Anal. Chem. 1086, 58, 1602-1607
1602
System Peaks in Liquid Chromatography: Their Origin, Formation, and Importance Shulamit Levin a n d Eli Grushka*
Department of Inorganic and Analytical Chemistry, The Hebrew University, Jerusalem, Israel
System peaks refer to the extraneous peaks, not dlrectly related to the InJected solutes, that appaar In the chromatck gram whenever the mdrllo phase con2alns more than one component. The tormatbn of these peaks can be explained In terms of the equUlbrium In the column and Its perturbatkm by the Injection of a sample In a solvent whose compodtlon is dlfferent from the mobile phase. The perturbation results in a relaxation process whose consequences are the desorption, or enhanced adsorptlon, of part or ail of the moMlephase componenkthat w e r ~prevkusty adeorbed on the statlonary phase. The system peaks are the product of the relaxation process. They are characterized by the fact that their capacRy ratios are Independent of the nature of the solutes Injected. However, their areas can be a strong function of the solutes. The dependence of the areas of the system peaks on the nature and amount of the solutes can yield a wealthofkrlormetkn such ascokrm void voltme, the amount of mobh-phase comqment adsorbed on the statbnary phase, and related parameters.
Quite frequently, upon injection of a sample into a mobile phase containing more than one component, the resulting chromatogram shows more peaks than the number of solutes in the sample. On the whole, these additional peaks, which will be called here system peaks, are regarded as nuisance, and the analyst will often search for ways to eliminate them from the chromatogram. These extraneous peaks, which can be visualized with the aid of an appropriate detector, are omnipresent whenever the mobile phase contains more than one component. They have been called system peaks (I,2), pseudo peaks (3),ghost peaks (4,5), eigenpeaks (6),vacancy peaks (7-9), induced peaks (IO),etc. They are frequently misunderstood and, consequently, misused. The name “system peaks” is perhaps most appropriate, since terms like eigenpeaks have the wrong connotation, and, as will be shown, there is nothing pseudo or ghostlike about them. Consequently, the term system peaks will be used in this paper to indicate peaks not directly attributed to the actual solutes in the sample. Several investigators have examined various aspects of system peaks (1-26). McCormick and Karger (9, as well as Scott et al. (9),considered these peaks as a phenomenon that deserved a special treatment. Other workers have even managed to obtain some useful information out of them (4, 6, 16, 17‘). Schill and his co-workers (19-2I), Bidlingmeyer et al. (22-25), and Deming and his group (IO)have discussed system peaks in conjunction with solute visualization in ion pair chromatography. Still, relatively little is discussed in the literature about the processes leading to the formation of system peaks (4-10, 27, 28) The system peaks contain a wealth of information about the thermodynamics (i.e., retention) and kinetics (i.e., broadening) processes that occur in the column. Thus, they can lead not only to a better understanding of the chromatographic process but also to a better evaluation of the nature 0003-2700/86/0358-1602$01.50/0
and amount of the solutes in the sample injected. It is the aim of the present communication to demonstrate the origin of such peaks and the multitude of information that can be obtained from them. Origin of System Peaks. When a mobile phase is introduced into a chromatographic system the equilibrium in the column is maintained as long as the chromatographic system is not perturbed. However, if the mobile phase is suddenly changed, or if a solute is injected into the column, then the equilibrium is disturbed. The perturbation can best be explained by the fact that the solutes enter the column moving with the velocity of the mobile phase and not with the equilibrium velocities dictated by the distribution between the stationary and mobile phases. At the onset of this perturbation, the chromatographic system begins immediately to relax to a new state of equilibrium. The relaxation can proceed in several ways: (1) The solute(s) can be preferentially solvated by one or more components of the mobile phase. (2) The injected sample, by virtue of being relatively devoid of (or richer in) one or more mobile-phase components, causes the redistribution of these components either from or to the stationary phase. (3) Interactions between the solutes and adsorbed mobile-phase components can cause a release, or further adsorption, of the component(s). The results of these events are that the injected zone, now perhaps broadened a bit, contains not only the solutes but also some or all of the mobile-phase components with concentration differing from the bulk mobile phase. More important, however, is the fact that the relaxation process is manifested in a decrease in the velocities of the solutes, and of the mobile-phase components whose concentrations are different from the bulk mobile phase, toward their characteristic velocities, ui, as defined by ~i = R ~ u where Ri is the equilibrium fraction of the ith species in the mobile phase and u is the velocity of the mobile phase. The net result is a chromatogram containing peaks for the solutes as well as for some or all of the mobile-phase components. A qualitatively similar chromatogram would have resulted from the injection of a sample containing the solutes and the appropriate mobile-phase components;the position of the peaks will be the same but not their areas or direction. System peaks are characterized by the fact that they have, in a given chromatographicsystem, constant k ’values irrespective of the sample injected. In spite of that, they can appear as positive or negative peaks, relative to the detector base line, and their areas can depend on the nature of the injected solutes. The above description leads to the conclusion that the chromatogram resulting from the relaxation process is actually an imprint of the equilibrium in the column before the injection. Thus, by the correct choice of the injected sample, extremely useful information can be obtained about the chromatographic column and the processes occurring in it. One such system, based on a previous work of ours (26), is reported here. It is composed of a reversed-phase column and a mobile phase containing acetate buffer, cupric acetate, and heptanesulfonate. The solutes included some amino acids, 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
mobile-phase components, and pure water. EXPERIMENTAL SECTION Instruments. The chromatographic system included the following components: Perkin-Elmer series 4 liquid chromatograph equipped with a 6-pL loop Rheodyne injection valve, Perkin-Elmer 85B spectrophotometric variable-wavelength detector having a 1.4-pL flow cell, and a W&W Model 600 recorder. The detector was operated at 235 nm. The column was a 25-cm Lichrosorb RP18 cartridge, held by a Hibar manual holder. It was thermostated with the aid of a homebuilt water bath (f0.5 "C). A precolumn, made in our laboratory from a mixture of Lichroprep Si-60 and Lichroprep C18 (Merck, Darmstadt), was connected between the pump and the injector. Material. The acetate buffer (0.1 M, pH 5.6) was prepared by using 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 the amino acids used and the sodium salt of heptanesulfonate were purchased from Sigma Chemicals. The water for the mobile phase and the samples was purified in our laboratory (26). Procedures. In all the studies, the mobile phase was 0.1 M, pH 5.6, acetate buffer, containing 5 X lo4 M cupric acetate and various concentrations of heptanesulfonate. Each time that a new mobile phase was introduced into the column, a period of about 20 min was allowed for equilibration. This was sufficient to establish the equilibrium in the column, as judged from the behavior of the k'values. Between changes of mobile phases, the chromatographicsystem, including the column, was washed with 100 mL of a solution made of 0.01 M citric acid in acetate buffer followed by 100 mL of methanol and water. RESULTS AND DISCUSSION A. Rationale f o r the Mobile Phase Used. The basic requirements from a chromatographic system that will produce system peaks are as follows: (1)The mobile phase should be complex, containing a t least two components. (2) The mobile phase should give a measurable response to the detector used. (3) A distribution of at least one mobile-phase component, between the stationary and the mobile phase, should occur. All these conditions are met by the choice of the present mobile-phase system and solutes. For one thing, the copper ions in the mobile phase have sufficient background in the UV range used for detection to allow the visualization of most solutes. In addition, complexation of the cupric ions by the solutes causes a change in the concentration of the free Cu(I1) ions, which can be monitored, and thus used for column equilibrium studies. Also, as was shown by Knox and Hartwick (30), by Deelder et al. (31,32), Cantwell (33),and others (34-39, the heptanesulfonate is adsorbed on the stationary phase forming a charged primary layer followed by a secondary charged layer of counterions. This provides opportunities to observe, directly or indirectly, interactions between the injected sample and the adsorbed mobile-phase components. The system peaks characteristic to the present mobile phase are shown in Figure 1. The figure consists of a group of chromatograms resulting from the injection of pure water (top) and various amino acids, a t identical concentration, each injected individually. The chromatogram due to the injection of water shows four peaks, marked A, B, C, and D. The rest of the chromatograms show the same four peaks plus an additional one that is due to the amino acid involved. Peaks A-D are the system peaks, and as expected, their retention times are independent of the nature of the solutes, whereas their areas are not. Of interest is the observation that the direction of the injected solutes peaks, i.e., negative or positive relative to the base line, is independent of their k' values relative to the k' values of the system peaks. This is the opposite of what Schill(l9-21), Solms (5),and others found. The reasons for the difference might be due to the nature of the interactions between the solutes and the present mo-
1603
L
time (min)
Chromatograms showing four system peaks. The system peaks are identified by the letters A, B, C, and D. The top chromatogram is the result of the injection of pure water. The bottom four chromatogramsare the results of the injections of ty~oslne,methionine, valine, and threonlne, respectively. In all cases, the mobile phase included acetate buffer (0.1 M, pH 5.Q 0.5 mM copper acetate, and 5.0 mM heptanesulfonate. Flgure 1.
"T
Heptanssulfonate conc.
[mMI
Figure 2. Dependence of the capacity ratios of tyrosine and system peaks B, C, and D on the concentration of the heptanesulfonatein the mobile phase. In addltion to the sulfonate, the mobile phase contained acetate buffer (0.1 M, pH 5.6) and 0.5 mM copper acetate.
bile-phase Components. As will be shown shortly, however, it appears likely that the concentration of the mobile-phase components can determine the direction of the peaks. The meaning of the chromatograms in Figure 1is rather important; they demonstrate that the system peaks are due to definite chemical species. The injection of the water is therefore quite revealing, since it points out to the mobile-phase components as the only possible source of the system peaks. The chemical species in the mobile phase are sodium ions, copper ions, acetate ions, heptanesulfonate ions, and water molecules. In principle, all of the species could result in system peaks. In fact, for reasons which will be discussed later, it was felt that system peak D is due to the sulfonate, system peak C is due to the Cu(I1) ions, system peak B is due to the acetate, and system peak A is due to water and, possibly, to sodium ions. B. Effect of Mobile-Phase Components Concentration on the Retention of System Peaks. While not affected by the nature of the injected solute, the retention factors were considerably influenced by the chromatographic conditions, such as temperature (26)or mobile-phase composition. An example is shown in Figure 2, where the influence of the heptanesulfonate in the mobile phase on the retention of the system peaks is depicted. The solute probe in this case was the amino acid tyrosine (0.4 mM) in water. As expected, the retention of the tyrosine increased with the increase in the concentration of the ion pair reagent in the mobile phase.
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
“T ZBI
t 6
e 15
+
d 1
6 iy
a.
Hept a n e s u l f o n a t e c o n c . 2
4
E
6
Hept anesulf onat e conc.
10
[mMl
Flgure 3. Behavior of the capacHy ratios of t h e arcylsunonate solutes
as a function of the concentratlon of heptanesulfonate in the mobile phase. The concentratlon of the solutes was 0.1 M in water. In addition to the heptanesulfonate,the mobile phase contained acetate buffer and copper acetate having the concentrations as Indicated in Figure 2. A, B, and C stand for heptane-, hexane-, and pentanesulfonate, respectively. System peak C, due to Cu(I1) ions, behaved in the same manner. However, k’of system peak D, which is attributed to the sulfonate, decreased rather noticeably. The retention of peak B, due to acetate, decreased slightly as well. The explanation for these behaviors is as follows. It was pointed out already, that the presence of the sulfonate in the mobile phase is accompanied by the adsorption of that compound on the reversed phase (30-37). The more sulfonate in the mobile phase, the greater is the amount adsorbed on the stationary phase. As a direct correlary, the concentration of the Cu(I1) ions, in the secondary layer of counterions, is also increased. The net effect, of course, is to increase the partition coefficient of Cu(II), as seen in the figure. Peak D, the sulfonate peak, represents a more complicated behavior. It was expected that the retention of that peak would be independent of the amount of sulfonate in the mobile phase. This is because the introduction of mobile phases containing increasing amounts of heptanesulfonate would cause reequilibrations in such a way as to keep the ratio of sulfonate concentrations in the stationary and mobile phases, constant, i.e., linear chromatography. If such was the case, then peak D (sulfonate) would have a constant retention time. The interpretation of Figure 2 is, therefore, that the adsorption isotherm of the sulfonate, at the concentration range shown in the figure, is not linear. Figure 3 lends credence to this argument. In this figure, the capacity ratios of pentanesulfonate, hexanesulfonate, and heptanesulfonate, injected as solutes, are shown as a function of the heptanesulfonate in the mobile phase. The behavior of the heptanesulfonate solute is almost identical with that of system peak D. The other two sulfonates behave similarly, insofar as their capacity factors decreased with increasing heptanesulfonate in the mobile phase. A possible reason for the nonlinearity is electrostatic repulsion in the adsorbed layer. The prospects of system peaks yielding important quantitative information about adsorption isotherms is now under further investigation. It should be pointed out that peak D could be observed only when the concentration of the sulfonate in the mobile phase was above 2 mM. Electrostatic effects may also be responsible for the slight decrease in t h e retention of system peak B, attributed to acetate. However, this behavior may also be an artifact due to errors in determining the k’ values of this peak. Undoubtedly, the increased amounts of adsorbed sulfonate change the volume of the stationary phase in a manner dis-
[mMl
Figure 4. Dependence of the area of tyrosine peak and of system peaks B, C, and D on the concentration of heptanesulfonate-in the
mobile phase. The rest of the mobile-phase components are as in Figure 2. The zero line is drawn for reference purpose only. cussed by Horvath and co-workers (27) and by Kovats (28). Therefore, the experimental measurements of the void time, to, are subjected to errors, which make the calculations of the capacity factors questionable. It should be mentioned here that system peak A was used as the to marker. However, the retention of peak A decreased as the concentration of the sulfonate in the mobile phase increased, a fact which might be due to the change in the phase ratio. Thus, the k’dependence of peak B, the acetate, is not completely understood, and further work on this point is needed. C. Dependence of the System Peaks’ Areas on the Sulfonate Concentration in the Mobile Phase. During the study it was observed that the areas of the system peaks changed with the amounts of the sulfonate in the mobile phase. Figure 4 shows the areas of peaks B-D and of tyrosine, which was the probe solute (0.4mM in water). The area of the amino acid was largely uneffected by the concentration of the sulfonate in the mobile phase. This is not surprising, since the only source of that solute is the injection, and in the absence of irreversible adsorption on the column, the amount that the detector sees is constant, irrespective of the amount of sulfonate. The same behavior was observed for all amino acids used in this study. The areas of the system peaks, on the other hand, did depend on the concentration of the sulfonate in the mobile phase. The pattern shown in Figure 4 repeated itself whether pure water or water with a solute was injected. Before discussing the data, a semantic point should be clarified. The zero line in Figure 4 represents the detector base line. Therefore,the lines in the figure show that the peak area and direction can change from a peak in the negative sense to the positive sense. It is difficult to state, unambiguously and in absolute terms, when the areas decrease or increase. However, for the sake of the present discussion, a change toward the positive direction will be taken as an increase in the peak area. Thus, under this convention, system peak C (Cu(I1) ions) increases in area as the concentration of the sulfonate in the mobile phase is increased. 1. Behauior of Peak C. The behavior of the copper-related system peak C can be explained as follows: A t very low concentrations of sulfonate in the column, the concentration of Cu(I1) ions in the counterion layer is small, in fact smaller than the concentration of Cu(I1) in the mobile phase. When the sample zone enters the column, the stationary phase sees a plug of mobile phase devoid of acetate buffer, of sulfonate, and of cupric ions. The copper ions will then be released to the mobile phase. The UV absorbance of this amount of copper ions is less than that of the mobile phase, and the result will be system peak C in the negative direction. This type of system peak is often referred to as a vacancy peak. As the concentrationof the sulfonate in the mobile phase is increased,
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8 , JULY 1986
the stationary phase is enriched by both the sulfonate and Cu(II) ions. When the injected sample enters the column more and more sulfonate and Cu(I1) ions are released and the area of peak C increases. Past a certain sulfonate concentration, which in the present study was about 0.8 mM, the amount of Cu(I1) ions released from the stationary phase absorbs UV radiation more than the mobile phase, and the system peak becomes positive. This variant of the system peak is often referred to as a displacement peak. 2. Behavior of Peak D . The behavior of system peak D (due to sulfonate) is contrary to expectation, since its area seems to decrease with increasing concentration of the sulfonate in the mobile phase. The contradictionis resolved when it is realized that a system peak represents the difference between the detector signal of the mobile phase and the zone containing the species responsible for the system peak. In the present set of experiments, the absolute level of the detector base line increased with increasing amounts of sulfonate in the mobile phase. Thus, even though the area of system peak D is becoming more negative, calibration curves showed that, in fact, the actual amounts of sulfonate released from the stationary phase increased with increasing concentrations in the mobile phase. Another apparent contradiction exists in view of the retention behavior of system peak D, shown in Figure 2. It is true that the capacity ratio of peak D decreases with an increase in the concentration of the sulfonate in the mobile phase. On the face of it, this should mean that the amount of sulfonate adsorbed is decreasing. However, in the actual experiment, the total amount of sulfonate in the system was increased, and therefore, the absolute amount of adsorbed sulfonate increased even though the ratio between the adsorbed and free sulfonate decreased. 3. Behavior of Peak B. System peak B (due to acetate) is negative, which can be explained as follows. It is evident from the k’value of peak B that the amount of acetate in the stationary phase is much less than the amount of acetate in the mobile phase. Since the source of the acetate in the zone of system peak B is the stationary phase, the amount of acetate in that zone, and its UV absorbance, will always be less than that of the mobile phase. However, it is not immediatelyclear why an increase in the mobile-phase concentration of the sulfonate should increase (i.e., make less negative) the area of system peak B. This question is now under investigation. D. Behavior of the System Peaks as a Function of the Amount of Injected Mobile-Phase Components. The previous section dealt with system peaks in the case where the concentration of one of the mobile-phasecomponents was varied. To more completely characterize the system peaks, attention will now be turned to studying the behavior of these peaks when various amounts of the mobile-phase components are injected into the column as solutes, while keeping a constant mobile-phase composition. These two studies actually complement each other, since in both cases the effects of the same components are examined. However, in the first case the thermodynamics of the system were allowed to vary, while in the next study they were kept constant. 1 . Effects of Injected C u ( I 0 Ions. When Cu(I1) ions in water were injected into the column, only peak C (due to Cu(I1) ions) showed a change; its height increased with increasing concentration of the injected cupric ions. Moreover, at zero Cu(II), Le., injection of pure water, system peak C is positive. The reason for that is as follows: When the pulse of injected water arrives at the head of the column, most, if not all, of the adsorbed acetate, and a large fraction of the adsorbed sulfonate, will be desorbed into the mobile phase. As a result, the copper ions in the counterion layer will also be resolvated in the mobile phase. Under the condition of the experiment,the capacity ratio of Cu(I1) ions is greater than
Conc. of
1605
InJected acetate [MI
-44
1
58
conc. o f
188
I 5m
inJected acetate
288
25b
[mMI
Figure 5. (A) Dependence of the heights of system peaks B and C on the concentration of injected acetate. (B) Dependence of the height of system peak D on the concentration of injected acetate. The mobile phase is described in Figure 1. The zero line is drawn for reference purpose only.
unity (k’= 1.38). Thus, the amount of released copper ions in the zone is greater than in the bulk mobile phase. Due to the large UV absorptivity, system peak C will be positive. When the injected sample contains Cu(I1) ions, the net effect will be to add this concentration of the injected sample to the cupric ions released from the stationary phase. Peak C will, therefore, increase with increasing concentration of the injected sample. This behavior, coupled with the behavior described above, provides the indication that peak C is due to the copper ions. The system peaks due to the acetate (peak B) and to sulfonate (peak D) are negative, since the UV absorbances of the relevant components in their zones are less than that of the bulk mobile phase. These peaks do not change their height, since the amount of acetate, or of sulfonate, released from the stationary phase is a function only of the volume of the injected sample and not of the Cu(I1) ions in it. It is important to stress here again that since the mobile phase remained constant throughout the experiment, the k ’values of all the peaks, including peak C, remained unchanged. 2. Effects ofznjected Acetate. The effect of the amount of injected acetate (dissolved in water), is different from above, as seen from Figure 5. With increasing acetate in the sample, peak C (Cu(I1) ions) decreases relative to the base line, whereas peak B (acetate) increases; see Figure 5A. The reason for the increase of the acetate system peak, peak B, is clear, and it provides the evidence identifying system peak B as indeed due to acetate ions. When pure water is injected, the adsorbed acetate at the head of the column will desorb as soon as the water zone reaches there. Since the capacity ratio of peak B is very small, the concentration of the released acetate is much less than that of the bulk mobile phase, and its UV absorbance is less than that of the bulk phase. Hence the peak initially is negative. As the amount of the injected acetate is increased,
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
.y.t.n
m $ c
Conc. of
m 18
v.y*tm 15
injected sulfonate
2B
p..t
D
psnk 0
25
InM
Dependence of the heights of system peaks B, C, and D on the concentration of injected heptanesulfonate. The mobile phase was as in Fgwe 1. The zero peak line is drawn for reference purpose Flgure 6.
only.
the amount of the desorbed acetate decreases, becoming zero when the injected concentration equals that of the mobile phase. Up to this point, the total s u m of the acetate in the system peak zone is such that its UV absorbance is still less than that of the mobile phase. Past that point, acetate from the injected sample will be adsorbed at the head of the column; still the amount of acetate in the system peak zone will be greater than that in the mobile phase, and the peak will be positive. The decreasing area of the copper system peak, peak C, is also clear in light of the above explanation. The amount of Cu(I1) ions in the counterion layer is intimately related to the amount of adsorbed acetate. I t was just shown that as the concentration of the acetate in the injected sample increases, the amount of acetate desorbed from the stationary phase into the entering sample zone decreases. Therefore, the amount of copper ions that will be released, accompanying the acetate, will also decrease, and the area of system peak C will decrease. Past a certain concentration of the injected acetate, copper ions will begin to be adsorbed at the head of the column upon the arrival of the sample zone. Further increase in the injected acetate will increase the amount of adsorbed acetate at the head of the column, and consequently more free cupric ions will be depleted from the injected zone, and peak C will become more negative. The unique concentration of the injected acetate that causes the copper-related system peak, peak C, to vanish is related to the capacity ratios of the acetate and Cu(I1) ions. The exact relationship will be dealt with in a forthcoming publication. For the present, attentions are called to the fact that the line describing peak B in Figure 5A crosses the x axis a t a higher injected acetate concentration than the line describing peak C. The behavior of the sulfonate system peak, peak D, shown in Figure 5B is more perplexing. I t is not clear why peak D should become less negative as the concentration of the injected acetate increases. Perhaps it is related to electrostatic interactions. If such is the case, it is possible that the behavior of system peak D can shed light on the dependence of the retention on ionic strength. However, the scale of Figure 5B should be noticed. More work is needed in order to understand the behavior of the sulfonate system peak. 3. Effects of Injecting Sulfonate. Figure 6 shows the effect of injecting increasing amounts of sulfonate dissolved in water. Peak B (acetate) was completely independent of the concentration of the sulfonate in the sample. The area of peak D increased, changing its direction from negative to positive. This behavior of the sulfonate system peak can be explained in exactly the same way as was done with the acetate system peak, peak B, when acetate was injected to the column. The cross point when peak D disappears from the chromatogram
occurs when the concentration of the injected sulfonate was close to that in the mobile phase, that is 5 mM. This, and previous discussions, point to the fact that system peak D is due to the sulfonate. Likewise, the explanations for the behavior of system peak C (copper ions), which decreased with increasing concentrations of the injected sulfonate, are the same as above. Here too, the concentration of the injected sulfonate that caused peak C to be zero is related to the capacity factors of the sulfonate and the copper ions. Attention is called to the fact that in the sulfonate case, unlike the acetate case shown in Figure 5A, the concentration of the injected sulfonate that causes peak C to be equal to zero is greater than the concentration that causes peak D to vanish. E. System Peak A. While it is relatively easy to attribute the chemical species belonging to system peaks B, C, and D, the identification of peak A is more problematic. Currently, it is thought to be due to water and, most likely, to sodium ions as well. Research under study is looking more closely at the chemical composition of this system peak. In any event, the chromatographic zone due to peak A absorbs UV radiation less than the mobile phase, thus appearing as a negative peak in all cases, except when the injected sample was high in sodium concentration. F. Calculation of the Amount of the Adsorbed Mobile-Phase Components. As was mentioned previously, the arrival of a small volume of pure solvent, at the head of a preequilibrated column, is accompanied by the desorption of some or all of the adsorbed components of the mobile phase. In the present case, most, if not all, of the acetate, a large portion of the sulfonate, and therefore practically all the copper ions will be resolvated at the head of the column upon injection of pure water. That release of mobile-phase components takes place in a volume very close to the injection volume, which in the present case was 6 wL. Thus, with the aid of the calibration-type curves shown in Figures 5 and 6, the amounts of adsorbed mobile-phase components can be calculated. An example for the calculation of this kind will be discussed in the case of copper ions. It was mentioned already that the injection of pure water results in a positive copper peak, peak C. The area under the peak was equivalent to the area that resulted from the injection of 6 WLof 0.7 mM copper acetate dissolved in the mobile phase. Therefore, the amount of copper ions, us, present in the counterion layer is about 42 nmol/6 pL of mobile phase. The concentration of the Cu(I1) ions in the mobile phase was kept constant a t 0.5 mM; that is, the amount of copper ions in the mobile phase, w,, is 30 nmol/the same volume unit as above. The ratio of the amounts WJW,, which is equal to 1.4, is, of course, the capacity ratio of copper ions. This value compares very favorably with the conventionally determined value of 1.38. The cardinal point of this discussion is that by using the area of the system peak, the capacity ratio can be obtained without having to know the void volume of the column. In fact, from the capacity ratio of the system peak, the void volume of the column can be calculated by using the conventional definition of k' in the following manner: to = t*/(1 + k? where to is the void time or the dead time and t R is the retention time of the component. This method of calculating to is very attractive as it circumvents all the well-known difficulties associated with the measurements of the void volume (27-29). Two main assumptions were made in this treatment: (1) the total desorption of the copper ions into the zone of the pure water and (2) the volume of this zone being very close to that of the injected sample. The validity of this approach to the measurements of the capacity ratios, and its extention to the determination of the void volume and therefore to adsorption isotherms, is now under intensive
Anal. Chem. 1988, 58, 1607-1611
investigation in our laboratory.
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(6) Melander, W. R.; Erard, J. F.; Horvath, Cs. J . Chmatogr. 1983, 282, 99Q
(7) &-&mlck, R. M.; Karger, B. L. J . Chromatogr. 1980, 799, 259. ( 8 ) Skis, K.; Krejcl, M. J . chrometogr. 1974, 9 7 , 161. (9) Scott. R. P. W.; Scott, G. C.; Kucera, P. Anal. Chem. 1972. 44, 100. (IO) Stranahan, J. J.; Demlng, S. N. Anal. Chem. 1982, 5 4 , 1540. (11) Hendrlx. D. L.: Lee. R. E., Jr.: Baust. J. G.: James, H. J . Chromatow. 1981, 270, 45. Barber, W. E.; Carr, P. W. J . Chromatogr. 1983, 260. 89. Barber, W. E.; Cam, P. W. J . Chrometcgr. 1984, 376, 211. Skelly, N. E. Anal. Chem. 1882, 54, 712. Herne, P.; Renson. M.; Crommen, J. Chromatograph& 1984, 79, 274. Hershcovltz, H.; Yarnltzky, Ch.; Schumuckler, 0.J . Chromatogr. 1982. 244. 217. .- .. .. . Vlgh, G.; Lehold, A. J . Chromtogr. 1984, 372, 345. M e n s , J. J . Llq. Chromatogr. 1982, 5 , 1467. Denkert, M.; Hackzell, L.; Schlll, 0.; Sjogren. E. J . Chrometogr. 1981, 278. 31. (20) Hacl&ell, L.; Schlll, G. Chromatographla 1980. 75, 473. (21) Hackzell, L.; Rydberg, T.; Schlll, G. J . Chrometogr. 1983, 282, 179. (22) Bldllngmeyer, B. A.; Deming, S. N.; Price, W. P., Jr.; Sachok, B.; Petrusek, M. J . Chromatogr. 1879, 786, 419. (23) Bldllngmeyer, B. A. J . chrometogr. Scl. 1980, 78, 525. (24) Bldlngmeyer, B. A.; Warren, F. V., Jr. Anal. Chem. 1982, 5 4 , 2351. (25) Sachok, B.; Demlng, S. N.; Bldllngmeyer, B. A. J . L l q . Chromatogr. 1982, 5 , 3 8 9 . (26) Grushka. E.; Levln. S. Anal. Chem. 1985. 5 7 , 1830. (27) Melartder, W. R.; Erard, J. F.; Horvath, Cs. J . Chromatogr. 1983, 282, 211. (28) Rledo, F.; Kovats, E. Sz. J . Chromatogr. 1982, 239, 1. (29) Krstulovic, A. M.; Colin, H.; Gulochon, G. Anal. Chem. 1982, 5 4 , 2436. (30) Knox, J. H.; Hartwick. R. A. J . Chromatogr. 1981, 204, 3. (31) Deelder, R. S.; Llnssen, H. A. J.; Konljnendljk, A. P.; Van de-Venne, J. L. M. J . Chromatogr. 1979, 785. 241. (32) Deelder, R. S.; Van den Berg, J. H. M. J . Chrometcgr. 1981, 278, 327. (33) Cantwell, F. F.; Poun, S. Anal. Chem. 1979, 57, 623. (34) Iskandaranl, 2.; Pletrzyk, D. J. Anal. Chem. 1982, 5 4 , 1065. (35) Scott. R. P. W.; Kucera. P. J . Chrometogr. 1977, 742, 213. (36) Melander, W. R.; Horvath, Cs. J . Chrometogr. 1980, 207, 211. (37) Kong, R. C.; Sachok, 6.; Demlng, S. N. J . Chromatogr. 1980, 799, 307.
CONCLUSIONS System peaks, which result from local departure from equilibrium at the head of the column, frequently can convey more useful information than the solutes themselves. Careful investigation concerning the origin and formation of the system peaks can lead to a better understanding of many fundamental chromatographic phenomena. It is clear from the present paper that system peaks, being the pictorial representation of the equilibrium in the column, yield information about adsorbed mobile-phase components. But more than that, the implications of the present work are that system peaks would be extremely useful (a) for the determination of the capacity ratios of solutes, (b) for the measurement of the void volume without having to resort to the actual determination of to, (c) for the estimation of the loadability of the column, (d) for the establishment of adsorption isotherms, (e) for better qualitative and quantitative characterization of the solutes, and (f) for the better designing of preparative columns, where nonlinear isotherms are common. While the mobile phase described here can be regarded as ion pair chromatography, the above discussions and descriptions hold true in general, since the phenomenon of system peaks is widespread. Thus, the statement made in the beginning of the paper that system peaks are not a nuisance is true indeed.
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LITERATURE CITED (1) Cassidy, R. M.; Fraser, M. Chrometographle 1984, 78, 369. (2) Dreux, M.; Lafosse, M.; Pequignot, M. Chromatographla 1882, 15, 653. (3) Fritz, J. S.;Gjerde, D. T.; Becker. R. M. Anal. Chem. 1980. 52, 1519. (4) Berek, D.; Bleha. T.; Pevna, 2. J . Chromatogr. Sci. 1976. 74, 560. (5) Solms, D. J.; Smuts, T. W.; Pretorlus, V. J . Chrometogr. Scl. 1871, 9. 600.
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RECEIVED for review December 3,1985. Accepted February 24, 1986.
Preparation of Chromatographic Supports of Variable Ligand Density Mary E. Landgrebe, Danlin Wu, and Rodney R. Walters* Department of Chemistry, Iowa State University, Ames, Iowa 50011
It was p0sslMe to prepare supports contalnhg “spacer arms” with terminal carboxylic acM groups, by refluxing dloCbonded slllca In a solution of a cycilc anhydride. Surface densities ranging from a few percent to complete monolayer coverage were obtained by varyhg the reactlon thne,temperature, and anhydrlde concentration. Cationsxchange chromatography of protelns on these supports Indicated that selectklty as well as retention was slgntficantly altered by changlng the surface density of exchange sites.
Studies of the chromatographic behavior of biomacromolecules on reversed-phase (1-5),hydrophobicinteraction (6-9), ion-exchange (5,10-13), and affiiity (14)columns have shown that multipoint adsorption greatly affects the retention and kinetic behavior of these molecules. It is also known that the specific activity of immobilized biomacromolecules is a function of the number of bonds per macromolecule (15-20). A simple method for varying the surface densities of the functional groups responsible for retention or immobilization 0003-2700/88/0358-1607$01.50/0
is needed to study these phenomena and to optimize chromatographic performance. A macromolecule may cover an area of approximately lo4 A2 on the surface of a support. When compared to the approximately 40 A2area covered by an alkyl chain in a commercial reversed-phase column, it is apparent that to avoid multipoint interactions one might want to prepare adsorbents with functional group surface densities of as little as 1% of the maximum value. Kopaciewicz et al. synthesized polyethyleneimine-based anion exchangers covering a 3-fold range of ligand density (13). They observed weaker retention of proteins on the low-coverage supports and higher recoveries of some hard-to-elute proteins. However, selectivity did not appear to change significantly with ligand density. In affinity chromatography, ligand density is often controlled by using limiting amounts of the activating agent. However, many such reagents, e.g., 1,l’-carbonyldiimidaole, are simply too reactive, and thus reproducibility and uniform distribution of active groups may be difficult to obtain (21). In this paper, cyclic anhydrides were utilized for the modification of hydroxyl-containing supports. Since cyclic 0 1986 American Chemical Society
