Chromatography.pdf

Preface

Due to their versatility and resolution,chromatographic separations of complex

mixtures of biologicals are used for many purposes in academia and industry.If

anything, recent developments in the life sciences have increased the interest

and need for chromatography be it for quality control, proteomics or the down-

stream processing of the high value products of modern biotechnology. How-

ever,the many “challenges”of present day chromatography and especially of the

HPLC of biomacromolecules such as proteins, are also present in the mind of

any practitioner. In fact, some of these latter were such hindrances that much

research was necessary in order to overcome and circumvent them. This book

introduces the reader to some of the recently proposed solutions.Capillary elec-

trochromatography (CEC),for example,the latest and most promising branch of

analytical chromatography,is still hindered from finding broader application by

difficulties related to something as simple as the packing of a suitable column.

The latest solutions for this but also the state of art of CEC in general are dis-

cussed in the chapter written by Frantisek Svec. The difficulty of combining

speed, resolution and capacity when using the classical porous bead type sta-

tionary phases has even been called the “dilemma of protein chromatography”.

Much progress has been made in this area by the advent of monolithic and relat-

ed continuous stationary phases. The complex nature of many of the samples to

be analyzed and separated in biochromatography often requires the use of some

highly specific (“affinity”) ligands.Since they can be raised in a specific manner

to many bioproducts,protein ligands such as antibodies have allowed some very

selective solutions in the past. However, they also are known to have some dis-

advantages, including the immunogenicity (toxicity) of ligands contaminating

the final products, or the low stability of such ligands, which prevents repeated

usage of the expensive columns. This challenge may be overcome by “molecular

imprinting”, a techniques, which uses purely chemical means to create the

“affinity”interaction. Finally we were most happy to have two authors from

industry join us to report on their experience with chromatography as a contin-

uous preparative process. Readers from various fields thus will find new ideas

and approaches to typical separation problems in this volume. Finally, I would

like to thank all the authors for their contributions and their cooperation

throughout the last year.

Lausanne,April 2002

Ruth Freitag

CHAPTER

Capillary Electrochromatography:

A Rapidly Emerging Separation Method

Frantisek Svec

F. Svec, Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.

E-mail: svec@uclink4.berkeley.edu

This overview concerns the new chromatographic method – capillary electrochromatography

(CEC) – that is recently receiving remarkable attention. The principles of this method based

on a combination of electroosmotic flow and analyte-stationary phase interactions, CEC in-

strumentation,capillary column technology,separation conditions,and examples of a variety

of applications are discussed in detail.

Keywords. Capillary electrochromatography,Theory,Electroosmotic flow,Separation,Instru-

mentation, Column technology, Stationary phase, Conditions,Applications

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Concept of Capillary Electrochromatography . . . . . . . . . . . 3

2.1 Electroosmotic Flow . . . . . . . . . . . . . . . . . . . . . . . . . 4

3 CEC Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . 8

4 Column Technologies for CEC . . . . . . . . . . . . . . . . . . . . 11

4.1 Packed Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4.1.1 Packing Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.2 Open-Tubular Geometry . . . . . . . . . . . . . . . . . . . . . . . 16

4.3 Replaceable Separation Media . . . . . . . . . . . . . . . . . . . . 22

4.4 Polymer Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

4.5 Monolithic Columns . . . . . . . . . . . . . . . . . . . . . . . . . 24

4.5.1 “Monolithized”Packed Columns . . . . . . . . . . . . . . . . . . 25

4.5.2 In Situ Prepared Monoliths . . . . . . . . . . . . . . . . . . . . . . 26

5 Separation Conditions . . . . . . . . . . . . . . . . . . . . . . . . 32

5.1 Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

5.1.1 Percentage of Organic Solvent . . . . . . . . . . . . . . . . . . . . 34

5.1.2 Concentration and pH of Buffer Solution . . . . . . . . . . . . . . 36

5.2

Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.3

Field Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Conclusions and Future Outlook . . . . . . . . . . . . . . . . . . 42

eferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Advances in Biochemical Engineering/

Biotechnology,Vol. 76

Managing Editor: Th. Scheper

F. Svec

Introduction

The recently decoded human genome is believed to be a massive source of in-

formation that will lead to improved diagnostics of diseases,earlier detection of

genetic predispositions to diseases,gene therapy,rational drug design,and phar-

macogenomic “custom drugs”. The upcoming “post-genome” era will then tar-

get the gene expression network and the changes induced by effects such as dis-

ease,environment,or drug treatment.In other words,the knowledge of the exact

composition of proteins within a living body and its changes reflecting both

healthy and sick states will help to study the pharmacological action of potential

drugs at the same speed as the candidates will be created using the methods of

combinatorial chemistry and high throughput screening. This approach is as-

sumed to simplify and accelerate the currently used lengthy and labor-intensive

experiments with living biological objects. To achieve this goal, new advanced

very efficient and selective multidimensional separation methods and materials

must be developed for “high-throughput” proteomics [1, 2]. The limited speed

and extensive manual manipulation required by today’s two-dimensional gel

electrophoresis introduced by O’Farrell 25 years ago [3] is unlikely to match the

future needs of rapid screening techniques due to the slow speed and complex

handling of the separations,and the limited options available for exact quantifi-

cation [4]. Therefore, new approaches to these separations must be studied [5].

Microscale HPLC and electrochromatography are the top candidates for this mis-

sion since they can be included in multidimensional separation schemes while

also providing better compatibility with mass spectrometry,currently one of the

best and most sensitive detection methods [6].

After several decades of use, HPLC technology has been optimized to a very

high degree. For example, new columns possessing specific selectivities, drasti-

cally reduced non-specific interaction,and improved longevity continue to be de-

veloped.However,increases in the plate counts per column – the measure of col-

umn efficiency – have resulted almost exclusively from the single strategy of

decreasing the particle size of the stationary phase. These improvements were

made possible by the rapid development of technologies that produced well-de-

fined beads with an ever-smaller size. Today, shorter 30–50mm long column

packed with 3 µm diameter beads are becoming the industry standard while

150–300mm long columns packed with 10-µm particles were the standard just

a few years ago [7].Although further decreases in bead size are technically pos-

sible, the lowered permeability of columns packed with these smaller particles

leads to a rapid increase in flow resistance and a larger pressure drop across the

column.Accordingly, only very short columns may be used with current instru-

mentation and the overall improvement, as measured by the efficiency per col-

umn,is not very large.In addition,the effective packing of such small beads pre-

sents a serious technical problem. Therefore, the use of submicrometer-sized

packings in “classical” HPLC columns is not practical today and new strategies

for increasing column efficiency must be developed.

Another current trend in HPLC development is the use of mini- and micro-

bore columns with small diameters, as well as packed capillaries that require

Capillary Electrochromatography: a Rapidly Emerging Separation Method

much smaller volumes of both stationary and mobile phases.This miniaturization

has been driven by environmental concerns,the steadily increasing costs of sol-

vent disposal, and, perhaps most importantly, by the often limited amounts of

samples originating from studies in such areas as proteomics. The trade-off be-

tween particle size and back pressure is even more pronounced in these minia-

turized columns. For example, Jorgenson had to use specifically designed hard-

ware that enabled operating pressures as high as 500 MPa in order to achieve an

excellent HPLC separation of a tryptic digest in a 25 cm long capillary column

packed with 1- m m silica beads [8].

In contrast to mechanical pumping,electroendoosmotic flow (EOF) is gener-

ated by applying an electrostatic potential across the entire length of a device,

such as a capillary or a flat profile cell.While Strain was the first to report the use

of an electric field in the separation of dyes on a column packed with alumina [9],

the first well documented example of the use of EOF in separation was the “elec-

trokinetic filtration”of polysaccharides published in 1952 [10].In 1974,Pretorius

et al.realized the advantage of the flat flow profile generated by EOF in both thin-

layer and column chromatography [11]. Although their report did not demon-

strate an actual column separation,it is frequently cited as being the foundation

of real electrochromatography. It should be noted however that the term elec-

trochromatography itself had already been coined by Berraz in 1943 in a barely

known Argentine journal [12].

The real potential of electrochromatography in packed capillary columns

(CEC) was demonstrated in the early 1980s [13–15]. However, serious technical

difficulties have slowed the further development of this promising separa-

tion method [16, 17]. A search for new microseparation methods with vastly

enhanced efficiencies, peak capacities, and selectivities in the mid 1990s re-

vived the interest in CEC. Consequently, research activity in this field has ex-

panded rapidly and the number of published papers has grown exponentially.

In recent years,general aspects of CEC has been reviewed several times [18–24].

Special issues of Journal of Chromatography Volume 887, 2000 and Trends in

Analytical Chemistry Volume 19(11), 2000 were entirely devoted to CEC and a

primer on CEC [25] as well as the first monograph [26] has recently also been

published.

Concept of Capillary Electrochromatography

Capillary electrochromatography is a high-performance liquid phase separation

technique that utilizes flow driven by electroosmosis to achieve significantly im-

proved performance compared to HPLC.The frequently published definition that

classifies CEC as a hybrid of capillary electrophoresis (CE) and HPLC is actually

not correct.In fact,electroosmotic flow is not the major feature of CE and HPLC

packings do not need to be ionizable. The recent findings by Liapis and Grimes

indicate that,in addition to driving the mobile phase,the electric field also affects

the partitioning of solutes and their retention [27–29].

Although capillary columns packed with typical modified silica beads have

been known for more then 20 years [30, 31], it is only now that both the chro-

F. Svec

Fig. 1. Flow profiles of pressure and electroosmotically driven flow in a packed capillary

matographic industry and users are starting to pay real attention to them. This

is because working with systems involving standard size columns was more

convenient and little commercial equipment was available for the micro-

separations. This has changed during the last year or two with the introduction

of dedicated microsystems by the industry leaders such as CapLC (Waters),

UltiMate (LC Packings), and 1100 Series Capillary LC System (Agilent) that

answered the need for a separation tool for splitless coupling with high resolu-

tion mass spectrometric detectors. Capillary µHPLC is currently the simplest

quick and easy way to clean up, separate, and transfer samples to a mass spec-

trometer, the feature valued most by researchers in the life sciences. However,

the peak broadening of the µHPLC separations is considerably affected by the

parabolic profile shown in Fig. 1 typical of pressure driven flow in a tube [32].

To avoid this weakness, a different driving force – electroosmotic flow – is em-

ployed in CEC.

2.1

Electroosmotic Flow

Robson et al. [21] in their excellent review mention that Wiedemann has noted

the effect of electroosmosis more than 150 years ago. Cikalo at al. defines elec-

troosmosis as the movement of liquid relative to a stationary charged surface un-

der an applied electric field [24]. According to this definition, ionizable func-

tionalities that are in contact with the liquid phase are required to achieve the

electroosmotic flow. Obviously, this condition is easily met within fused silica

capillaries the surface of which is lined with a number of ionizable silanol groups.

These functionalities dissociate to negatively charged Si–O – anions attached to

the wall surface and protons H + that are mobile.The layer of negatively charged

functionalities repels from their close proximity anions present in the sur-

rounding liquid while it attracts cations to maintain the balance of charges.This

leads to a formation of a layered structure close to the solid surface rich in

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: