Anal. Chem. 1995, 67, 1737-1741
Use of System Peaks in Liquid Chromatography for Continuous On=LineMonitoring of Chemical Reactions Nira Mizrotsky and Eli Grushka* Department of Inorganic and Analytical Chemistty, The Hebrew University, Jerusalem, Israel
System peaks, which occur in chromatography with multicomponent mobile phases, can be used for the continuous on-linemonitoring of chemical reactions. The mobile phase reservoir is the chemical reactor, and the mobile phase, which contains the reaction mixture, is recycled continuously through the column. At predetermined times, a neat solvent, normally the solvent of the reaction, is injected. The system peaks that result reflectthe status of the reaction. We describe here the use of system peaks to follow the progress of two reactions: titration of aniline and hydrolysis of aspirin. In the case of aniline titration, we obtained a titration curve by plotting the capacity ratio of the aniline system peak versus the acid volume added during the titration. The experimental endpoint agreed very well with the theoretical equivalence point. A plot of the capacity factor as a function of mobile phase pH behaves similarly to such plots in conventional chromatography. In the hydrolysis of aspirin, we followed the progress of the reaction by monitoring the formation of salicylic acid via its system peak. The data allowed us to calculate the apparent hydrolysis rate constant. The value obtained is in excellent agreementwith literature values. The introduction of a multicomponent mobile phase into a chromatographic column results in a redistribution of the components between the mobile and stationary phases. This process of mobile phase components redistribution continues until a state of thermodynamic equilibrium is reached in the column. During the redistribution process, the composition of the mobile phase entering the column differs from the composition of the phase leaving it. After attainment of equilibrium, the composition of the mobile phase leaving the column is identical to that of the phase entering it. When a perturbation, whose composition is different from the mobile phase, is injected into the column, the equilibrium is disturbed. Since the chromatographic system always aspires to be in a state of thermodynamic equilibrium, a new process of redistribution immediately begins to take place as the column relaxes to a new equilibrium stage. The result of this relaxation process is the appearance of system peaks. The fundamental importance of system peaks was recognized by many workers in chromatography. Possibly the first discussion on system peaks and their origin was by Solms et al.,l who attributed these peaks, which they called ghost peaks, to displacement effects. Scott and colleagues,2who referred to system peaks as vacancy peaks, used a plate model to describe the formation (1) Solms, D. L.; Smuts, T. W.; Pretorius, V.J Chromatogr. Sci. 1971,9,600. (2) Scott, R P. W.; Scott, C. G.; Kucera, P. Anal. Chem. 1972,44,100.
0003-2700/95/0367-1737$9.00/0 0 1995 American Chemical Society
of such peaks. Berek and co-workers (ref 3 and references therein) attributed the formation of system peaks to solvation processes that occur in the column. Redo and Kovats4v5examined the theoretical background of system peaks formation. Melander et al.? as well as Knox and Kaliszan? have also examined the process of system peaks development in binary mobile phases to gain a better estimation of the column's void volume. Levin and Grushka8-'0 showed that system peaks can be utilized to calculate chromatographic quantities such as void volumes and capacity factors. Schill and co-workers (e.g., refs 11and 12 and references therein) analyzed system peaks resulting from indirect detection in ion pair chromatography. Golshan-Shirazi and Gui~chon'~ examined numerically the formation of system peaks in linear chromatography. More recently, Levin and colleagues characterized the chromatographic behavior of enantiomers in chiral liquid chr~matographyl~ as well as the distribution of several hydroxylated benzenes15via system peaks. The papers cited above dealt mainly with fundamental aspects of system peaks and their formation. Several of the above papers hinted that system peaks can be utilized for practical purposes. For example, Scott et al.2indicated that system peaks can be used in conjunction with recycle chromatographyto monitor chemical reactions. Yet, surprisingly little can be found in the literature about the direct application of system peaks for analytical purposes. Westerlund and his group, in a series of papers,16-20 describe the use of a system peak as a means of generating in situ a local mobile phase gradient for the sharpening of an analyte (3) Berek, D.; Bleha, T.; Pevna, 2.]. Chromatogr. Sci. 1976,14,560.
(4)Redo, F.; Kovats, E. ]. Chromatop. 1982,239, 1. (5) Redo, F.; Kovats, E. In Theoretical Advancement in Chromatography and Related Techniques; Dondi, F., Guiochon, G., Eds.; NATO AS1 Series; Kluwer Academic Publishers: Dordrecht, 1992; pp 211-226. (6) Melander, W. R; Erard, J. F.; Horvath, Cs.]. Chromatogr. 1983,282, 229. (7) Knox,J. H.; Kaliszan, R J Chromatop, 1985,349,211. (8)Levin, S.; Gmshka, E. Anal. Chem. 1986,58, 1602. (9) Levin, S.; Gmshka, E. Anal. Chem. 1987,59,1157. (10) Levin, S.; Grushka, E. Anal Chem. 1989,61,2428. (11) Hackzell, L.; Rydberg, T.; Schill, G. J Chromatogr. 1983,282,179. (12) Arvidsson, E.; Crommen, J.; Schd,G.; Westerlund D.]. Chromatogr. 1989, 461,429. (13) Golshan-Shuazi, S.; Guiochon, G. Anal. Chem. 1990,62,923. (14)Levin, S.; Abu-Lafi, S. Chirality 1994,6,148. (15) Levin, S.; Abu-Lab, S.; Golshan-Shirazi. S.; Guiochon, G. J. Chromatogr., in press. (16) Sokolowski, A; Fomstedt. T.; Westerlund, D. ]. Liq. Chromatogr. 1987, 10,1629. (17) Fomstedt, T.; Westerlund, D.; Sokolowski, A J Liq. Chromatogr. 1988, 11, 2645. (18) Fomstedt, T.;Westerlund, D.; Sokolowski, A ]. Chromatogr. 1990,506, 61. (19) Fomstedt, T.; Westerlund, D.; Sokolowski, A ]. Chromatogr. 1990,535, 93. (20) Fomstedt, T,; Westerlund, D.]. Chromatogr. 1993,648,315.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995 1737
I
,I Mobile phase reservoir & Reaction chamber
Figure 1. Schematic representation of the closed-loop HPLC used in this study.
zone in the column. More recently, we have shownz1that the size of system peaks is directly related to the concentration of the compounds responsible for these peaks. We have demonstrated that, with mobile phases comprising the mixtures to be analyzed, the injection of a neat solvent, or of controlled concentrations of the components of interest, gives quantitative information about the concentration of these components in the mixtures. Hence, system peaks can be used to monitor, in a continuous fashion, the levels of various components in a flowing stream. Phillips and McIlWrickz2described the use of system peaks (although they referred to them as vacancy peaks) in gas chromatography to monitor the products of a catalytic reaction. Their experimental setup included a catalytic reactor column connected to a conventional GC column that in turn was connected to the detector. The carrier gas plus reactant was passed through the reactor, the column, and the detector. Reaction occurred in the catalytic reactor. The products plus any unreacted starting material entered continuously the analytical column. Injection of pure carrier gas directly into the GC column, bypassing the reactor column,yielded system peaks due to all the components, products, and reactants in the mobile phase. The experimental design of Phillips and McIlwrickz2was openended in the sense that the effluents, once past the detector, were vented to the atmosphere. Therefore, they used system peaks for monitoring the final products of the reactions. They could not monitor, in an easy fashion, the progress of the reaction. In our experiments, we used a closed system in which the liquid chromatograph was a part of the chemical reactor. As a result, we were able to monitor the reaction progress in real time. In the present work, we utilize system peaks to continuously monitor on-linethe progress of two chemical reactions. First, we describe monitoring the titration of aniline with HC1. Second, we discuss the hydrolysis of aspirin to salicylic acid. EXPERIMENTAL SECTION Instrumentation. Figure 1 shows schematically the experimental system. The liquid chromatograph used in this study was a Perkin-Elmer series 4 (Norwalk, 0equipped with a 2@pL loop Rheodyne injector valve (Contati, CA), a Perkin-Elmer 85B variable wavelength W-vis detector having a l.4pL flow cell, and either a D-2000 Merck Hitachi (Darmstadt, Germany) or a BarSpec Chrom-A-Set (Rishon Lezion, Israel) integrator. The detector was operated at (a) 254 nm for the titration of aniline (21) Mimotsky, N.; Kristol, L.; Gtushka, E. j . Chromatogr., in press. (22) Phillips, C. S. G.; McIlwrick, C. R Anal. Chem. 1973,45, 782.
1738 Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
and (b) either 273 or 298.5 nm for the hydrolysis of aspirin. The column was a 12.5-cm Lichrosorb RP18 cartridge, held by a Hiber manual holder. The column was thermostated with the aid of a homebuilt water bath. The chemical reaction chamber was usually a conical glass flask. The reaction chamber was also the mobile phase reservoir. In the titration of aniline study, a Mettler (Essex, England) Delta 320 pH meter was used to monitor the pH of the mobile phase. The pH electrode was immersed in the mobile phase in the reservoir. Materials. Two different mobile phases were used in the study. (A) For the titration of aniline, the mobile phase was an aqueous solution containing KCl (0.01 M) and aniline (1 x M). Aniline was purchased from Aldrich Chemicals Vel Aviv, Israel). The mobile phase was titrated with HCl during the experiments. (I3) For the hydrolysis of aspirin, the mobile phase was a phosphate buffer, pH 7 (0.01 M NaHzP04 and 0.01 M Nar HPO4), containing various concentrations of aspirin and/or salicylic acid. Aspirin and salicylic acid were purchased from Merck (Darmstadt, Germany). Procedures. (A) Titration of Aniline. The initial concentration of aniline in mobile phase A was 0.001 M. The pH of the solution was adjusted to 7 with NaOH. While circulating through the column and back to the reservoir, mobile phase A was titrated with 0.1 M HCl. After each addition of an HCl aliquot, the mobile phase was allowed to equilibrate for 30 min before the injection of mobile phase solvent (0.01 M KC1 solution). (B) Hydrolysisofhpirin. The initial concentration of aspirin in mobile phase B was 1 x M. The buffer reservoir was heated to 50 "C. Every 30 min, neat buffer was injected into the column. Two types of calibration curves were prepared for the aspirin hydrolysis study. (1) Aspirin and salicylic acid were injected separately into the column. Plots of peak height versus concentration were used as the calibration curves. (2) Two mobile phases, one containing aspirin and the other salicylic acid, were introduced into the column. The concentration of each compound was increased in a step function. A calibration curve was obtained by plotting the increase in the baseline signal as a function of the appropriate compound concentration. RESULTS AND DISCUSSION The two chromatographic systems under consideration are complicated since the mobile phases contain many components. It is well known that the adsorption isotherm of one component can be affected by the presence of other componants in the column (viz. refs 23 and 24 and references therein). Therefore, the concentrations of aniline or of aspirin and salicylic acid in the mobile phase were kept as low as possible to m i n i i e competitive equilibria and yet to allow a significant detector signal. Titration of Aniline. The e s t reaction chosen to demonstrate the ability of system peaks to monitor reactions was an acidbase titration. Such a reaction is simple in the sense that there is a rapid equilibrium between the protonated and the unprotonated species, e.g., a weak base B and its conjugated acid BH+. Several groups have investigated the pH dependence of the retention of acids and bases in conventional chromatography (viz. (23) Poppe, H. J. Chromatogr. 1993,656, 19. (24) Guiochon, G.; Golshan-Shirazi, S.; Katti, A. M. Fundamentals of Preparative and Nonlinear Chromatography; Academic Press: Boston, 1994.
lime lminl
0
5
10
15
20
25
30
3.09
V
3.
A
3.45
t
vh 7 7 9
lo 5
0
25
I
1
"
0
Figure 2. Aniline system peaks at different pH values. The pH value of each chromatogram is indicated next to the appropriate trace.
"
1
"
"
"
2
"
"
'
3
4
5
6
7
8
Volume of HCI (mL)
Figure 4. Chromatographic titration curve of aniline obtained frrom the aniline system peak.
>
0
I
Y I
'0
t 0 ' ' ' 0
1
" " " " " " " 2
3
4
5
6
7
8
PH
Figure 3. Aniline system peak K values as a function of pH. Volume of HCI (mL)
refs 25-27). In these studies, the pH of the mobile phase was changed in a discontinuous manner by changing the mobile phase. The solutes, acids or bases, were injected into the column at each new mobile phase. In the present work, the pH of the mobile phase, which included the compound to be analyzed, was changed continuously on-line. For our study, we chose aniline as the analyte since it can be easily observed with the detector and it can be titrated in a pH range commensurate with silica based columns. The resulting chromatogram is simple since it has only system peaks. In the pH range of the titration, from p 7 to 3.09, aniline changes from the practically un-ionized form to the almost completely protonated ion. Therefore, the retention time of the aniline system peak should be the longest at pH 7 and should decrease as the titration proceeds to pH 3.09. Figure 2, gives an overlay of the chromatograms obtained during the titration. The expected retention behavior is observed. Figure 3 shows a plot of k' versus the pH of the mobile phase during the titration. The behavior of the capacity ratio is well understood in terms of the aniline ionization as just discussed. Similar shaped curves are observed in conventional chromat ~ g r a p h y . ~ The ~ - ~ ~simple acid-base ionization equilibrium considerationsthat explain the behavior of k' as a function of pH in conventional chromatography (e.g., ref 28) are valid for the present case as well. A plot of the capacity ratio, k', versus HCl volume gives a typical acid-base titration curve (see Figure 4). A titration fist derivative plot, Figure 5, allows us to estimate the endpoint of the titration at 3.7 mL of HCl. The calculated equivalence point should occur (25)Graffeo, A P.;Karger, B. L Clin. Chem. 1976,22,184. (26)Horvath, Cs.;Melander, W.; Molnar, I. Anal. Chem. 1977,49,142. (27)Fong, G. W.IC;Grushka, E. Anal. Chem. 1978,50,1154. (28) Karger, B. L.; Wage, J. N.; Tan&, N. In High Perfrmance Liquid Chromatografihy,Vol. 1;Horvath, Cs., Ed.; Academic Press: New York, 1980; p 113.
Figure 5. First derivative plot of the titration curve in Figure 4.
at 3.8 mL of HCl. The agreement between the experimental value and the theoretical one is very good. Clearly, chromatography will not replace current titration equipment. The aim of this experiment was to examine the validity of our assertion that system peaks can be used to follow the progress of chemical reactions. The results discussed thus far demonstrate that our contention is correct. Hydrolysis of Aspirin. Another example of the use of system peaks to monitor chemical reactions is the hydrolysis of aspirin to salicylic acid. This hydrolysis reaction was studied previously mainly by E d w a r d ~ and ~ , ~Garrett,3l ~ who used spectrometric methods in their analyses. More recently, Sunada and coll e a g u e ~investigated ~~ the hydrolysis of aspirin using HPLC to analyze the contents of the reaction mixture off-line. In the present study, we circulated mobile phase B containing aspirin and, as the hydrolysis progressed, salicylic acid through the column. Every 30 min, an aliquot of neat buffer was injected, resulting in two system peaks, one belonging to aspirin and the other to salicylic acid. As the reaction progresses, the salicylic system peak increases (becomes more negative, see Figure 6). The aspirin system peak does not seem to change much during the course of the reaction because of (a) its smaller absorptivity at the wavelength used and (b) the constantly changing baseline due to the formation of salicylic acid. Since salicylic acid absorbance is greater than that of aspirin, the baseline of the chromatogram increases continuously. From the increases in the salicylic acid system peak, we can follow the progress of the reaction. Using the salicylic acid (29)Edwards, L. J. Trans.Faraday SOC.1950,46, 723. (30) Edwards, L J. Trans. Faraday SOC. 1952,48, 696. (31)Ganett, E. RI.Am. Chem. SOC.1957, 79, 3401. (32) Sunada, H.; %a, K; Ogawa, S.; Arakawa, E.; Masuyama, T.; Hara,IC;Otsuka, A Chem. Pharm. Bull. 1985,33,2158. Analytical Chemistty, Vol. 67, No. 10, May 15, 1995
1739
k
2"
Table I.Rate Constant Values for the Hydrolysls of Asplrln Memsund at Two Different Wavelengths Using Two Methods of Calculatlona
0 min
wavelength (nm)
mode of calculation
rate constant (day-')
273 273 298.5 298.5
baseline absorbance system peaks baseline absorbance
3.36 3.68 3.14 3.23
system peaks
In all cases the temperature of the hydrolysis was 50 "C. The
reaction pH was 7.
/-
0
5
IO
Figure 6. Chromatograms of the the aspirin hydrolysis reaction at several times into the reaction.
mentioned previously, the change in the system peak of aspirin is not as pronounced as the increase in the salicylic acid system peak. Therefore, calculation using the aspirin system peaks is much more prone to errors and was not used in this study. Both the reactant and product absorb UV radiation. At the two wavelengths used in the study, salicylic acid has the higher absorbance. Therefore, the baseline increases continuously with time as more and more salicylic acid is produced. That increase in the baseline can be utilized directly to measure the reaction rate constant. In this mode of operation, chromatography is not essential, and the chromatographic system fullills the function of the spectrophotometer. Since the absorbance of the baseline is the sum of the two individual absorbances, the following expression can be written:
-
(3)
C
I
-1.50
0.00
0.10
0.20
0.30
0.40
0.50
Time (day)
Figure 7. Plot to obtain the aspirin hydrolysis rate constant. The correlation coefficient of the fitted straight line is 0.992.
where At is the total absorbance, E is the molar absorptivity, and b is the detector cell path length. The rest of the parameters are as in eq 1. The relevant be values were obtained from the appropriate calibration curves. Equations 1 and 3 form a set of two equations with two unknowns, ,C, and C d at any time in the reaction. The solutions are
calibration curve type 1 (see Experimental Section) as well as the expression for mass conservation in eq 1, the concentration of both components can be obtained from the system peak:
(4)
and
where C indicates concentration, and the subscripts asp and sal signify quantities belonging to aspirin and to salicylic acid, respectively. denotes the initial concentration of aspirin in the mobile phase. The hydrolysis reaction rate constant can be calculated from the data. Assuming a first-order r e a c t i ~ n ,the ~ , ~ ~ The hydrolysis rate constant can be calculatedfrom either of these equations; eq 4 is used to plot - C&/empl versus the following expression can be written: reaction time or eq 5 is employed to plot ln(CasplcsJ as a function of time. The values of the rate constant calculated using the baseline approach are very close to those calculated from system peaks. Table 1compares the results obtained using both approaches. where tis the time, and k is the observed rate constant. Therefore, a plot of the ln term on the left-hand side of eq 2 versus the In view of the results using the drift in the baseline, it might reaction time yields a straight line whose slope is the rate constant. be argued that system peaks are not needed for monitoring the The hydrolysis rate constant, obtained directly from the slope of reaction or for calculating parameters such as rate constants. If the line in Figure 7, is 3.23 day-', which is in excellent agreement baseline variations are used, then a simple spectrophotometer with with literature value^^-^^ for similar conditions. a flow cell will sufiice. Such an argument might be true with Similar results should be obtained from the aspirin system simple systems, as in the present case with aspirin and salicylic acid. However, when the reaction involves more than two peak, which should decrease with time. However, as was
csp
ln[(csp
1740 Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
components, the baseline approach may be too complicated and not accurate enough. Moreover, when the reactants and products have similar absorbances, the change in the baseline is negligible, and the system peaks approach is the method of choice. With complex chemical reactions, system peaks have an advantage over the baseline approach since each peak can be related to a component in the mobile phase. To further check the validity of the approach, the measurements were repeated at two wavelengths: 273 nm, where both aspirin and salicylic acid absorb, and 298.5 nm, where only salicylic acid has an appreciable absorbance. At both wavelengths, the system peaks approach and the baseline method were used. The results are shown in Table 1. The agreement between all the values is excellent. CONCLUSIONS
As mentioned previously, the adsorption isotherms of the mobile phase components can be interdependent. To reduce the influence of competitive and coadsorptions, low concentrations of some mobile phase components were used. Under these conditions, the size of system peaks due to these minor components is a function of their concentration, and the system peaks
can be used for monitoring the concentrations of the components in the mobile phase. Future work is planned for developing a chromatographic system in which the concentrations of the compounds of interest are appreciable. A practical application of system peaks is to follow the course of a chemical reaction in a continuous on-line manner. The reaction takes place in the mobile phase reservoir, and the content of the reservoir is recycled through the column. In this paper, we give two examples of continuous on-line monitoring of chemical reaction: titration and hydrolysis. In both cases, the results are in excellent agreement with the expected values. System peaks allow us to follow the progress of the reaction and to calculate parameters such as the hydrolysis rate constant. The results obtained with system peaks agree very well with results obtained using conventional chromatography or other independent techniques such as spectroscopy. Received for review November 3, 1994. Accepted February 28, 1995.@ AC941069Q @Abstractpublished in Advance ACS Abstracts, April 1, 1995.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
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