Preparative high-performance liquid chromatography - Analytical

Instrumentation

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

reparative Iigh-Performanee iquid Chromato&aphy Since 1969 t h e rapid development of high-performance (“modern”) liquid chromatography ( H PLC) has established this technique as a major tool for analytical chemical characterization. Complementing the widely used gas Chromatography, H P L C enjoys certain unique advantages. I t is applicable to a wider range of compound types, particularly Donvolatile or thermally unstabla materials. In liquid chromatography inany separations are easily accoraplished which otherwise would be very difficult because t h e two phases allow for more selective interaction of sample molecules. Also, separations iire enhanced in HPLC because intermolecular interactions are more effective a t the lower temperatures used. A particular advantage of H P L C s t h e ease of re-

IBiochemicals Department. *Central Research and Cievelopment Department,

instrumental

in synthesis, or f

Resolution

Separation Speed

Sample Capacity

Figure 1. Interrelationship of goals in chromatography

covering samples. Separated fractions are simply collected by placing a n open container a t t h e outlet of the detector attached to the chromatographic column. T h e theory and practice of analytical HPLC have been adequately summarized in several monographs ( 1 - 4 ) , However, in recent months interest has increased in t h e use of HPLC as a preparative technique. Discussions of this topic are beginning t o appear in t h e literature [e.g., see ( 5 ) ] Frequent. ly, 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. However, larger quantities of purified materials may be needed as standards, synthesis intermediates, testing materials, and so forth. Preparative HPLC is a n 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 t h e parameters in the chromatographic system must be drastically adjusted t o 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 opiimized only a t t h e expense of t h e other two. Alternatively, a combination of two goals can be optimized a t t h e expense of the third. In analytical HF’LC, speed and resolution are the desired goals; capacity usually is compromised. On t h e other hand, preparative separations require high sample capacity, and some of the separation speed and/or resolution often must be sacrificed t o achieve this goal.

ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 12, OCTOBER 1’375

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Strategy for Preparative Separations In analytical separations, column resolution R, can be described by the expression:

Selectivity

(ii) Capacity

0

10-5

10-4 g COMPONENT/ g ADSORBENT

10-2

10-3

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 -

.

1

I

I

0.5 mg/g 2.5mg/g

I

I

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 describe t h e isolation process rather than defining the quantity of material isolated. However, we define preparative HPLC as the isolation of significant amounts of pure compounds from mixtures using large-diameter columns operated in an overloaded condition. Figure 2 illustrates the difference between analytical (also “scaleup”) and preparative HPLC as herein defined. These plots show that analytical separations by liquid-solid chromatography (LSC) typically are car1104A

ried out with sample weights of lo% decrease as t h e sample weight is increased; separation efficiency similarly decreases ( 7 ) .T o enhance throughput, most preparative HPLC separations are made using samples of >>1mg/g of adsorbent with the column in an “overload” condition.

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

iiii) Efficiency

(See Glossary for definitions of symbols and terms used in this paper.) By use of this equation, the effect of t h e chromatographic parameters on an analytical separation can be predicted. However, with preparative HPLC the situation is different. The commonly accepted quantitative relationships 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., 1 mg solute/ g adsorbent), both CY and h’ are drastically decreased, and t h e effect of velocity 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 behavior of such a system. and trial-anderror experiments are frequently necessary to obtain the desired purified product. Adjusting peak retention by varying the strength of the mobile phase (Le., t h e ability to elute the solute) is the initial step in obtaining the required resolution. The mobile phase is selected to obtain some retention (usually h’ = 1-10) for the compounds of interest in the mixture. If a satisfactory analytical 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 history of the sample is known. an analytical separation should be developed to serve as “pilot” for the preparative work.

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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 6. Maximization of preparative yield ( 70)

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

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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 it passes through the column compared to the same component a t smaller k’. Thus, a larger sample weight may be used a t higher k’ for increased throughput for a single run. In addition, adjusting the mobile phase strength to retard elution increases resolution compared to a separation with rapid elution (e.g., k‘ = N 1).Optimum conditions for maximum sample throughput per unit of time have not yet been determined and may involve a compromise between k’ values and the weight of sample injected. After adjusting the k’ value of the peak (or peaks) to be isolated, resolution may have to be increased to enhance 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 increase the selectivity factor cy, which describes the retention of the desired product relative to t h a t of the nearest contaminant. Selectivity usually is increased by optimizing the composition of the mobile phase to provide a greater spacing between the peaks. As shown in Figure 4, much larger samples can be used for a separation with greater spacings between the peaks of interest (high cy value), because the


column can be overloaded more heavily before peak overlap becomes a problem. As described in Equation 1, resolution also can be improved by increasing N , the plate count of the column. However, the practical limit for increasing N is dictated by slaveral factors-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 preparative LC are illustrated in Figure 5 . These involve ( I )a single major component in the mixture, (2) two closely similar main components (e.g., isomeric 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 [Figure 6 (C)]. At this point the purified component can be collected in amounts t h a t mlay be adequate for some purposes. However, if more material is needed the column can be overloaded. Even though the peaks are overlapping, a “heart-cut” [Figure 6 (D), cross-hatched portion] will produce a highly purified component in excellent yield, with a greater throughput of purified cornponent per unit of time.

pq I

x

I .

I

TIME

-

I

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

2

1

CYCLES

3

5

4

6

I

TIME

COLLECTED FRACTIONS

-

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

CHOLESTERYL PHENYLACETATE (ONE GRAM1

.

STEP CHANGE to CH,CI* (0.1%MeOH) STEP CHANGE to 50150

c6/cH2c12

(0.lo/~ MeOHl I

INJECT RETENTION TIME (MINUTES)

A different approach may be required 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 t h e cross-hatched front and back “wing” portions of the overlapping peaks gives t h e desired purified materials. However, if t h e initial sample is in short supply, the overlapping middle portion A B can be recovered and, after concentration, reinjected into the column for further collection of t h e purified component “wings”. T h e rechromatography or recycling of this overlapped center portion can be carried out manually or with an automatic switching system ( 1 1 , 12). Automatic recycling is particularly useful in exclusion (size) separation

+

chromatography or in other separations where all sample components elute rapidly ( 1 2 1. However, automatic 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 feasible since t h e volume of mobile phase required for elution exceeds the column void volume because of increased band broadening during each recycle. Thus, t h e end of one cycle begins to intrude on t h e beginning of t h e next, resulting in a cross-contamination rather than increased component purity. T h e increase in resolution afforded by each recycle operation is shown in Figure 8 where the cross-hatched portion of the sample is automatically rechromatographed through t h e column. It is important to note, however, t h a t if the retention of the overlapping peaks in this separati’onhad been increased t o about k ’ = 5-7 (rather t h a n the k’ < 1 used), the resolution of the two components would have been almost equivalent to that obtained in t h e fourth recycle. Thus, by optimizing k’, this preparative isolation probably could have been accomplished in a single run without the need for recycle. Another preparative procedure should be employed when t h e desired material is a minor or trace component as in situation (3) of Figure 5. T h e trace material should be significantly enriched by overloading t h e column after resolution is optimized. First, fractions are collected in the elution region expected for t h e desired trace component. T h e collected fractions are analyzed; the fractions containing t h e 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 t o increase sample throughput and/or the convenience of the preparative HPLC separation. Figure 9 shows the isolation of a steroid using a step-gradient technique. This particular sample, soluble a t effective concentrations only in a moderately strong solvent, was injected into the colurrin initially operated with a mobile phase of low strength. T h e s-trength of the mobile phase then was; increased in two steps so t h a t one gram of the desired component eluted in an optimum h’ range with the required re.;olution.

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)


1107A

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In next month's I N S T R U M E N T A 11 of this article will present the experimental conditions for preparative high-performance liquid chromatography.

TION, Part

Glossary

H: Height equivalent of a theoretical plate, equal to LIN (cm) (included in selling price)

k': Solute capacity factor; equal to total amount of solute in stationary phase divided 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 , = 2it,, - t d i t u , + t u 2 ) t o :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 liquidsolid Chromatography

Literature Cited Figure 10. Flow-programmed separation of aniline derivatives Column, as in Figure 2; mobile phase. 0 5 % methanol (v/v) in cyclopentane (50% HPO-saturated): sample, 50 mg/ml ( 5 )

HPLC

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Flow programming can reduce the time needed for a preparative separation if peaks of interest are widely separated. As illustrated in Figure 10, a n early eluting aromatic amine was preparatively 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 collect 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 analytical 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 isolate from overlapping hands frequently is possible with Snyder's simple standard resolution curve system, which assumes that the two overlapping bands have approximately equal detection sensitivities (14. 25)


1108A


(1) J . J. Kirkland, Ed., "Modern Practice of Liquid Chromatography," CViley-Interscience, New York, N.Y.. 1971. (2) N.Hadden, F. Baumann, F. MacDonald, M. Munk, R Stevenson, D. Gere. F. Zamaroni, and R. Majors, "Basic Liquid Chromatography," Varian AeroeraDh. 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, "Introduction to Modern Liauid Chromatography," Riley-Interscience. New York. N.Y., 1974. ( 5 ) .J. J . DeStefano, in "Introduction t o 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, Manual, "Solving Problems with Modern Liquid Chromatography," American Chemical 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 R a t e r s Associates Technical Bulletin, AN-130. Oct. 1973. (14) L. R.'Snyder, J . Chromatogr. Sci., 10, 200 (1972). (15) L. R. Snyder, ibid.,p 369. (16) E. I. d u Pont de Nemours & Co., Instrument Products Division, Liquid Chromatography Applications Lab Report, 73-03, 1973.

Preparative high-performance liquid chromatography - Analytical

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