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

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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
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

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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
 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

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1

mM
4r 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.
 TABLE I
Amino Acids Isolated f?om Hydrolysate of Bovine Serum Albumin by Chromatography on Ion Exchange Resins

Analytical datat Specific rotationt

Amino acid Amount Yield* C H N From literature

isolated
Ash Found
,&U- ,Mcu- Refer-
Found Found lated Found lated Value eme No.
_
w. per cent ter cd >er Gf% w cenf er cent w cent Per celltP er cent degrees degrees
Lysine monohydrochlo- 335 82 39.6 39.4 8.3 8.3 15.4 15.3 0.51 [a]:: = +25.0 [a],” = +23.0
ride
Histidine monohydro- 115 84 34.5 34.4 5.8 5.8 20.1 20.1 0.00 [a]:: = +13.6 [a]; = +13.8 (20)
chloride monohydrate
Arginine monohydro- 110 61 34.2 34.2 7.1 7.1 26.4 26.6 0.00 [a],” = +27.2 [a]; = +27.6 (21)
chloride
Glutamic acid hydro- 250 48 32.7 32.7 5.6 5.5 7.7 7.6 0.00 [a]; = +31.2 [a]; = +32.0 (22)
chloride
Aspartic acid 205 74 36.3 36.1 5.5 5.3 10.6 10.5 0.00 [a]:: = +25.3 [a]:: = t-25.2 (22)
Proline 95 51.9 52.2 7.7 7.9 12.1 12.2 0.32 [a]; = -84.2 [a]‘d = -85.0 (21)
Cystineg 80 ii,, 29.9 30.0 5.0 5.0 11.5 11.7 0.00 [a]:: = -121 [cx]; = -220 (23)
Valine 65 43 51.3 51.3 9.5 9.5 11.8 12.0 0.00 [a]; = +27.4 [a]; = +27.4 (22)
Leucine 240 77 54.8 54.9 9.7 10.0 10.5 10.7 0.00 [a],” = +15.2 [cY]Z: = +15.9 (22)
Threonine 95 64 40.1 40.3 7.8 7.6 12.0 11.8 0.36 [a]; = -28.9 [a]; = -28.3 (22)
Serine 85 79 34.6 34.3 6.5 6.7 13.2 13.3 0.21 [a]; = +15.1 [a]; = +14.8 (17)
Alanine 110 69 40.4 40.4 7.7 7.9 15.8 15.7 0.00 [Ix]; = +14.7 [a]; = +14.5 (23)
Glycine 35 76 31.9 32.0 6.5 6.7 18.4 18.6 0.07
Isoleucine 45 68 55.1 54.9 9.8 10.0 10.7 10.7 0.90 [aI’d = $41.3 [a]; = 440.8 (24 1
Tyrosine 94 73lI 59.6 59.6 6.1 6.1 7.6 7.7 0.00 [a]; = -6.6 [a]; = -7.0 (25)
Phenylalanine 2,5-di- 210 43 37.6 37.4 3.1 3.1 2.9 2.9 0.00 [a]; = -36.8*’ [a]: = -35.1 (261
bromobenzenesulfo-
nate
-

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 * Recoveries from 2.54 gm. of protein (on a moisture- and ash-free basis), calculated with the data given by Stein and Moore (8)
for bovine serum albumin.
t Corrected for ash content and expressed on an ash-free basis.
$ Determined throughout at the same concentrations as in the corresponding references, and based on the free amino acids.
Recalculation of some of the literature values has been necessary.
$ S found, 26.9 per cent; calculated, 26.7 per cent.
/I Material obtained by direct crystallization (Fig. 1).
1 Corresponds to quantity obtained by direct crystallization plus that obtained from the chromatogram after rechromatography
(Fig. 1). The two samples gave identical specific rotations and analyses.
** Specific rot,ation determined on a sample of the free amino acid derived from the sulfonic acid 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

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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
 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 suspensionis stirred at 60-70” on the
steam bath for 5 hours. After the cooled suspensionhas 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 excessalkali is removed by ten

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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 suspensionis 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 ceasesafter 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.
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.

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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
 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

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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.

Volumes to be mixed
Buff ei-* Final pHt
2 M acid 1 P NHaOH
-

0.2 nr ammonium formate

“
3.08
cc.
375
I- cc.
200
0.2“ L( 3.40 263 200
0.2” “ <‘ 4.10 138 200
0.2“ ‘I acetate 4.48 220 200
0.2“ ‘( “ 5.00 147 200
0.2 IL it ‘I 5.46 114 200
0.4 I‘ “ “ 5.60 228 400
0.5 <‘ “ “ 6.80 260 500
-
* 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
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

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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 possiblewith 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 assembliesof 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
 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

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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
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

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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.
 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

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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.
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.

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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.

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 ISOLATION OF AMINO ACIDS BY
CHROMATOGRAPHY ON ION
EXCHANGE COLUMNS; USE OF
VOLATILE BUFFERS
C. H. W. Hirs, Stanford Moore and William H.
Stein
J. Biol. Chem. 1952, 195:669-683.

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