Instrumentation
J. J . D e S t e f a n o 1 a n d J . J . K i r k l a n d 2 Experimental Station E. I. du Pont de Nemours & Co. Wilmington, Del. 19898
Preparative High-Performance Liquid Chromatography In P a r t I of this article (1) which was last month's INSTRUMENTATION feature, the philosophy of preparative high-performance liquid chromatogra phy was presented. In P a r t II the nec essary experimental conditions will be discussed. Optimizing the preparative system for sample capacity involves different operating conditions than normally used for analytical separations. These differences are exemplified by com paring typical operating parameters listed in Table I. Columns
In preparative separations, column sample capacity usually is increased
1 Biochemieals Department. - Central Research and Development. Department.
Table I. Typical Operating Parameters for LC Parameter
Analytical
Column diameter Column packing
2—5 m m i.d. l O u m totally porous or 30 μητι superfi cially porous (pellicular) ~ 1 ml/min
> 8 m m i.d. 3 0 - 6 0 jzm totally porous
0.5 cm/sec
>S0.1 cm/sec
< 2 0 0 μΙ High
> 1 0 0 0 μ! Low
< 0 . 5 mg
> 1 0 0 mg
Mobile phase flow rate Mobile phase velocity Sample volume Detector sensiti vity required Wt of injected sample
by using columns of larger internal di ameter (2). T h e plate count (or effi ciency) of LC columns generally in creases as column internal diameter is increased, as illustrated in Figure 1 for dry-packed columns of a 30-μιτι totally porous adsorbent (3). This effect ap-
COLUMN INTERNAL DIAMETER , cm Figure 1 . Effect of column internal diameter on plate count, Ν Columns, 50 cm long, with 1 0 ml/min
parently involves a complicated inter action between particle size, column internal diameter, column length, and packing structure of the chromato graphic bed. Chromatographic theory suggests t h a t there would be no effect of column diameter on plate count if the sample were placed as a sharp pulse on homogeneously packed col umns. However, in practice, packing inhomogeneities occur because of wall effects and the sizing of particles in the chromatographic bed. These im perfections appear to be less signifi cant in larger diameter columns (i.e., up to at least 2.3 cm i.d.). Separations with large diameter columns frequent ly are superior to those obtained with narrower bore columns, provided the same ratio of sample weight to crosssectional area is maintained. T h e practical upper limit of column i.d. has not yet been established, but equipment and separations using 8-cm i.d. columns have been reported (4). Preparative columns are most com monly made of stainless-steel tubing which can operate up to about 3000 psi. However, large-diameter glass col umns are available for use at pressures below about 200 psi.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER
1975 ·
1193 A
Column Packings Since the preparative separation is to be optimized primarily for sample capacity, it is particularly important t h a t a totally porous support be used. Columns of superficially porous or pellicular supports generally have a sample capacity less t h a n about onefifth of totally porous particles of the same type. Although milligrams of pu rified components have been conve niently isolated with columns of pelli cular packings, it is inconvenient (and costly) to isolate gram amounts by this approach. Some high-capacity totally porous packings which are useful for preparative separations by the four LC techniques are listed in Table II. Whenever feasible, preparative sep arations should be attempted by liq uid-solid (adsorption) chromatogra phy (LSC). This method provides the
greatest flexibility and convenience for separating complex mixtures at high resolution with good sample throughput and moderate cost. By ap propriate selection of the mobile phase, adsorption chromatography can be used for many compound types [e.g., see (5) for materials t h a t have been separated with adsorbents by TLC]. In addition, LSC permits the use of mobile phases which generally have higher solubility for many com pounds, allowing higher sample loadability and throughput. Solvents gener ally used for LSC also are easily re moved after isolation of the desired peaks. Because of the general utility of the method, most of the discussions in this paper are directed toward the LSC approach. However, preparative separations also have been made with the other H P L C methods—exclusion, liquid-partition, and ion exchange
[e.g., see (6) for a liquid-liquid prepar ative separation]. Although no systematic study of the particle size of totally porous particles for preparative H P L C has been made, the 30-60-,um range appears to have advantages. In our experience, a $2,000 for a single 25 X 2.3-cm i.d. column, if commercially available. An alternative to large-diameter columns of
