Instrumentation
J. J. DeStefano 1 and J. J. Kirkland 2 Experimental Station E. I. du Pont de Nemours & Co. Wilmington, Del. 19898
Preparative High-Performance Liquid Chromatography Since 1969 the rapid development of high-performance ("modern") liquid chromatography (HPLC) has established this technique as a major tool for analytical chemical characterization. Complementing the widely used gas chromatography, HPLC enjoys certain unique advantages. It is applicable to a wider range of compound types, particularly jionvolatile or thermally unstable materials. In liquid chromatography many separations are easily accomplished which otherwise would be very difficult because the two phases allow for more selective interaction of sample molecules. Also, separations are enhanced in HPLC because intermolecular interactions are more effective at the lower temperatures used. A particular advantage of HPLC is the ease of re-
1 Biochemicals 2
Department. Central Research and Envelopment Department.
Tentative leerrtiticatioo , by instrumental methods Positive identification by instrumentai methods. Confirmation of struc* ture by chemical tactions Jse as analytical standard, r» synthesis, or for :estintj
100
Figure 1 . Interrelationship of goals in c h r o m a t o g r a p h y
covering samples. Separated fractions are simply collected by placing an open container at the outlet of the detector attached to the chromatographic column. The theory and practice of analytical HPLC have been adequately summarized in several monographs (1-4). However, in recent months interest has increased in the use of HPLC as a preparative technique. Discussions of this topic are beginning to appear in the literature [e.g., see (5)]. Frequently, only milligram quantities of purified materials are needed for the identification of unknowns by instrumental and chemical means. Analyticalscale separations often suffice for this purpose, as suggested in Table I. How ever, larger quantities of purified materials may be needed as standards, synthesis intermediates, testing materials, and so forth. Preparative HPLC is an effective and convenient technique for isolating the desired amounts in very high purity. Previous discussions of preparative
HPLC usually follow the same experimental theme as described for analytical HPLC, except that larger diameter columns are recommended for higher sample capacity. We view this as an oversimplified approach. For many preparative separations the parameters in the chromatographic system must be drastically adjusted to obtain the required amounts of purified material conveniently and in a reasonable time. As indicated in Figure 1, the three main goals of any chromatographic system—resolution, separation speed, and sample capacity—are interrelated. Usually one goal can be optimized only at the expense of the other two. Alternatively, a combination of two goals can be optimized at the expense of the third. In analytical HPLC, speed and resolution are the desired goals; capacity usually is compromised. On the other hand, preparative separations require high sample capacity, and some of the separation speed and/or resolution often must be sacrificed to achieve this goal.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975 ·
1103 A
Strategy for Preparative Separations
In analytical separations, column resolution Rs can be described by the expression: Rs = 4
(« " 1) (i) Selectivity
CTTv) (ii) Capacity
Figure 2. Effects of solute weight Upper curve, solute capacity factor, k'; lower curve, column plate height, H; column, 50 X 1.09 c m i.d. with 1 0 % (w/w) H 2 0 on 35-75-μηη Porasil A; mobile phase, chloroform ( 5 0 % H 2 0-saturated); pressure, 200 psi; mobile phase velocity, 0.25 c m / s e c ; sample, diethylketone in chloroform (6)
Figure 3. Effects of mobile phase velocity and solute weight on resolution Mobile phase and column as in Figure 2; sample, diethylketone in chloroform; plots show mg of total sample injected per gram of packing (6)
The phrase "preparative liquid chromatography" often is used to de scribe the isolation process rather than defining the quantity of material isolated. However, we define prepara tive HPLC as the isolation of signifi cant amounts of pure compounds from mixtures using large-diameter col umns operated in an overloaded con dition. Figure 2 illustrates the differ ence between analytical (also "scaleup") and preparative HPLC as herein defined. These plots show that analyt ical separations by liquid-solid chro matography (LSC) typically are car
ried out with sample weights of 10% decrease as the sample weight is increased; sepa ration efficiency similarly decreases (7). To enhance throughput, most pre parative HPLC separations are made using samples of » 1 mg/g of adsor bent with the column in an "overload" condition.
1104 A · ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
x
*
·ν1/2
(1)
(iii) Efficiency
(See Glossary for definitions of sym bols and terms used in this paper.) By use of this equation, the effect of the chromatographic parameters on an analytical separation can be pre dicted. However, with preparative HPLC the situation is different. The commonly accepted quantitative rela tionships involving chromatographic resolution Rs no longer apply when large samples grossly affect column equilibrium since all three terms of the resolution equation (Equation 1) are altered as the degree of overload varies. In Figure 3 the resolution of a column in a nonoverloaded condition (e.g.,
