Instrumentation
J. J. DeStefano' arid JI. J. Kirkland2 Experimental Station E . I. du Pont de Nemours & Co. Wilmington, Del. 19898
Preparative HighmPerformanee Liqulid Chromato&aphy In Part I of this article ( 1 ) which was last month's I N S T R I M E N T A T I O N feature, the philosophy of preparative high-performance liquid chromatography was presented. In Part I1 the necessary experimental corlditions will be discussed. Optimizing the preparative system for sample capacity involves different opwating conditions than normally used for analytical separations. These differences are exemplified by comparing typical opera tinp parameters listed in Table I Columns
In preparative separations, column sample capacity usudly is increased
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Table 1. Typical Operating Parameters for
LC
Column diameter Column packing
Mobile phase flow rate Mobile phase velocity Sample volume Detector sensitivity required Wt of injected sample
(pellicular) - 1 ml/min
0.5 cm/sec
2 liters are desirable because of the large volume of mobile phase used. Pumping systems with very high-pressure capabilit y usually are not required in prepara-
tive HPLC, since columns of larger particles (higher permeability) are used; pump ratings of 1000-3000 psi are adequate. The precision and accuracy of pumps are not critical, since analytical interpretation of the chromatogram usually is not required. Reciprocating and pneumatic (or hydraulic) amplifier pumps are preferred because of their capability for higher pumping rates and continuous solvent output. “Mini”-pneumatic amplifier pumps are particularly convenient and are relatively inexpensive. Some of the reciprocating pump systems which are available in commercial LC instruments can only deliver a maximum of -10 ml/min (the lower end of the desired preparative flow rate range). Although this limitation does not preclude their use for preparative applications, longer separation times often must be accommodated, Pulsations from certain pumps which disturb the detector baseline in analytical runs are not a serious disadvantage in preparative separations. Highly sensitive detectors usually are not required in preparative HPLC as solute concentrations generally are quite high. These high concentrations can cause problems, since frequently it is difficult to determine whether the overlapping of peaks is due to an overloaded column or a nonlinear detector response. For example, the trace for the ultraviolet photometric (UV) detector in Figure 6 suggests essentially no resolution of two components. However, the response of the less sensitive refractive index (RI) detector shows a good separation, indicating
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
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Figure 6. Detector-overload effects (22)
that the capacity of the column has not yet been overloaded by the Sample. Although the UV detector is useful, the RI detector appears to be more generally suitable for preparative separations. Sometimes a combination of both detectors may be required for the accurate monitoring of peaks. The possible anomalies in using only a UV detector are illustrated in Figure 7 . Chromatogram A was obtained with a UV detector on 1 gram of an impure sample containing a pair of isomeric compounds. Preparative isolation of an apparent major peak 1, believed to be one of the desired components, gave 39% recovery of the injected sample. Approximately 34 and 23% by weight eluted before and after this peak, respectively, even though only very minor components were indicated later in the chromatogram. Rerunning the sample with both the RI and UV detectors produced chromatogram B. Collection of the major components detected by RI showed that the cross-hatched peaks 2 and 3 were the desired isomers, representing a total of about 66% by weight of the injected sample. Thus, the UV detector suggested peak 1 as a major component, although it was only a minor impurity with a retention time very similar to that of the desired constituents. Using both the UV and RI detectors increases the probability that the peaks of interest and possible interfering contaminants will be properly identified. Spectrophotometric detectors also are very useful for preparative separations. These devices are more versatile than single-wavelength UV photometers and have the unique capability to operate in the 210-230-nm region, 1202A
which allows the detection of many compounds not sensed a t longer wavelengths. Another advantage of spectrophotometric detectors is in the “detuning” of the wavelength from the solute absorption maximum, to decrease the sensitivity of the measurement and reduce the potential for detector overload. Other detectors for HPLC are available, but for preparative work they should be nondestructive of the sample. Transport (often called flame ionization) detectors are useful, since only a small fraction of the total column effluent is destroyed in the detection process. These devices respond to all compounds containing carbon in the reduced state. Unfortunately, present commercial devices are costly, inconvenient, and highly operatordependent. Other considerations in equipment for preparative HPLC are different from those normally required for analysis. Extra-column effects are less of a problem with large-diameter columns because of their relatively large internal volumes. Therefore, low dead-volume tubing and fittings are not as critical. Detectors should not be made with very narrow-bore tubing which severely limits the flow of mobile phase, since any restriction causes considerable back-pressure a t the flow rates needed in preparative HPLC. RI and UV detectors designed with larger bore tubing (e.g., 0.75 mm i.d.) are
available from some manufacturers. Alternatively, a stream splitter on the exit of the large-diameter column can supply a small fraction of the effluent to an analytical detector. Manual collection systems usually are adequate for isolating one or a few separated components. However, automatic collectors are convenient when a large number of components must be collected in a single run. T o conserve mobile phase, solvent systems can be equipped with a switching valve which allows uncontaminated mobile phase to either be recycled back to the pump reservoir or collected for purification and reuse. Comprehensive systems have been devised so that the mobile phase automatically is repurified and returned to the solvent reservoir in a “closed-loop” operation (23,2 4 ) . Sample injection in preparative HPLC is an important but poorly understood operation. Introduction of the sample at a point in the center of the column bed inlet apparently results in greater column efficiency; however, all the column packing does not interact with the sample, and localized overloading is a problem. Loading the sample across the entire column cross section permits better use of the total column packing with reduced column overload. Further studies of sampling effects in largediameter columns are needed. Large sample aliquots can be conve-
Figure 7. Detector anomaly in preparative isolation Column same as in Figure 2, B except: mobile phase, 1.5 % isopropanol in pentane; flow rate, 10.7 ml/min: velocity, 0.06 cm/sec; sample, 15 ml of 17 mg/ml mixture in mobile phase; RI detector, 64 X RlUFS ( 12)
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
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niently introduced with a valve without interrupting the flow of the mobile phase. Injection with a syringe normally is by the stop-flow technique whereby the sample is introduced a t atmospheric pressure; then pumping is started. Very large volumes can be delivered with a syringe by septum injection, but difficulties can occur (e.g., pieces of septum plugging the inlet). Alternatively, large sample volumes can be conveniently injected with a syringe through a high-pressure valve in a stop-flow mode (12). The volume of sample which can be introduced into the column will depend on column internal diameter, sample solubility, and the mobile phase-stationary phase combination. In a nonoverloaded column condition, this volume generally should not exceed about one-third of the volume of the earliest peak of interest. However, when the column is overloaded, this relationship no longer holds and larger volumes are tolerated. Samples of 5-20 ml or larger frequently can be used with a 100 X 2.3-cm i.d. column. I t is apparent that the equipment for preparative HPLC is not as critical as for analysis, and less sophisticated and generally lower-cost systems can be used satisfactorily. Conclusions
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Existing knowledge of preparative HPLC techniques suggests that certain approaches different from those normally used for analysis are desirable for obtaining significant amounts of purified components: Use liquid-solid chromatography, if possible. Good analytical separations are important as “pilot” runs. Use wide-diameter columns for increased sample throughput per unit time. Columns up to a t least 2.3 cm i.d. provide excellent resolution. Use totally porous packings and higher stationary phase loadings to optimize sample capacity. Particles of 30-50 pm are a good compromise between column efficiency, operating pressure, and cost. For higher resolution with porous particles, use lower mobile velocities. For highest sample throughput per unit time, use the highest velocity permitting adequate resolution. Overload columns to increase sample throughput (but with decreased resolution). Isolation of “heartcut” portions of peaks will improve purity but decrease yield. If feasible, adjust the retention of the compound to be isolated to higher k’ values (e.g., k’ 5 5 ) to optimize both sample loading and column resolution. Use larger sample volumes of more dilute solutions to minimize column overload a t the inlet. Use continuous pumping systems
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
(e.g., reciprocating or pneumatic amplifier pumps); pressures above 3000 psi rarely are required. Sensitive detectors usually are not necessary; dual UV and RI detectors and transport detectors generally are satisfactory. Samples are more conveniently introduced with a valve, but syringe injection can be used. Use low viscosity mobile phases and higher temperatures if sample permits. Acknowledgment
We are grateful for the useful discussions with P. E. Antle and the use of his chromatograms for Figures 2 and 7 (Part 11). The helpful comments of L. R. Snyder on this manuscript are much appreciated. Literature Cited
(1) J . J. DeStefano and J. J. Kirkland, Anal. Chem., 47,1103A (1975). (2) J. J. DeStefano. H. C. Beachell. J . Chromatogr. Sci., 8,434 (1970). (3) J. P. Wolf 111,Anal. Chem., 45,1248 1197.11.
(4)-E.Godbille and P. Devaux, J . C h r o m a togr. Sei., 12,564(1974). ( 5 ) E. Stahl, Ed., “Thin-Layer Chromatography,” 2nd ed., Springer-Verlag, New York. N.Y.. 1969. (6) F. E.Rickett, J . Chromatogr., 66,356 (1972). (7) J.F.K. Huber, Chimia (Aaran), Suppl. 24-35 (1970). (8) J . J . Kirkland, J . Chromatogr. Sei., 10, 593 (1972). (9) R. E. Majors, Anal. Chem., 44,1722 (1972). (10) J. J. Kirkland, in “Gas Chromatography 1972,”p 39,S.G. Perry, Ed., Applied Science Publ., Essex, England, 1973. (11) L. R. Snyder and J. J . Kirkland, “Introduction to Modern Liquid Chromatography,” Chap. 8, Wiley-Interscience, New York, N.Y., 1974. (12) P. E. Antle, E. I. du Pont de Nemours & Co., Wilmington, Del., unpublished studies. 1974. (13) Waters Associates, Technical Bulletin AN 72-120,1972, (14) J. J. Kirkland, Ed., ‘‘Modern Practice of Liquid Chromatography,” Wiley-Interscience, New York, N.Y., 1971. (15) L. R. Snyder and J. J. Kirkland, “Introduction to Modern Liquid Chromatography,” Wiley-Interscience, New York, N.Y., 1974. (16) J. J . Kirkland and L. R. Snyder, Manual, “Solving Problems with Modern Liquid Chromatography,”American Chemical Society, Washington, D.C., 1974. (17) L. R. Snyder, in “Modern Practice of Liquid Chromatography,” Chaps. 4,6,J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971. (18) E. Stahl, Chem. Ztg., 82,323 (1958). (19) H. Schlitt and F. Geiss, J . C h r o m a togr., 67,261 (1972). (20) L. R. Snyder, ibid.,92,223 (1974). (21) L. Rohrschneider, Anal. Chem., 45, 1241 (1973). (22) Waters Associates, Technical Bulletin D5028,July 1973. (23) W. H. Pirkle and R. W. Anderson, J . Org, Chem., 39,3901 (1974). (24) W. H. Pirkle and M. 8. Hoekstra, ibid.,p 3904.
