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Anal. Chem. 1985, 57, 1830-1835
Reversed-Phase Liquid Chromatographic Separation of Amino Acids with Aqueous Mobile Phases Containing Copper Ions and Alkylsulfonates Shulamit Levin and Eli Grushka* Department of Inorganic and Analytical Chemistry, The Hebrew University, Jerusalem, Israel
A new isocratic method for the chromatographic separation
offree amino aclds without any pretreatment or derivatlzation is described. An aqueous solutlon of copper acetate, alkylsulfonate, and acetate buffer Is used as the mobile phase In conjunctlon with a conventlonal reversed-phase column. Detection Is carrled out at 230-240 nm. Up io 18 amino acMs can be separated In 30-40 min. The influences of Ionic strength, temperature, chain length, and concentratlon of the aikyrsunonate were Investigated. The last two parametersare shown to prodde powerful means of selectlvtty manlpulatlons.
The separation of amino acids by liquid chromatography has been pursued by many investigators in various fields of science. The obvious importance of that separation problem, especially in the area of protein chemistry, is exemplified by the large number of publications on the topic; see for example the reviews in ref 1-4. The analysis of amino acids presents the chromatographer with a challenging task due to two main difficulties. (1) Most of the amino acids are not amenable to easy detection with conventional detectors. Thus, frequenctly these solutes undergo some sort of either pre- or postcolumn derivatization procedure. (2) k a group, the amino acids cover a very wide range of polarities, making isocratic analysis, at best, very difficult. There is not, as of yet, a simple direct method which can meet the following demands: be isocratic, require no or very little pretreatment of the solutes, have acceptably low detection limits, be reproducible and quantitative. Recently Grushka et al. (5)described a new approach to the separation of free amino acids using reversed-phase chromatography and conventional HPLC equipment. In this approach the analysis of amino acids was accomplished by the in situ formation of their copper complexes. The present paper reports some new developments in the isocratic separation of free amino acids. In order to achieve the separation, the mobile phase is made up of a sodium acetate buffer (pH 5.6) containing cupric ions and alkylsulfonate ion-pair reagent. The rationale behind these additives is as follows: Cupric ions are known to form charge-transfer complexes with amino acids. The complexes absorb UV radiation with a maximum around 235 nm and a molar absorptivity of about 6000 (for the 1:l complex). In the chromatographic system described previously (5) and here, the complexes are formed in the column upon injection of the amino acids. In addition to affecting the detectability of the solutes, the presence of the cupric ions alters the selectivity of the system. While in general the presence of Cu(I1) ions causes an increase in the k’values of the amino acids, the retention of some of the polar solutes is still too short. To overcome this problem hydrophobic ion-pair reagents, such as the alkylsulfonates,are added to the mobile phase. Indeed, as will be shown here the combination of cupric ions and alkylsulfonates offers the analyst a powerful tool in order to control the separation of amino acids. Other workers have used copper ions, either as a component of the stationary phase (6, 7)or of the mobile phase (8-IO), 0003-2700/85/0357-1830$01.50/0
for controlling the retention of amino acids. The important role of Cu(I1) ions in the resolution of amino acids enantiomers is very well documented (e.g., ref 11-14). For detection purposes cupric ions were used electrochemically (15-17)and spectrophotometrically in the visible range (9). Similarly, alkylsulfonates and other hydrophobic ions were used to separate amino acids on reversed-phase columns (18-26). Most of the above works have some practical difficulties in obtaining reproducible and quantitative results. Moreover, very few of them can produce isocratic chromatograms of over 15 amino acids. The unique feature of the present research is the concurrent use of both copper ions and an alkylsulfonate as mobile phase constituents. The synergistic effects of these two additives help to overcome most of the problems encountered with the use of either one and to answer some of the demands outlined above. EXPERIMENTAL SECTION Materials. The acetate buffer was prepared in our laboratory with analytical grade sodium acetate and acetic acid. The pH was monitored with the aid of a Beckman pH meter. Analytical grade cupric acetate was obtained from Mallinckrodt. All amino acids, as well as the sodium salta of 1-hexanesulfonate,l-heptanesulfonate, and 1-octanesulfonate,were purchased from Sigma Chemicals. Water for the mobile phases and sample preparation was deionized and then purified by Seral purification system. All solutions and samples were filtered through a 0.3-pm Millipore filter. Instrumentation. The chromatographic system included the following components: Perkin-Elmer Series 4 liquid chromatograph; Rheodyne injection valve; Perkin-Elmer 85B spectrophotometric variable wavelength detector with 1.4-pL flow cell. The chromatograms were recorded on a W&W Model 600 recorder. The column temperatures were kept constant f0.1 O C using a circulating water bath with a Julabo thermostat. The column used was a Merck Lichrosorb RP18 cartridge, packed with 7-pm particles, held by a Hibar manual holder. The cartridge dimensions were 250 X 4 mm. A Merck 4 X 4 mm guard cartridge was connected to the column using a zero dead-volume union. A precolumn, made in our laboratory, was connected between the pump and the injector. It was refilled once a month with a mixture of Partisil 10 silica gel and Partisil 10 ODs-3. Solutes. To demonstrate the influence of each of the chromatographic variables, the following amino acids were chosen as sample solutes: aspartic acid (Asp),glycine (Gly),histidine (His), lysine (Lys), arginine (Arg), valine (Val), methionine (Met), threonine (Thr), hydroxyproline (Hyp), asparagine (Asn), and tyrosine (Tyr). These amino acids cover a wide range of polarities and hydrophobicities, and they represent all types of side chains. The amount of injected amino acids was 2.4 nmol for all but the most retained solutes where 10 nmol were introduced to the column. RESULTS AND DISCUSSION Two very practical aspects of the work must be described before the results of the study are detailed and analyzed. These aspects are important in so far as they point to the line of reasoning leading to the development of the present mobile phase buffer and to the solution of problems that can po0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
tentially destroy the separation. Choice of Buffer, Our previous study (5) exploited the effects of Cu(I1) ions without controlling the ionic strength of the mobile phase. However, it soon became apparent that ionic strength is a very important factor affecting the retention and the reproducibility of the system. To overcome these difficulties,buffered mobile phases should be used. Selecting a compatible buffer and the correct pH can prove to be a time-consumingaffair, since the presence of cupric ions in the mobile phase imposes severe limitations on possible buffer candidates. The first, and perhaps the most problematic, limitation is that of pH. The complexation of Cu(II) by amino acids, and consequently chromatographic retention (5),is more favorable at high pH. However, at pH >6 cupric hydroxide will precipitate out, unless some chelating agent, which is capable of strongly complexing copper ions, is present in the mobile phase. On the other hand the complexation should not be too strong, for then amino acids chelation would greatly diminish and the benefits of cupric ions will be lost. In an attempt to overcome these problems, the basicity of the mobile phase was raised with organic buffers, or additives, such as triethylamine, capable of chelating copper ions. These efforts were unsuccessful for two main reasons: (1) the extremely long equilibration times required to reach constant retention and stable detector base line; (2) the inability to completely eliminate precipitation of CU(OH)~The second problem lies in possible precipitation of the metal ions by the anions of the buffer solution. For example, phosphate buffer, which normally is the buffer of choice for the separation of amino acids and of nucleotides, is completely unusable when copper ions are also present in the mobile phase. Finally, the presence of Cu(I1) excludes the possible use of other strongly UV absorbing species, such as nitrates, as mobile phase additives, since then the detector signal may be swamped. As a result of these deliberations it is felt that an acetate buffer at pH 5.6 is the best compromise available. Problems Associated with Use of Cu(I1) Ions. Over and above the buffer issue, there are several other chemical and technical difficulties which can be attributed to the presence of cupric ions. These are as follows: (1) Exposed metal parts of the system, such as tubings, pump pistons, and check valves, can be subjected to chemical attack by the copper ions in the mobile phase. The extent of corrosion is a function of the mobile phase pH, of the type of other additives, of the stainless steel quality of the pump parts and tubings, and of their condition, i.e., the presence of fractures, pits, etc. Although this is a well-known problem, there is almost no discussion of it in the chromatographic literature, save by some equipment manufacturers who suggest avoiding the use of copper ions altogether. The corrosion difficulties can be greatly minimized if the following actions are taken: The mobile phase flow should not be stopped for long periods of time. The system, including the column, should be rinsed thoroughly with water and methanol whenever it will not be used for a while, e.g., overnight. At least once a week the system should be washed with 0.01 M EDTA in a 0.1 M acetate buffer. (2) The column efficiency and peak symmetry begin to deteriorate after a certain period of usage with Cu(I1) ions. The reason for the loss in efficiency is not immediately clear, but it is related probably to copper uptake by the stationary phase silanols. A wash with EDTA solution seems to be the best solution since columns undergoing such treatment exhibit pre-copper efficiencies. The use of EDTA in conjunction with metal additives has been mentioned in the chromatographic literature but rarely described in detail. For example, Feibush et al. (27)have used EDTA for extracting Cu(I1) ions from column packing. In
201
1831
P
5
sulphonate conc.
(mM1
Flgure 1. Dependence of the capacity ratios of some amino acids on
the concentration of heptanesulfonate. The mobile phase also contained 0.1 M acetate buffer (pH 5.6) and 5 X I O 4 M Cu(11) ions. The temperature was 30 O C . the present work it was found that a wash with EDTA is advantageous in solving many copper associated problems. The reasons are quite obviously related to the strong chelating capability of EDTA. It removes small amounts of copper from the entire system, even from parts where slow accumulation of Cu(I1) ions occurs, such as on flow cell lenses and in pump check valves. Most important, and perhaps more impressive, is the effect of EDTA on restoring column efficiency, probably achieved by the quantitative removal of Cu(I1) ions from the free silanol groups. Frequent treatment with EDTA extended the lifetime of the columns to equal those of conventional reversed-phase systems. In addition, EDTA was found to be essential in the studies of different sulfonates and/or of different concentrations of the ion-pair reagents. Without it, reproducible results were difficult to obtain. Effects of Alkylsulfonates. As mentioned above, increasing the retention of the solutes by manipulating the pH of the mobile phase proved unsuccessful for various reasons. Consequentally, efforts were made to find an additional modifier which will be able to influence the retention of the amino acids. The copper complex of the amino acids, which is assumed to be in a 1:l ratio in the range of concentrations employed in this study, is probably positively charged. Thus,introducing an anionic species such as an alkylsulfonate should enhance the retention via an ion-pair mechanism. Sulfonates are frequently the additives of choice in order to influence the retention of positively charged solutes (28). As mentioned in the introductory section, ion-pair chromatography has been applied to the analysis of amino acids-but not to their copper complexes. Figure 1 shows that when heptanesulfonate is added to the copper-containing mobile phases, the retention of the test amino acids can vary substantially. Similar behaviors were found when hexane- or octanesuifonate were used as the ion-pair reagent: the capacity factors of the amino acids increased with increasing amounts of the sulfonates in the mobile phase. In all cases the Cu(I1) concentration was 5 x M and the buffer concentration was 0.1 M. The concentration of the sulfonates varied from zero to 0.005 M or to 0.01 M. The isotherm type behavior shown in the curves here is similar to that found in systems not containing cupric ions (19,20,24,26). It should be pointed out, however, that with the exception of the work by Kraak et al. (24) the dependence of amino acid retention on the sulfonate concentration has not been thoroughly studied.
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
II :
l2I
91
1
C
12
A
9
3
n n n Figure 2. Dependence of the capaclty ratlos of some amino acids on the sidechain length of the aikylsulfonates. Concentrations of the ion-pair reagents are (a) 0.2 mM, (b) 0.4 mM, (c) 2 mM, and (d) 5 mM. All other conditions are as given in Figure 1. n
With the exception of Asp, whose capacity factor was very close to zero in all cases, the k’values of all amino acids used as test solutes increased with the concentration of the sulfonates. The increase in k’values is particularly noticeable in the case of the basic amino acids, especially with Lys and Arg. This is not surprising in view of the strongly positive nature of these solutes. The overall increase in the retention, coupled with the extreme changes in k’values of the solutes mentioned above, makes the early part of the chromatogram less crowded, and thus more attractive for the separation of amino acids. The extent of retention increase is a function not only of the alkylsulfonate concentration but also of the alkyl length. This is amply demonstrated in Figure 2, in which the capacity ratios of several amino acids are plotted vs. the alkyl chain length for several sulfonates concentrations. Irrespective of the ion-pair reagent concentration, all k’values increased with chain length. Not surprisingly the increase in much more pronounced with the basic amino acids. It is interesting to note that the slopes of the lines are a function of the sulfonates concentration. This is the result of the fact that the dependence of the capacity ratios on the concentration of the individual alkylsulfonates is not parallel. The discussions above lead to the important observation that retention data, such as shown in Figures 1and 2,can be used to establish window diagrams (29),in terms of both the sulfonate concentration and alkyl length, for resolution optimization. The selectivity changes that accompany the manipulation of the quantity and quality of the ion-pair reagent, offer the analyst an important tool in maximizing the separation of amino acids. The synergistic effect of the copper ions and the alkylsulfonate in the mobile phase was mentioned, but not elaborated upon, previously. Table I justifies the claim that these two additives together act synergistically. In this table, the capacity factors of the amino acids are given for the following mobile phases: (1)acetate buffer plus 5 X M Cu(II), (2) acetate buffer plus 5 mM heptanesulfonate, and (3) acetate buffer plus Cu(I1) and sulfonate at the concentration levels just indicated. With the exception of histidine, the solutes are retained longer with the mobile phase containing both additives. The fact that His acts differently is not surprising since it is known to form a tridentate, rather than a bidentate, complex with copper. The retention processes in an ion-pair system can be related to either ion pair formation in the mobile phase, a dynamically
Table I. Comparison of k’Values of the Test Solutes in Three Mobile Phasesa solute GlY Asn HYP Val
LYS TYr His Met Arg
5
mM C7S030.09 0.11 0.28 0.44 1.31 1.42 1.93 0.87 6.78
0.5
mM Cu(I1) C7SO; 0.03 0.05 0.21 1.16 0.17 3.8 0.35 1.95 0.55
+ Cu(I1)
0.35 0.52 0.61 3.85 5.19 9.87 1.65 6.0 33.5
In each case the mobile phase contains 0.1 M acetate buffer in addition to the indicated additives. C7S03- means heptanesulfonate.
formed ion exchanger on the stationary phase, or a combination of both (viz. ref 18,30). The dynamic exchanger can operate in the following manner. Some of the alkylsulfonate will be adsorbed on the reversed phase due to hydrophobic interactions. Because of the negative sulfonate moiety, a fraction of the cupric ions will be localized on the stationary phase. The amino acid solutes can interact with these Cu(I1) ions and are therefore retained in the column. As the alkyl part of the sulfonate increases, so will its hydrophobicity. A larger fraction of the ion-pair reagent will then be extracted onto the reversed phase. As a consequence, the amount of copper ions localized on the stationary phase will be larger, providing more interaction sites for the solutes. The net effect will be an increase in the retention. The data in Table I and Figures 1 and 2 tend to support the dynamic ion exchange retention process. At present, however, these arguments are rather speculative, and further work is needed in order to unambiguously assign the retention process. Over and above controlling retentions and selectivities,the sulfonates play an important role in achieving very quick equilibration and stabilization of the system. Without the ion-pair reagent, day-to-day reproducibility and base line stability were rather difficult to obtain. The addition of any of the three sulfonates used in this study to the mobile phase drastically alleviated these difficulties. The exact mechanism by which the sulfonates improve the stability of the column is not known. It can be attributed, perhaps, to the adsorption of the sulfonates on the reversed phase, thus hindering the
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
1833
ET
6
k
1
4
2
J
3.6-83
3.1E-05
3,s-05
3.d-83
3.4t-83
1/ T
(MI Flgure 3. Dependence of the capacity ratios of some amino acids on the acetate concentration. The pH of the buffer is 5.6. The mobile phase contained 1 X lo-' M Cu(I1). The temperature was 40 O C .
Figure 4. Dependence of In k' on 1/T for some amino aclds. The eluent was an acetate buffer (pH 5.6) containing 5 X M Cu(I1) Ions and 5 mM heptanesulfonate. The line marked C refers to the behavior of a system peak.
access of copper ions to surface silanols. Effects of Ionic Strength. The above studies were done over a wide range of concentrations, and therefore ionic strength effects may strongly influence the retention of the solutes. To ascertain the contribution of ionic strength to the capacity ratios, the effects of changing the concentration of the acetate buffer in a mobile phase containing only the buffer and cupric ions were studied. Figure 3 shows the capacity ratios of some amino acids as a function of the buffer concentration, from 5 mM to 0.5 M. The amounts of copper ions were kept constant at all buffer concentrations. Although the behavior of the k'values is typical (viz., ref 20 and 241, and in general well understood, several points should be made. The decrease is most noticeable at low concentrations of the acetate where the drop in the capacity ratios is quite rapid. At higher concentrations the change in k' is much more moderate. For that reason the sulfonate studies described above were carried out at a buffer concentration of 0.1 M. In this manner, ionic strength effects due to the sulfonate concentration changes will be kept to a minimum. The effect of the ionic strength, as shown in Figure 3, is similar for all amino acids studied. This fact is of theoretical importance when one is attempting to isolate the various interactions between the additives and the solutes. Effects of Temperature. Since the many equilibria controlling the retention in the present system should be temperature dependent, it was decided to briefly investigate this parameter. Figure 4 gives the effect of the temperature on the capacity ratios of some test solutes with a mobile phase M Cu(I1) ions, and containing 0.1 M acetate buffer, 5 X 5 mM heptanesulfonate. The decrease of In k'with increasing temperatures, as shown in Figure 4, is quite typical of reversed-phase systems. The slopes of the lines, and consequentally the enthalpy of transfer of the amino acids, seem to follow neither the capacity ratios order nor any subgrouping of the solutes. Thus,retention in this chromatographicsystem is not controlled by enthalpy effects, at least not in a simple manner. Undoubtably this is related to the fact that the nature of the different amino acid-Cu(I1) ions-sulfonate complexes are not equivalent. If true, the data in the Figure 4 have some interesting ramifications in the qualitative analysis of amino acids, in that the temperature dependence of the retention can aid in the identification of the solutes. It should be noted in passing the changing the temperatures in the absence of the ion-pair reagent resulted in changing retention times and in fluctuating detector base lines. The
Abs
ionic strength
MOBILE PHASE O O l M acetate b u f f e r ( p H 5 6 ) 4xIO'+H CuAc2 BxIO-" M heptonesulphonote COLUMN R P 18, 2 5 O x 4 m m 7u
FLOW R A T E . P m l / m i n TEMP, 45'C
17
16
,
I
10
' I /Icu
I/
I
20
I
30
I
I
40
1
I
50
MIN D
IA
Flgure 5. A chromatogram of a mixture of amino acids. The 0.01 acetate buffer (pH 5.6)contalned 4 X lo4 M Cu(I1) ions and 8 X 10"' M heptanesulfonate. The temperature was 45 O C . The detector sensitivity was 0.16 AUFS. The solutes are (1) Asp, (2) Giu, (3)Gly, (41 Ser, (5) Asp, (6) Ala + Gin, (7) Hyp, (8)Thr, (9) His, (10) a-Abu, (11) Pro, (12) Val, (13) Nvi, (14) Met, (15) Tyr, (16) Ile, (17) Leu, and (18) Arg. Peaks A, C, and D refer to system peaks.
presence of the sulfonates improved the situation greatly, to the point where temperature can be used routinely to manipulate the analysis of amino acids. Typical Chromatograms. The above results can lead to the separation of a large number of amino acids. For example, Figure 5 shows a 45-min separation of 18 acids, using a mobile phase containing acetate buffer, Cu(I1) ions, and heptanesulfonate, run at 45 O C . The elution order is as expected according to the hydrophobicity and charge of the amino acids (32). The grouping of the peaks, depending on the polarity of the solutes, should be noted. The acidic amino acids elute very fast, while the basic ones can remain in the column for a long time. In fact, under the chromatographic conditions shown in Figure 5, Arg has one of the longest retention times of all solutes. Only phenylalanine and tryptophan (not shown in the chromatogram) had higher k'values. The observation made before about the strong dependence of the basic amino acids retention on the type and concentration of the sulfonate should be kept in mind, since it could provide the means of further shortening of the analysis time in the future. The chromatogram shown in Figure 6 demonstrates the effect of the alkylsulfonate length on the retention. With hexanesulfonate, higher concentration of the reagent and lower temperature are needed in order to resolve the polar amino
1834
ANALYTICAL CHEMISTRY, VOL. 57,NO. 9,AUGUST 1985 MOBILE P H A S E : 0 0 1 M b u f f e r ocelote
4 ~ 1 0 .M~ C u A c p 0.004M hexonesuiphonote COLUMN. RP18. 25014mm, 7u FLOW R A T E : Z m I / m i n
T E M P 30'C
hapt anatulphonntr 2
1
Figure 8. A chromatogram of a mixture of amino aclds. The 0.01 acetate buffer contained 4 X M Cu(I1)ions and 4 mM hexanesulfonate. The temperature was 30 O C . The detector sensitivity was 0.16 AUFS. The solutes are (1)Asp, (2)Glu, (3)Gly, (4)Ser, (5)Asn + Ala, (6) Gln, (7) Hyp, (8)Thr, (9)His, (10)cy-Abu, (11)Pro, (12)Val -I-Nvl, (13)Met, (14)t y r , (15)Arg, (16) Ile, and (17)Leu. Peaks A, C, and D refer to system peaks.
acids. On the other hand, the retention of the hydrophobic solutes, under these conditions, increases and the analysis time is longer than that with heptanesulfonate. The two chromatograms demonstrate the versatility of the method: the type of alkylsulfonate can be chosen according to the desired retention times and resolutions of amino acids subgrouping. The peaks of the very retained solutes are rather broad, a fact which leads to poorer detection limits of these amino acids. The present paper does not deal with the quantitation of the amino acids, and thus the peak broadening did not present a serious problem. This point will have to be dealt with in future work when detection limits and minimum detectable amounts are studied. However, it was found that very linear calibration curves can be obtained with the present system, provided that the in column concentration is less than that of the copper ions. For example, valine was injection in the concentration range of 4 X to 0.001 M, and the resultant peak height followed the equation height = 3.87
+ (1.72 X 105)C
where C is the concentration. In cases where the solute concentration exceeds that of the copper ions, the calibration curves are no longer linear, and the retention becomes a function of that concentration. System Peaks. The negative peaks in the chromatogram are of great importance for several reasons. From a practical point of view, it is clear that these peaks can interfere with either the quantitation or identification of the amino acids. From a theoretical point of view, however, they can be of great value in helping to study the retention processes which occur in this complicated system (1432). Negative peaks are part of what is known as "system peaks". Although such peaks are well-known, they are rarely discussed (21,32)in the literature. There are four system peaks in the present case, of which three are indicated by the letters A, C, and D in Figures 5 and 6. These are due most likely to the acetate, copper ions, and sulfonate. While the exact assignment of the system peaks, and their dependence on the experimental conditions, are currently under investigation, we would like to present here some initial results as to the nature of one of the negative peaks. Figure 7 shows the dependence of the capacity ratio of system peak C on the concentration of each of the sulfonates. The first obvious observation is that for a given concentration the k'value increases with the chain length of the ion-pair
hexanaaulphonnt a
3
6
9
12
s u l p h o n a t e c o n c . ImMI Flgure 7. Dependence of the capacity ratio of system peak C on the concentration of the three sulfonates. The acetate buffer eluent contained, in addition to the sulfonates, 5 X loa4M Cu(I1) ions.
reagent. The second point to observe in the figure is the continuous, albeit not linear, increase in the capacity factors with the alkylsulfonate concentrations. The exact reason for the increase, which is probably related to the formation of the Cu-sulfonate pair on the stationary phase, is now under study.
CONCLUSIONS To summarize it can be said that the improved method described here is quite promising at the present stage. The combination of the various parameters, chemical as well as chromatographic, as a means of tuning the selectivities adds a significant tool to the analysis of free amino acids. Once the effects of other parameters, such as pH, Cu(I1) concentration, and organic modifiers, are ascertained, then further advances will be feasible, especially with respect to the analysis time. It is in this direction that our research is now aimed. Registry No. Asp, 56-84-8; Glu, 56-86-0; Gly, 56-40-6; Ser, 56-45-1;Am, 70-47-3; Ala, 56-41-7; Gln, 56-85-9; Hyp, 51-35-4; Thr, 72-19-5;His, 71-00-1; a-Abu, 1492-24-6;Pro, 147-85-3;Val, 72-18-4;Nul, 6600-40-4;Met, 63-68-3;Tyr, 60-18-4;Arg, 74-79-3; Ile, 13-32-5; Leu, 61-90-5; Cu, 7440-50-8; CuAc2, 142-71-2; heptanesulfonic acid, 60586-80-3;acetic acid, 64-19-7; octanesulfonic acid, 3944-72-7; hexanesulfonic acid, 13595-73-8. LITERATURE CITED (1) Pfeifer, R. F.; Hill, D. W. "Advances In Chromatography"; Glddlngs, J. C., Grushka, E., Cazes, J., Brown, P. R.. Eds.; Marcel Dekker: New York, 1983; Vol. 22, p 37. (2) Willlams, A. P. "HPLC in Food Analysis. Food Science and Technology"; Macrae, R., Ed.; Academic Press: New York, 1982; p 285. (3) Hearn, M. T. W. "Advances in Chromatography"; Vol. 20, Glddings, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1982; p 1. (4) Kuster, T.; Niederwleser, A. "Chromatography Part B"; Heftmann, E., Ed.; Elsevier Scientific: Amsterdam, 1983; p 1. (5) Grushka, E.; Levin, S.;Gilon, C. J . Chromatogr. 1982, 235, 401. (6) Foucault, A.; Rosset, R. J . Chromatogr. 1984, 3 1 7 , 41. (7) Gimpel, M.; Unger, K. Chromafographis 1983, 17, 200. (8) Sampson, B.; Barlow, G. B. J. Chromatogr. 1980, 183, 9 . (9) Chlnnick, C. C. T. Analyst (London) 1981, 106, 1203. (10) Kok, W. Th.; Brlnkman, U. A. Th; Frei, R. W. J . Chromatogr. 1983, 256. 17. (11) Davankov, V. A,: Kurganov, A. A,; Bochkov, A. S. "Advances in Chromatography"; Giddlngs, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1983; VoI. 22, p 71. (12) Gil-Av, E.; Tishbee, A,; Hare, P. E. J. Am. Chem. SOC. 1980, 102, 5115. (13) Gilon, C.; Leshem, R.; Grushka, E. J . Chromatogr. 1981, 203, 547. (14) LePage, J. N.; Llndner, W.; Davles, G.; Seitz, D. E.; Karger, B. L. Anal. Chem. 1979, 51, 433. (15) Alexander, P. W.; Maltra, C. Anal. Chem. 1981, 53, 1590. (16) Kok, W. Th.; Hanekamp, H. B.; BOS, P.; Frel, R. W. Anal. Chlm. Acta 1882 .- - -, 142 . .- , 31 - .. (17) Alexander, P. W.; Haddad, P. R.; Low, G. K. C.; Maitra, C. J. Chromatogr. lS81, 209, 29.
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(27) Feibush, 6.;Cohen, M. J.; Karger, B. L. J . Chromatogr. 1983, 282, 3. (28) Hearn, M. T. W. “Advances In Chromatography”; GLddlngs, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York,
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RECEIVED for review January 9,1985. Accepted April 8,1985.
Peak Compression Effects in Ion-Pair Reversed-Phase Liquid Chromatography of Substituted Benzamides Lars B. Nilason* Department of Bioanalytical Chemistry, Research and Development Laboratories, Astra Lakemedel AB, S-151 85 Sodertdlje, Sweden
Douglas Westerlund Department of Analytical Pharmaceutical Chemistry, Uppsala University Biomedical Center, P.O. Box 574,S-751 23 Uppsala, Sweden
Peak compression effects have been studied in Ion-pair reversed-phase iiquld chromatography of substituted benzamldes. The sample Is injected in a soiutlon of an organic anion. A zone wlth a depletion of one of the mobile phase components is created by this organlc anion. The peak for an analyte which coeiutes with the depleted zone wlii be extremely narrow: chromatographic efficlencles corresponding to >lo6 plates/m have been obtalned. A retention model for this effect is proposed.
Reversed-phase liquid chromatography is widely accepted
as a tool for the separation and quantitation of compounds in complex mixtures. One of the main features is the possibility of optimizing the separation in a simple way by varying the mobile phase composition, e.g., the nature and amount of organic modifying solvent, the pH, the ionic strength, and the nature and concentration of additives such as complexforming or ion-pairing agents. In many cases the injected sample is dissolved in the mobile phase in order to minimize base line disturbances. When a sample with a composition deviating from the mobile phase is injected, the established column equilibria are disturbed and anomalous peaks, called ghost peaks (11,vacant peaks (21,or induced peaks (3),may appear. Such a peak is due to a migrating zone with a deviating concentration of one of the mobile phase components. Peaks of this kind are often regarded as complications or curiosities (1,2).Normally the mobile phase is made up of components not registered by the detector in use and the induced peaks are thus not observed. However, in the so-called UV-visualization liquid chromatography, the mobile phase contains a UV-absorbing component and the UV-trace is monitored (4-10). In such a system the injection of components which interact with the UV-absorbing component in the mobile phase will give rise 0003-2700/85/0357-1835$01.50/0
to two kinds of peaks, The first kind involves one peak for each interacting component in the sample, and the second kind originates from the elution of an excess (positive peak) or a deficiency (negative peak) of the detectable mobile phase component. These peaks are usually called system peaks (4, 5). The UV-visualization technique has been applied to quantitative determinations (8-10). This paper reports on a study, where UV-transparent zones are used to enhance the chromatographic efficiency for UVabsorbing compounds. The analyks, substituted benzamides, were injected dissolved in an acidic buffer containing a high concentration of an organic anion, alkyl sulfate or alkylsulfonate. The acidic mobile phase contained a tertiary amine as a cationic ion-pairing agent. The injection of the organic anion gave rise to a migrating zone with a deficiency of the cationic mobile phase component. A sufficiently high concentration of the injected anion gave a totally depleted zone and an extremely compressed peak was observed for the analyte that coeluted with this depleted zone. With this method it was possible to obtain peak compression for a selected component in the sample by a careful choice of the composition of the mobile phase and of the injected solution. Injections of ion-pairing agents have earlier been used to influence the retention properties of the column. The ionpairing agents have then been injected before or after the injection of the sample and for quite different purposes. Stranahan et al. (11)used pulsed injections of octanesulfonate to influence the separation of substituted anilines and Berry and Shansky (12)used a similar approach to)obtain qualitative information on the charge of the injected components.
EXPERIMENTAL SECTION Chemicals. The sodium salts of pentane-, hexane-, and oc: tanesulfonic acid were obtained from Eastman Kodak Co. (Rochester, NY). The sodium sal& of hexyl and octyl sulfate were 0 1985 Amerlcan Chemlcai Society
