Anal. Chem. 1990,62,2557-2565
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Displacement Chromatography Applied to Trace Component Analysis Roswitha Ramsey, A. M. Katti,'J a n d Georges G ~ i o c h o n * - ~
Department of Chemical Engineering and Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600,and Division of Analytical Chemistry, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
Displacement chromatography has been used prknarlly for the Isolation of relatively large quantitles of materials In preparatlve scale Separations. We show that It also offers advantages for the enrlchment of trace components. Durlng displacement development, slgnlflcant compression of the trace component bands occurs. This enrlchment Is studied both experlmentally and theoretically. The theoretlcal model Is based on the solution of the mass balance equatlons for nonllnear chromatography, assuming competltlve Langmulr Isotherms. The system studied experlmentally consisted of parts-permlllion levels of 8-naphthylamlne and an Inpurity of the naphthylamine as the sample and dlethyl phthalate as the dlsplacer. The band profiles of the trace components were monitored by fluorescence detection whlle the dlsplacer was monitored by UV absorbance. Wavelengths were chosen such that the profiles of the sample and the dlsplacer could be monltored Independently. Trace enrlchment by band compression was achleved by Increasing the displacer concentratlon. Experlmental results show very narrow bands at enhanced concentratlons as compared to the relatively broad Gaussian-shaped profiles observed In llnear elutlon chromatography. The experlmental results are In agreement with theoretlcal predlctlons of peak shape.
INTRODUCTION Over the last 40 years, trace analysis has progressed as a result of improvements in the sensitivity of available detectors and through the development of new specific detectors. During this time, the chromatographic separations necessary for identification and quantitation of trace components have been conducted in the linear mode. This approach offers the advantage that retention times remain constant, independent of sample composition. Many new difficult problems arise, however, which cannot be solved by the classical procedures combining sample pretreatment or cleanup, chromatographic separation, and on-line detection. The sensitivity is insufficient and complex multistage procedures are required to enhance detectability. Recent results in the theory of nonlinear chromatography may be useful for addressing some of the special problems encountered in trace analysis. In a previous paper, we have shown how small amounts of a trace component may be extracted from a mixture by preparative chromatography using the overloaded elution mode (1). When the main component of a mixture elutes after the trace component of interest, interaction may develop between their bands. In order for this to take place, the sample size and the concentration of the main component *Author to whom correspondence should be sent. Current address: Ciba Geigy, Basel, Switzerland. *Department of Chemical Engineering. 3Department of Chemistry. 0003-2700/90/0362-2557$02.50/0
must be large enough for the nonlinear displacement effect to occur. This effect pushes the trace component band forward and at the same time significantly increases its concentration. The trace component may be concentrated and efficiently extracted, in spite of its long, thin tail,characteristic of the L-shape profiles of strongly displaced first component bands in nonlinear elution chromatography. The recovery yield can exceed 95% and the concentration of the trace component in the recovered fraction can be increased by orders of magnitude ( I ) . In other paper (Z), we have studied theoretically the squeeze effect that takes place in overloaded elution when a trace component is eluted between two main components. We have shown that the nonlinear effects can be strong and result in very strange band shapes. The phenomenon is difficult to harness for practical analysis. In this context, displacement chromatography appeared attractive for trace analysis. Preliminary calculations (3) have suggested that considerable concentration of trace components may be observed in this mode. The trace component bands are significantly narrower than in linear elution chromatography and their apparent efficiencies may exceed 100 times the natural column efficiency. At the same time, the band height may be increased more than 10-fold. Since displacement chromatography can accommodate much larger samples than elution, trace component bands will contain much more material than in linear elution chromatography. If selective on-line detection is possible, the detection limits may be decreased a hundred to a thousand fold or more. Thus, displacement chromatographycould simplify current methods for trace analysis or enable the determination of previously undetected trace components. Recently, the elution of narrow peaks of trace impurities in the trypsin digest of a recombinant protein was reported in separations performed by displacement chromatography ( 4 ) . Displacement was used rather than conventional elution to obviate the small samples which can be accommodated by on-line liquid chromatography/mass spectrometry (LC/MS) with narrow bore packed columns. Between the expected peptide bands, very narrow peaks, with widths less than the scan time of the mass spectrometer, appeared. These peptides are probably the digest of impurities in the recombinant protein. These remarkably sharp peaks are the first experimental report of the trace concentration effect just discussed (3). The aim of the present work is to present a feasibility study of this method, to illustrate some of its potential problems, and to present solutions that are reasonably easy to carry out. It should not be forgotten, however, that in any case, a displacement analysis takes comparatively longer to develop and is slower to carry out than the equivalent elution analysis. The proper displacer has to be selected and its concentration optimized. After the analysis is finished, the column has to be regenerated and the displacer washed before another analysis can be started. Only if the advantages compensate for these drawbacks will this method be successful. @ 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
THEORY Displacement chromatography was developed by Tiselius (5) and recently adapted to high-performance liquid chromatography by Horvath (6). The theory of displacement chromatography has been established within the framework of the ideal model by Glueckauf (7), by Aris and Amundson (81, and more recently by Helfferich and Klein (9).Since the ideal model assumes an infinite column efficiency, this assumption permits the prediction of the characteristics of the isotachic train but not of the exact individual profiles of the band boundaries obtained in real columns where band broadening due to dispersion occurs. The calculation of these individual band profiles has been carried out by Katti and Guiochon (10) using the semiideal model of chromatography which takes into account the finite nature of column efficiency. Progressive changes in the band profile during the formation of the isotachic train (10-12) are accurately predicted. A study of the effect of the kinetics of mass transfer across the chromatographic column has shown that the isotachic train cannot form when the rate constant is too low (13). In ideal chromatography (Le., infinite column efficiency), the isotachic train that forms is made of a series of rectangular bands. The bands elute in increasing order of the free energy of interaction between that component and the stationary phase. The height of each band depends exclusively on the equilibrium isotherm of the corresponding component, the isotherm of the displacer, and the displacer concentration. The band height is given by the interaction of the isotherm of that component and the operating line. The operating line connects the origin and the displacer isotherm at the displacer mobile phase concentration chosen. It is important to note that the concentration height of a component band in the isotachic train of the displaced mixture is independent of the amount of sample injected, as long as an isotachic train forms. The width of that band, on the other hand, is proportional to the amount of the component in the sample. I. Behavior of Trace Components in Displacement Chromatography. For a real column, an infinite concentration gradient is impossible: perfectly rectangular bands do not form, so band boundaries are slightly diffuse. The concentration discontinuities or shocks are replaced by shock layers, whose thickness is a function of the height of a transfer stage, Le., of the height equivalent to a theoretical plate. Although the band of a trace component cannot be rectangular, it is considerably narrowed by the displacement effect. Each side of the elution profile of the trace component band will be a shock layer and this may correspond to a considerable concentration effect (3). The band profile results from a steady-state equilibrium between the influence of the thermodynamics (nonlinear equilibrium isotherms), which tend to produce vertical band boundaries, and the kinetics (finite rate of mass transfers, axial dispersion), which tend to relax the concentration gradients. As long as the resulting peak height is small compared to the theoretical height of the band in the isotachic train, the peak height will be proportional to the amount of the corresponding compound in the isotachic train. Finally, there are usually several main components and several trace components, in working with complex mixtures. Theory predicts that when multiple trace components have isotherms between two major components, these traces will coelute in the mixed zones between the two main component bands and will not be resolved from each other. Calculations of the individual band profiles can be performed by using numerical solutions of the semiideal model of chromatography (3,10,14). They permit prediction of the band profiles provided the column efficiency under linear
Table I. Isotherm Parameters Used for the Calculation of the Displacement Chromatograms (Figures 1 to 4)’’
component first impurity
main component second impurity displacer
bi, mL/mg
a:
0
2.68 3.34 4.19 4.84
0.0025 0 0.0039
k:
tR,it s
1.043 1.30 1.63 1.88
368 414 473 519
“Other parameters: L = 25 cm, to = 180 s, 0 = 0.72, HETP = 0.007 cm. *In milliliters of mobile phase per milliliter of stationary phase. conditions and the equilibrium isotherms of the components involved are known. Previous results have confirmed the validity of the model (15). Usually, the competitive Langmuir isotherm is taken as a first approximation. It is written UjCj
4i = 1
+ jc= 1b j C j
where qi and Ci are the concentrations of the ith component in the stationary and mobile phases at equilibrium and ai and bj are numerical coefficients characterizing each component. The summation extends to all components of the mixture and to the displacer. However, for a trace component, k, the corresponding term, bkck, can be neglected as long as Ck remains small. In displacement chromatography, the isotherm of a trace component is not linear as it depends on the concentrations of the major components eluted before and after the trace band. Equation 1 permits the determination of the band profile by integration of the system of mass balance equations for the different components of the mixture (3). 11. Calculated Displacement Chromatograms of Trace Components. As an example, Figures 1to 4 show the elution profiles of two trace components eluted before and after a main component band in displacement chromatography. In the calculations, we have used the numerical values of the isotherm parameters determined in our experiments for the displacer (see Table I) and the efficiency of the column used (3600 theoretical plates). The main component band profile and the trace component peaks are shown on separate figures because of the different concentrations scales. In Figures 1-4, the amount of the main component increases from 20 to 200 mg, while the amounts of the two trace components remain constant and equal to 0.01 mg each, their concentrations actually decreasing from 500 to 30 ppm. Other calculations have shown that the trace component profiles are not affected by a reduction of their concentration by a factor of 1000 (3). In both Figures l a and 2a, the isotachic train is formed and for the two trace component peaks, the profiles are the same in Figures l b and 2b. Only the distance between the two bands (i.e., the width of the main component band) is different. The band of the second component has an apparent efficiency of 16OOOO theoretical plates while the column simulated has only 3600 theoretical plates. The squeeze effect is important, resulting in a band that is 7 times higher and 7 times narrower than the peak obtained under linear conditions for the same amount of trace component. The squeeze effect is less important on the first eluted trace component whose front is free to diffuse, experiencing no nonlinear effects due to interaction with another component. Its band has an apparent efficiency of only 56 OOO plates, and is about 4 times narrower and 4 times taller than the natural band, obtained under linear conditions for the same amount of sample. In Figure 3a, the sample size is 100 and the displacement train has not developed completely. The front of the main component band is higher than the isotachic band height. The
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
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