J. J. DeStefano‘ and ,I. J. Kirkland* Experimental Station E. I . du Pont de Nemours 8 Co. Wilmington, Del. 19898

Instrumentation

reparative Iigh-Performanee iquid Chromato&aphy

Since 1969 the rapid development of high-performance (“modern”) liq- uid chromatography ( H PLC) has es- tablished this technique as a major tool for analytical chemical character- ization. Complementing the widely used gas Chromatography, HPLC en- joys certain unique advantages. I t is applicable to a wider range of com- pound types, particularly Donvolatile or thermally unstabla materials. In liquid chromatography inany separa- tions are easily accoraplished which otherwise would be very difficult be- cause the two phases allow for more selective interaction of sample mole- cules. Also, separations iire enhanced in HPLC because intermolecular in- teractions are more effective a t the lower temperatures used. A particular advantage of HPLC s the ease of re-

IBiochemicals Department. *Central Research and Cievelopment De-

par tment ,

instrumental

in synthesis, or f

Resolution

Separation Sample Speed Capacity

covering samples. Separated fractions are simply collected by placing an open container a t the outlet of the de- tector attached to the chromatograph- ic column.

The theory and practice of analyti- cal HPLC have been adequately sum- marized 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 ) ] . Frequent- ly, only milligram quantities of puri- fied materials are needed for the iden- tification of unknowns by instrumen- tal and chemical means. Analytical- scale separations often suffice for this purpose, as suggested in Table I. How- ever, larger quantities of purified ma- terials may be needed as standards, synthesis intermediates, testing mate- rials, and so forth. Preparative HPLC is an effective and convenient tech- nique for isolating the desired amounts in very high purity.

Previous discussions of preparative

HPLC usually follow the same experi- mental theme as described for analyti- cal HPLC, except that larger diameter columns are recommended for higher sample capacity. We view this as an oversimplified approach. For many preparative separations the parame- ters in the chromatographic system must be drastically adjusted to obtain the required amounts of purified ma- terial conveniently and in a reasonable time. As indicated in Figure 1, the three main goals of any chromato- graphic system-resolution, separa- tion speed, and sample capacity-are interrelated. Usually one goal can be opiimized only a t the expense of the other two. Alternatively, a combina- tion of two goals can be optimized a t the expense of the third. In analytical HF’LC, speed and resolution are the desired goals; capacity usually is com- promised. On the other hand, prepara- tive separations require high sample capacity, and some of the separation speed and/or resolution often must be sacrificed to achieve this goal.

Figure 1. Interrelationship of goals in chromatography

ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1’375 1103 A

Strategy for Preparative Separations

In analytical separations, column resolution R, can be described by the expression:

I I I I

0 10-5 10-4 10-3 10-2 g COMPONENT / g ADSORBENT

Figure 2. Effects of solute weight Upper curve, solute capacity factor, k‘; lower curve, column plate height, H; column, 50 X 1.09 cm i.d. with 10% (w/w) H20 on 35-75-pn Porasil A: mobile phase, chloroform (50% H20-saturated): pressure, 200 psi; mobile phase velocity, 0.25 cm/sec: sample, diethylketone in chloroform (6)

0.025 mg/g ‘X

-x- 0.5 mg /g

1 . 2.5mg/g

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 “scale- up”) 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 <1 mg/g of adsorbent. In this range, peak retention times (measured in capacity factor h’ values) and column efficiency change little with sample size. The column can be considered in an “over- load” condition when h’ values for sample peaks show >lo% decrease as the sample weight is increased; sepa- ration efficiency similarly decreases ( 7 ) . T o 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.

Selectivity

( i i ) i i i i ) Capacity Efficiency

(See Glossary for definitions of sym- bols and terms used in this paper.)

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 R, 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., <0.5 mg of solute/g of adsorbent) is significantly dependent on mobile phase velocity u; both CY and k’ remain essentially constant during the separa- tion, and only the plate count N is af- fected by u. However, when the col- umn is overloaded (e.g., >1 mg solute/ g adsorbent), both CY and h’ are drasti- cally decreased, and the effect of ve- locity on 3’ (and resolution) becomes relatively minor. Therefore, maximum throughput per unit of time is achieved by deliberately overloading the column with sample and using the highest mobile phase velocity which provides adequate resolution. Since a quantitative chromatographic theory has not been developed for columns in an overload condition, empirical data are often needed to describe the be- havior of such a system. and trial-and- error experiments are frequently nec- essary to obtain the desired purified product.

the strength of the mobile phase (Le., the ability to elute the solute) is the initial step in obtaining the required resolution. The mobile phase is select- ed to obtain some retention (usually h’ = 1-10) for the compounds of interest in the mixture. If a satisfactory ana- lytical separation has previously been made on a sample, the mobile phase used in that study can be a guide for the preparative separation. If no his- tory of the sample is known. an ana- lytical separation should be developed to serve as “pilot” for the preparative work.

By use of this equation, the effect of

Adjusting peak retention by varying

1 1 0 4 A ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975

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ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975 1105A

Figure 4. Importance of separation factor 01 in preparative separations (9)

Figure 5. Typic.al situations encountered in preparative liquid chromatography ( 70) (1) Desired cornponent present as single major peak. (2) Two lor more major components. (3) Minor component ik desired compound

Whereas k’ values of 2-5 normally are sought in analytical separations, larger values (e.g., k’ > 5 ) can be used in preparative LC to enhance sample loadability (8). At a larger k’ value the average concentration of the solute is significantly lower as i t passes through the column compared to the same component a t smaller k’. Thus, a larg- er sample weight may be used a t high- er k’ for increased throughput for a single run. In addition, adjusting the mobile phase strength to retard elu- tion increases resolution compared to a separation with rapid elution (e.g., k‘ = N 1). Optimum conditions for maxi- mum sample throughput per unit of time have not yet been determined and may involve a compromise be- tween k’ values and the weight of sam- ple injected.

After adjusting the k’ value of the peak (or peaks) to be isolated, resolu- tion may have to be increased to en- hance sample loadability. Howwer, increased resolution frequently will be limited by convenience and expense. After optimizing k’ the most effective way of improving resolution is to in- crease the selectivity factor cy, which describes the retention of the desired product relative to tha t of the nearest contaminant. Selectivity usually is in- creased by optimizing the composition of the mobile phase to provide a great- er spacing between the peaks. As shown in Figure 4, much larger sam- ples can be used for a separation with greater spacings between the peaks of interest (high cy value), because the

Figure 6. Maximization of preparative yield ( 70)

column can be overloaded more heavi- ly before peak overlap becomes a problem.

As described in Equation 1, resolu- tion also can be improved by increas- ing N , the plate count of the column. However, the practical limit for in- creasing N is dictated by slaveral fac- tors-the length of column that is available or can be prepared, particle size, the cost of the packing material, and the pressure required.

The three separation situations most commonly encountered in pre- parative LC are illustrated in Figure 5 . These involve (I) a single major com- ponent in the mixture, (2) two closely similar main components (e.g., isomer- ic structures), and (3) very minor or trace components. In situation (1) the preparative isolation of a single major peak is best handled using the scheme pictured in Figure 6. Starting with the analytical separation [Figure 6 (A)], resolution is enhanced [Figure 6 (B)] using the approaches just described. Then the sample weight is increased until the peaks begin to overlap [Fig- ure 6 (C)]. At this point the purified component can be collected in amounts tha t mlay be adequate for some purposes. However, if more ma- terial is needed the column can be overloaded. Even though the peaks are overlapping, a “heart-cut” [Figure 6 (D), cross-hatched portion] will pro- duce a highly purified component in excellent yield, with a greater throughput of purified cornponent per unit of time.

1106A ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975

I pq I . x

I I

TIME - Figure 7. Recovery of two incompletely resolved components ( 10)

CYCLES 1 2 3 4 5 6

I COLLECTED FRACTIONS

TIME - Figure 8. Preparative separation of cannabinol diacetates by recycle Column, 8 ft X 318 in., Porasil C, 35-75 pm: mobile phase, dichloromethane-acetonitrile (99.510.5): sample size, 150 mg ( 131

CHOLESTERY L PHENYLACETATE

(ONE GRAM 1 .

STEP CHANGE

(0.1% MeOH) to CH,CI*

STEP CHANGE to 50150

(0.lo/~ MeOHl c6/cH2c12

I

INJECT

RETENTION TIME (MINUTES)

A different approach may be re- quired for the two major, close-eluting components shown in situation (2) of Figure 5 , As illustrated in Figure 7 , if sufficient sample is available, direct collection of Components A and B in the cross-hatched front and back “wing” portions of the overlapping peaks gives the desired purified mate- rials. However, if the initial sample is in short supply, the overlapping mid- dle portion A + B can be recovered and, after concentration, reinjected into the column for further collection of the purified component “wings”. T h e rechromatography or recycling of this overlapped center portion can be carried out manually or with an auto- matic switching system (11 , 12).

Automatic recycling is particularly useful in exclusion (size) separation

chromatography or in other separa- tions where all sample components elute rapidly ( 1 2 1. However, automat- ic recycling usually is not practical with components eluting a t k’ > 2. When sample components have k ’ values N 2, only 1-2 recycles are feasi- ble since the volume of mobile phase required for elution exceeds the col- umn void volume because of increased band broadening during each recycle. Thus, the end of one cycle begins to intrude on the beginning of the next, resulting in a cross-contamination rather than increased component pu- rity.

The increase in resolution afforded by each recycle operation is shown in Figure 8 where the cross-hatched por- tion of the sample is automatically re- chromatographed through the column. It is important to note, however, tha t if the retention of the overlapping peaks in this separati’on had been in- creased to about k ’ = 5-7 (rather than the k’ < 1 used), the resolution of the two components would have been al- most equivalent to that obtained in the fourth recycle. Thus, by optimiz- ing k’ , this preparative isolation prob- ably could have been accomplished in a single run without the need for recy- cle.

Another preparative procedure should be employed when the desired material is a minor or trace compo- nent as in situation (3) of Figure 5. The trace material should be signifi- cantly enriched by overloading the column after resolution is optimized. First, fractions are collected in the elution region expected for the desired trace component. The collected frac- tions are analyzed; the fractions con- taining the trace component then are pooled, concentrated, and reinjected for a final purification of this now major constituent, using the approach described for situation (1) in Figure 5.

Other techniques such as gradient elution (solvent programming) and flow programming sometimes can be used to increase sample throughput and/or the convenience of the prepar- ative HPLC separation. Figure 9 shows the isolation of a steroid using a step-gradient technique. This particu- lar sample, soluble a t effective concen- trations only in a moderately strong solvent, was injected into the colurrin initially operated with a mobile phase of low strength. The s-trength of the mobile phase then was; increased in two steps so tha t one gram of the de- sired component eluted in an opti- mum h’ range with the required re.;o- lution.

Figure 9. Preparative isolation of cholesteryl phenylacetate Column, Spherosils XOA 400 (two 50 X 2.3-cm i.d. columns in series): mobile phase, step-gradient (two changes) dry hexane to CH& (0.1 % MeOH); flow rate, 30 ml1min: pressure, lOOCl psi; temperature, ambient: detector, UV, 254 nm, 0.32 AUFS ( 16)

ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975 1 1 0 7 A

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Figure 10. Flow-programmed separa- tion of aniline derivatives Column, as in Figure 2; mobile phase. 0 5 % methanol (v /v ) in cyclopentane (50% HPO-satu- rated): sample, 50 mg/ml ( 5 ) Reprinted with permission of publisher

Flow programming can reduce the time needed for a preparative separa- tion if peaks of interest are widely sep- arated. As illustrated in Figure 10, an early eluting aromatic amine was pre- paratively isolated with the column first operated a t a relatively low flow rate for high resolution. Following the collection of this first peak, the flow rate was significantly increased to col- lect the much later eluting second peak with adequate resolution hut greatly decreased separation time.

Following the final collection of the fraction of interest. the purity should be measured by high-efficiency ana- lytical HPLC or another appropriate technique. If the isolated component is not of the desired quality, it can he rechromatographed. Prediction of the purity and recovery of a particular iso- late from overlapping hands frequent- ly is possible with Snyder's simple standard resolution curve system, which assumes that the two overlap- ping bands have approximately equal detection sensitivities (14. 25)

In next month's INSTRUMENTA- TION, Par t 11 of this article will present the experimental conditions for preparative high-performance liq- uid chromatography.

Glossary

H: Height equivalent of a theoretical plate, equal to LIN (cm)

k': Solute capacity factor; equal to total amount of solute in stationary phase divid- ed by total amount of solute in mobile phase; calculated by it, - t , ) / t ,

L : Column length (cm)

N: Column plate number (number of theo- retical plates); calculated by N = 16(t./t,, l 2 P': Solvent polarity index

R,y: Resolution function: calculated by R , =

to: Retention time for unretained solute (solvent "front") (sec)

t,: Retention time for a given band (sec)

t,, : Baseline band width in time units (sec)

u : Mobile phase velocity (cm/sec)

a: Separation factor; a = kJk: e o : Solvent strength parameter in liquid- solid Chromatography

2it,, - t d i t u , + t u 2 )

Literature Cited

(1) J . J. Kirkland, Ed. , "Modern Practice of Liquid Chromatography," CViley-In- terscience, New York, N.Y.. 1971.

(2) N. Hadden, F. Baumann, F. Mac- Donald, M. Munk, R Stevenson, D. Gere. F. Zamaroni, and R. Majors, "Basic Liquid Chromatography," Varian Aero- eraDh. Walnut Creek. Calif.. 1971.

( 3 r P', R. Brown. "H,igh Pressure Liquid Chromatography, Academic Press. New York, N.Y., 1973.

(4) L. R. Snyder and J. J . Kirkland, "In- troduction to Modern Liauid Chroma- tography," Riley-Interscience. New York. N.Y., 1974.

( 5 ) .J. J . DeStefano, in "Introduction to Modern Liquid Chromatography," by L. R. Snyder and J. J. Kirkland. Chap. 12 . LViley-Interscience, New York. N.Y.. 1974.

(6) J. J. DeStefano and H. C. Beachell. J . Chromatogr. Sci., 10,654 (1972).

( 7 ) L. R. Snyder, Anal. Chern., 39,698 (1967).

(8) K . J. Bombaugh and P. W. Almquist, Chrornatographia, 8, 109 (1975).

(9) J . J. Kirkland and L. R. Snyder, Manu- al, "Solving Problems with Modern Liq- uid Chromatography," American Chemi- cal Society, Washington. D.C., 1974.

(10) G. J. Fallick. Amer. Lab. , 5 (8), 19 (1973).

(11) K. J. Bombaugh and R. F. Levangie, J Chromatogr. Sci., 8,560 (1970).

(121 R. A. Henry. S. H. Byrne, and D. R. Hudson, ibid., 12,197 (1974).

(13 1 Raters Associates Technical Bulletin, AN-130. Oct. 1973.

(14) L. R.'Snyder, J . Chromatogr. Sci . , 10,

(15) L. R. Snyder, ibid., p 369. (16) E. I. d u Pont de Nemours & Co., In-

strument Products Division, Liquid Chromatography Applications Lab Re- port, 73-03, 1973.

200 (1972).

1108A ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975