic acid, alanine, tyrosine, methionine, and isoleucine in 2.4 ml. of 1 S hydrochloric acid and niaking up to 10 ml. with 0.1 A‘ hydrochloric arid. By making the amino acid solutions thus 0.02 Ai in 0.1 S hydrochloric acid, complete dissolution of the relatively insoluble amino acids such as cystine and tyrosine was effected, and bacterial contamination was inhibited. JVhen a neutral solution was required (as for application t o a single dimensional buffered paper) a portion was neutralized to bromothymol blue with 1 iV sodium hydroxide. and applied t o the paper before the cystine and tyrosine had time to crystallize.

I n the case of butanol-acetic acid chromatograms, the Rf’s were identical whether the amino acids were applied a t p H 1.0 or p H G to 7, except for those of alanine and glutamic acid which xere a little lower Tvhen neutralized. The chromatograms were run by the descending method on Whatman S o . 1 paper for 16 to 24 hours in a Chromatocab Model B 250 cabinet 27 x 18.5 X 25 inches (University -4pparatus Co., 2229 NcGee hve., Berkeley 3, Calif.). One-microliter aliquots (equal to 0.02 micromole of each amino acid) of solutions A and B were applied 2 inches from the solvent level, Tvith the aid of a Carisberg (Lang-Levy) constriction pipet ( 5 ) . The microliter pipets meritivned in this paper were all of this type, and n.ere obtaine 1 from EIerr Pedersen, the Carlsberg Laboratory, GI. Carlshergvej 10, Copenhagen, Valby, Denmark. The chromatogram \vas not improved b y equilibration (2 hours) in the cabinet prior to introduction of the butanol-acetic acid, as has sometimes been recommended. The papers were sprayed with 0.1% ninhydrin in ethyl alcohol (10) containing 5 % collidine ( I $ ) , and the color was developed by brief heating (1 t o 3 minutes) over a hot plate. Under these conditions the amino acid spots she\\- up in a variety of different colors, which vary with the temperature and length of time of heating, v i t h the paper used, and with the purity of the collidine. For this reason no attempt is made t o list the colors. HoiTever, no two neighboring spots in mixtures d and E have quite the same color, and this is particularly helpful in identifying such closely adjacent pairs as aspartic acid and glycine and phenylalanine and leucine. Finally, the papers were sprayed with 1% copper nitrate in ethyl alcohol ( 4 ) ,causing the spots to appear salmon pink on a pale blue-green background. This ( a ) prevented fading on storage (Q), ( b ) gave better contrast when photographed or contact-printed, and (c) prevented subsequent soiling of the chromatogram by fingerprints and other extraneous ninhydrin reacting materials (since chelated amino groups do not react with ninhydrin). Phenol and m-Cresol. I n order to separate the four pairs of amino acids unresolved by butanol-acetic acid, phenol saturated with water appeared to be the most desirable solvent. However, as the experience of many other workers has shown, this system gives the best results only xhen buffered by some means, and for thip purpose ammonia has generally been used. Recently, i\Ic>Farren( 7 ) employed aqueous buffers for this purpose, showing that both the phenol and the paper must be separately buffered. In the authors’ euperience, McFarren’s buffered papers

V O L U M E 25, N O . 3, M A R C H 1 9 5 3 are better than those run with phenolammonia for the fallowing reasons:

cyl

H,r

ASP

GI" olu

Thr AI^

1. The R i s are more reproduoihlc. 2, There is no oxidative d,soo]oration of the phenol 3 The spots are more compact. 4. The phenol runs faster and more cleanly (mimmal "brawn fingem" in the front). 5. Controlled p H adjustment is possible for inducing particular separations

The authors' Rt's have been somewhat lower than those reported by MeFarren, rtlthough the sequence has in general remained the same. This can probably be ascribed to the fact that the chromatographic equipment in this laboratory consists of large (20 X 36 X XG inches) wooden cabinets lined with pnrtlffin. Under these conditions the degiee of saturation of the bo.: with water vapor 1s undoubtedly less than in McFarren's small, paper-lined chambers, even though two 22 5 X 18 25 inch sheets of TVhatman No 4 paper wet with buffer (saturated with the phenolic phase) were hung in the oahmet with each run. in addi-

TV

397 TWO-DIMENSIONAL CHROMATOGRAMS

The most satisfactory system has been found to be butsnolacetic acid-water, followed by 1 to 1 m-cresol-phenol, p H 9.3 borate buffer, run on Whatman No. 52 paper (an acid-washed paper, with considerable wet strength). A photograph of such 5 chromatogram is shown in Figure 3, N n with a synthetic mi\ture of the 20 natural amino acids in equimoleeular proportions.

e

2 Y

2 c Y

2 2

a I,

d $ 4

2

-2

1

Val

hi.*

BUTANOL-ACETIC ACID-WATER (4 j:5)-

rigured.

.-

Use ot 1:l rn-L.resol-1'henol

Figure 1. Single Dimensional Chromatograph) of Natural Amino Acids

Figure 2.

Use of rn-Cresol Alone

tion to the usual tray of aqueous phase at the bottom. Another consequence of the greater air-to-buffer ratio in these experiments has been the effect of carbon dioxide in lowering the pH of the more alkaline (pH > 10) buffers. Also, the chromatography room w m not thermostatically controlled, but the fluctuations in temperature during a run have only occasionslly been sufficient to &use "heading" of the spots (mainly in the tyrosine-phenylalanine area). Roam temperature was 22" to 25" C. The buffers used by the authors have been somewhat simplified hy omission of potassium chloride, and they have found it more convenient to make up the phenolic-rich layer a8 a single phase by adding buffer to the phenol, to a paint just short of saturation. Finally, it has been found that a more satisfactory distribution of amino acids is achieved by using suitable mixtures of phenol and cresol.

1

HI

Figure 4.

Use of Phenol Alone

With buffered moresol alone for the second dimension (Figure 2 ) , the area between aspartic acid and alanine is unduly cronded compared with the area between valine and phenylalanine, whereas with buffered phenol alone for the second this effect is reversed (Figure 4). A 1-to-1 mixture of rn-cresol and phenol gives an equitable distribution of the amino acids over the paper. When the butanol-acetic acid run is made second, on a paper already buffered from a prior cresol-phenol run, a less satisfactory chromatogram results (Figure 5 ) . Removal of water and acetic acid by the buffer results in considemb1;- I n w r

398 Rl's and some distortion of the spots. Also, after treatment with ninhydrin, 8evemI colored fronts frequently CPOSB the paper at IowRj's, presumably resulting from chromatography of the buffer. To emphasize the value of buffering the phenolic phase, Figure 6 shows a chromabgram run under the standard conditions (as in Figure 3), but omitting the buffers completely. Heading and merging of the spots are evident, and, in addition, the pattern i8 not satisfactorily reproducible. The distribution of amino acids is also a function of the paper used, and Figure 7 shows a system suitable for Whatman No. 1 paper-namely, butanolacetic acid, 2 to 1 nz-cresol-phenol, p H 8.3 borate buffer. The spots are not as compact as on No. 52 paper, but the system is included because of the common use of No. 1paper for chramatographic work. Finally, using the routine described in the experimental section below, chromatograms similar to those shown zbove[are made in 40 hours-Le., 1day and 2 nights.

E

BUTANOL-ACETIC ACID-WATER (4: 1 :51-3

Figure 6 . Developed as i n S t a n d a r d Chromatogram (Figure 3) b u t w i t h Buffer O m i t t e d

Figure 5. Buffered rn-Cresol-Phenol Run First

Two microliters each of mixtures A and B and 2 microliters of 0.02 M asparagine and glutamine were applied to a spot 4 inches from each edge of 22.5 X 18.25-inch sheet of Whatman Xo. 52 Figure 7.

Modified System for W h a t m a n No. 1 Paper

oollidine, and 15 ml. of glacial aoetic acid. While still wet (cf. 11) i t was held 2 t o 3 inches above a hot plate (in a hood or a well ventilated room), until color development was complete ( 1 to 3 minutes). Under these conditions spots of the following relativelv reoroducible colors are observed: histidine, tyrosine, and

acids, biue-bude.

How-eve;, the spot from isoleucine 7s more

had the "composition 30 grams of ?wcresol plus 15 grams Phenol plus 7.5 ml. of p H 8.3 borate buffer (300 ml. of 0.1 '14 boric acid plus 60 ml. of 0.1 N sodium hydroxide). PROCEDURES FOR PROTEIN HYDROLYSIS

The experimental procedure employed in this laboratory utilizes 1 mg. of protein, but can be used satisfactorily down to 0.1 to 0.2 mg., if necessary. h ehromatogram of a bovine Eerum albumin hydrolyzate prepared in this manner is shown in Figure 8.

V O L U M E 25. N O . 3, M A R C H 1 9 5 3

399

Approximately 1 mg. of the protein was weighed into a 2-inch length of borosilicate glass tubing (G mm. in outside diameter, 4 mm. in inside diameter), sealed a t one end. One hundred microliters of constant boiling (20%) hydrochloric acid (glass distilled) were pipetted in, and the tube was evacuated and sealed lit ha8 been shown by Jacobsen ( 5 ) that humin formation is catalyzed by trace metals in hydrochloric acid]. The sealed tube was then heated in an oven a t 150' C. for 6 hours [it was shown with preparation E of adrenooorticotroDic hormone

,

with 100 $. of 0.38 N barium hydroxide a t 150" C.for 1.5 hours in a sealed tube, On cooling, 20 PI. of 1.9 N sulfuric acid were added, the precipitated barium sulfate was centrifuged, and the pFocedure was continued as above. It was useful for the detection of tryptophan but was not used routinely owing to extensive decomposition of serine, threonine, cystine, and arginine. PHOMGRAPHIC DETAILS

The photographs of chromatograms (Figures 2 to 8)were taken by a combination of reflected and transmitted light on Kodalith Ortho, Thin Base, Type I1 film, developed with D-85 developer (film) and printed on Eastman AZO (1-4)paper (using D-72). Since the film was relatively insensitive to the yellow-brown spots due to asparagine, proline, and tryptophan, these areas received extra treatment with Farmer's Reducer. The best photographic records were, however, obtained in color on Eastman Ektachrome, but they unfortunately cannot be reproduced in this journal. For making routine records of ohromatogram, the authors employ direct contact prints on- Kodagraph . Standard contact paper, - toprint is reproduced in Fig AC

The authors wish to express their gratitude to C . K. Li, in whose laboratory this work was carried out, for his generous encouragement and support at all stages of the investigation. They would also like to thank Leon Messier for his helpful cooperation with the photography. Thanks are due to Armour and Co. for a gift of bovine serum albumin. Figure 8. Chromatogram of Bovine Albumin Hydrolyzate 14 miomgrams of NHn-N epp1i.d;

Serum LITERATURE ClTED

(1) Block, R. J.. LeStrange, R., and Zweip. G.. "Paper Chromatopraphy." New York, Academic Press, Ino., 1952. (2) Consden, R.. Gordon. A. H.. and Martin, A. J. P., Bioehem.J., 38,224 ( 1944). (3) Jaoobsen, C:.F.,Compt. rend. frav. lab. Cc&berg, S h . chim., 26, 463 (194II1 .,. (4) Kawerau, E>,,and Wielmd, T., Nature, 168,77 (1951). (5) Levy, M., 17omp. mnd. t m a . lab. C d s b e 78, S h , chim., 21, 101

run 16 hours in each

dimon-ion To revsel methionine spot 40 miorograms of NHI-N mnst be(app1ied

(ACTH) (6) that 3 hours a t 150" C. was sufficient to hydrolyze all peptides; after 6 hours the amino acid pattern was unchanged; after 12 hours considerable loss of serine and threonine was observed and, after 24 hours, destruction of these amino acids was almost complete]. The seal was opened and the acid was evanarated over phosphorus pentoxide and Dotassium hvdroxide

~~~~~~

~~

,..~.

._- .....

and 200 PI. used per assay) was assayed for amino nitrogen by the quantitative ninhydrin method of Moore and Stein (8). The volume of solution (5to 10 PI.), calculated to contain 1micromole (14 micrograms) of amino nitrogen, was then applied to the paper in 2-SI. portions, drying between addition6 with a current of warm air from a hair drier. Alkaline hydrolysis was carried ant by heating 1 mg. of protein

(1936).

Li, C. H., J . Am. Chsm. Soc., 74, 2124 (1952). (7) MoFarren. E.F.,ANAL. CHEM.,23, 168 (1951). ( 8 ) Moore. S., and Stein, W. H.. J . Bid. Chhem., 176,367 (1948). (9) Partridge, S. M.. Biochm. J., 42, 238 (1948). (10) Patton, A. R.,and Chism, P., ANAL.CHEM..23, 1683 (1951). (11) Thompson, J. F., Zaeherius. R. M., and Steward, F. C., Plant Phusiol., 26, 375 (1951). (12) Woiwod. A. J., I . Gen. Micwbiol., 3, 312 (1949). (6)

RP_OEIVED for review September 12. 1952. Aceeptod December 12, 1952. Supported in part by the Rockefeller Foundation, New York, and the U. 8. Publio Health Servioe, Nhtiond Institutes of Health.

Displacement Spebctrophotometry I

ROBERT HOUSTC )N HAMILTON Temple University Schoolof Mezdicine, Philadelphia 40, Po.

I-

U T H E usual absorption photometry

of solutions, light of restricted wave length incident on the photocell or phototube is set to reed unit intensity after passing through a cell containing solvent or solvent plus the amount of impurities in reagents (blank). Then, light intensity being maintained constant, an identical cell is substituted, containing solvent plus the lighh absorbing molecules whose concentration is t o be determined. The decrease in light intensity is noted and the concentration of the salute is calculated from the absorption produced by known concentrations. The same results can be obtained by the addition to the light

".

I

. ..

"

.

patn 01 layers of solution O t constant thickness. Such addition can be accomplished by removal of a piece of plate glass immersed in the solution. For a given solution and wave length the effect of the glass plate itself on light trannsmittance will be eonstant. Either of two procedures c m be followed: (1) With the glass plate immersed a t right angles to the light beam, light intensity is set to read 100% (unity). The plate is then removed, and light intensity is read after removal. (2) Light intensity is allowed t o remain such that transmittance is close t o unity (between 80 and loo%), and transmittance is read exactly, before and again after removal of the glass plate. The difference in the