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CHROMATOGRAPHY OF AMINO ACIDS ON STARCH COLUMNS.SOLVENT MIXTURES FOR THE FRACTIONATION

OF PROTEIN HYDROLYSATESBY STANFORD MOORE AND WILLIAM H. STEIN

(From the Laboratories of The Rockefeller Institute for Medic al Research, New York)(Received for publicatio n, October 8, 1948)

In previous communications (l-3) procedures have been described forthe quantitative separation of amino acids by chromatography on starch.The present paper is concerned with the extension of these techniques toinclude most of the amino acids commonly found in protein hydrolysates.In the earlier experiments n-butyl alcohol-benzyl alcohol solvents contain-ing about 15 per cent water were employed to separate phenylalanine,leucine, isoleucine, methionine, tyrosine, and valine. In all alcohol-watersolvents these are among the fastest moving amino acids on starch columns.Preliminary experiments had indicated (1) that the amino acids withslower rates of travel could be eluted successfully from the column by theappropriate choice of acidic solvents of higher water content. Many typesof solvents have subsequently been investigated in order to arrive at aconvenient system for the fractionation of protein hydrolysates.

The effluent concentration curves shown in Figs. 1 and 2 give the resultsobtained with two of the solvent mixtures which have proved most useful.The synthetic mixture of amino acids chromatographed corresponded incomposition to a hydrolysate of bovine serum albumin. The effluent fromthe column was collected in a series of 0.5 cc. fractions on an automaticfraction-collecting machine (2). The amino acid concentration in eachfraction was determined by the photometric ninhydrin method previouslydescribed (3). For the curve in Fig. 1, the column is started with asolvent composed of n-butyl alcohol, n-propyl alcohol, and 0.1 N HCl inthe proportions of 1:2: 1. After the emergence of aspartic acid, the ratesof travel of the amino acids remaining on the column are increased by ashift of solvent to 2:l n-propyl alcohol-O.5 N HCl. In this experiment,the first six amino acids are incompletely separated, and a chromatogramrun with 1: 1 :O. 288 n-butyl alcohol-benzyl alcohol-water (2) is sti ll re-quired for resolution of these components.

The curve in Fig. 1 gives quantitative values for proline, threonine,aspartic acid, serine, glycine, ammonia, arginine, lysine, histidine, andcystine. Glutamic acid and alanine appear as a single peak. These twoamino acids can be separated by the chromatogram illustrated in Fig. 2.The solvent in this case is composed of tert-butyl alcohol, set-butyl alcohol,

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54 CHROMATOGRAPHY OF AMINO ACIDSand 0.1 N HCl in the proportions of 2: 1: 1. Thus, by the use of threecolumns it is possible to separate from one another the eighteen constit-uents most commonly encountered in acid hydrolysates of proteins. The

;E:E 2,0 Leucine,+t fsoleuane

^ rine Ammonia :yI 0i3.55.3 242 136 149 163 119

Effluent cc.FIG. 1. Separation of amino acids from a synthetic mixture containing seventeen

amino acids and ammonium chloride. Solvents, 1:2: 1 n-butyl alcohol-n-propyl al-cohol-0.1 N HCl, followed by 2:l n-propyl alcohol-O.5 N HCI. Column, 13.4 gm. ofstarch (anhydrous) ; diameter, about 0.9 cm.; height, about 30 cm. Sample, about3 mg. o f amino acids. A is a small artifact peak (see the text).

tLeucine +Isoleucine

Valine a:zP Threonine +Aspartic acidAlanine p,17.5 31 38 46 81 91 112

ffl uent cc.FIG. 2. Separation of glutamic acid, alanine, and other amino acids from a syn-thetic mixture containing eighteen components. Solvent, 2: 1: 1 tert-butyl alcohol-set-butyl alcohol-O.1 N HCl.following experimental section describes he procedure employed to obtainresults of the type shown in Figs. 1 and 2. The discussiondeals with someof the considerations introduced by the presence of additional components

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S. MOORE AND W. H. STE IN 55

in the mixture being fractionated and outlines the results obtained by theuse of other solvent combinations. The results of analyses of hydrolysatesof /%lactoglobulin and bovine serum albumin are given in the followingpaper (4).

EXPERIMENTALPreparation of Column-The potato starch column is poured as pre-

viously described (2) .l Unless otherwise specified, starch columns 0.9 cm.in diameter and about 30 cm. in height have been used. The procedurescan be scaled up proportionately for columns up to 8 cm. in diameter.After the starch has settled to constant height, the excess butyl alcohol isremoved and a 1: 1 mixture by volume of n-propyl alcohol-O.5 N HCl isplaced on the column. The solvent is run through the column under apressure of 8 cm. of mercury overnight, and the pressure is then raised to15 cm. The solvent flow is continued until 0.5 cc. samples (neutralized)of the effluent and the influent solvent both yield the same color valuewhen analyzed by the ninhydrin method. Starch contains small quantitiesof ninhydrin-positive material which are extracted by acidic alcohol sol-vents. The use of a propyl alcohol-HCl mixture with a 50 per cent watercontent serves to clean out the column fairly rapidly. When the numberof cc. of the effluent required to yield a ninhydrin-negative column havebeen determined for a given batch of starch, the prescribed number of cc.can be used in the preparation of subsequent columns. For the samplesof starch tested thus far, 55 cc. of the 1: 1 solvent have proved adequatefor columns 0.9 X 30 cm.2 When the column is ninhydrin-negative, thesolvent mixture is changed to that to be used in the chromatographicanalysis.3 After the new solvent has been run through overnight at 15cm. pressure (20 to 25 cc. of effluent), the column is equilibrated and readyfor use. Columns may be left in contact with solvents of low acidity,

i For work with acidic so lvents , the delivery tip of the chromatograph tube can bepulled down so that a drop of effluent colle cts therein. In this manner ammoniafrom the air is prevented from reaching the inner walls of the tip. Beveled tips arestil l required on tubes which will be used with water as the solvent. If a beveled tipis used with acid ic solvents, the inside section, up to the sintered plate, must be rinsedwith a stream of the solvent before the column is placed on the fraction collector.A pipette, the end of which has been bent to form a U, is used for the rinsing.2 It is poss ible to wash large amounts of starch at one time with the propyl alcohol-HCl solvent, thus avoiding the preliminary washing each time a column is poured.Th is procedure is not recommended, however, sinc e sam ples of starch washed anddried in the laboratory have been found not to give as uniform colum ns as the un-treated comm ercial material (2).

3 The solvents employed in these investigations have been prepared from n-butylalc oho l (reagent grade, Merck) and n-propyl alco hol , see-butyl alco ho l, and tert-butyl alcohol (c.P. grade, Columbia Organic Chemicals Company, Inc., Columbia,South Carolina). Red istillation prior to use has not been found necessary.


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S. MOORE AND W. H. STEIN 57silicone film. Glassware coated with a silicone f ilm is repellent to waterand to the water-miscible alcohols such as propyl alcohol and tert-butylalcohol. For the present experiments al l the sets of photometer tubes(3) for use with the fraction collector have been coated with Dri-film No.9987 (General Electric Company, Schenectady, New York), which is amixture of organochlorosilanes. For polymerization on glass, the Dri-filmis applied as a 5 per cent (by volume) solution in chloroform (reagent grade).The coating of the tubes should be carried out in a hood and gloves shouldbe worn. The glassware is first cleaned in chromic-sulfuric acid cleaningsolution, thoroughly rinsed, dried at 110, and allowed to stand at roomtemperature for 1 hour. When sets of 200 tubes are being coated, 200 cc.of the Dri-film solution are prepared. A sheet of filter paper or a towel isplaced on the bottom of each test-tube rack (3). The first ten tubes arefilled about half full with the filming solution. Each tube is emptiedrapidly over a flask or beaker, causing the solution to flow over the upperwalls of the tube, and set to drain inverted in the rack. The filming solu-tion is used over again for the treatment of 200 tubes. The racks are leftat room temperature overnight. The tubes are then returned to the up-right position and each rack is baked for 2 to 3 hours in an air oven at150-180. This procedure has given more durable silicone films than thoseobtained by applying the Dri-film in vapor form or by the use of less con-centrated solutions of the coating agent. Control of the relative humidityat which the filming is conducted has not proved necessary.

The f ilm has no effect on the optical properties of the tubes as measuredin the Coleman junior spectrophotometer. The silicone-coated tubes havemaintained their water repellency during constant use for periods of about6 months, at the end of which time recleaning and refilming have beennecessary. The film is remarkably resistant to boiling water, alcohols, oracids, but is readily destroyed by alkali or cleaning solution. The coatingis also rendered ineffective by ordinary soap, but Duponol C has beenfound to have no injurious effect. The washing procedure for the coatedphotometer tubes, therefore, is different from that previously described(3). After each set of ninhydrin analyses, the tubes are rinsed with waterin racks of 50 and scrubbed with a brush (e.g., E. Machlett and Son, NewYork, catalogue No. A-7-870) which has been dipped in a 0.2 per centsolution of Duponol C. The brushing is necessary to remove the ring ofmaterial that is sometimes deposited on the walls of the tubes. If thisdeposit resists removal by brushing, it is an indication that the tubes needrefilming. An aluminum rod notched to fit the rim of the tube is usefulfor holding the individual tubes in position in the rack while they are beingbrushed. The brush employed should be reserved for this purpose andkept out of contact with ordinary soap. No evidence of any scratching of

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58 CHROMATOGRAPHY OF AMINO ACIDSthe photometer tubes by this cleaning procedure has been observed, butcare should be taken to insure that no metal parts of the brush make con-tact with the walls of the tubes. The tubes are rinsed several times withdistilled water and dried in an oven at 110.

To prevent creeping of the solvent on the tip of the chromatograph tubeand the glass funnel of the fraction collector, these items are also given asilicone coating. The tip of the chromatograph tube is cleaned with a hotmixture of HNOs and HzS04 and coated by dipping the lower portion of thetube in the Dri-film solution, contamination of the sintered glass plate beingavoided. The funnel of the fraction collector is coated both inside andoutside.

In order to be certain of the proper setting for the impulse counter when awater-repellent tip is used, it is necessary to redetermine the drop size (2)more frequently than is required with an untreated funnel. For the sol-vents referred to in Fig. 1, the drop sizes have been so nearly the same thata single impulse counter setting has been used throughout the experiment.The use of acidic solvents requires precautions against the uptake ofammonia from the air by the effluent fractions during the period they arestanding on the automatic fraction collector. The ninhydrin method em-ployed to analyze the effluent can readily detect 0.1 y of ammonia per cc.If no preventive steps are taken, tubes containing 0.5 cc. of 2: 1 propanol-0.5 N HCl, left overnight open to the laboratory air or on the fractioncollector, may pick up enough ammonia to give a positive reading of 0.10optical density unit in the ninhydrin analysis. This uptake may be vir-tually eliminated by lining the inside surface of the cover of the fractioncollector with filter paper impregnated with citric acid. Large sheets offilter paper are cut to fit the cover and taped in position. A 2 per centsolution of citric acid in ethanol is brushed onto the surface. With thefraction collectors in use in this laboratory, the ammonia problem has beenincreased by the liberation of ammonia from the bakelite parts of themachines. It was not appreciated for some time that hexamethylene-tetramine is used in the manufacture of many samples of bakelite and thatthe material, as a result, may contain appreciable quantities of ammonia.Samples of bakelite can readily be tested for ammonia liberation as de-scribed earlier (2). If the test is positive, the citric acid solution must beapplied to al l the bakelite parts of the fraction collector, including thephototube housing. Commercial models of the fraction collector are cur-rently being built with special ammonia-free bakelite, which eliminatesthis source of contamination.

In work with acidic solvents, the cotton packing around the stem of thechromatograph tube is also treated with citric acid. When the tubes areremoved from the machine, they are stoppered with corks which have

4The Technicon Company, 215 East 149th Street, New York 51.

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S. MOORE AND W. H. STE IN 59previously been shaken with the alcoholic citric acid solution and air-dried. Corks thus treated have been satisfactory for a year or more.Rubber stoppers have proved unsuitable.

Contamination with ammonia can also occur during the handling ofthe solvents. The lips of all storage vessels should be wiped before use.Care must be taken to avoid any liquid contact between the solvent andthe rubber stoppers on the top of the column and the top of the separatoryfunnel. The glass should always be wiped dry before the insertion of thestoppers. It is important that the need for reimpregnation of the coveron the machine be checked periodically by placing test samples of the2: 1 n-propyl alcohol-O.5 N HCl solvent on the machine overnight. Theninhydrin readings should be no higher than those of control tubes whichhave remained stoppered prior to analysis.

In performing a chromatogram of the type referred to in Fig. 1, asolvent change is made about half-way through the experiment. Theeffluent fractions should, if possible, be analyzed each day to provide acheck on the progress of the experiment and to furnish a basis for estimat-ing the exact point at which the solvent change should be made. In anexperiment such as that shown in Fig. 1, it is desirable to shift the columnto the second solvent mixture during the emergence of aspartic acid. Thispoint can be predicted fairly accurately by multiplying the position of thereadily identif ied proline peak by 1.6. The change point is usually reachedat about 83 cc. of the effluent and can be predicted from the position ofone of the earlier peaks, if necessary. If the change of solvent is scheduledto occur at an inconvenient hour, the column can be slowed down byrunning it under lower pressure without affecting the results. At the timeof the change of solvent, the separatory funnel is removed and the liquidabove the starch in the chromatograph tube is withdrawn before the addi-tion of the new solvent.

For the experiment illustrated in Fig. 1, the solvent shift occurs on aboutthe 3rd day, and the completion of the experiment, through the emergenceof cystine, requires about 7 days of continuous operation on the fractioncollector.When a column is shifted from one solvent to another, a specific series ofchanges occurs in the composition of the effluent. In the example shown inFig. 1, the init ial solvent contains 25 per cent water and is 0.025 N withrespect to HCI. The second solvent contains 33 per cent water and isabout 0.17 N with respect to HCl. The e&rent attains the higher watercontent of the second solvent when a volume of solvent equivalent to thatretained by the column has passed through the starch. The increasedwater content, which appears at about 6 cc. after the solvent change, servesto increase the rates of travel of the amino acids. If the solvent shift hasbeen made too early, the latter part of the aspartic acid curve will be

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60 CHROMATOGRAPHY OF AMINO ACIDSdistorted. Since asymmetrical peaks frequently indicate the presence ofmore than one component, it is preferable, in order to avoid ambiguity, toarrange for the emergence of the higher water concentration after theaspartic acid curve is down to the base-line.

The increase in HCl concentration, however, to 0.17 N, occurs sharplyat about 14 cc. after the solvent has been changed. The HCI thus has aretention volume, in the terminology of Tiselius (5), of about 6 to 8 cc.The rise in the HCl content of the effluent in Fig. 1 occurs just at the begin-ning of the serine peak. Although a change in acid concentration is notcapable of distorting the serine peak significantly, it is desirable from theanalytical standpoint to have the change occur before the amino acidemerges.

The peak A in Fig. 1 is an artifact which occurs at the point of increaseof the water content of the e&rent. This small peak represents a transientrise of only 0.02 to 0.04 optical density unit in the blank and indicates thatthe starch column has been thoroughly freed of ninhydrin-positive materialin the preliminary washing procedure. It is indicative also of the adequacyof the silicone film on the tip of the chromatograph tube and the funnel.In earlier experiments, before these parts of the glassware were coated, arelatively large artifact peak was usually obtained at position A. A con-trol experiment with a strong solution of methyl red in the acidic solventdemonstrated that, during a 1 to 3 day run on unfilmed glassware, a smallamount of solid material was deposited around the outside of the tip of thefunnel as a result of creepnig and evaporation of the solvent. Similarly, adeposit of some of the solute could be seen around the periphery of theflowing stream of the effluent within the funnel. When the solvent wassubsequently shifted to one of higher wat,er content, and consequentlydifferent surface properties, some of this residue was redissolved andemerged as the artifact peak. A similar experiment with glassware ren-dered hydrophobic by a silicone fi lm showed no residual deposit of methylred on any part of the tip or funnel.

The shift from one solvent to another, after a sample has been added to achromatogram, has proved practical only with solvents that are misciblewith water in all proportions. When an attempt has been made to shifta butyl-benzyl alcohol column to a propyl alcohol-water solvent, drop-lets of water have formed at the interface, thus destroying the efficiencyof fractionation (2).

Analysis of Efluent Fractions-The concentration of amino acid in theeflluent fractions is determined by the photometric ninhydrin method (3).For the 0.5 cc. fractions, 2 cc. of the ninhydrin reagent are used. Thesolvents possessing a total acidity of 0.025 N or less do not require neu-tralization. Samples of the 2: 1 n-propyl alcohol-O.5 N HCl mixture, how-ever, must be neutralized just before the addition of the reagents. For

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9. MOORE AND W. H. STEIN 61routine work, a burette tip of appropriate size can be prepared to deliver0.10 cc. of alkali per 2 drops. A rack of 50 tubes can conveniently bemoved along underneath a burette dripping at a constant rate. The rackshould be shaken by hand after the addition of the alkali. The concentra-tion of NaOH (about 0.8 N) is adjusted so that, in the titration of testsamples, 2 drops leave the fractions slightly acidic. The amount of alkaliadded should be such that an additional 0.1 cc. of 0.1 N NaOH is requiredto render the samples alkaline to phenolphthalein. One purpose of keepingthe samples slightly acid is to avoid loss of ammonia from the NH&lpeak.After a solvent shift, as in Fig. 1, it is necessary to locate the effluentfraction at which the increase in acid concentration occurs. A smalldrop of 0.02 per cent phenolphthalein in ethanol is added to the twenty-eighth fraction after the time of change of the solvent on the top of thecolumn. Depending upon whether this tube is or is not rendered alkalineby 1 drop of the approximately 0.8 N NaOH, the tubes ahead or after itare treated similarly until the point is determined at which al l subsequentfractions require 2 drops of the alkali. The subsequent additions are madewithout use of the indicator. By this procedure a few of the fractionsaround the change point may be overneutralized. No significant errorshave been observed when the increase in acid concentration occurs duringthe first two or three fractions containing serine, but as already men-tioned, it is preferable to have the change occur earlier.

For an experiment such as that illustrated in Fig. 1, every effluent frac-tion is analyzed until after the emergence of glycine. From that point on,analysis of every other fraction is sufficient. If the first chromatogram onan unknown sample shows areas in which there are no peaks, such as thelong valley between tyrosine and proline in Fig. 1, the number of analysesrequired in a duplicate experiment can be reduced by omitting some of thefractions. If the chromatogram is being run for the determination of onlyone or two amino acids, the rest of the curve can be neglected. The solventmixture referred to in Fig. 2 is usually employed only to separate glutamicacid and alanine, and generally the first 45 cc. of effluent are collected as afore fraction before the column is placed on the fraction collector. Ifaccuracy to the last few per cent is not important, the amount of ninhydrinrequired can be halved by the use of only 1 cc. of ninhydrin solution per0.5 cc. sample.

The choice of blanks against which the amino acid peaks are read iscrucial for maximum accuracy in the integration of the curves. In manyinstances the average blank tube for the base-line of the effluent curvecan be readily determined in the manner previously outlined (2). In thefirst part of Fig. 1, there are blank tubes ahead of leucine and in the valleysbefore proline and threonine. The proline peak, reddish yellow in color,

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S. MOORE AND W. H. STE IN 63reference tubes, the correction is made by adding 0.03 unit to the appro-priate fractions before integration of the peaks.The variations in the blank and the need for the use of these corrections,however, mean that the accuracy of integration of the peaks after thesolvent shift in Fig. 1 is, as a rule, not as satisfactory as that obtained inchromatograms developed with a single solvent mixture.

TABLE INinhydrin Color Yie lds from Amino Ac ids in Organic Solvent Solu tions

Determined on 0.5 cc. samples of 0.35 mM solutions of the amino acids. Heatingtime 20 minutes. The samples in 2:l n-propyl alcohol-O.5 N HCl were neutralizedwith 0.1 cc. of about 0.8 N NaOH prior to analysis.

Compound

Leucine ............Isoleucine ........Phenylalanine ....Valine ............Methionine .......Tyrosine. ..........Proline ............Glutamic acid. ... .Alanine. ...........Threonine ........Aspartic acid. ... ..Serine............Glycine. ...........Ammonia. .........Arginine ...........Lysine. ............Histidine .........Half cystine .......

. .. . .. .. . . .. .. .. .

Color yield on molar bas is relative to leucin e in water, read at 570 mu

-

1:2: 1 n-butyl alcohol-n-prop ylalcohol-O.1 N HCl (d*5 =0.X62)

0.991.000.851.011.000.860.05 (0.27 at 440 rnp)1.021.020.940.89

2: 1: 1 lert-butylalcohol-sec.butylalcohol-o.1 N HCl(a= = 0.858)1.001.020.851.020.860.051.021.00

-2:-

-

0.940.980.98 cu.*0.971.141 .Ol0.54

* For accurate ammonia determinations the factor should be checked with aknown NH&l solution run at the same time as the unknown (cf. (3)).Calculations-The procedure for integration of the curves has been out-

lined earlier (3). When only every other effluent fraction is analyzed(i.e., ammonia through cystine, Fig. l), satisfactory recoveries are obtainedby doubling the usual summation (cf. (3), Table V6). For the relativelyvolatile solvent mixtures referred to in Figs. 1 and 2, the entire 0.5 cc.5 Table V (3) contains an error. In the third line of the integration belowTable V, read Sum of Fractions 37-40 and 45-47 for Sum of Fractions 37-42 and

46-47.

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64 CHROMATOGRdPHY OF AMINO ACIDSsample evaporates during the 20 minute heating in the water bath. Forunneutralized samples, the calculated correction factors for 5, 10, and 15cc. of diluent (cf. (3), Table III) become 0.230, 0.216, and 0.212. Forsamples which have been neutralized with 0.10 cc. of NaOH, the factorsare 0.232, 0.218, and 0.212. In the integration of the curves, the summa-tions of the uncorrected amino acid concentrations are routinely multipliedby one-half the above factors (cf. (3), Table V5). The whole factors areused for the conversion of the analytical results to leucine equivalents inplotting the curves for publication and in the determination of ninhydrincolor yields on standard solutions. If only 1 cc. of the ninhydrin solutionis used per 0.5 cc. sample, the evaporation loss is about 0.62 cc. The factorsare 0.193, 0.196, and 0.199 for unneutralized fractions and 0.196, 0.198,and 0.199 for samples neutralized with 0.1 cc. of NaOH.

The ninhydrin color yield for each of the amino acids has been determinedin the solvent in which it emerges from the column. The yields given inTable I should be checked under the users experimental conditions (3).It is convenient to prepare 2 mM standard solutions which are diluted toabout 0.35 mM for analysis. The blanks consist of 0.5 cc. aliquots of thesame sample of solvent.

Use of the color yield of 0.27 for proline at 440 rnp is the same as mul-tiplying the leucine equivalents by 3.7, as previously described (3). Forpublication, the proline curve has been corrected, whereas the other peakshave been left in terms of leucine equivalents.

In most instances, the choice of fractions to be included in the integrationof a given amino acid peak is evident from the graph of the results. Inthose cases in which the valley between two peaks does not fal l to the base-line, one-half of the quantity of amino acid represented by the low point ofthe valley is assigned to each peak. This procedure has given satisfactoryintegrations when the valley is less than half the height of the smaller ofthe two peaks. In the present experiments, no pairs of peaks have beenencountered which required an attempt to apply the method of calculationfor overlapping components used in the case of tyrosine and valine in thebutyl-benzyl alcohol solvent (2).

In the experiment shown in Fig. 2, proline overlaps glutamic acid. Theentire glutamic acid curve is read at 570 rnp, and the integration subse-quently corrected for the contribution of proline, which has a color yieldof only 0.05 (relative to leucine as 1.00) at this wave-length (cf. (3)).

Quantitative Analysis of Synthetic Mixtures-The results obtained by theintegration of the curves in chromatograms of the type shown in Fig. 1are summarized in Table II. The synthetic mixture of amino acids cor-responded in composition to an acid hydrolysate of bovine serum albumin.Cysteine was omitted since, as will be shown later (4), it was found not to


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S. MOORE AND W. H. STE IN 65be present in protein hydrolysates that had been repeatedly evaporated todryness in order to remove excess HCI.The separation of phenylalanine from leucine plus isoleucine is notsufficient to permit fully reliable division of the peaks. Since leucine andisoleucine are usually present in by far the larger quantity, the percentagerecovery may be fairly accurate for these two amino acids. The phenyl-alanine values, although more variable, are frequently accurate to f5 percent. If the column is loaded more heavily, however, as is sometimes

TABLE IIRecovery of Amino Acids from Known Mixture C ontaining Eighteen ComponentsSolvents, 1:2:1 n-butyl alcohol-n-propyl alcohol-O.1 N HCl followed, after theemergence of aspartic acid, by 2:l n-propyl alcohol-O.5 N HCl (cf. Fig. 1).

Constituent Amountpresent -

Leucine-isoleucine ...............Phenylalanine ...................Valine-methionine-tyrosine. ......Proline. .........................Glutamic acid*-alanine ...........Threonine .......................As partic acid *. ..................Serine ...........................Glycine ..........................Ammonia ........................Arginine .........................Lysine ...........................Histidine ........................Cystine ..........................

mg.0.3640.1650.3540.1360.5150.2010.2670.1180.0510.0240.1430.3020.0940.133

99.4 99.5 101.594.8 96.1 94.899.6 101 o 100.199.7 97.8 100.095.2 94.6 96.897.5 101.0 102.093.5 94.1 94.7

100.0 99.8 101.299.1 100.5 101.0

102.0 99.5 104.597.7 102.8 105.096.3 103.0 99.599.7 104.6 97.489.5 102.7 101.5

Al l con stitue nts ................ 2.867 97.3 99.3 99.6-

Average

100.395.2100.2

99.295.5

100.294.1

100.3100.2102.0101.899.6100.6

97.9--98.7

* When the value for glutam ic acid is corrected for the 7 per cent low recovery dueto esterification, the recoveries for glutam ic acid plus alanine become 100.2, 99.7,and 101.7 per cent. The aspartic acid recoveries, which run 6 per cent low, may besimilarly corrected to yield the figures 99.4, 99.9, and 100.8 per cent. The total re-coveries, on this basis, beco me 98.6, 100,8, and 101.0 per cent.

desirable in order to attain higher accuracy in the analysis for the basicamino acids, the resolution of leucine plus isoleucine and phenylalaninebecomes less satisfactory than that indicated by Table II. Valine, me-thionine, and tyrosine are integrated as a group. On an unknown solu-tion, the principal calculation of value for these combined peaks is theestimation of the total amino nitrogen in leucine equivalents.

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66 CHROMATOGRAPHY OF AMINO ACIDSProline and threonine emerge as well defined peaks before the solvent

shift and are recovered quantitatively. Adjacent to them, however, arethe peaks for glutamic acid plus alanine and aspartic acid for which, itwill be noted, the recoveries are low. It has been found that the yieldsof glutamic and aspartic acids are low in this solvent as a result of esterifica-tion. With unknown mixtures, the aspartic acid values obtained by in-tegration are divided by 0.94 to give corrected figures.

The procedure which has been outl ined for the establishment of theblank after the solvent shift permits quantitative recoveries to be obtainedfor the peaks emerging after the change to 2: 1 n-propyl alcohol-O.5 NHCl.

The results obtained in the separation of glutamic acid and alanine with2: 1: 1 tert-butyl alcohol-set-butyl alcohol-O.1 N HCI are summarized in

TABLE IIIRecovery of Glutamic Acid , Alanine , and Other Amino Ac ids from Synthetic

MixtureSolven t, 2:l:l terl-butyl alcoho l-set-butyl alcohol-O .1 N HCI (cf. Fig. 2). Th e

mixture contained eighteen compon ents (cf. Tab le II).Amountpresent

w.Leucine-isoleucine ............... 0.373Phe nylalan ine. .................. 0.169Valine-methionine-tyrosine. ...... 0.363Glutam ic acid . .................. 0.426Ala nine .......................... 0.102

T

-

Chromato- Chromato- Chromato-gram 474 gram 543 gram 481 Average

99.0 100.8 99.9101.6 103.6 102.6100.6 104.4 102.5

96.3 97.8 100.2 98.197.3 101.3 97.5 98.7

-I------

Table III. Esterification of glutamic acid is neglig ible in this solvent mix-ture, as evidenced by the essentially quantitative recovery of the aminoacid. The chromatogram also provides an alternative determination ofphenylalanine, which is well separated in this case. If methionine isabsent, the column can yield quantitative values for tyrosine and valine.In most instances, the column has been used only for the separation ofglutamic acid and alanine. The valley-does not fall to the base-line, andit sometimes is necessary to reduce the load on the column in order toobtain adequate resolution.

Accuracy of Chromatographic Analysis-In general, the chrnmatographicprocedure on starch columns is capable of yielding recoveries of 100 f3 per cent. The average recoveries for the components of the syntheticmixture used in the chromatograms summarized in Tables II and III arewell within this range. The deviations which do occur appear to be random


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S. MOORE AND W. H. STE IN 67and cancel out, in part, in the calculation of the total recovery for the sumof the amino acids, which is almost invariably accurate to fl per cent.In any given experiment, however, a number of factors operate to reducethe accuracy of the analysis for one or more of the constituents. Theamount of a given amino acid present is the principal variable. When amixture contains ten or twenty components, it is probable that a loading forthe column which is optimum for some will not be the most favorable forall of the amino acids. When the optical density readings on the peakpoints of a curve are as ow as 0.20, a variation of 0.01 unit in the blank cancause an error of 10 per cent in the integration. Some of the peaks inte-grated for Table II fall into this category. The accuracy of the recoveriesindicates that, in practice, the averaging of a series of blanks usuallyestablishes he base-line to considerably better than 0.01 optical densityunit. But the determination is on a sounder basis f the load on the columncan be increased to give a peak reading of 0.50 to 1.00 optical density unit.An increase in the total load, however, as has already been mentioned,can have an adverse effect upon the degree of resolution obtained in thecase of components present in relatively large amounts. An attempt toobtain an adequate picture of the composition of a mixture in a singlechromatogram will usually require a compromise on the question of theoptimum load for the column. If the emphasis s on the determination ofonly a few specific constituents of the mixture, the load can be adjusted togive maximum accuracy for these amino acids. In the case of low peaks,it should also be possible to obtain increased accuracy by using 4 times thepresent sample size on a column 2 cm. in diameter, if 2 cc. effluent fractionsare collected and concentrated to 0.5 cc. before analysis.

DISCUSSIONIdentification of Amino Acid Peaks-A discussionhas already been given(2) of the general measures which can be taken to assist in the identifi-cation of a peak on an effluent concentration curve. The problems as-sociated with the interpretation of the results obtained with unknown mix-tures were enumerated for the butyl-benzyl alcohol experiments (2) andapply with added emphasis to the present curves. The positions of the

peaks shown in Fig. 1, together with the points of emergence of a number ofadditional amino acids6and related compounds, have been summarized inTable IV. The absolute value for the position of a peak is not as useful anaid in identification as it was in the case of simpler chromatograms. Asin the earlier experiments, the relative positions of the peaks are highlyreproducible. The same general pattern has been obtained routinely on6 We are indebted to Dr. A. Killer and Dr. D. D. Van Slyke for a sample of hy-

droxylysine, to Dr. H. T. Clarke for a sample of cysteic acid, and to Dr. H. Borsookfor a sample of or-aminoadipic acid.

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68 CHROUTOGRAPHY OF AMINO ACIDSboth synthetic mixtures and protein hydrolysates. In a given experiment,however, all the peaks may emerge somewhat faster or slower than in-dicated by Table IV. Shifts of as much as 10 per cent have been obtained.These deviations can result from a number of causes,among which may bementioned small variations in the amount of starch introduced during thepouring of the column, slight differences in the composition of the solventmixtures, and errors in the adjustment of the size of the fractions collected.

TABLE IVOrder of Emergence of Amino Acids and Related CompoundsDetermined on columns 0.9 X 30 cm. prepared from 13.4 gm. of starch (anhy-drous), developed with 1:2:1 n-butyl alcohol-n-propyl alcohol-O.1 N HCl and shifted

to 2:l n-propyl alcohol-O.5 N HCl at 83 cc.Compound (cf. Fig . 1)

Leucine-isoleucinePhenylalanineValineMethionineTyrosineProlineGlutamic acid-alanineThreonineAspartic acidSerineGlycineAmmoniaArginineLysineHistidineCystine

Positionof peakcc.

13.516.524262852597582100

106117136149163179

Additional compounds

DiiodotyrosineTryptophanoc-Amino-n-butyric acidLu-Aminoadipic acidCysteic acidTaurineHydroxyprolineSarcosineCitrullineEthanolamineAsparagineGlucosamineHistamineOrnithineHydroxylysine

Positionof peakcc.

12.51838416474808498.5102121126

160176180

The exact point of the solvent shift, of course, affects the positions of thepeaks after aspartic acid. These variations mean that, in a given chromato-gram, a peak emerging at 163 cc., for example, cannot be stated to occurat the histidine position, unless t has been placed either by reference to thesequence of the other peaks from the sample or by observance of the riseof the peak at this position after the addition of known histidine.Considerable variations have also been observed in the absolute positionsof the peaks in Fig. 2. The deviations are believed to result from varia-tions in the moisture content of the samplesof tert-butyl alcohol from whichthe solvent has been prepared. If proline is present, its characteristiccolor in the ninhydrin reaction serves to identify the beginning of theglutamic acid peak.


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S. MOORE AND W. H. STE IN 69The relative positions of the peaks are fully reproducible only if the

column has not been overloaded. The amounts of each amino acid usedfor Fig. 1 are low enough so that the column is capable of yielding fairlysymmetrical effluent curves. As the load of a given component is increased,a point is reached at which the peak in question begins to show a steepfront, indicative of a non-linear isotherm. The tail portion of the peak isidentical in position and slope with the right-hand half of the peak in Fig.1, but the increased load will have advanced the point of maximum, con-centration 1 to 3 cc. ahead of its former position. If this trend is extendedby increasing the load to 10 to 20 times the present level, the position ofthe advancing front is markedly moved ahead. In general, a 2-fold in-crease over the amounts given in Table II does not lead to significant dis-tortion of the peaks, but, as already mentioned, the degree of increase inthe load which is tolerable will depend upon the objectives of the givenexperiment.The second column of Table IV gives the positions of some amino acidsand related compounds not covered by Fig. 1. As the number of possiblecomponents in a mixture is increased, the problems of identification aremultiplied, and no general solution can be offered. By the use of addi-tional solvent mixtures, a number of the overlaps in Table IV may be re-solved. Diiodotyrosine emerges with leucine and isoleucine, but can bedifferentiated on a butyl-benzyl alcohol chromatogram (2). Tryptophancoincides with phenylalanine in the solvent mixture referred to in Table IV,but can be determined with 0.1 N aqueous HCl (cf. Fig. 3). In acid hy-drolysates of proteins the problem seldom arises, since tryptophan is usu-ally decomposed during the hydrolytic process (2). Either cu-amino-n-butyric acid or ol-aminoadipic acid, if present, would appear as a new peakmidway in the valley between tyrosine and proline. Cysteic acid has beenfound to give a clearly defined peak on the right side of the curve for glu-tamic acid plus alanine. Taurine is indistinguishable from threonine inthis solvent, but moves ahead of glutamic acid in 2: 1: 1 tert-butyl alcohol-set-butyl alcohol-O.1 N HCl. Hydroxyproline travels at a rate similar tothat of aspartic acid. Although hydroxyproline cannot be determined inthis solvent mixture, its color yield is only 0.03 at 570 mp, and unless pres-ent in unusually large quantities, it will not interfere with the estimation ofaspartic acid. Citrulline and ethanolamine are slightly to the left and theright, respectively, of serine. The presence of either of these substanceswill be manifested by a broadening of the peak in the serine position.Glucosamine, if present, would appear as a peak midway between ammoniaand arginine. Ornithine and hydroxylysine both coincide with cystine.With protein hydrolysates, therefore, the maximum possible amount ofcystine present should be calculated from the total sulfur minus the meth-ionine sulfur. If the amount of ninhydrin color in the cystine position ex-

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i0 CHROMATOGRa4PHY OF AMINO ACIDSceeds that allowed by the calculation, the presence of additional com-ponents in the cystine range is indicated.The fact that D-, L-, and m-amino acids travel at the same rates onstarch columns (2) has been checked in the present experiments with theL and DL forms of proline, glutamic acid, alanine, threonine, aspartic acid,and serine.

Behavior of Cysteine-When a freshly prepared solution of cysteine hy-drochloride is added to a column with 1: 2 : 1 n-butyl alcohol-n-propyl al-cohol-0.1 N HCl as the solvent, the amino acid is gradually oxidized tocystine and no cysteine peak is obtained in the effluent. No ninhydrin-

Phenyl-&nine

Effluent cc.FIG. 3. Separation of tryptophan, with 0.1 N HCl as solvent, from a syntheticmixture containing eighteen components.

positive material emerges rom the column until after the shift of solventto 2:l n-propyl alcohol-O.5 N HCl. In the range of arginine a long flatzone begins and continues up to the position of cystine, where a definitepeak occurs. The absorption maximum of the material in this broad zoneis at 570 rnp, whereas the absorption maximum of cysteine is at 470 rnp(3). The acidity of the initial solvent is thus insufficient to maintaincysteine in the reduced state. When this amino acid is allowed to standin the 1:2: 1 solvent at atmospheric pressure, about 45 per cent of thecysteine is oxidized in 24 hours. The rate of oxidation on the column isprobably accelerated by the increased amount of air in the solvent whichenters the column under 15 cm. pressure.Cysteine, if present in a sample of amino acids applied to the column,would interfere with the determinations of the basic amino acids. In the


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8. MOORE AND W. 11. STEIN 71chromatographic work with protein hydrolysates, however, the presence ofcysteine has not, thus far, been detected (cf. (4)).Cysteine is fairly stable in the more strongly acid solvent, 2: 1 n-propylalcohol-O.5 N HCl. If the column is run from the beginning with thissolvent mixture, a cysteine peak (absorption maximum 470 rnp) is obtainednear the position of threonine (cf. Fig. 4).

Separation of Tryptophan-The behavior of tryptophan on a column runwith aqueous 0.1 N HCl has been referred to previously (1,2). The columnfor this purpose is poured in butyl alcohol and washed with 1: 1 n-propylalcohol-O.5 N HCl as usual before being shifted to 0.1 N HCl. The curveobtained with the synthetic bovine serum albumin mixture to which tryp-tophan had been added is given in Fig. 3. The first peak contains mostof the components of the mixture. Only the aromatic amino acids areappreciably retarded, and tryptophan emerges as a completely separatedpeak. The color yield of tryptophan in the neutralized 0.5 cc. samples hasbeen 0.72 (3) and the recoveries from the chromatogram have been 100 f3 per cent.

EsteriJication in Acidic Solvents-The fact that the amino acids shouldnot be allowed to stand in an acidic alcoholic solvent before the sample isplaced on the column has been noted previously (2). Aspartic and glu-tamic acids are the only amino acids which have shown measurable esteri-fication on the starch column during the course of the present experiments.The degree of ester formation is a function of the amount of water in thesolvent mixture, the KC1 concentration, the nature of the alcohols, and thetime of contact. In the chromatograms with 1: 2: 1 n-butyl alcohol-n-propyl alcohol-O.1 N HCl the recoveries of aspartic and glutamic acidshave been 6 and 7 per cent low, respectively. The percentage loss is in-dependent of the amounts of the amino acids present. If the column isdeveloped from the beginning with 2:l n-propyl alcohol-O.5 N HCI (cf.Fig. 4), the losses of these two amino acids are 10 and 20 per cent. On acolumn developed with n-butyl alcohol-17 per cent 0.57 N HCl (2), therecoveries are low by 20 and 30 per cent.If the synthetic mixture of amino acids is allowed to stand for 1 week inthe 1:2: 1 solvent before the sample is placed on the column, two smalladditional peaks appear ahead of leucine plus isoleucine in Fig. 1. Theyield of glutamic acid is about 20 per cent low and that of aspartic acidabout 10 per cent low. Al l other components are quantitatively recovered.The small amount of esterification which occurs during the usual chromato-graphic experiment is not manifest in any way other than in the reductionof the yields of aspartic and glutamic acids. The esters, as they are con-tinuously formed, move fairly rapidly through the column and doubtlesscontribute some ninhydrin color to al l the effluent fractions preceding the

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72 CHROMATOGRAPHY OF AMINO ACIDSglutamic and aspartic acid peaks. The quantity of ester is so small, how-ever, and is distributed over so many fractions that the increase in nin-hydrin color for any given fraction is almost imperceptible.

It has already been noted that in the mixture of secondary and tertiaryalcohols used for the separation of glutamic acid and alanine (Fig. 2) esteri-fication appears to be negligible.

Studies on Other Solvent Mix~res-In the chromatographic separation ofthe faster moving amino acids described in the previous communication(2), neutral water-immiscible organic solvents such as n-butyl alcohol andbenzyl alcohol were used with columns 30 cm. in height. In order to elutesome of the slower moving amino acids from such columns, inconvenientlylarge effluent volumes are required. As the concentrations in the effluentbecome more dilute, the analytical accuracy is decreased. By the use ofthese same solvents with shorter columns (7.5 cm. in height), proline,alanine, and threonine can be eluted satisfactorily (1). The number ofeffective plates in a column, however, or the potential resolving power ofthe chromatogram is proportional to its length, and consequently it ispreferable to use the longest column compatible with convenient laboratoryoperation. In order to attain satisfactory rates of travel for the slowermoving amino acids on columns 30 cm. in length, a variety of solvent mix-tures have been studied.

Neutral n-propyl alcohol-water mixtures were investigated on starchcolumns poured in butanol, washed to constant blank with the neutralsolvent, and treated with S-hydroxyquinoline (2). With 2 : 1 n-propyl al-cohol-water, a curve was obtained which was similar to the first portion ofFig. 1, except that glutamic acid and aspartic acid were not present as dis-crete peaks but were spread out in a long low plateau extending from 60to 100 cc. of the effluent. The other amino acid peaks emerged somewhatahead of their positions in Fig. 1, threonine being at 51 cc. The last peak,which emerged at 71 cc., was composed of both serine and glycine. Therealso appeared, between proline and alanine, a large artifact peak whichproved to result from ninhydrin-positive material eluted from the starchby the HCl in the amino acid sample. It was found that a small amountof either HCl or NaCl, when added to the top of the column, was capableof liberating material containing amino nitrogen, which moved down thecolumn as a discrete zone and emerged as an irregular peak just ahead ofthe alanine position. The 2 : 1 n-propyl alcohol-water experiment provideda possible determination of proline, alanine, and threonine. The presenceof the artifact peak and the unsatisfactory behavior of the acidic aminoacids were marked disadvantages.Glutamic acid and aspartic acid were obtained as normally sharp peaksin the alanine-threonine range when 0.25 N acetic acid was substituted

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74 CHROMATOGRAPHY OF AMINO ACIDSamino acids emerged at greater effluent volumes as the amount of water inthe solvent was decreased, this retardation was accompanied by a broaden-ing and flattening of the peaks when the water content was reduced belowabout 30 per cent. Thus, 3: 1 n-propyl alcohol-water gives a curve inwhich a peak emerging at a given effluent volume is slightly lower andbroader than its counterpart in a 2: 1 solvent. If the water content isreduced from 25 to 20 per cent, a comparison of the peaks emerging at thesame effluent positions shows those in the 4: 1 solvent to be about halvedin height and doubled in width. A further reduction in the amount ofwater, to 12 per cent, causes the amino acids to emerge at a fairly steady

88 1.5d.rl3 1.0:..A

0.5

255 30 35365 49.5 56.5 67.5 03 97 115Effluent cc.

FIG. 4. Separation of amino acids on a chromatogram carried out with 2:ln-propyl alcohol-O.5 N HCI.low concentration level devoid of discrete peaks and valleys. Similareffects are noted with n-butyl alcohol when the water content is reducedbelow 15 per cent. Combinations of n-propyl and n-butyl alcohols, asused in the 1:2: 1 n-butyl alcohol-n-propyl alcohol-O.1 N HCl solvent,permit mixtures to be employed which have water contents intermediatebetween 15 and 30 per cent without there being manifest any undesirablebroadening of the peaks.It may prove desirable for some purposes to run the column from thebeginning with 2:l n-propyl alcohol-O.5 N HCI, instead of employing asolvent of lower water and HCI content for the first part of the curve.The results of such an experiment are shown in Fig. 4. The resolution ofthe faster moving amino acids is less satisfactory than in Fig. 1. Thelossesof glutamic and aspartic acids as a result of esterification are greater,as already noted. The fact that ammonia and arginine emerge together

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S. MOORE AND U. H. STE IN 75is a disadvantage. Nevertheless, the solvent may have some utility forscreening work. A general picture of the composition of a mixture ofamino acids is obtained in a 4 day experiment, instead of the 7 days re-quired to obtain the results shown in Fig. 1.

Many experiments with acidic solvents other than the ones alreadydescribed were carried out in an attempt to increase the resolution of theamino acids in the proline-aspartic acid range. Usually a preliminaryexperiment was performed with the synthetic serum albumin mixture.Inspection and integration of the curves were frequently sufficient to elim-inate a given solvent from further consideration. Some of the solventswere investigated in greater detail with simpler mixtures of amino acids.The only combination found which would completely separate glutamicacid and alanine was 3: 1 tert-butyl alcohol-0.1 N HCl. Because of itsviscosity, this solvent gives excessively slow flow rates on the starch columnsand has not been used routinely. The incorporation of 25 per cent sec-butyl alcohol in the mixture has given a satisfactory flow rate and reason-ably good separation of the two amino acids. Various other mixtures of0.1 N HCl with set-butyl alcohol, tert-butyl alcohol, isobutyl alcohol, iso-propyl alcohol, methyl cellosolve, and butyl cellosolve were tried. Themixtures did not offer any general advantages over the solvents referred toin Figs. 1 and 2.

In the present experiments, emphasis has been focused on solvents con-taining no non-volatile acids or salts which would tend to complicate thepossible isolation of constituents from the effluent. A few chromatogramshave been run with buffered solutions and with non-volatile acids. In2:l n-propyl alcohol-O.5 N HsP04, the results were fairly similar to thoseshown in Fig. 4. In 2: 1 n-propyl alcohol-O.5 N trichloroacetic acid, thebasic amino acid peaks were advanced to positions on top of the componentsin the alanine-glycine range. No advantages in the proline-aspartic acidrange were afforded by the use of 2: 1 n-propyl alcohol-O.5 N monochloro-acetic acid. With 2:l n-propyl alcohol-O.2 N citric acid the peaks weremarkedly broadened and resolution was inferior.

When buffered solutions are used on starch columns, sharp peaks areobtained with both the acidic and basic amino acids. In 2: 1 n-propylalcohol-O.2 M citrate buffer, pH 5, the curve was similar to that in Fig. 1,except that glutamic acid and aspartic acid were shifted to the right.Glutamic acid emerged at a position on top of serine and glycine and wasfollowed by the aspartic acid peak. The chromatogram was not con-tinued to cover the basic amino acid range. When a citrate buffer of pH 4was used, the basic amino acids were moved up to give an overlapping zonewith glycine, serine, ammonia, and the acidic amino acids. In 3:2 n-propyl alcohol-O.08 M citrate buffer, pH 8, the relative rates of travel of the

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76 CHROMATOGRAPHY OF AMINO ACIDSbasic amino acids were further increased to give a heavily bunched groupin the center section of the curve. Solvents that are much more alkalinethan pH 8 cannot be used with starch. With 0.1 N NaOH, the starchat the top of the column swells and gelatinizes in the presence of the strongalkali.

Thus, both organic acids and the.citrate buffers of pH 4 and 8 increasethe rates of travel of the basic amino acids relative to the monoaminoacids, thereby increasing the probability of overlaps in the chromatogram.The use of HCI possesses the advantage that minimum rates of travel forthe basic amino acids are obtained, placing them in a region to the right ofglycine.

SUMMARYChromatographic fractionation of amino acids on starch columns has

been extended to include most of the common constituents of proteinhydrolysates. The principal solvent mixture which has been used is 1: 2: 1n-butyl alcohol-n-propyl alcohol-O.1 N HCl followed, after the emergenceof aspartic acid, by 2: 1 n-propyl alcohol-O.5 N HCl. In experiments withsynthetic mixtures containing seventeen amino acids and ammonia, thiscombination of solvents yields in a single chromatogram a curve whichincludes al l the components, with a few overlaps. For analytical work,about 2.5 mg. of the amino acid mixture are required per chromatogram.Integration of the curves has given quantitative recoveries for proline,threonine, aspartic acid, serine, glycine, ammonia, arginine, lysine, his-tidine, and cystine. Glutamic acid and alanine emerge together but canbe resolved in a separate chromatogram with 2:l: 1 tert-butyl alcohol-set-butyl alcohol-O.1 N HCl. The six most rapidly moving componentsare partially resolved and have been separated, as previously reported, oncolumns run with 1: 1: 0.288 n-butyl alcohol-benzyl alcohol-water for thedetermination of phenylalanine, leucine, isoleucine, methionine, tyrosine,and valine. Thus, by the use of three starch columns it is possible toseparate from one another al l the eighteen components.

The average recoveries in duplicate or triplicate determinations havebeen 100 f 3 per cent. The positions of emergence of some of the lesscommonly occurring amino acids and related compounds have been de-termined. Tryptophan, although not usually present in acid hydrolysates,presents a special case and can be determined on a column developed withaqueous 0.1 N HCl. If desired, a variety of other solvents, includingneutral, acidic, and buffered solvent mixtures, can be used satisfactorilywith starch columns.

The authors wish to acknowledge the assistance of Miss Enid Mellquistand Mr. H. R. Richter in the performance of this work. I

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S. MOORE AND W. H. STE IN 77BIBLIOGRAPHY

1. Moore, S., and Stein, W. H., Ann. New York Acad. SC., 49, 265 (1948).2. Stein, W. H., and Moore, S., J. Biol. &em., 176, 337 (1948).3. Moore, S., and Stein, W. H., J. Biol. Chem., 176, 367 (1948).4. Stein, W. H., and Moore, S., J. Biol. Chem., 176, 79 (1949).5. Tiselius, A., in Anson, M. L., and Edsall, J. T., Advances in protein chemistry

New York, 3 (1947).


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