Water Elution Chromatography of Amino Acids on Ion Exchange Materials DONALD L. BUCHANAN Veterans Administration Hospital, West Haven, Conn., and Department o f Biochemistry, Yale University, New Haven, Conn.
ROMAN T. MARKIW Veterans Administration Hospital, West Haven, Conn.
b Successful chromatography of amino acids has been performed on the free acid and base forms of a number of ion exchange resins with water as the only eluent. As predicted by theory, resins of appropriate acid or basic strength differentially retard amino acids and allow separations to occur. Although a complete scheme of separating all of the common protein amino acids has not evolved, the data indicate that this might be achieved if resins with improved physical properties can be prepared. Some of the experiments have practical applications. The feasibility of producing a resin column of desired acid strength by mixing strong and weak acid resins and the possibility of automatic assay of effluent by conductometric methods are demonstrated. Data are presented on the effect of column loading.
A
of both resin and solute are the primary determinants of the affinity these materials have for each other. Strongly acid resins donate protons and a positive charge to strongly or weakly basic solutes. Strongly basic solutes accept protons even from weakly acid resins. I n all these instances and in the analogous interactions between basic resins and acid solutes, resin and solute easily acquire opposite charges and are firmly held together. When the resin and solute are both weak there is only a small tendency to exchange hydrogen ions; both substances remain urichnrged and the unbound solute is free to move. Thus \Then an aqueous solution of nionoaniiiiomonocarhox~lic acids is poured onto a column of a sulfonated polystyrene resin in the hydrogen form, the amino acids are retained a t the top and only an excessively large amount of water would then wash through. K i t h carboxylic acid resins, the same kind of mixturo easily passes through the column. I t seemed likely that with watc,r as the sole eluent the free acid and base forms of resins of weak and intermediate acidity or basicity might separate CIDITY ASI) BASICITY
1400
ANALYTICAL CHEMISTRY
amino acids and other substances by allowing them to migrate through columns at differential rates. A communication by Thompson and Morris (13) that appeared while this work was in progress supports this hypothesis. The experiments described here further demonstrate the feasibility of this approach. Although a scheme for resolving all the amino acids of an acid hydrolyzate of protein has not been attained, some of the separations do have practical applications. The data are of interest in pointing out the possibility of developing further useful methods. EXPERIMENTAL
Resins. Coniniercially available resins and donated samples of experimental resins were used. s o m e were obtained pulverized or in fine beads, while others n-ere initially in the form of large beads or granules. I n the latter instances, the material was ground in a Kiley cutt'ing mill and recycled through the mill until i t viould pass a 100- or a 140-mesh screen. Some of the resin was graded by differential settling (6). All acid resins were n-ashed first with 2M sodium hydroxide and then with 4h' hydrochloric acid until sodium free (flame test) and then with water. Except in the instances indicated, basic resins were first treated with normal hydrochloric acid. washed with normal sodium hydroxide until chloride free (acidic silver nitrate), and then washed nith water. These procedures ensure the conversion of cation exchange resins to the hydrogcn form and weak anion exchangers to thc frw base form. The sources and other details of individual resins appear with thr rcwlts. Equipment and Column Operation. Columns \yere glass t u h w of several sizes with sintered plates and T fittings. F o r chromatograph)- a t lowered or elevated temperatures, water was circulated through jacketed columns. I n most cases gravity provided adcquate flow, but pressure u p t o 1 abm. was appliod when necessary. Standard time actuated or drop counting fraction collectors w r e used. Details of the procrdures were described in conjunction with carrier displacement chromatographj- ( 2 ) .
Detection Methods. Eluted amino acids were detected by paper spot tests nith ninhydrin (8) and identified by standard paper chromatography. Quantitative measurement of eluted solutes was performed by: weighing the fractions after evaporation of water, titrimetry, ultraviolet absorbance, and colorimetry nith ninhydrin (6). Preliminarj. experiments have deinonstrated the possibility of measuring amino acid concentrations by automatic recording of the conductivity of the column effluent after it has come into contact with insoluble salts of. copper. Amino acids solubilize these salts. forming cationic complexes (12). The resulting solutions h a w a much greater conductivity than thoFe containing only amino acids. A plastic and platinum microconductivity cell was constructed according to a sketch by Partridge and Westall (IO). Effluent from the column first passed through a 2-cn1. oegment (5-mm. diameter) of cellulose impregnated with basic cupric carbonate or cupric phosphate. The solution then passed through the conductivity cell, which was in a Wheatstone bridge circuit with a recorder. An increase in conductivity caused the recorder to respond positively, although the deflection was not linear with concentration and the response on a molar basis differed among various amino acids. ABBREVIATIONS
The following abbreviations are used: Asp. aspartic acid; Ser, serine; Ala, alanine; Leu. leucine; Glu, glutamic acid; Pro, proline; Thr, threonine; Gly, glycine; T'al, valine; Tyr, tyrosine; Try$ tryptophan; Phe, phenylalanine; Met. methionine; Ileu, isoleucine; Cys, cystine; Lys. lysine; .4rgj arginine; His. histidine; CAI, carboxymethylcellulose : DEA4E. diethylaminocthylccllulosc. RESULTS
Eccausc no complete echenie of separation has evolved, the data will be grouped according to the type of resin tested. Some of the results show only partial separations or spreading and trailing because of slow exchange rates or rapid flow. Such data are included
because they show tendencies toward separation, which may lead to more useful procedures if resins with more desirable physical characteristics can be prepared. Sulfonated Polystyrene Resin. Because t h e d a t a of Moore and Stein ( 7 ) show that taurine and urea separate and run far ahead of the carboxylic amino acids on Dowes 50 (The Don. Chemical Co., hlidland, Mich.) when they are eluted by buffer a t p H 2.2, chromatography of these substances \vas conducted on the hydrogen form of this resin with water alone. The results, plotted in Figure l , a , show a \vide separation of 100 mg. of each substance. Taurine is retarded somewhat by the resin, as is shown in Figure l , b , n-hcw 2.0 mmoles of HC1 are well wparated from 2.4 mmoles of taurine. C'cinsidered with the data cited ( 7 ) . this result indicates that a mixture of cysteic acid, glycerophosphoethanolamine, phosphoethanolamine, taurine, and urea might possibl!. separate on sulfonated resin columns by Tvater plution. Phosphonic and Phosphonous Acid Resins. Two phosphonic acid resins were tested. T h e first: Salcite X219, a n experimental resin (National .\luminate Corp., Chicago), was arailnhlc in a fine powder and in limited quantity. When aspartic acid, serine, alanine, and leucine were added: the fii,st two compounds emerged separately, followed by considerable ninhydrin negative effluent (Table I, A ) . C'ollrction was discontinud because of tlic extremely slow flow rate; alanine :ind leucine Jvere left on the column. A larger column of an analogous but co:i~'scr experimental resin (100- to 110niesh) Duolite C-63 (Chemical Process Co.. Redwood City, Calif.), was t c d e d with several mixtures i'rablc I, B, C, D. and E). Aspartic acid separated froni alanine, B, but not completely from ,swine, C. blaninr separated froni lcucint'. B, which reniainrd on the column. From the patterns in general, Duolitc) C-63 seemed less acid than S d c i t e X-219 and the spreading and t i d i n g of amino acid zones wert greater. Hcating the column to 50' c. improved the r~solving power to somci extent ( ( m i p a r e II and C). .I shorter column ot I h o l i t c C-63. E , wa. prrparcd from niatcrinl that had .et1 a 140-mesh s ~ r ~ w i'Tlic' . rolumn rvsolvfd aspartic acid, +ritw. and alan i i i c , Iwtter than had tlw l o n g ~ rcdunin of ro:irwr resin, I:, but n1:mitic. :ind scrinc, TI ( w l i c i t :is \vel1 scpar.:itt.cl ai on S:tlcitc S-219. .\, nhere & y ) i t ( >tlic sninller size. of t h column t1lt.r~ was a larger wlurnr of ninhydriil niigative offluent follou ing wrine. It i> possi1)lc that a Iicxtctl c d u m n of cwtJfully gratl(d C-63 i,(+iii might allow iiir~luiwparations to 1 K' I)cirformed. ~
6
11
30-
11
3
c
\
Furine
5oc1
40-
0; 20-
E lo-
Tau r i ne
1
1
,I ~
6.
a.
Urea
Experiment -4
Q,
n
I
:L2-
l
-0.1
C
0
Column Length, Resin and Added Volume, and Amino Acids Temp., C. Nalcite Y-219" 28 cm., 23 ml. iphosphonic acid) Asp, Sei, Ala, T , m . 22 "-24 10 mg. ea O
Fraction Number 1-56 57-64 65-128 129-200 201-260
Effluent Amino Volume, Acids 111. 0
0
1 70 37 I00 112
0
90
Asp Ser
Duolite C-63* 105 em., 140 ml. (phosphonic acid) Asp, Ala, Leu, 100 22"-24" mg. ea.
1-26 27-77 78-144 145-2004-
0 iisp 0
460 765 980 820
Duolite C-63*
1-74 75-109 110-280 28 1-360
0 ASP .4sp, Glue
470 1 70 810 360
105 cm., 140 ml. 220-240
mg. ea.
Duolite C-63*
105 mi., 140 ml.
.Asp, Glu, Sei, Thr, Pro, Gly, Ala. T'al. Leu. Met, 100 mg.
ea.
E
Diiolite C-63d
50
28 mi., 23 ml.
Asp) Ser, Ala, 10
mg. ea.
229-240
F
E E
Chromatography of Selected Amino Acids on Phosphoric and Phosphonous Acid Resins in Hydrogen Forms
Asp, Glu, Pro, Thr, Ser, 100
D
c
\
' I "
Discontd. B
3
.6
L i,.
I
Table I.
-20
Diiolite C-63*
Scr, Thr, 100 mg. ea.
105 cm., 140 ml.
0"
1-:11 32-40 41-45 46-70 71-80 81-86 87-150 151-li4 175-
1-32 33-51 52-90 91-119 120-200 2011-240 241-360 360-
Ala Discontd.
Ser, Thr, Pro 0 Asp
ASP, Glu Glu, Ser, Thr Thr. S w Gly' - Gly, Ala
Ala 0
+
324 98 55 270
1i n
_ ~ .
70 770 260
Discontd 0 Asp
Ser
0
Ala 0 0
Ser, Thre 0
58 115 85 245 , . .
1320 720 ..
G
Duolite C-6zh 93 em., 130 ml. 1-8 0 120 (phosphonic 9-24 . h p , Ser 245 acid) 25-160 Asp, Ser, Ala 3800 Asp, Ser, Ala, Leu, 22O-24O 161-200 Ala, traces Leu GOO+ 100 ing. ea. Discontd. Very fine rwin and slow flow rate (0.5ml./hour); fractions collected manually and not uniformly timed. Coarse resin (100 to 140 mesh) and fast flow (60 ml./hour). c Boundaries between aspartic acid- serine and glutamic arid-threonine not sharply demarcated. Resin finer than b (through 140 mesh less "fines"); flow rate 15 ml./hour. No separation.
VOL 32, NO. 11, OCTOBER 1960
1401
70
-
-
'GI u t a rni c acid
0
2
30 laninc
-
x
:: 20 -Aspartic =
50-
5
Q,
n
3
acid
-
c
\
d xE
-
P- Alanine
1
tube #
3 4 HOURS
5
6
7
Figure 3. Elution diagram of selected amino acids (0.4 lmole each) on Amberlite XE-89, 100 to 140 mesh, hydrogen form
Figure 2. Elution diagram obtained by water elution of 100 mg. each of three amino acids on Amberlite IRC-50 (CG 50 Type I), hydrogen form Column 140 cm., 130 ml.; fractions 1 1 ml., 20 rnin.1 eluted solutes weighed in each fraction
Because both serine and threonine moved a t moderate rates through this resin, an attempt was made to separate them a t 0' C. as had been previously done on Dowex 50 saturated with 1isopropylpyrazole ( 2 ) ,but no separation
2
Column 1 IO cm., 15 ml.; segment (3 cm., 1 mi.) o f cellulose impregnated with CuCO+Cu(OH)n placed between column and conductivity cell; measurement conductometric (see text)
was evident, F. Aspartic acid, serine, alanine, and leucine failed to separate well, G, on an experimental phosphonous acid resin, Duolite C-62 (Chemical Process Co.). All amino acids emerged after a smaller effluent volume than had
been noted with the C-63 resin, indicating that the acidity of this resin is less than that of either of the two phosphonic acid resins tested.
Carboxylic Acid Resins. Two of
Table II.
Experiment
.4
Chromatography of Selected Amino Acids on Amberlite IRC-50 in Hydrogen Form a t Room Temperature
Resin and Added Amino Acids CG 50-Type I a (IRC 50)
Column Length and Fraction Volume Number 100 cm., 110 ml.
Tyr, Try, 20 mg. ea. B
Phe, 100 mg. CG 50-Type II* Ser, Thr, Ala, Gly, Val, Met, Ileu, 1 mg. ea.
C
CG 50-Type IIIc Asp, Glu, Pro, Ser, Thr, Ala. Glv. Val. Met. Leu. 100'mg: ea. Cys, 10 mg.
550 cm., 75 ml.
600 cm., 450 ml.
1-11 12-24 15 16-23 24-29 30-50 1-190 191-197 198-212 213-221 222-285 286-306 307-360 361-362 363-422 423-540 1-141 142-146 147-149 150-156 157-159 160161-169 170-192 193 194-202 203-220 221-231 232 ~~
233-288
a
b
Effluent Amino Volume, Acids Ml . 0
Tvr
Tyr, Phe
Phe 0
Try 0
Ser Ser, Thr Thr Ala, Gly 0
Val 0
Met Ileu 0
ANALYTICAL CHEMISTRY
75
240 66.0 2.3
5,1
2.3 21.7 7.0 18.0 0.7 19.3 28.0 436
Asp 11 Asp, Glu 6 Glu. Ser 15 Thi. Ser 6 2 Thrj Gly 19 Gly, Ala, Thr Gly, Ala 48 2 Cys, Ala, Pro, Val 19 Cys, Pro, Val Pro, Val 38 Val 22 Val, Met, Met 2 Sulfoxide Met, Met Sulfox- 112 ide 350 Leu
289-464 Coarse resin 20 minutes/fraction. Resin particles 50 to 100 microns in diameter, 8 minutes/fraction. Resin particles 25 to 50 microns in diameter, 30 minutes/fraction.
1402
110 36 12 90
these were tested, the first being Amberlite CG-50 (chromatographic grade of IRC-50. Rohm & H a a s Co., Philadelphia) The three aromatic amino acids separated moderately well on this rcsin a t room temperature (Table 11, A). Chromatography a t 70" C. gave almost identical results and after recrystallization the recoveries (dry weight) of the three aromatic amino acids were: 71% of 100 mg. of phenylalanine, 707, of 20 mg. of tyrosine, and 78Ycof 100 mg. of tryptophan, each pure by paper chromatography. I n preliminary trials, there was a slight tendency for some of the nonaromatic, a-amino acids to separate on 100-em. columns and a m i x t h e of glutamic acid, $alanine, and ?-aminobutyric acid was easily resolved (Figure 2 ) . X mixture of 1 mg. each of several neutral amino acids was partially resolved on a very long, narrow column of finer IRC-BO (Table, IT, B). Valine: methionine. and isoleucine emerged individually and were -a-ell separated from the pair, alanine-glycine, which in turn was separated from serine and threonine; even these were partially resolved. Trailing and spreading were prominent, making it unlikely that this resin would be very useful for analytical purposes. The potential use of this type of resin for preparative chromatography was t'ested on an even longer column, 20 feet, 1 cm. in diameter, and packed with still finer material; this made the flow rate per cross-sectional area much
DUOLlTE
A- 4
Emm I 20
47J
I
-[
LYS?
20
I
maJ?EI m m L EI?2
to
I
I do
8'0
6'0
DEA ClDlTE
i L
'
I
METHIONINE THREONINE
J
1
SERINE
do
4'0
BO
I00
3
DOWEX
1
LEUCINE
METHlO&INE T H R t ONiNE
SERINE
II
I
4b
Id0
8'0
6'0
AMBERLITE
I
I Ad A N I N E GLYCINE
4b
VALIkE
A
PROLINE
r
2%
I&
IR- 45
AMBERLITE
:&?Em A
ALANINE
ab
6%
1I
XE-168
-
I
I I
f
__
I
PHENYLALANINE
._
~.
DlETHYLAMlNOE THYLCELLULOSE
I
Milliliters
Figure 4. Elution diagrams of 10 amino acids (see Amberlite XE-168) on six weakly basic resins and on DEAE Columns, 1 5 mi., 2 5 cm.; 10 amino acids (2.5 mg. of each) a d d e d in 0.5 mi. o f solution; positions o f basic amino acids on Deacidite superimposed from d a t a obtained on larger column of Deacidite (see Figure 5); positions d e t e m i n e d by p a p e r chromatography
slon.er. The column mas much more heavily loaded than the narrower column and more amino acids w r e added, but the separation was nearly as good (Table 11, C). S e x t to be tested was an experimental resin. designated -linherlite XE-89 when in coarse beads and XE-112 when ground to 325 mesh (both from Rohm 8r Haas Co.). ThP resin is an acrylic polymer of lower cross linkage than IRC-50. -4ccordiiig to data furnished by t h r company it is more acid (apparent pK, 5.2) than Xmberlite IRC-50 (apparent pK, 6.0). Because the degree of cross l i n k a p of acrylic polymers is related in a systematic way to the apparent dissociation constants ( d ) , it can be wtimaterl that SE-89 is approximately iyc cross-linked while IKC-50 is 25 to 30% cross-linked. In the first trixls the flow through XE-112 was extremely s l o ~so , XE-89 w-as ground to 100- to 140-mesh and this material was utilized in subsequent tests. The data of Table 111 show that a 100cni. column of this material tends to separate the neutral and acidic amino acids in a pattern nearly identical to that wen in displacc~neritchromatog-
Table Ill.
raph?. on sulfonic resin columns (9). Separation of a selected groui) of amino acids is easilj- achieved with this resin (Figurr 3). The tracing showed peaks corrcsponding to each amino acid added, but anomalous peaks appeared with alanine and isoleucinr, possibly the result of two specics of metal-complex ions. Carboxymethylcellulose. 4 solution (1 mi.) of 10 nig. of each protein amino acid esccpt tyrosine and ( J,stine n-as added to a short (11 cm., 30 ml., 3.5 grams d r y \\.eight) column of C M in the hydrogen form (Brown Co., Berlin, S.€ I . ) , iifter thc column had befn n-ashcti \vith 140 nil. of v a t e r t h e e fflu eii t 1)ec a m ninhydrin 11 eg a t ive . The basic. amino acid? remaiiied 011 the column : histitlinr~ is disl)l:icc4 with 0.1M Iiydrazinc ;inti then lysiiii. :ind argiiiini. \r-(>rc hrought off with 0.1.1.2 aninioni:i. -\ loiigc,r c*olumn 186 cm., 91 nil.) uf this niatr~inlf a i l d to ploducc any ~1):11xtiim of ;i niisture of two :iridic :ind niiic nitutr:d aliplixtic :mincl a d s . W e a k Base Resins and DEAE. The free base forms of sever:+l \vi'akly liasii. rpsins \vore teFtetl, first x i t h a mistrirc, of 10 neutral amino acids. lhix elution patterns, t1i:igranini~d in F i g u r ~ 1, indicate that thc hasic strengths of these rcsins diffcsr considerably. With Duolite -4-4(Chemical Process Co.) and Amberlite IR-45 (Rohm &: Haas Co.), t h e stronger two of these weakly basic resins, even grolinp (pK2, 10.6) was
Separation of Selected Amino Acids on the Hydrogen Form of a Low Cross-Linked Acrylic Polymer at Room Temperature
Resin and Added Amino Acids XE-112'
Column Length and Volume
Fraction Sumber
100 em., 100 ml.
1-36 37-41
0
45-55
0
.4sp, Ser, Ala, Leu, 100 mg.
ea.
XE-8gb Asp, Glu, Ser, Thr, Ala, GI!-
Pro, Val, Met, Leu, Ileu, 10O'mg. ea
100 cm.,
100 ml.
54-67 68-80 81-120 121-125 126-200 1-20 21-22 23 24-25 26-27 28
Effluent Amino Acids Asp
Volume,,
iri. 60 7
8
Se! Ser, .Ala
12 11
Ala 0
34
T,?il
0 Asp
Glu, Asp Glu. Ser Ser>T h r Ser, Thr Ala.$ Gly Ala, Gly Val, Ala, Gly Ya12Pro, Gly
3 63 70 8 4
8 8 4
29--30 8 4 31 8 32-33 12 1-al, Gly, Cys 34-36 Val, Cys 8 37-38 8 Ileu, Ileu 39 Leu, Ileu, Met 105 40-6G Leii) Ileu 31 G7-74 Through 325 mesh, fines removed by repeated sedimentation, 20 minuteslfraction, room temperature; flow rate diminished during experiment. b Ground and dry sieved t o 100 to 150 mesh, 20 minutesjfraction. Cys, 10 mg.
0
VOL. 32, NO. 1 1 , OCTOBER 1960
1403
:I
AMBERLITE
m
r-*Ianine
'
is
*
4'0
*
DUOLITE
IR-45
Bo A
DEA CIDITE
ma
\ c d
*
io
.
0.0
-4
I
HISTIDiNF
t
I
1
E
Leucine
10
L
,tube #
Figure 5. Elution diagram obtained by water elution chromatography of 100 mg. each of /?-alanine and leucine on Deacidite, 100 to 140 mesh Column 95 ml., 1 1 5 cm.; fractions 8 ml., 20 min.; eluted solutes weighed in each fraction
Table IV. Separation of /?-Alanine and Leucine on Free Base Forms of W e a k Anion Exchange Resins at Room Temperature
(Each column 15 ml. and 25 cm. long; 1.25 mg. of each amino acid added in each test) Tube Amino Resin N0.s Acid Duolite 1-14
1-50 51-70
0 p-Ala Discontd.
Amberlite IR-45
1-30 31-
0 Traces @-Ala
Amberlite IR-45b
1-19 20-25 25-65 66-100
Discontd.
Deacidite
1-20 21-26 27-36 37-50
0
p-Ala 0
Leu Discontd. 0
@-Ala 0
Leu
Amberlite X E 1 6 8
1-15 16-21 22-24 25-34
0 p-Ala 0
Dowex 3
1-19 20-26 27-30 31-50
&Ala @Ala, Leu Leu
Amberlite XE-58
1-19 20 21-33 24-33
a
b
Leu 0
0 @-Ala
6-Ala, Leu Leu
All fractions 0.5 ml. Resin used as received (see text).
1404
ANALYTICAL CHEMISTRY
considerably retarded. With the weakest of these basic resins, Amberlite XE-58 (Rohm & Haas Co.), all of the nonaromatic neutral amino acids emerged promptly. DEAE (Brown Co.) allowed phenylalanine to wash through but tryptophan was retained. Dowex 3 (The Dow Chemical Co.) and Deacidite (The Permutit CO., Yen- York City) gave the best separations, but the former seemed to allow more trailing and spreading, especially with leucine. When the basic amino acids were included in a mixture, a separation on a larger column of Deacidite showed that arginine and lysine emerged even more promptly than proline but that histidine was retarded (Figure 4). The potentiality of this resin for preparative scale separations is displayed in Figure 5 where 100 mg. of /%alanine are eluted ahead of 100 mg. of leucine on this same larger column. The six resin columns of Figure 4 were then tested with the monohydrochlorides of the basic amino acids, each added individually with removal of chloride from the resin with normal sodium hydroxide betneen runs. The results (Figure 6) showed the resins to have essentially the same order of basic strength as they had with the neutral amino acids. All resins readily allowed arginine to pass, but Amberlite IR-45 and Duolite A-4 retarded lysine. Deacidite allowed arginine and lysine t o pass rapidly through but retarded histidine. When thc three basic amino acids were added simultaneously instead of individually to this column of
Figure 6. Elution diagrams of basic amino acids on resin columns of Figure 4 Each amino acid ( 4 mg. of monohydrochloride) a d d e d and eluted individually; positions determined b y ninhydrin spot tests
Deacidite, histidine was retarded a little less. It seemed that the best combination for separating the basic amino acids would be Deacidite to hold back histidine, and IR-45 to retard lysine. However, when 100 mg. of each of these basic amino acids were added to a 20-cm., 35-m1. column of Deacidite, histidine promptly came through mixed with the other two amino acids. The elution pattern of histidine x a s then found to be related to the quantit'y of the amino acid present (see column loading experiments). I n Table IV are data on the separation of b-alanine and leucine on the same group of resin columns. The order of apparent basic strength of the resins is only slightly different from that of the previous tn-o series. Before regeneration .kmberlite IR-45 behaved as a weaker base than after being treated with NaOH. It is probable that when the resin is used untreated carbonate ties up the more strongly basic groups. Considerable carbon dioxide is evoked on acidification of the untreated resin. I n Table V data are given from experiments n-here larger quantities of neutral amino acids were put through Deacidite and DEAE. It' was necessary to elute the acidic amino acids from DE.1E with acetic acid. Mixed Base Resin. When the mixture of 10 neutral amino acids of Figure 4 was placed on a 15-ml., 28cm. column of a resin with both quaternary and tertiary amine groups, Permutit A (The Permutit Co.), even proline, t,he amino acid least firmly
IO. o
t IO0
m
ELUTION
""I
VOLUME
imli Figure 8. Elution diagrams showing effect of loading on elution of smaller quantities of histidine from Deacidite (100 to 140 mesh)
1
c
ELUTION
cmlJ
VOLUME
Columns 15 ml., 25 cm.
Figure 7. Elution diagrams showing effect of loading on elution of histidine from Deacidite (through 140 mesh) Columns all 15 ml., 25 cm.; histidine monohydrachloride monohydrate (quantities shown) a d d e d in 1 ml.; histidine determined colorimetrically with ninhydrin
bound, failed t o emerge with 70 mi. of water. Column Loading Experiments. Because t h e larger quantity of histidine \vas not held back 011 tmheDeacidite column, experiments were conducted t o test t,hc effect of loading. Quantities of histidine varying bptween 2 and 100 mg. werc p u t t'hrough 3, series of five identical columns of this resin. T h e effluent from each colunin \vas collcctcd in 1-nil. fractions and after appropriate tiilut,ioii the aniino acid conccxntration was colorimc~tricdlyn i t ~ ~ s urcd with ninhydrin (6). Thc amino ticid conccntrations of the efflucxnt solutions arc! plotted logarithmicnlly in Figure 7 . K i t h larger (1uantitic.s of solute thtb pc~ik voncentration emerged much soonvr than with lowcf conccntrations. The roncentration c u r w s were very s k e w d whrn columns viere owrloadcd. h i t ) assumed a symmftrical configuration a t the l o w s t concmtrations. In the overloaded range it is evident that aftw the peak has rmergcd t h r (wxwitration of solute in the c>ffluc>nt i.c i n t l ~ pendmt of the initial hac-ling. To determine whether further lo\wring of concentration has an additional influence on the position of the e1utc.d peak. an espcrinient was performfd with n d y preparrd columns and quantities of histidine between 0.5 and 4.0 mg. (Figure 8). The peak concentration came somewhat later for the 2.0than for the 4.0-mg. loading, but below
2.0 nig. there was no appreciable change in position and the peaks retained their symmetry. Alore lightly packed columns of coarser resin proh-
Table
V.
ably account for the difference in positions between these peaks and those of Figure 7 . Similar esperinients were performed with alanine on ground Amherlite XE-89 and with phenylalanine on Amberlite IRC-50. I n both instances, the addition of l e s amino acid to the
Chromatography of Amino Acids on Free Base Forms of Deacidite and DEAE a t Room Temperature
Resin and Added Amino Acids Dencidite 100 to 140 mesh Ser, Thr, Pro Gly, Ala, Val Ileu, 1Iet. 100 mg. ea.
Column Length and Volume 115 em., 95 ml.
Fraction Numbe; 1-13 14-15 16 17-21 22-3 1 32-33
I*:ffluent Amino
Acids
31-37 38-40
41-130 90 cm., 95 ml. A r g Lys, "is, Ser, Pro, Ala, Gly, Thr, Val, Leu, Glu, Met, Asp, 10 mg. ea
Cys, I mg.
1-1 3 11-17 18 1R-20
21 22-24 25-40
4 1-80 SI 4N.l 91-!)4 95-80 I G1-805 396-397
398-400
410-436 13i-440
* 0 . M acetic acid added a t fraction 371 and 361 acetic arid at fraction 420
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I
5
- A
4io
ELUTION
VOLUME
(m1. ) Figure 9. Elution diagrams showing effect of loading on elution of alanine from Amberlite XE-89 ( 1 00 to 140 mesh) Columns 15 mi., 25 cm.; alanine a d d e d in 1 ml. in quantities shown and determined colorimetricolly with ninhydrin
55
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tz'w
M LLIL
bo MIL-
16bo
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ZbO
i 'ERS
Figure 10. Elution diagrams of 2 amino acid solutions from Amberlite XE-89 (100 to 140 mesh) mixed with Dowex-50 W (X4) (200 mesh) Ratio XE-89 to Dowex 50: column 1, pure XE89; 2, 1 O O : l ; 3, 50:l; 4, l 0 : l ; 5, 5:l; positions of amino ocids determined b y ninhydrin spot tests Ten milligrams each amino acid
column was associated with a delay in the emergence of the peak. Results obtained with alanine are shown in Figure 9. A symmetrical peak was not obtained wit,h only 0.5 mg. of alanine on XE-89, and in results not shown, 2 mg. of phenylalanine failed to give a symmetrical peak on an equal-sized column of Amberlite IRC-50. M i x e d Resin Experiments. Because successful chromatography depends on local equilibria between mobile and immobile phases it seemed reasonable t h a t t'wo unrelated solids could constitute t h e stationary phase, each equilibrating with the ambient solution and thereby with each other. T h e apparent acid strength of a n immobile phase consisting of a weak acidic resin could thus ho increased by tlie addition of strongly nc.idic resin. This idea was testcd with several proportions of D o w s 50, Aniberlite ,YE-89, and Amberlite IRC-50 in the three possible paired ronibinations. 811 experiments showed :i gradual transition in the chromatographic behavior as the mixture of resins was altered. The bcpt demonstration of the cffect is shown in Figure 10 where diagrams are shown of the elution patterns of two aniino acid solutions on colunins of mised Amberlite XE-89 and Dowex 50. I n both cases i t is evident that adding the sulfonic acid resin gives wider Eeparations, but also causes greater spreading of eluted amino acids. 1406
ANALYTICAL CHEMISTRY
DISCUSSION
Results of the present study substantiate the theoretical consideration advanced by Davies (3) and emphasized by Partridge and Brimley (8, 9 ) , that amino acids emerge from a resin column in an order almost predictable by their relative acid or basic strengths. As in other types of chromatography on ion exchange resins. the sequence of amino acids emerging from a column seems to be influenced slightly by molecular size and somewhat more by aromaticity ( I ) . These considerations, nolv amplified by considering the acid or basic strength of the resin, have proved useful in selecting resins and conditions most likely to promote separation. Some of the water elution esperiments of Thompson and Morris ( I S ) were carried out on a sulfonic acid resin in the sodium form and othcrs on a quaternary base resin in the chloridt. form. I n this instance where in both eases a strong acid is already stoichiometrically neutralized by a strong base, the separations of acidic amino acids on Dowex 50 and basic amino acids on Dowes 1 w r e attributed to the Donnan equilibrium effrct in ion exclusion. The differential mobility of a, 3, 7 , and basic amino acids on Dowex 1 in the chloride form mas also considered by them to be the result of different proportions of positively charged amino acid molecules. If ion exclusion is to bc held responsible for a separation, the
substance first off the column should appear when the effluent volume approximates the void volume. Any solute that appears when or after the effluent volume equals or exceeds the volume of the column cannot have been excluded. From this it can be inferred that in the experiments of Thompion and Morris the acidic amino acids irere excluded from Dowex 50 in the sodium form and the basic amino acids were excluded from Dowes 1 in the chloride form. A11 of the monoaminomonocarboxylic acids came off both columns after the effluent considerably exceeded the void volume. These substances must therefore have entered the resin. Because our preliminary experiments with Dowex 1 in the chloride form and D o w x 50 in the sodium form showed that detectable quantities of chloride and sodium ion were eluted from thcse resins with m a l l amounts of both a- arid palanine. there is evcn some ion exchange. Separations with water are not simply due to ion esclusion although this mechanism undoubtedly plays a role. Because there was some elution of mineral ions from the neutralized forms of the strong acid and base resins, t,heir use was abandoned in this study and only free acid and bas(. forms were used. Several advantages to water elution
chromatography seem obvious. Automatic monitoring of column effluents by available methods are necessarily coniplicatcd by the prcsence of the eluting solute, usually present in much higher concentration than the substance being separated. Successful water elution chromatography might make possible the detection and measurement of eluted peaks by a simple physical means such as a refinement of the conductivity mcthod demonstratd here, ultraviolet absorbance, p H measurement, or possibly others. -4s pointed out by Thompson and Morris ( I S ) , the chemical mildness to which the separating substances arc subjected might be advantageous with labile compounds. I n prcparative work the ease with which the purified material c a n be recovered cannot bc escet,ded by any ot,her method of chromat,ogrLipliy. In large scale separations, such as those that might be carried out indust~rially. the elimination of reagcnt cost \vould bc a n important factor. X disadvantage of this typt, of chromatography as dewloped so far, lies in its lack of versatility. From available data it ~vouldbe diffiidt to devise a uwful schcme for tlie scyaration of all the protclin amino acids on a single column or even by an orderly succcssioii of diffc,rent columns. If the isolation of ccvtniii individual amino acids is intendcd, a successful mctliod (mild cert a i n l ~ i. i o be ~ drvelopcd. P u t of tlir difficulty ill pcrforniing prcy:trntivc, c-hromatography n i t h these techiliqrics lit's in the e f h t of column loatling t i n tht' iiosition of peaks. 'The ~licwdslin~icof clution p d s that oceurs xhenever a relatively abundant solute, is present and wliich is h ~ l dhack to some degree by tlw resin is adequately t~sp1:iiiii~d by a tlit~orc~tical ant1 c o i i d i w t i o n of self-sl~ar~~t~riirig self-(liffiising boinidariw ( 2 1 ) . Froni
the data presented here i t seenis probable that the position of a n y skewed peaks would advance if more solute were present and retard if less material were put on the column. This uncertainty in the posit'ion of a peak Tould give rise to inconstant results with different mixtures and would cause the most trouble when separations are incomplete. This type of difficulty is encountered in other types of preparative chromatography but in some, such as carrier displacement chromatography ( I ) , it is less conspicuous. Many of the resins tested seemed to have acidic or basic properties in the desirablt, range but behaved sluggishly, perhaps because of high cross linkage. Xmberlite IRC-50 is such a material. .A carboxylic resin of lower cross linkage, rlnibcrlite SIal p i m p c ~ t i t ~Es. periment,s dcsc:ribetl here intlicut,c, that only a few s w h resins would hr~nec~led; columns of an>. rcquisitcs acidity or basirity could be prq)Lirid b>- mising. Hon-ever, tlie mising of ver!. -trong and w r y w : t k a ( d resins in iiixiiy proportions would wcni g e n t ~ t l ltlisadvan~~ tagcous i i i that the rrcalc resin xould act niainlj, as a nicchaniixl rlilucnt or filler and little of its t~wliangr~ c.al)ac.ity v-ould bc utilized. -1 scrics of acidic (cation) csc~huiigi~rcyitis with suitnble physical propCrtiw :ind app:iwnt pK v ~ l u o s bitn.c,cm 1 antl 6, pcrlixps a t int(n:ils uf :ipprosim:itc~ly 1 to 2 p K units. antl a similar 5erit.r of basic (anion) psclimigc resins with a n upparent pI< range bctwccw 8 and 12 n-oultl prolxihly allow useful chromnto-
graphs of nearly all organic electrolytes. These resins should have relatively low cross linkages to allow rapid exchange of dissolved solutes, and high mechanical rigidities to allow useful flow rates even when finely divided. If all the resins now available had this combination of properties, both series would be nearly complete. ACKNOWLEDGMENT
The authors are indebted to the Rohm & Hans Co.for generous gifts of the Amberlite resins XE-89, XE-168, IR-45. XE-112, and XE-58. The Kational Aluminate Corp. kindly donated a sample of Xalcite X-219 and the Chemical Procws Co. furnished the Duolite rwins C-62. C-63, and A-4. LITERATURE CITED
(1) Biic:hanan. 11. L . ,
A s . 4 ~ .CHEar.
31,
833 (1959). (2) Buchanan, 1). I,, J . Biol. Cheni. 229, 211 (195ij. (3) Davies, C. l V . j Biochem. .I. 45, 38 (1949). ( 4 ) Fisher, 5 . F.,Kunin, R., J . Pliys. Chem. 60, 1030 (1956). ( 5 ) Hamilton. P. B..ANAL.Cr-mar.30, :314 (1958). ( 6 ) IToore, S., Stein, IT. H., J . R i d . Chem. 176,367 (1948). ( 7 ) Ibid., 211, 803 (1954). 18) Pai.tridw. S. 11.. Brimlev. 11. C.. Biociiem.2.'49, 153 11931). " (9;) Ihid., 51, 628 (1952). (11.)) Partridge, S. M., Kestall, I!. G., \ -
'
Ibid., 44, &l8(1949).'
(11) Iteichcnberg, D., "Ion Exchangers in Organic :ind Biochemistry," C. Calmon and T. R . E. Krrssman, eds., p. 90, Interscience, S c w York, 1957. (12) Spies, J . R., Charnliers, D. C., J . B i d . Cherri. 191,79ti !1951).
(13) Thompson, J. F., Iloi-ris, C:. ,J.> Arch. Biochern. Biophys. 8 2 , 380 (1959).
RECEIVEDfor review Ilarch 24, 1960. hccepted .July 22, 1960. LVork was slipported i n part by the V.s. Pulilic Hralth Pcrvice.
Stable Diazo Salts for Chromatographic Spray Reagents IRWIN A. PEARL and PATRICIA F. McCOY
The Institute of Paper Chemistry, Appleton, Wis.
b Commercially produced stable diazo salts Of a number of aromatic amines provide readily available spray reagents for locating and identifying phenolic compounds and aromatic amines on paper chromatograms,
lized diazo salts. The colors produced were noted before and after subsequent spraying with saturated sodium so,utions. A great variety
Twenty phenolic compounds were spotted on paper and sprayed with water solutions of 30 different stabi-
combin~tions- These cOmmercial sa'ts appear to be stable for chromategraphic spray use for extended periods.
of colors were noted for the various
I
5 A i'(wnt pwliminary coniniuiiit~at'ion (9) the use of Fast Red Salt GG, a comnic,i,rial!y avai1aI)le staldizcd diazo salt of p-nitroaniline, as a spray reagc>nt for plienolic compounds related t o wood cheniistqr, was reported. The advantages noted for its use over the classical procedure of diazotization of the p-nitroaniline immediately hefore
VOL. 32, NO. 1 1 , OCTOBER 1960
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