Coupled Anion and Cation Exchange Chromatography of Complex Biochemical Mixtures Charles D. Scott, Dennis D. Chilcote, and Norman E. Lee Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 Previously, high-resolution separation of the molecular constituents of body fluids has been achieved by cation exchange chromatography and by anion exchange chromatography separately. Hundreds of constituents can be separated by these techniques, even though the sample contains multiple types of ion and neutral components, but the separation time has been too excessive for wide use. A separation concept using coupled anion and cation exchange columns gives higher resolution with up to 17% more UV-absorbing chromatographic peaks being resolved from a 0.15ml urine sample if the two columns are eluted separately but simultaneously after the initial loading step. A routine analysis for over 100 UV-absorbing molecular constituents in body fluids can be made in 14 hours compared to 24 hours for the single anion exchange column separation. Design of the system and operational problems are also discussed.
EXTREMELYVALUABLE chromatographic separation systems that use a single separation medium have been developed for use in the analysis of complex biochemical mixtures such as body fluids. These systems include, for example, cation exchange columns for the analysis of ninhydrin-positive compounds in body fluids ( I ) and anion exchange columns for the analysis of UV-absorbing constituents (2, 3). In both cases, ion exchange columns of very high resolution (requiring very small ion exchange resin beads and high operating pressures) are required t o separate mixtures that may include a variety of ionic and neutral compounds. Although such systems separate well in excess of 100 molecular constituents each from a small urine sample, the separation time is quite long [in some cases, >60 hr ( I ) ] . Also, a single sorption medium is not necessarily effective for separating all of the groups of compounds present. In other schemes, the analysis of many molecular constituents in body fluids has been achieved by a series of separate separation steps in which a gross separation into several groups or classes of compounds with similar properties is made and then the individual components in each group are further isolated for the actual quantification. Such a technique was effectively used by Horning and Horning ( 4 ) in establishing the metabolic profiles in human body fluids by means of gas chromatographic analyses of several groups of compounds previously separated by ion exchange or solvent extraction. In this case, even though the final separation by gas chromatography is relatively fast and amenable t o automation, considerable manual handling may be required between separation steps and a relatively long time for the total sequence may be necessary. (1) P. B. Hamilton, in “Handbook of Biochemistry. Selective Data for Molecular Biology,” H. A. Sorber, Ed., The Chemical Rubber Co., Cleveland, Ohio, 1968, p B-47. (2) C. D. Scott, Clin. Chem., 14, 521 (1968). (3) C. D. Scott, R. L. Jolley, W. W. Pitt, and W. F. Johnson, Amer. J . Clin. Pathol., 53, 701 (1970). (4) E. C . Horning and M. G. Horning, J . Chromafogr. Sci., 9 , 129 (1971).
A possible method for circumventing the weak points of both of these separation schemes is the use of multiple sorption columns, placed in sequence, that contain different types of separation media. Rather than isolating the individual column eluates, each effluent is fed directly into that of a succeeding column in series without interruption. Such a n approach has been investigated for the chromatographic separtion of the UV-absorbing constituents of body fluids, particularily human urine. CONCEPTS
All of the discussions will center around the separation of the UV-absorbing constituents of body fluids as demonstrated by a previously developed high-resolution anion exchange chromatographic system (2, 3). The system, “the UVanalyzer,” is characterized by relatively long columns (up to 150 cm), very small ion exchange resin beads (5 to 15 p in diameter), and high operating pressures (up to 5000 psi). Sorption phenomena other than simple ion exchange contribute to the separation scheme in ion exchange columns; however, to take advantage of more than one ion exchange mechanism, one must include more than one ion exchange medium. Although any number of different types of ion exchange resins could be used, we will restrict our discussion here to a simple two-column system that includes a strongly acidic cation exchanger and a strongly basic anion exchanger. Several operational variations can be envisioned. For example, columns could be operated as entirely separate systems, with the various fractions being collected and processed between each step. However, to take advantage of the high resolving power of the separation system with maximum automation, an alternative method would be to place the columns in series and carry out the separation without processing the eluate of individual columns. Commercial installations for water treatment have used such an arrangement of columns, and similar schemes for somewhat different applications have been recently suggested for chromatographic use by Snyder (5) and Lijamaa and Hallen (6). Elution conditions that are compatible with all of the separation media must be used with the sequential-column system, and the design of the system must take advantage of the separating power of each medium. For example, the early chromatographic peaks from the anion exchange separation of a complex biochemical mixture will include neutral species, cations, and weakly sorbed anions. If a cation exchange column is placed in series with the anion column, the early part of the chromatogram is simplified (fewer, well-separated peaks) because of the retarding effect of the cation exchange resin on the cations; however, the cations may then be eluted in a n area of the chromatogram previously containing separated anions and, in turn, cause a resulting decrease in the apparent resolution at that point. (Used in this context, the term resolution means the total number of detectable chromatographic peaks.) An additional scheme, which may potentially yield a much higher resolution, could conceivably result in a significant (5) L. R. Snyder, J . Chromatogr. Sci., 8, 692 (1970). ( 6 ) J. J. Liljamaa and A. A. Hallen, J. Chromatogr., 57, 153 (1971). ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
85
reduction in separation time. In this scheme, the anion and cation exchange columns are initially used in series during the period when the sample mixture is being loaded on the columns and the neutral species are being separated (probably by surface sorption effects). Then, after the loading phase (when the remaining components are more or less segregated on each of the two columns), the columns can be eluted separately. If both columns are eluted simultaneously, it may be possible to decrease the overall separation time significantly. Further, if common eluent conditions can be used for the parallel elution step, a single eluent pump and gradient generation system can be used to supply both columns a t the same time. However, provision must be made for adjusting the flow resistance in each of the columns to give a predetermined flow rate. In addition, simultaneous elution will require two detection systems, one of which will not be used during the loading step. To automate such systems, it will be necessary to have effective, multiport, switching valves that operate a t high pressures.
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Automated and semiautomated liquid chromatographic systems operating a t pressures in excess of 1000 psi are now widely used. The UV-analyzer, as modified for this study, is such a system, and it is designed to operate as a n automated gradient elution chromatograph containing the following major components: the separation section consisting of a closed tubular column packed with the ion exchange resin; a n eluent storage and gradient elution preparation section; a high-pressure eluent pump equipped to deliver the eluent to, and force it through, the separation column; a sample injection valve for introducing the sample to the column ; and a flow photometer for detecting and quantifying the separated constituents in the column eluate. Automated data acquisition and processing may also be used. Additional details have been given ( 2 , 3). For operation with two columns in parallel, it was necessary to add a high-pressure switching valve and a second UV-photometer (Figure 1). The requirements of high-pressure operation affect the design and operation of the eluent delivery, sample introduction, and separation systems; however, since high-pressure liquid chromatography is becoming so commonplace, special attention will not be given t o high-pressure design except where a new and unique component or operating technique is used. Columns. The ion exchange columns, designed for operation at pressures up to 5000 psi, were fabricated from nominal l/s-in. 0.d. type 316 seamless stainless steel tubing. Final column dimensions of 0.22 cm i.d. and a n overall length of 150 cm were used. This overall column length was the same as that used for the previous high-resolution separation by a single anion exchange column ( 2 , 3). Although combined column lengths of the anion and cation columns were maintained a t 150 cm, tests were made in which the lengths of the individual columns were varied from 25 to 125 cm in 25-cm increments. Tests for comparison were made by using a full-length anion exchange column. Closely sized (12 to 15 p ) ion exchange resins were obtained from Bio-Rad Laboratories. The anion exchange resin, Aminex A-27, was a polystyrene-divinylbenzene copolymer matrix with quaternary ammonium active sites, and the cation exchange resin, Aminex A-6, had a similar matrix with sulfonic acid active sites. Columns of these resins were packed dynamically by rapidly displacing a thick slurry of the appropriate resin from a reservoir into the column (7). Eluent System. Gradient elution chromatography was used in which the concentration of acetate ion in the eluent (an ammonium acetate-acetic acid buffer at p H 4.4) was (7) C . D. Scott and N. E. Lee, J . Chuomatogr., 42, 263 (1969). 86
CONSTAYT TENPER3TURE CIACULAT3R
Figure 1. Coupled column chromatography with the possibility of simultaneous elution after sample loading
slowly increased from 0.015M to 6M. Either a nine-chambered gradient box or a n automated system using coupled reservoirs of varying cross-sectional area was used to form the eluent gradient, and a positive displacement piston pump (A MilRoyal D pump manufactured by the Milton Roy Co., or a Model LS-30 Pulsafeeder manufactured by the Lapp Insulater Company) was used to transport the eluent to the high-pressure column and force it through the column and detector. Operating pressures up to 3500 psi were used routinely in this system. Sample Introduction and Column Switching. A six-port sample injection valve capable of operation up to 5000 psi was used to introduce a sample of 0.15 ml of reference urine into the eluent stream just prior to the column (8). This valve operates by first allowing a n external sample loop to be filled a t ambient pressure; then, when the internal port connectors are reoriented by turning the valve handle, the contents of the sample loop are swept into the eluent stream and thence onto the column (Figure 2). The operation of the chromatograph is not interrupted by the sample introduction, which constitutes the beginning of the run. The same type of valve modified for use as a switching valve was used to allow automated operation of the coupled system. The two columns were first operated in series; then, after a loading phase, a simple reorientation of the interior port connectors by turning the valve handle (either manually or mechanized) changed the operation to parallel elution with a common eluent supply and different detection systems (Figure 2). Detection Systems. Although the concepts described here may have general utility, they were tested in a system specifically developed for analyzing for the UV-absorbing constituents of body fluids. The detection system was comprised of one or two miniaturized flow photometers that operated with a dual beam (for referencing the inlet stream against the column eluate) and a t two wavelengths continually in the ultraviolet (254 nm and 280 nm). These photometers, specifically designed for use with liquid chromato(8) C. D. Scott, W. F. Johnson, a n d V . W. Walker, A i d . Biochem., 32, 182 (1969).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
to 6M for the next 210 ml, and then a final 21 rnl of 6M buffer. The same total eluent volumes were used for both single-column and coupled-column tests. Thus, when the coupled columns were eluted in parallel, the supply of eluent was exhausted much more rapidly (because of the effective doubling of the total eluent flow rate), run times were significantly decreased, and the individual columns were subjected to a more rapidly changing buffer concentration. The column temperature was programmed to be at ambient during the first 5 hr of operation (26 i 2 "C), thereafter increasing to 60 "C for the remainder o f the run. Evaluation of the sequential-column concept with well defined, simple mixtures would be a somewhat easier task. However, since our ultimate objective is to develop the capability for separating and quantifying the molecular constituents of body fluids on a routine basis, separation of the components of urine (the most complex body fluid) appeared to provide a useful test for the practical value of the proposed column operation. Therefore, 0.15 ml of a reference urine was used as the sample in each test. This reference urine, a composite from several normal subjects, has been studied many times previously. The usual anion exchange separation yields about 90 chromatographic peaks (10).
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