ISOLATION OF AMINO ACIDS BY CHROMATOGRAPHY ON ION EXCHANGE COLUMNS; USE OF VOLATILE BUFFERS

BY C. H. W. HIRS, STANFORD MOORE, AND WILLIAM H. STEIN

(From the Laboratories of The Rockefeller Institute for Medical Research, New York, New York)

(Received for publication, November 14, 1951)

The separation of 0.1 to 0.5 mg. quantities of amino acids for analytical purposes on columns of Dowex 50 has been described in a previous publi- cation (1). The present communication is concerned with the use of larger ion exchange columns for the isolation of 50 to 300 mg. quantities of the components of a mixture.

Amino acids and related compounds may be isolated in several dif- ferent ways by ion exchange chromatography, each method possessing advantages for certain purposes. The system of high resolving power developed for analytical work (1) can also be used for isolation experi- ments, if the non-volatile buffer salts employed as eluants are removed from the amino acids after chromatography by appropriate cycling of the effluent over cationic or anionic exchange resins. Amino acids can be eluted from sulfonat,ed polystyrene resins by 1.5 to 4 N concentrations of HCl (2), the eluant in this case being removable by simple evaporation.’ Another possibility, and the one used in the present work, is to employ as eluants ammonium formate and acetate buffers which can be removed from the effluent by sublimation. The use of ammonium buffers has permitted the development of a mild procedure applicable in principle to the isolation of peptides or other substances which might be labile if exposed to extremes of temperature or pH.

The three aforementioned procedures are based upon the principles of elution analysis. Displacement development on ion exchange columns, as investigated by Partridge a,nd his associates (4-7), has already been demonstrated to be a very effective preparative method, particularly when sufficient quantities of each amino acid are available for isolation. In elution analysis the highest resolving power is obtained when small quan- tities of amino acids are chromatographed, a characterist.ic which can be a virtue or a limitation, depending upon the amount of sample available for study and the degree of resolution required. The displacement de- velopment procedure, on the other hand, reaches its best efficiency when the column is fairly heavily loaded.

1 The resolving power of the HCl system may be improved if longer columns poured in sections (cf. (1)) are employed. The utility of this system has already been demonstrated (3).

669

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670 ISOLATION OF AMINO ACIDS

Elution with ammonium formate and ammonium acetate buffers has been applied to the isolation of the amino acids from an acid hydrolysate of 2.5 gm. of bovine serum albumin. This protein was chosen as a test substance since it possesses an amino acid composition which is known (8) and which is also fairly typical of tissue proteins in general. The scheme of fractionation employed is given in Fig. 1. Columns 7.5 cm. in diameter were used for each chromatogram. The scheme was first worked out on an analytical scale with simple synthetic mixtures and columns of am- monium Dowex 50 0.9 cm. in diameter. The relative positions of the amino acid peaks on the ammonium Dowex 50 columns are very similar to those obtained with the sodium buffer system, although the resolving power is less. With sodium Dowex 50, all the amino acids in a protein hydrolysate can be separated from one another in a single chromatogram. With columns of ammonium Dowex 50, on the other hand, several overlaps are obtained, necessitating rechromatography of those primary fractions which contain more than one amino acid. The separations shown in Fig. 1 are the result of chromatograms operated at a room temperature of about 25”. Higher operating temperatures increase the resolving power somewhat, as was found to be the case with sodium Dowex 50 columns. With ammonium Dowex 50, however, the improvement is not sufficiently great to obviate the need for rechromatography of some of the peaks. Hence room temperature operation was adopted as the simplest procedure. The presence of 40 per cent ethanol in three of the eluants for rechroma- tography accomplishes the separation of those amino acids which emerge together when simple aqueous eluants are used.

For the isolation of a given component, the fractions corresponding to a single amino acid peak in Fig. 1 were pooled, brought to pH 6.5, concen- trated to dryness, and the ammonium buffer removed by sublimation. The residue was recrystallized to yield the amino acid in analytically pure form.

A few points in connection with Fig. 1 merit special mention. The first chromatogram yields the three basic amino acids well separated from one another. The use of buffers of pH 5 to 7, rather than NHIOH (Block (9)), has the advantage that the elution is quantitative. With alkaline eluants it has not been possible to obtain complete recovery of the basic amino acids from cation exchange resins (1). To separate the acidic amino acids, Fraction A from the first chromatogram is rechromatographed on the acetate form of the weakly basic anion exchanger, IRIB (10, 11). If glutamic and aspartic acids are not removed at this stage, they will partially overlap threonine and proline, respectively, on the succeeding 120 cm. column of Dowex 50.

The yield, the elementary analysis for C, H, and N, and the optical rotation obtained for each amino acid isolated are given in Table I. On

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C. H. W. HIRS, S. MOORE, AND W. H. STEIN 671

the average, 66 per cent of each amino acid present in the original protein (methionine, tryptophan, and cysteine excepted) has been isolated in ana-

Protein Hvdrolvsate

cr-l .c I bwex-3u 13 cm.1

bfpf53,OZMAcetate ---+fc;~~~l+pH 6.fJO.5~ Acetate -4

1

mM 4r

nM

AThreonine Alanine

FIG. 1. Isolation of amino acids by chromatography on ion exchange columns. An acid hydrolysate of bovine serum albumin (2.5 gm. on columns 7.5 cm. in diame- ter) was fractionated. Ammonium formate and acetate buffers were used. Amino acid concentrations are given in mu per liter and effluent volumes in liters. The

fractions included within the markers were pooled and worked up together.

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TABL

E I

Amino

Ac

ids

Isolat

ed

f?om

Hy

droly

sate

of

Bov

ine

Seru

m

Albu

min

by

Ch

rom

atogr

aphy

on

Io

n Ex

chan

ge

Resin

s

Amino

ac

id

Lysin

e m

onoh

ydro

chlo-

rid

e Hi

stid

ine

mon

ohyd

ro-

chlo

ride

mon

ohyd

rate

Argin

ine

mon

ohyd

ro-

chlo

ride

Glut

amic

acid

hy

dro-

ch

lorid

e As

parti

c ac

id

Proli

ne

Cysti

neg

Valin

e Le

ucine

Th

reon

ine

Serin

e Al

anin

e Gl

ycine

Is

oleu

cine

Tyro

sine

Phen

ylalan

ine

2,5-

di-

brom

oben

zene

sulfo

-

nate

Am

ount

iso

late

d Yi

eld*

C

H N

From

lite

ratu

re

Ash

Foun

d Fo

und

Foun

d ,&

U-

lated

M

cu-

lated

Fo

und

,

w ce

nt P

15

.4

Value

Re

fer-

me

No.

e

w.

335

per

cent

ter

cd

>er G

f%

82

39.6

39

.4

w ce

nf

er

cent

8.3

8.3

_ tP

er

cell

15.3

er c

ent

degr

ees

degr

ees

0.51

[a

]::

= +2

5.0

[a],”

=

+23.

0

115

84

34.5

34

.4

5.8

5.8

20.1

20

.1

0.00

[a

]::

= +1

3.6

[a];

= +1

3.8

(20)

110

61

34.2

34

.2

7.1

7.1

26.4

26

.6

0.00

[a

],”

= +2

7.2

[a];

= +2

7.6

(21)

250

48

32.7

32

.7

5.6

5.5

7.7

7.6

0.00

[a

]; =

+31.

2 [a

]; =

+32.

0 (2

2)

205 95

80

65

24

0 95

85

110 35

45

94

21

0

74

ii,,

43

77

64

79

69

76

68

73lI

43

36.3

36

.1

5.5

5.3

10.6

10

.5

0.00

[a

]::

= +2

5.3

51.9

52

.2

7.7

7.9

12.1

12

.2

0.32

[a

]; =

-84.

2 29

.9

30.0

5.

0 5.

0 11

.5

11.7

0.

00

[a]::

=

-121

51

.3

51.3

9.

5 9.

5 11

.8

12.0

0.

00

[a];

= +2

7.4

54.8

54

.9

9.7

10.0

10

.5

10.7

0.

00

[a],”

=

+15.

2 40

.1

40.3

7.

8 7.

6 12

.0

11.8

0.

36

[a];

= -2

8.9

34.6

34

.3

6.5

6.7

13.2

13

.3

0.21

[a

]; =

+15.

1 40

.4

40.4

7.

7 7.

9 15

.8

15.7

0.

00

[Ix];

= +1

4.7

31.9

32

.0

6.5

6.7

18.4

18

.6

0.07

55

.1

54.9

9.

8 10

.0

10.7

10

.7

0.90

[a

I’d

= $4

1.3

59.6

59

.6

6.1

6.1

7.6

7.7

0.00

[a

]; =

-6.6

37

.6

37.4

3.

1 3.

1 2.

9 2.

9 0.

00

[a];

= -3

6.8*

’

[a]::

=

t-25.

2 (2

2)

[a]‘d

=

-85.

0 (2

1)

[cx];

= -2

20

(23)

[a

]; =

+27.

4 (2

2)

[cY]Z

: =

+15.

9 (2

2)

[a];

= -2

8.3

(22)

[a

]; =

+14.

8 (1

7)

[a];

= +1

4.5

(23)

[a];

= 44

0.8

(24

1 [a

]; =

-7.0

(2

5)

[a]:

= -3

5.1

(26 1

Anal

ytica

l da

tat

Spec

ific r

otat

iont

-

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* Re

cove

ries

from

2.

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gm.

of

prot

ein

(on

a m

oistu

re-

and

ash-

free

basis

), ca

lcula

ted

with

th

e da

ta

given

by

St

ein

and

Moo

re (8

) for

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vine

seru

m

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in.

t Co

rrecte

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as

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nten

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d ex

pres

sed

on

an

ash-

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. $

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e sa

me

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entra

tions

as

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th

e co

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g re

fere

nces

, an

d ba

sed

on

the

free

amino

ac

ids.

Re

calcu

latio

n of

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me

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lues

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$ S

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ater

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t cr

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(Fig.

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. 1

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ined

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llizat

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plus

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at

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atog

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af

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atog

raph

y (F

ig.

1).

The

two

sam

ples

ga

ve

iden

tical

sp

ecifi

c ro

tatio

ns

and

analy

ses.

**

Spec

ific

rot,a

tion

dete

rmine

d on

a

sam

ple

of

the

free

amino

ac

id

deriv

ed

from

th

e su

lfoni

c ac

id

salt.

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674 ISOLATION OF AMINO ACIDS

lytically pure form. Tryptophan is decomposed upon hydrolysis by acid, and cysteine, if present in the hydrolysate in addition to cystine, is oxi- dized on the chromatogram and does not give a separate peak (1). Methi- onine was not isolated in crystalline form partly because it is present in such small amount (0.8 per cent) in serum albumin (12). A fraction con- taining it as the only amino acid was obtained, however. The optical rotations reported in Table I show that, with the exception of cystine, the pure L antipodes of the amino acids were obtained in every case. The

FIG. 2. Assembly for the chromatography of amino acids on large columns of ion exchange resins. A, B, and C, stop-cocks; P, tube for attachment to air pressure regulator; R, 3 gallon Pyrex bottle; S, No. 12 rubber stopper; T, solvent inlet tube; D, coarse porosity sintered glass disk; E, tube leading to the top of the column.

observed 50 per cent racemization of cystine during 20 hours of acid hydrolysis can be expected on the basis of the extent of racemization known to occur (13) when this amino acid is refluxed with 6 N HCl.

Procedure

Preparation of Columns-The chromatograph tubes (from the Scientific Glass Apparatus Company, Bloomfield, New Jersey) were made from standard Pyrex tubing possessing an outside diameter of 8 cm. Semiball joints are used at both the top and the bottom, as shown in Fig. 2. To minimize strain, pads of rubber sheet 4 mm. thick are inserted between the large ball joint clamps and the glassware. The length of the tubes (exclusive of the joints) should be about 15 cm. longer than the height of

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C. H. W. HIRS, S. MOORE, AND W. H. STEIN 675

resin to be employed in them. Four tubes are required for the present experiments, two 30 cm., one 75 cm., and one 135 cm. long.

1 kilo of Dowex 50 (250 to 500 mesh), as used in the previous experi- ments (l), will suffice for the preparation of about a 20 cm. vertical seg- ment of a column 7.5 cm. in diameter. In preparing the resin for use, water redistilled in glass is used throughout the following procedure. The resin (1 kilo) is added slowly with stirring to 3 liters of 2 N NaOH. When the evolution of heat has subsided, the suspension is stirred at 60-70” on the steam bath for 5 hours. After the cooled suspension has settled for about an hour, the supernatant fluid is decanted or siphoned off. The resin is washed twice by decantation with 4 liter portions of water, and left over- night in 3 liters of 2 N NaOH. Most of the excess alkali is removed by ten decantations with 3 liter portions of water. The resin is filtered on a Biichner funnel and washed with water until the filtrate is nearly neutral. The sample of Dowex 50 used in this work was contaminated with a small quantity of unsulfonated large bead polymer. It is possible to remove this material by suspending the resin in 4 liters of water and passing the suspension through several layers of cheese-cloth. The resin is again fil- tered and 1 liter of 4 N HCI allowed to percolate slowly and evenly through the filter cake, followed by 8 liters of 2 N HCl, washed through with care for the purpose of completely removing the last traces of sodium ion from the walls of the funnel and from the resin. A final wash with several liters of distilled water is needed to bring the filtrate almost to neutrality. The Dowex 50 is stored in this slightly moist form until required.

For the preparation of the columns, each kilo of moist resin is sus- pended in 2.5 liters of 4 M NHhOH, stirred until the initial evolution of heat has diminished, allowed to cool to room temperature, transferred to a Biichner funnel, and washed with 3 liters of distilled water (turbidity may appear in the filtrate). About 2 liters of the buffer to be employed in the column are allowed to percolate through the filter cake, after which the wet material is removed from the funnel and suspended in 1.5 liters of buffer preparatory to pouring the column. The suspension is stirred intermittently for 2 hours to remove air bubbles, and then poured into the chromatograph tube in portions in the manner already described for the preparation of the sodium Dowex 50 columns (1). The columns described in this paper were all poured in approximately 15 cm. sections. The resin should settle evenly to yield a column with a level surface. To complete the process of preparation, the buffer is passed through the column at a rate of 200 cc. per hour until the effluent and influent have the same pH. During the initial stages of this process, there is leakage of a small quantity of resin through the plate, which ceases after about 2 hold-up volumes of buffer have passed through the column. The hold-up volume of the columns 7.5 cm. in diameter is about 200 cc. per 15 cm. of height.

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676 ISOLATION OF AMINO ACIDS

The anion exchanger employed for the separation of aspartic and glu- tamic acids was Amberlite IR-4B obtained in a finely ground form (200 to 320 mesh), designated as XE-59, for which we are greatly indebted to Dr. James C. Winters of the Rohm and Haas Company, Philadelphia.

For the preparation of the column, 800 gm. of the resin are suspended in 3 liters of 2 N acetic acid. After stirring for 2 hours, the resin is suc- cessively washed slowly on a Biichner funnel with 3 liters of 2 N acetic acid, 3 liters of distilled water, and 4 liters of 0.2 M ammonium acetate buffer at pH 5.0. The resin is suspended in 1 liter of the buffer and the suspension allowed to stand for 2 hours to eliminate air bubbles. The column is poured as described above and washed with buffer until the effluent and influent have the same pH.

In the present work with large columns, a time-operated fraction col- lector (Technicon Company, 215 East 149th Street, New York 51) has been employed. The drop-counting model (1, 14), which is much to be preferred for. analytical work, can also be used with the 7.5 cm. columns if desired. When a time-operated collector is used, it is necessary to main- tain a uniform rate of solvent flow through the column for long intervals. Satisfactory performance has been obtained by the use of the air pressure regulating equipment, already described (I), in conjunction with the ar- rangement shown in Fig. 2. The solvents are stored at bench level in 3 gallon Pyrex bottles, and forced by air pressure to the necessary height at the column head. By initial adjustment of the air pressure, any desired flow rate can be maintained irrespective of the volume of liquid in the reservoir. The 3 hole rubber stopper in the neck of the bottle must, of course, be clamped tightly in place. With this equipment, the fraction size has been constant to about f3 per cent. In order to ascertain the effluent volume at which the peaks (Fig. 1) emerged, the average fraction size was determined by measuring the volume of about two tubes per 100.

In the operation of t,he equipment, enough solvent is first added over the top of the resin so that the vertical inlet tube I dips below the surface, thereby maintaining permanently fluid continuity with the solvent in the reservoir. The stop-cocks (A and B) at the reservoir are then closed, whereas the stop-cock C at the column head is left open, while a steadily increasing air pressure is applied at P to raise the solvent to the top of the column and to start it flowing into the inlet tube. When the latter is filled, a slight increase of pressure suffices to displace the air remaining in the top of the tube, whereupon the upper stop-cock C is closed. During the subsequent adjustment, should a decrease in air pressure be required to reach the desired flow rate, the excess pressure in the reservoir air space may be vented through stop-cock A.

The quantities of acid and base needed to prepare the various buffers

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C. H. W. HIRS, S. MOORE, AND W. H. STEIN 677

are given in Table II. In the preparation of all of the buffers, water redistilled in all-glass equipment was used to insure the isolation of ash- free samples of amino acids.

Preparation and Addition of Sample-The sample of bovine serum al- bumin used in the present work is an Armour preparation (ISo. 51) with moisture content 6.74 per cent, ash 0.53 per cent. The hydrolysis is carried out with 200 cc. of 6 N HCl per gm. of protein, as already described (14). The hydrolysate from 2.7 gm. of this preparation (2.54 gm. on an ash- and moisture-free basis) is freed of as much excess HCl as possible

TABLE II

Preparation of Ammonium Formate and Acetate Buffers

Redist.illed reagent formic and acetic acids, and ammonia freshly distilled into water, were used. The quantities given are in each case diluted to a volume of 1 liter with water or, if called for (Fig. l), with water and redistilled ethanol in such amounts that the final solution contains 40 per cent ethanol by volume.

Buff ei-*

0.2 nr ammonium formate 0.2“ L( “

0.2” “ <‘

0.2“ ‘I acetate 0.2“ ‘( “ 0.2 IL it ‘I

0.4 I‘ “ “ 0.5 <‘ “ “

Final pHt

3.08 3.40 4.10 4.48 5.00 5.46 5.60 6.80

-

-

Volumes to be mixed

2 M acid 1 P NHaOH

cc.

375 263 138 220 147 114 228 260

I- cc.

200 200 200 200 200 200 400 500

* The molarity given for the buffer refers to the ammonium concentration. t Determined as described previously (1).

by being repeatedly concentrated to dryness under reduced pressure. It is finally diluted with 100 cc. of distilled water, filtered through a layer of Celite to remove the humin, and the filtrate concentrated to dryness. The almost colorless crystalline residue is dissolved in distilled water and the solution made up to a volume of 60 cc. The samples to go on a column 7.5 cm. in diameter can be varied in volume from 20 to about 150 cc. The pH of this solution was about 2. In order to avoid disturbance of the resin at the column surface during the manipulations of sample addition or solvent change, a 7 cm. circle of filter paper is centered on top of the column. The sample is allowed to sink into the 15 cm. ammonium Dowex column (Fig. 1) in 10 to 15 cc. portions without application of external pressure, and rinsed in with 25 cc. of solvent. The samples to be added

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678 ISOLATION OF AMINO ACIDS

to the other columns are each obtained from preceding chromatograms. The requisite effluent fractions are pooled in each case, and freed of buffer in the manner outlined in the next section. Prior to rechromatography, the fractions are dissolved in the buffer to be used on the column in ques- tion. The mixture of phenylalanine and tyrosine is added to the 60 cm. column at the close of the isoleucine experiment, the buffer of pH 4.5 being used without prior equilibration of the column in this case.

During the operation of the 60 and 120 cm. columns, air held under pressure in the solvent may slowly escape as small bubbles from the solu- tion at the column surface and in the first 3 to 4 cm. of the column itself. This process has not been found to be detrimental to the performance of any given chromatogram, but, prior to the reuse of the column, such air bubbles are eliminated by stirring with a glass rod the first 5 or 6 cm. of the resin beneath the surface and allowing it to settle.

Collection and Analysis of Efluent Fractions-The columns were suitably mounted over a fraction collector provided with a 30 minute reset timer. The rate of flow through the columns was adjusted so as to be 120 to 150 cc. per hour, and the effluent was collected in 20 to 23 cc. fractions. A detergent is not used in the ammonium buffers and the optimum rate of flow is less than was possible with the sodium system. Changes of solvent are made with these large columns in a manner completely analogous to that previously described (1). For the first chromatogram shown in Fig. 1, solvent changes occur after 7.2 and 10 liters of effluent have been collected, and the single change indicated for the 120 cm. column is made after 16 liters.

The analysis of aliquots from the effluent fractions by means of the ninhydrin procedure (15) requires the preliminary removal of the am- monium salts, since ammonia reacts positively in the ninhydrin method. For this purpose, 0.05 cc. aliquots of effluent are pipetted from every second or every fourth fraction into matched photometer tubes with the aid of the pipetting device previously described (15). The size of the aliquot may be increased or decreased, depending upon the load placed on the column. The photometer tubes are placed in Pyrex vacuum desiccators (22 cm. in diameter), the stop-cock assemblies of which have been replaced by standard taper joints bearing 20 mm. tubes which connect to a large trap cooled with solid COZ. The desiccators are totally enclosed (top and bottom and sides) by specially constructed electric heating mantles and are warmed to an internal temperature of 40-50” while being evacuated continuously by an oil pump. After 16 hours (overnight) the tubes are allowed to cool, and the residue is analyzed with 1 cc. of the ninhydrin reagent. The blank reading usually falls between 0.10 and 0.15 optical density units. Chloride ions present in the sample will be responsible for

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C. H. W. HIRS, S. MOORE, AND W. H. STEIN 679

a positive ninhydrin reaction in one or two fractions at the point of emer- gence of the first hold-up volume, because NH&l is not removed by the sublimation procedure. The chloride is present as part of peak A (Fig. 1) and is removed on the subsequent IR-4B chromatogram.

After completion of a run, the 60 and 120 cm. Dowex 50 columns are ready for reuse after a shift of the solvent back to that with which the columns were started. The 15 cm. Dowex 50 column can be similarly treated unless inorganic salts were present in the original mixture chroma- tographed. Inorganic cations may adhere to the resin in the 15 cm. column, and can be removed by extrusion of the resin which is treated with 2 N hydrochloric acid as detailed under the preparation of the resin for column use. The IR-4B (XE-59) column is regenerated after each run, since it binds the inorganic anions, mainly chloride, originally present in the amino acid mixture. For this purpose, the exchanger is washed on a Biichner funnel with 5 liters of 2 N ammonium hydroxide solution, after which it is converted to the acetate form with 2 N acetic acid and equili- brated with ammonium acetate buffer, pH 5.0, in the manner already indicated.

Isolation of Amino Acids from Efluent-The fractions comprising a given amino acid peak are combined, and the collecting tubes each rinsed out twice by passing 20 cc. volumes of water (redistilled in glass) through the particular sequence of tubes in question. The solution so obtained (500 to 2000 cc.) is evaporated under reduced pressure to a volume of about 50 cc., diluted with about 500 cc. of water, and again evaporated to a volume of 50 cc. under reduced pressure to remove the excess free acid originally present. The evaporations may be effected conveniently in an apparatus of the type described by Craig, Gregory, and Hausmann (16). The concentrate is neutralized to pH 6.5 by careful addition of 1 M NHdOH solution and washed with ten 20 cc. rinses of water into a 2 liter round bottomed flask provided with a male 71/60 standard taper joint. The flask is attached to the condenser bulb of the rotating evaporator (16) with an adapter, and the solution taken to a thick syrup under reduced pressure. At this point slight agitation frequently causes the syrup to crystallize, especially on cooling. In this event, a small quantity of water is added to render the mixture mobile. The thick suspension is now swirled while the flask is evacuated (through a rubber tube) with an oil pump, protected by a cold trap. A semicrystalline solid film can thereby be deposited uniformly over the sides of the flask. Before proceeding with the sublimation, it is essential to dry the film of buffer salt thoroughly to prevent subsequent spattering. For this purpose, the flask is attached to a freeze-drying manifold equipped with an efficient pump and evacuated for 3 to 4 hours. Thereafter, the adapter is replaced with one carrying

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680 ISOLBTION OF AMINO ACIDS

a water-cooled finger type condenser, which reaches to within 4 cm. of the bottom of the flask and has a diameter of about 30 mm. The flask is enclosed in a spherical heating mantle and evacuated on the freeze-drying manifold through a side arm on the adapter. After evacuation, the cur- rent through the mantle is set to warm the flask to 30-40” (outside tem- perature), under which conditions the ammonium formate or acetate in the film readily passes over onto the condenser, to which it adheres as a hard deposit of large crystals. Depending on the uniformity of thickness attained in spreading the film, the sublimation is complete in 15 to 20 hours for ammonium formate, and in less than 12 hours for ammonium acetate.

The amino acids are left as a clean, white deposit, with the exception of histidine, which forms a tan-colored residue on the sides of the flask. The amino acids are dissolved out of the flask with 10 to 20 cc. of warm water (dilute HCl for cystine and tyrosine). Occasionally, some Dowex 50 particles may have been carried through the procedure. These, and small quantities of dust and fibers, are removed by filtering the solution through a 2 mm. layer of Celite. The solutions are decolorized with acid-washed charcoal (20 to 40 mg.) at this stage, if necessary. In most instances the filtrate was concentrated to 1 to 3 cc. and the amino acid crystallized by the addition of 1 to 5 cc. of ethanol. For aspartic and glutamic acids, 0.5 cc. of glacial acetic acid was also added. Glutamic acid was converted to the hydrochloride for analysis.2 Proline was taken to dryness, dis- solved in 3 cc. of ethanol, and crystallized by the addition of 3 cc. of ether. The basic amino acids were dissolved in 2 N HCI, taken to dryness, redis- solved in 2 to 5 cc. of warm ethanol by the addition of a few drops of water, and the monohydrochlorides precipitated by the addition of about 0.5 cc. of pyridine. Histidine monohydrochloride was recrystallized from 1 cc. of water with the addition of 1 cc. of ethanol. Arginine monohydro- chloride was recrystallized from 0.3 cc. of water with the addition of 0.2 cc. of ethanol and 1.2 cc. of ether.

Phenylalanine contained a trace of tyrosine from the preceding peak. This pair of amino acids is one of the most difficult to separate on Dowex 50 columns (1). A pure derivative of phenylalanine was obtained by crys- tallizing the 2,5-dibromobenzenesulfonic acid salt (17). To recover the free amino acid, the salt (200 mg. in 50 cc. of water) was passed through a 0.9 X 6 cm. column of IR-4B (XE-59) acetate, and the column washed with 70 cc. of water.

* Glutamic and aspartic acids are not completely separated on the 15 cm. IR-4B column and a small overlapping area is discarded in working up the fractions. The separation could be rendered fully complete, if desired, by using a slightly longer column.

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C. H. W. HIRS, S. MOORE, AND W. H. STEIN 681

Valine, as first isolated, contained some cystine, as would be expected from the curve in Fig. 1. The resolving power of the ammonium Dowex 50 column is not quite sufficient to accommodate the whole of the broad cystine peak between the glycine plus alanine and the valine peaks. The position of cystine is extremely sensitive to pH (1). At pH 3.43, with the ammonium buffers, cystine partially overlaps glycine plus alanine. The selection of pH 3.40 was made to insure clean removal of cystine from alanine and glycine. The separation of cystine from valine could be ac- complished by rechromatography of the pair at a somewhat higher pH, under which conditions cystine moves well ahead of valine. In the present instance, the valine was conveniently purified for analysis by submitting the mixture to a nine funnel counter-current distribution, by the method of Craig et al. (18), with 50: 50 n-butyl alcohol-see-butyl alcohol-aqueous 5 per cent HCI. In this solvent system, the partition coefficients for valine and cystine are 0.43 and 0.06, respectively.3 This difference in partition coefficient is sufficiently great so that in one nine funnel dis- tribution half of the valine may be recovered cystine-free from a sample initially contaminated with cystine to the extent of 10 per cent.

Tyrosine and cystine, because of their very slight solubility in water, also crystallize directly at an early stage of the chromatography, as in- dicated in Fig. 1. Soon after the protein hydrolysate was added to the first chromatogram, a uniform band of white crystalline material separated in the Dowex 50 column about 4 cm. from the top. This band, pre- sumably cystine and tyrosine, gradually disappeared before the emergence of any amino acids in the effluent. When the tubes containing peak A (Fig. 1) were allowed to stand overnight at room temperature, or preferably at 4”, cystine crystallized in about 50 per cent yield. The crystals, after filtration and washing with 15 cc. of hot water, gave the correct elementary analysis (Table I). Similarly, pure tyrosine crystallized from the tubes of the second peak in Fig. 1. The tubes were allowed to stand at 4” for 3 days before the crystals were filtered. The remaining amounts of ty- rosine and cystine obtained from the later chromatograms (Fig. 1) were re- crystallized by solution in aC1 and neutralization with ammonium acetate.

The amount of protein hydrolysate taken for fractionation depends upon the purpose of the experiment and the relative quantities of amino acids contained in the hydrolysate. In the present work, twice as large a sample (5 gm.) could probably have been employed without serious sacri- fice of resolving power. Overloading the column will result in broad and irregular peaks, and consequently poorer resolution of those amino acid peaks which are close together. In general, for optimum results the load

3 We are greatly indebted to Dr. W. E. Hausmann for furnishing us with this unpublished information.

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682 ISOLATION OF AMINO ACIDS

per individual amino acid should not exceed 0.05 mM per sq. cm. cross sectional area of the column.

For peaks which are close together in Fig. 1, the resolution can be in- creased by using longer columns. For example, the favorable recovery of isoleucine in Table I was possible because of the small amount of methi- onine present in bovine serum albumin. If there were more nearly equal concentrations of the two components, a longer column would have been required.

The authors wish to acknowledge with appreciation the assistance of Mr. S. Theodore Bella, who performed the microanalyses reported in this paper.

SUMMARY

A chromatographic fractionation procedure is described which permits the isolation of about 100 mg. quantities of amino acids from protein hydrolysates. Its relationship to other chromatographic methods for the isolation of amino acids is discussed. Four columns of ion exchange resins are employed. A 7.5 X 15 cm. column of Dowex 50 is used to separate the basic amino acids, a 7.5 X 15 cm. column of Amberlite IR-4B (XE-59) to separate the acidic amino acids, and two Dowex 50 columns, 7.5 X 120 cm. and 7.5 X 60 cm., to separate the neutral amino acids. Elution is effected with ammonium formate or acetate buffers in the range, pH 3 to 7. The buffers are removed by sublimation at 40”, and the residual amino acids are readily recovered in crystalline form. The fractionation scheme was applied to the isolation of the amino acids present in an acid hydrolysate obtained from 2.5 gm. of bovine serum albumin. All the amino acids (methionine excepted) were obtained in analytically pure form in an average yield of 66 per cent. The pure L antipode was re- covered in each case, with the exception of cystine, which was about half racemized during the hydrolysis.

BIBLIOGRAPHY

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SteinC. H. W. Hirs, Stanford Moore and William H.

VOLATILE BUFFERSEXCHANGE COLUMNS; USE OFCHROMATOGRAPHY ON ION

ISOLATION OF AMINO ACIDS BY

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