Relationship between Retention Volume and Elution Condition on the Cation Exchange Chromatography of Primary Amines Fumiko Murakami Department of Chemistv, Faculty of Science, Kyoto University, Kyoto, 606, Japan
Distribution coefficients of primary mono- and diamines in catlon. exchange chromatography are presented. Elution parameters influence the distribution coefficients of the amines. Dissociated amines are eluted from a column in order of carbon chain length of the molecules. Undissociated aliphatic amines have little interaction with the resin on the column. However, monoamines containing an aromatic group are adsorbed on the resin throughout the pH range employed. An equation which includes the distribution coefficients, dissociation constants and counter ion concentrations, is derived and applied to the chromatography of the primary amines.
and of the diamine
K [H']X -t ([Na']
where
390
XI = D,,, X, = X,[Na'],
X, = XZ[Na'l2
and XI is the selectivity coefficient between [Na+] and the first dissociated amine and X2 is that between [Na+] and the second dissociated amine. [Na+] is the concentration of exchangeable sodium ion in the resin. In this experiment, the acid dissociation constants of the amines, K 1 and K z at 25 "C were taken from the literature ( 8 , 9 ) . The selectivity coefficient between [Na+] and [H+] is expressed as X N ~ ~ and is reported by Bonner and Pruett ( 1 0 ) to be 1.2 on the cation resin Dowex 50. Characteristic parameters of the amines X I , X2 and X3 were determined experimentally.
Methods for the chromatographic separation of primary mono- and diamines on a cation exchange column using an amino acid analyzer have been previously reported ( 1 - 4 ) . Retention volume of the amines was remarkably affected by the pH and sodium ion concentration of the eluents. For aromatic amines, Chu and Pietrzyk reported the relationship between distribution coefficient and pH conditions of eluents in adsorption chromatography on Amberlite XAD2 (5). Cantwell and Pietrzyk determined the distribution coefficient of weakly basic amines on a strong cation exchange resin by a batch equilibration method (6). Also, the dependence of elution volume of analgesic drugs and disubstituted benzens on resin cross-linking for ion-exchange resins has been reported (7). This paper describes the effects of eluent composition on volume distribution coefficients, D,, for primary monoand diamines in ion exchange chromatography. In ion exchange chromatography of ionic compounds, a net charge of a compound dominates the chromatographic behavior of the compound and p H of the eluent controls protonation conditions of solutes and selectivity of the ion exchange resin. Since D, can be expressed as a function of distribution coefficients of the dissociated state of the compounds, in the case of diacidic bases on a cation exchange resin, D , is expressed by the following equation,
where K1 and KS are the acid dissociation constants of the protonated bases, BH22+ and BH+, respectively. D ,", D and D,, are the volume distribution coefficients for non-, first, and second dissociated compounds, respectively. The D vo value represents a non-ionic adsorption force of a compound on the stationary phase. The values of D,, and D,, may be given as a function of concentration of hydrogen and of sodium ions [Na+] on an ion exchange ions [H+] equilibrium. Then, a correlation between D ,and elution conditions is expressed on the chromatography of primary mono- and diamines as follows. In case of the monoamine:
[H']'X + X,~H[Ht])2
EXPERIMENTAL The amines were purchased from Nakarai Chemicals Ltd., Kyoto, and Tokyo Chemical Co., Tokyo. The other chemicals were obtained from Nakarai Chemicals Ltd., Kyoto, and Wako Pure Chemicals, Co., Osaka. A Hitachi amino acid analyzer, Model KLA-3 (Hitachi Ltd., Tokyo), was employed throughout this work. Chromatography of the primary amines was carried out on a cation exchange resin, Hitachi custom resin No. 2611, a t a flow rate of 30 ml/hr and a column temperature of 50 O C . To study the effect of pH and counter ion concentration, either pH or sodium ion concentration of eluents was held constant and the other parameters were varied. First, the chromatography of amines was carried out a t a constant sodium ion concentration of 0.4M using buffers of pH from 2 to 11.3. The composition of the buffers is shown in Table I. The pH was adjusted using a 6M hydrochloric acid solution. Below pH 9, the pH of the eluent was adjusted a t room temperature and above pH 9 adjusted in a water bath a t 50 "C. Second, the amines were chromatographed using eluents with various sodium ion concentrations ranging from 0.2 to 1.OM a t pH 8.0. Ninhydrin color development was measured a t 570, 640, and 440 nm and recorded on an amino acid analyzer. The experimental D , values of the amines were calculated from retention volumes, V R , according to an equation, D, = ( V R - V M ) / X ,where V M is the hold-up-volume. The hold-up volume was determined from the retention volume of cysteic acid, which is eluted without retention on the column. X is column bed volume.
RESULTS AND DISCUSSION Effects of pH on the Distribution Coefficients. The log D ,values of several aliphatic monoamines, methyl, ethyl, n- propyl, isobutyl, n- butyl, isoamyl, and n- amylamine on the cation exchange resin column were measured a t various pH values. The results are plotted in Figure 1. In the acidic region, the pH is well below the pK value of the amine by more than two units, and most amine molecules are present in a dissociated state. Within this region, the
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
Table I. Composition of Eluting Buffer PH
2 .o
4.0
s .o
6 .O
7 .O
Citrate ( M ) Borate ( M ) NaCl ( M )
0.05
0.05
0.05
0.05
0.05
NaOH (At') [Na'] (M)
...
0.25
...
0.4
...
0.25
... 0.4
...
...
0.25
0.25
0.4
0.4
...
...
...
0.20 0.05 0.4
retention volumes of the compounds were not affected by pH of the eluents. In the alkaline region, concentration of the unionized amine increased with increasing pH of the eluents. The retention volumes of the amines decreased with increasing pH, and at high pH approached the hold-up volume. Therefore, the D , values of each amine approached zero. The curves in Figure 1 were drawn according to Equation 1, which was a function of [H+] at a given sodium ion concentration. Each term becomes dominant as a function of D ,according to pH. The values of X I and Xz for monoamines were determined according to the following procedures. Chromatography of given compounds was carried out twice using two different p H eluents successively. For example, for methylamine, buffers of p H 4.0 and 9.0 were employed and the D , value for each experiment was determined. Each D ,value was substituted into Equation 1 and the simultaneous equations were solved to get X I and X Z values for the compound. X1 values of aliphatic monoamines in Equation 1 were very small, and therefore these values can be neglected. The relation between D , values and pH of the eluent is classified into three modes according to pH ranges. First, when pH is well below pK1, K i in Equation 1 is negligible in comparison with [H+],and the value of log D , is independent of pH. Second, when pH is well above pK1, K1 is large compared with [H+],and the curve represents a straight line with a slope of -1 unit. Finally, in the pH range near pK1, there is a curved region on the graph. The experimental results are plotted on the same figure, and it can be observed that experimental points fit well on the curves drawn from Equation 1. Figure 2 shows a relationship between log D and pH for monoamines containing an aromatic group; tyramine and phenethylamine. The curves drawn according to Equation 1 fit well on the plots of the experimental data. Parameters for amines containing an aromatic group were also obtained by the method mentioned above. The X Ivalue was larger than zero. These compounds were eluted more slowly than aliphatic compounds with comparable carbon chain length. Above pH 10, the D , values of the amines containing an aromatic group are constant and the curves become independent of pH, becoming horizontal at log D , = log
7.5
8 .o
9 .o
9.9
11.3
10.6
...
...
...
...
...
...
0.05 0.25 0.05
0.05 0.25 0.05 0.4
0.05 0.25 0.05
0.05 0.25 0.05
0.025 0.30 0.05
0.025 0.30 0.05
0.4
0.4
0.4
0.4
0.4
20
t
0
1
2
4
6
8
1
0
1
2
PH
Figure 1. Effect of pH on log D, of monoamines. Each plot represents an experimental value and the curves are drawn according to Equation 1 (see text). Curve (1) methylamine. (2) ethylamine, (3) npropylamine. (4) isobutylamine. (5)nautylamine, ( 6 ) isoamyiamine
1
Q
Z
i
6
8 PH
l
O
1
2
Figure 2. Effect of pH on log D, of cyclic monoamines. Each plot and curve have the same meaning as in Figure 1. Curve (1) tyramine, (2) phenethylamine
X 1. Several diamines, ethylenediamine, 1,3-propanediamine, putrescine, and cadaverine, were chromatographed and log D ,values of the amines were plotted against pH of the eluent as shown in Figure 3. For each diamine, three suitable D , values were substituted into Equation 2 and three parameters XI,XZ,and Xa,were obtained by solving these three simultaneous equations. Curves for the diamines also agreed fairly well with the experimental data. In the acidic region, log D , values are independent of pH, and the values in this flat region were log X?/[Na+I2from Equation 2. In the alkaline region, log D , values decreased with increasing pH. When the two pK values for a diamine are sufficiently different, the curve has a flat region between the two pK values or a shoulder
i
0
. ,
2
L
6
8
1
0
1
2
PH
Figure 3. Effect of pH on log D, of diamines. Each plot and curve is the same as in Figure 1. Curve (1) 1,3-propanediamlne, (2) putrescine, (3) ethylenediamine. (4) cadaverine
at the pH near pK2. As the difference between the two pK values of each diamine became smaller the shoulder of the curve was less pronounced. Effects of Sodium Ion Concentration on t h e Distribution Coefficients. Several primary amines were chro-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
391
r- "*
c2
CL
r5
'i
C _ I _ L L i L
pNa
Figure 4. Effect of sodium ion concentration of log D, of monoam-
ines.
0
a2
0.4
9.6
OB
PNa
Eluent, pH 8.0 borate buffer, (1) methylamine, (2) ethylamine, (3) allylamine, (4) n-propylamine. (5)isobutylamine. (6) n-butylamine, (7) isoamylamine, (8) n-amylamine
Figure 6. Effect of
sodium ion concentration on log D, of diamines.
Straight lines represent a slope of 2. Eluent: pH 8.0 borate buffer, (1) ethylenediamine, (2) 1,3-propanediamine,(3) putrescine, (4) cadaverine
L
0
02
0.L
0.6
PNa
Figure 5. Effect of sodium ion concentration on log D, of cyclic mo-
noamines. Straight lines represent a slope of 1. Eluent: pH 8.0 borate buffer, (1) histamine, (2) tyramine, (3) phenetylamine
matographed with eluents of various sodium ion concentrations a t fixed pH. Volume distribution coefficients of amines increased with decrease in the sodium ion concentration of the elution buffers. In Figure 4,log D , values of the monoamines; methyl, ethyl, n- propyl, allyl, isobutyl, n- butyl, isoamyl, n- amylamine, at pH 8.0 were plotted us. pNa (-log[Na+]). Curves in Figure 4 were drawn for each amine according to Equation 1, substituting the parameters from the results of the pH effects. When XI values of these amines are negligible and [Na+] is much larger than XN,~[H+]in the bracket of the second term in Equation 1, then the curve is a straight line with unit slope. The intercept of the curve increased as the length of the carbon chain increased. When D , values of these amines were replotted us. the reciprocal of sodium ion concentration of the eluents, the curves became straight lines passing through the origin and having various tangents (2). The slope became greater with increasing length of the carbon chain. For some monoamines containing an aromatic or heterocyclic group, the plots are not unit slope a t a low pNa range as shown in Figure 5 . The D, - l/[Na+] plot of these compounds shows a linear relationship and the curves across the ordinate at D ,= X Aliphatic diamines were chromatographed at pH 8.0 and a relationship between log D , and pNa was studied. The results are shown in Figure 6. The plots for 1,3-propanediamine, putrescine, and cadaverine fit a straight line with a slope of 2, and have different intercepts that increase with the carbon chain length as expected from Equation 2. At this elution condition, the experimental values of ethylene392
diamine, however, deviated from a slope of 2 and the theoretical curve is seen to be curved at a lower pNa range. In Equations 1 and 2, parameter X I represents the extent of the non-ionic interaction between resin matrix and the unionized compound. X1 values for aliphatic amines are practically negligible, while the amines containing an aromatic or heterocyclic group have large X I values, as shown in Figure 2. Therefore, amines containing an aromatic or heterocyclic group were eluted from a column more slowly than the aliphatic amines of equal carbon number. The parameters X2 and Xs refer to the ionic interaction between the resin and ionized state of the compounds. In acidic conditions, the D ,value and the parameters X 2 and X 3 in the equations increase according to the length of carbon chains in the solute alkyl group. These parameters for amines containing a branched chain group are smaller, and these amines elute faster than the straight chain isomer. At a given pH, both Equations 1 and 2 are functions of sodium ion concentration and log D.-pNa curve is obtained according to these equations. When the dissociated monoamines and first dissociated diamines are in the majority, Equations l and 2 are represented as the first order equation for [Na+], and the graphs show a unit slope. At the pH range in which the second dissociated amines are in the majority, the graph of diamine is expected from Equation 2 to show a slope of 2. And when pH is high enough, it is expected from the Equations that most of the amine molecules are undissociated and log D will not be affected by the sodium ion concentration of the eluent. Therefore, the log D ,-pNa curves are expected to be horizontal lines. In these extreme cases of dissociation, all log D,-pNa curves represent straight lines and experimental results were fit satisfactorily to the theoretical curves. When the pH of the eluent is near the pK value, the amine molecules are the mixture of two or three stages of dissociation; then equations cannot be simplified and log D v-pNa curves became more complicated. In the present studies, Equations 1 and 2 can be used to predict the experimental results with reasonable accuracy. The results indicate that the dissociated form of an aliphatic amine molecule can penetrate into the ion exchange resin matrix and has hydrophobic interaction with the resin matrix a t the carbon chain. Egashira et al. ( 1 1 ) reported that the dissociated orgainic acids were absorbed on the anion exchange resin (Cl- form) a t low ion concentra-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
tion, and had an opposite effect a t high ion concentration, although in their work such an effect was not observed for cation exchange chromatography of amines. In conclusion, it appears that the correlation discussed above is very helpful to predicting D , values and obtaining the best separation conditions.
ACKNOWLEDGMENT The author expresses her thanks to H. Hatano, S.Egashira, and S. Rokushika for helpful discussions and suggestions for this work.
LITERATURE CITED
(2) H. Hatano, K. Sumizu, S. Rokushika, and F. Murakami, Anal. Biochem., 35, 377 (1970). (3) F. Murakami, S.Rokushika, K. Sumizu. and H. Hatano; Bunseki Kagaku, 19, 1664 (1970). (4) S. Rokushika, S . Funakoshi. F. Murakami, and H. Hatano. J. Chromatogr., 56, 137 (1971). (5) C. H. Chu and D. J. Pietrzyk, Anal. Chem., 46, 330 (1974). (6) F. F. Cantwell and D. J. Pietrzyk, Anal. Chem., 46, 344 (1974). (7) P. Larson, E. Murgia, T. Hsu, and H. F. Walton, Anal. Chem.. 45, 2306 (1973). (8) Saul Patai, Ed., "The Chemistry of the Amino Group" Interscience, New York, N.Y.. 1968. (9) D. D. Perrin, Ed., "Dissociation Constants of Organic Bases in Aqueous Solution," IUPAC compilation, London, 1965. (10) 0. D. Bonner and R. R. Pruett, J. Phys. Chem., 61, 326 (1957). (11) S. Egashira, H. Nakasuka, and Y. Takeyama, Bunseki Kagaku, 14, 636 (1965).
RECEIVEDfor review May 13, 1974. Accepted November 4, 1974.
(1) H. Hatano, "Methods of Amino Acid Analysis," Kagaku-dojin, Kyoto, 1964, p 126.
Reactant ions in Negative Ion Plasma Chromatography Glenn E. Spangler and Charles 1. Collins
U S .Army Mobility Equipment Research & Development Center, Fort Belvoir, Va. 22060
With zero air as the carrier gas, experimental evidence is presented which supports the possible identification of the negative reactant ions in plasma chromatography as Ozand singly hydrated (H20)02-, (H20)0H- ions. in the presence of chlorine, CI- ions appear at the same location in the ion mobility spectrum as the (H20)OH- ions while in the presence of NO and ammonia, NOz is formed to allow contributions from NO2-. interchange reactions studied between CO2 and the (H20)02- ions to form CO4- illustrate how mobility vs. mass assignments are affected by reversibly fast reactions. Similar results are also obtained for the fast reactions describlng ion cluster reactions with water. When laboratory air is introduced into the reaction region, (H20),0H- ions are generated in favor of (H20),02- ions and, depending on environmental conditions, NO2- ions are present. Contrlbutions from the C04- ion are felt negligible.
Plasma Chromatography (trade name originated by the Franklin GNO Laboratories) has been developed in recent years to detect and identify trace constituents of organic compounds in gaseous mixtures at atmospheric pressure. A recent review of the technique has been provided ( I ) . Detection is achieved by subjecting trace molecules to ionmolecule reactions, the product ions of which are subsequently analyzed by ion mobility drift tube spectrometry ( 2 ) .The reactions have been described as bimolecular and are governed by ( 3 , 4) d nD _ _ -- kNn,
dt
- cyn+n,
(1)
where n is the product ion concentration, n R is the reactant ion concentration, N is the neutral sample molecule concentration, n + is the total positive ion concentration, k is the bimolecular reaction rate constant, and cy is the recombination coefficient. k depends on the nature of the reactant ions, the properties of the trace molecules being analyzed (e.g., electronegativity), temperature, etc., but should not exceed 10-10-10-9 cm3 molecule-1 sec-' under
thermalized conditions since this rate corresponds to the number of close encounters between ion and molecule per second with 100% ionization efficiency (5). Typically kN
