Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 21) ppt

21

TRENDS IN PREPARATIVE HPLC

Ernst Kuesters

21.1 INTRODUCTION

Directly from its beginning—now 100 years ago, when Michail Tswett devel￾oped the principles [1, 2] with the isolation of chlorophyll—chromatography

has always been a preparative technology, and its value in producing com￾pounds of high purity cannot be overemphasized. It was Paul Karrer [3] who

stated very early “. . . it would be a mistake to believe that a preparation puri￾fied by crystallization should be purer than one obtained from chromatographic

analysis. In all recent investigations chromatographic purification widely sur￾passed that of crystallization.” and Leslie Ettre, although not distinguishing

between analytical and preparative separations, denoted chromatography as

“the separation technique of the 20th century” [4]. From a historical point of

view, the beginnings of preparative isolation of natural compounds were cum￾bersome. For example, it is reported [5] that six years of work and processing

of 30 tons of strawberries was needed to finally obtain 35mL of an oil, the

essence of the fruit. This situation changed dramatically in the 1960s with the

theoretical understanding of the chromatographic process, the development

of high-performance liquid chromatography, and the synthesis of highly selec￾tive stationary phases. As a result of these improvements, the isolation of

natural compounds with preparative chromatography on production scale

(e.g., drug substances from fermentation processes) is still state of the art, even

after 100 years.

Today, preparative HPLC has also become a powerful technology in phar￾maceutical development and production either for isolation of impurities, for

937

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto

Copyright © 2007 by John Wiley & Sons, Inc.

chromatographic purifications, or as part of a scale-up process and subse￾quently has been reviewed in a lot of monographs [6–10]. The term prepara￾tive amount thus covers the range from milligram quantities (amounts for

structure elucidation, analytical characterization, toxicology, or reference

material) to large-scale production of tons of intermediates and drug sub￾stances. The separations therefore can be performed on all types of columns,

starting from analytical ones up to production scale columns with 1-m i.d and

several meters in length. Typical applications are summarized in Table 21-1.

The success of preparative HPLC on a production scale has been made pos￾sible because of significant improvements made in several areas like (i) column

technology (today, mainly compressed columns are used), (ii) packing mate￾rials (pressure stable spherical particles with high homogeneity, either non￾chiral or chiral), and (iii) the understanding of the nonlinear process in

preparative HPLC (overloaded conditions) which resulted in new methods to

determine the adsorption isotherms and which consequently led to new con￾cepts like displacement chromatography and simulated moving bed (SMB)

chromatography, where the knowledge of such adsorption isotherms is a pre￾requisite for the design of the corresponding separation process.

The aim of this chapter is to highlight current developments in these various

fields of preparative HPLC, with particular emphasis on applications that have

been developed at Chemical & Analytical Development at Novartis Pharma

AG. Drug substance purifications from biological and synthetic sources are

presented, along with the separation of chiral and/or achiral molecules on

chiral stationary phases and typical isolations of by-products. Special attention

is given to the determination of adsorption isotherms and their interplay with

respect to the layout of chromatographic processes as well as the choice of

938 TRENDS IN PREPARATIVE HPLC

TABLE 21-1. Order of Magnitude and Purpose of Purified Amounts Obtained from

Preparative Chromatography

Amount of

Stationary Amount of

Column Type I.D. (mm) Purpose Phase (g) Product (g)

Analytical 1–5 Isolation of reference 0.2–3 0.0002–0.003

substances (MS or

NMR)

Analytical— 5–10 Starting materials 0.003–25 0.003–0.1

semipreparative for toxicology

Semipreparative 10–40 Intermediates for 25–100 0.1–5

—preparative lab synthesis

Pilot plant 100–300 Manufacturing of 100–1000 20–5000

drug substances

for pharmaceutical

development

Production 300–1,500 Manufacturing of 1,000–4,000,000 kg-tons

trade products

technology. The applications have been selected in such a way that a broad

variety of technologies like multiple injection, recycling, displacement, and

SMB chromatography is covered. On-line detection tools have to fulfill other

demands in preparative chromatography than in analytical chromatography.

A special section has been devoted to this aspect below, and an instrument

that was developed in-house is presented.

21.2 METHOD DEVELOPMENT IN PREPARATIVE HPLC

Since chromatography scales up linearly and independently from the selected

technology (rationales when making a choice will be given later on), the

column containing the stationary phase is still the heart of the system. Method

development will therefore always start with the selection of the best station￾ary and mobile-phase composition to achieve an optimum in productivity,

which does not necessarily mean an optimum in selectivity. For example, a high

selectivity of α > 10 has been obtained for the enantiomeric separation of β￾blocking agents like pindolol using amylose- or cellulose-derived stationary

phases, but the poor solubility of the racemates in the mobile phase (hexane/2-

propanol mixtures) will never result in an economic separation process. This

situation can be significantly improved by (i) solvent switch and (ii) adding of

bases or acids, which leads to higher solubility and productivity, although the

selectivity decreases. Figure 21-1 shows the separation of the enantiomers of

pindolol under different conditions [11, 12]. Even though the addition of TFA

clearly results in very distorted isotherms, the situation from the point of view

of the preparative separation is much improved, with the throughput increas￾ing from 322 to 860g of racemate per kilogram of chiral stationary phase per

day. Nevertheless, as a rule of thumb, in most cases higher productivities have

METHOD DEVELOPMENT IN PREPARATIVE HPLC 939

Figure 21-1. The effect of mobile-phase additives on pindolol on Chiralcel-OD

(analytical column). Mobile phase: (a) Methanol/diethylamine = 99.9/0.1, 20°C. (b)

Hexane/ethanol/trifluoroacetic acid = 60/40/0.5, 40°C. (c) Conditions as for (b), but

25-mg load. (Reprint from reference 12, with permission.)

been obtained under separation conditions where high selectivities have been

identified.Therefore, in parallel, parameters like solubility of the sample in the

mobile phase, capacity of the stationary phase, stability, and work-up of

product containing fractions have to be determined. Once a robust system has

been developed, the possibilities of scale-up (solubility of sample, stability of

product in mobile phase, work-up, etc.) are investigated in the next step. And

finally the adsorption isotherms are measured as a guide to the appropriate

and economic technical realization on pilot plant or production scale.

21.2.1 Optimization of Selectivity

The first step, the search for an appropriate chromatographic system, can be

explored with the aid of analytical columns or even more easily in the case of

straight-phase chromatography with thin-layer chromatography (TLC). In the

case of chiral separations with chiral stationary phases (CSP), a quick survey

of separation strategies is provided by using electronic databases like Chir￾base in advance. Since each type of column overloading will result in a loss of

separation, the method development should start with the search for a suffi￾cient peak resolution Rs

. Under analytical conditions, the peak resolution Rs

is the result of the interplay of selectivity or separation factor α, retention time,

and column performance according to equation (21-1):

(21-1)

where a is the separation factor (selectivity) = k2

/k1 for k2 > k1

; k1 and k2 are

the capacity factors of substance 1 and 2, respectively; and N is the plate

number.

A rough estimation nicely highlights the contribution and importance of a

well-developed separation factor. Whereas changes in k from 3 to 5 only

improve the peak resolution by 10.7% and a doubling of N by 41.4%, the

increase of selectivity from 1.2 to 2.2 will result in an improvement of 83.3%.

Since in most cases the technical parameters like particle size and pressure

are given and used under optimum conditions, the search for high selectivity

cannot be overemphasized.

The main parameters to optimize the separation factor and peak resolu￾tion, respectively, are as follows:

• Appropriate stationary phase (which not only seeks for the appropriate

polarity of the material; the “same stationary phase” from different sup￾plier may have a significant influence on the selectivity because of differ￾ences in the manufacturing process).

• Appropriate mobile phase (which includes the choice and composition of

solvents, additives, and pH value).

R

k

k

S = − ( ) N

( ) +

1

4

1

1

a

940 TRENDS IN PREPARATIVE HPLC