polymer coatings as stationary phases in high-performance liquid chromatography

368 trends in analytical chemisty, vol. 71, no. 10, 1992

Polymer coatings as stationary phases in high-performance liquid chromatography

Michael Hanson and Klaus K. Unger Mainz, Germany

Modern biochromatography demands highly sophisticated packing materials in terms of biocompatibility, (substrate-) selectivity and recovery. Polymers can be designed in a wide variety and therefore deliver solutions to specific chromatographic problems. Thus, tailor- made polymer coatings are an alternative to the classical chemically bonded stationary phases.

Introduction

Over the last decade polymer coatings became increasingly important as stationary phases for liquid chromatography (LC). Although wall-coated capil- laries have been applied for a long time in gas chromatography, up to the mid-eighties had not been considered as competitive stationary phases in LC, due to their slow mass transfer properties [ 1,2]. How- ever, one has to trade off mass transfer advantages of chemically-bonded, conventional stationary phases against a better selectivity or biological recovery. Taking into account that most of the applied polymeric stationary phases have a film thickness in the range of a monolayer up to 5 nm [3], it is obvious that differences in mass transfer velocity between modern polymeric and other stationary phases have become more increasingly negligible. Physico-chemical approaches to investigate the characteristics and advantageous parameters of polymer coatings have also helped to optimize their chromatographic performance [3-51. This article gives an overview of modern polymer coatings in LC and complements the review by Schomburg [6]. We discuss new, tailor-made stationary phases, according to their application in biochromatography.

Types of polymer coatings

There are several variants in immobilizing polymers on supports. Fig. 1 shows one of the most

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common coating variants in chromatography, which is generated by physisorption of reactive prepolymers with well-defined chemical composition on the support and subsequent immobilisation by thermal treatment or y-radiation. The procedure was developed by Schomburg and co-workers [ 1,7,8] on porous silica and alumina. Hanson et al. [3] have opti- mized this technique and applied it to non-porous, microparticulate silica, thereby combining polymer- selectivity with support-efficiency. This method is advantageous, especially in biochromatography, since it allows the design of stationary phases with well-defined properties, such as hydrophobicity and denaturation potential against polypeptides. The potential value of mild polymeric stationary phases that provide high biological recoveries has been dis- cussed elsewhere [9,10]. Furthermore, chromatogra- phers can design tailor-made polymers, without re- striction of the surface properties of the support material, so that basically any support may be coated (Table 1). In addition, the following advantages should be mentioned: The rententivity of the stationary phase can easily be varied by coating the surface with variable amounts of prepolymer before immobilization. Also, more than one polymeric layer can be deposited. The multilayers obtained show special mixed polarities [ 11. However, in this so-called “dry” or bulk polymerisation, some oligomers may behave like non-wetting liquids on the support surface, so

that the support material is not completely encapsu- lated during the crosslinking procedure. This results

RETAINED J POLYMER COATING

ANALYTE

Fig. 1. Polymer coating without chemical bond to the support surface (generated by physisorption of reactive prepolymers and subsequent thermal immobilization).

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trends in analytical chemistv, vol. 17, no. 70, 7992 369

TABLE 1. Support material in liquid chromatography

Support material References IPS

Inorganic Silica 2, 3, 8-12, 16, 28, 29,

56-60,62-69 Glass 50,51 Alumina 8, 28, 46-48, 76 Zirconia-coated silica 76 Titania 76 Magnesia 76 Vitreous carbon 37

Organic Polystyrene Sepharose Agarose Dextran

52,53 21,55,81 15,47 21

Fig. 3. Flexible polymeric or oligomeric ligands stationary phase).

(as

proteins by ion-exchange chromatography, as ._ _ _ _ _ de-

in inhomogeneous coatings and may lead in some cases to a poor liquid chromatographic performance.

An alternative is a chemically-bond coating (Fig. 2), where the polymer layer is connected to the surface by functional or reactive groups, such as vinyl, ect. [7,11]. The polymerisation may be carried out in situ in a monomer or oligomer solution, in which the support material is dispersed and the re- sulting, chemically-bound precursor is subsequently crosslinked. Alternatively, an externally synthesized prepolymer with functional groups is subjected to react with the support surface, thereby generating an “anchored coating”. This type of polymer coating is considered to be more homogeneous, but it is limited to the surface chemistry. Therefore, inorganic supports, except for silica, are barely described in the literature when this method is reported.

A further polymeric modification is the so-called tentacle type (Fig. 3), where the polymer (or oligomer) does not encapsulate the bead. This stationary phase is useful for mild separation or purification of

scribed by Mtiller and co-workers [12-141. Horvath et al. [15] and Chang and An [ 161 reported on a hydrophobic interaction chromatography (HIC) variant where long, flexible side chains are capable of surrounding the proteins and thereby interacting only with their hydrophilic and/or charged outer surface, so that the ternary or quatemary structure of proteins is maintained.

Desilets et al. [17] and Sing et al. [ 181 described supports which are modified with polymeric non- ionic surfactants, such as polyoxyethylene (Fig. 4). Proteins are no longer retained on such supports at moderate or low ionic strengths and can be desorbed by a decreasing ionic strength gradient as applied in HIC. Polyoxyethylene-type non-ionic surfactants in aqueous solution can be retained on reversed-phase supports by their alkane tails, leaving the compara- tively polar parts of the surfactant molecule exposed to an aqueous mobile phase. In contrast to a covalent grafting of the polymer, in this case the surface properties are reversibly modified. Although the in-

NON-IONIC POLYMERIC 1 SURFACTANT

RETAINED ANALYTE 1

REVERSED-PHASE

IRT

RETAINE ANALYT FUNCTIONAL GROUP

POLYMER COATING

Fig. 2. Polymer coating with chemical bond to the Fig. 4. Stationary phase modified with polymeric non- support surface. ionic surfactants.

370 trends in analytical chemisty, vol. 11, no. 70, 1992

SMALL RETAINED MOLECULE 1

I UNRETAINED LARGE PROTEIN

SEMIPERMEABLE POLYMER

RETENTIVE STATIONARY PHASE

IRT

Fig. 5. Stationary phase with polymeric diffusion barrier.

teractions between non-ionic surfactant and reversed-phase support are non-covalent and the HIC mobile phases contain no surfactant, the modified columns are described as stable enough to be used repeatedly [18].

Drug analysis of blood samples is one of the current challenges in chromatography. In most cases a sample pretreatment is necessary to overcome the fouling of the packing materials caused by irre- versible adsorption of denaturated plasma proteins. A highly sophisticated solution to this problem is the concept of “restricted access” packings [ 17,191, which allows the penetration of the retentive site of the stationary phase with small molecules, while the large plasma proteins are excluded by an external hydrophilic network. Hence, biological fluids containing low-molecular-weight analytes may be in- jected directly. The hydrophilic shielding (Fig. 5) with an appropriate polymer is described in depth by Feibush and Santasania [20].

Antibodies can be bound to a hydrophilic polymer phase, which in addition to providing mild conditions

ANTIBODY, 1 POLYMERIC SPACER

ANTIGEN 7 I I

SUPPORT

Fig. 6. Hydrophilic polymeric spacer (e.g. in HPLAC).

for the binding of proteins, also acts as a spacer and allows a better sterical accessibility [21]. The antibodies also cover the negatively-charged silanol groups of silica supports [22], thus avoiding non-specific adsorption due to the silanols of the surface, which act as weak ion-exchangers. Due to their ex- cellent hydrodynamic behaviour, it is not surprising that polysaccharides are a first choice [23,24]. Fig. 6 depicts a model of this variant, which shows similari- ties with the tentacle type; however, the active site is terminal.

Mosbach and co-workers [25,26] developed an alternative procedure for the preparation of substrate- selective polymers. A monomer mixture containing a large proportion of crosslinking units is polymerized in the presence of a free substrate, which acts as a template during the polymer-forming process. A non-covalent attachment of the substrate to the monomeric units is formed (i.e. ionic, hydrogen bond, hydrophobic, charge transfer, etc.), comple- mentary to the different sites of the substrate. After polymerization and removal of the substrate (i.e., washing, hydrolization, etc.), an imprint of the template is formed within the polymer matrix (Fig. 7). These polymer coatings are capable of specific substrate recognition, due to their imprinted interacting sites.

Support materials

Basically any support material, inorganic or organic, can be coated with a polymer, as shown in Table 1. However, inorganic oxides such as silica or alumina are preferred because of their high rigidity, which provides a stable and efficient column packing. The lack of rigidity of organic gels can be com-

POLYMER, rlMPRlNTlNG

.. . ........ ........ .............. . : : .. ... ..: ... ,.::,., .::: .. :, ,: ..... ........ ................... .:: :c:::::: :.: : : .... . :: :. ...

...................... .................... .:

RETAINED J ... ......... ............................ . ................... ............................ . .................. .......... .............. ............ .), ........ ......................

ANALME .:;:I: :; cSfjFPO,Z,T .)> .: ..> .......... ::::::. .:.:.:.:.:.:.:: ........ .: : :.: : :.::, : :.:: : : : : : ............. ..::. .. ........ :. ........... ::::. .-::, ..... :...:I. .. :.:, :.: . . ,,: :: .,,.: :: ....... : ........... .: :, ........ ‘: .... .. .: ....

l INTERACTING UNITS (e.g. hydrophobic, eleCtrO-

static etc.1 OF MONOMERS AND SUBSTRATE

Fig. 7. Substrate selective polymer phase with imprinted interacting sites.

trends in analytical chemistry, vol. 11, no. 10, 1992 371

TABLE 2. Polymers used in liquid chromatography RP = Reversed-phase; IEC = ion-exchange chromatography; HIC = hydrophobic interaction chromatography; AC = affinity chromatography; SEC = size-exclusion chromatography; NP = normal phase

Polymer HPLC-mode References

Polysiloxanes

Polysiloxanes with chiral sectors

Polybutadienes

Polybutadiene- copolymers

Polyacrylates Polyacrylates with

chiral selectors Polyacrylates with

thermotropic liquid crystalline side groups

Polyethylenimines

IEC 28,29 RP, IEC 9, 10,41

Chiral 41

RP IEC

Polystyrenes RP Toluene-polymer RP Polyethylene oxides HIC Polyethyleneglycols HIC, AC

Polyvinylimidazole Polyvinylpyrrolidones

Polymethylglutamate Polypyrroles Polyamides Polyamines Polyvinyl alcohols PolyoxyalkyleneglycoIs Polyether-copolymer

with active ligands Polysaccharides

-with active ligands -with chiral selectors

Proteins (or fragments)

RP RP, HIC, SEC, NP RP RP, IEC RP RP HIC HIC

AC SEC AC Chiral AC, chiral

Poly(crown-ether) Poly(crown-vinyl) Inorganic coating (Alumina, Zirconia)

RP

Chiral RP

RP RP

IEC 47

1,2,4,5, 778, 34, 38, 50,51, 57, 63, 71

40

1, 3, 5,8, 46, 48,56

30,31 12,49, 62,67, 69,76 11 58 55 15, 16, 81

35 66, 72, 78

59 33,37 42,77 68,79 60,61, 70 17, 18, 54

27 65 21,23,24 74,75 43, 45, 47, 73, 80 36 39

pensated for by their (in most cases) high biocompatibility, a prerequisite for biotechnological processes. Therefore, the choice of the most suitable support depends on the application.

Polymers

A wide variety of polymers as stationary phase in LC are mentioned in the literature. Synthetic poly-

mers and biopolymers can both be used as stationary phases. Table 2 shows a selection of polymers used in LC: In general, prepolymers are synthesized externally - not in situ - and then polymerized onto the support surface. In order to guarantee reproducibility, polymers of well-defined composition have to be employed. This is basically no problem for polymers consisting of the same monomer units. However, a “polymer coating design” includes means such as copolymerization [9,10,27-291 or subsequent introduction of functional groups or ligands [21,23, 24,30,3 11. Therefore, the knowledge of co-polyer co- efficients [32], liganddensities and reactivities is essen- tial.

Conclusions

As we have shown in this article, polymer coatings are highly competitive stationary phases for LC and may be applied for substrate selective purification processes as well as for challenging analytical applications. Especially biotechnological applications on the process scale require materials which include mechanical stability as well as biocompatibility. Al- though the synthesis of such packing materials may not always be without difficulties, the charac- terization of the polymers by means of modern spec- troscopic methods is a tool to control their reproducibility.

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Dr. Michael Hanson and Professor Klaus K. Unger are at the lnstifut Nr Anorganische Chemie und Analytische Chemie of the Johannes Gutenberg UniversitSt, J. J. Bechenveg 24, W-6500 Mainz, Ger- many.

Automatic determination of physico-chemical parameters by the flow-rate gradient technique

J. Marcos, A. Rios and M. Valciircel C6rdoba, Spain

There is a pressing need to expand the scope of continuous flow techniques, particularly to some unusual applications where normal flow injection analysis modes and completely continuous procedures are useless. In this con- text, the flow-rate gradient technique plays a major role, because it enables automation and greater speed than the time-consuming and tedious batch procedures. The automatic determination of physico-chemical parameters is one case in point: its broad range of possi- bilities makes fertile ground for the application of the flow-gradient technique with interesting analytical results.

Introduction

The flow-rate is a key variable in continuous-flow analytical systems, and is customarily kept at a constant, optimal value throughout the analysis. How- ever, some recent approaches involve altering the flow-rate during the analytical process, i.e. changing the hydrodynamic conditions of flow systems [ 11. Methodologies which use variable flow-rates clearly

0165.9936/92/$05.00

open new prospects for expanding the potential and applications of unsegmented-flow systems operated under constant conditions.

Flow-rate gradients (Q, in ml/min2) are characterized by continuous changes in the flow-rate (q, in ml/min) according to a linear function:

q=at+b (t = time; a = slope; b = intercept)

or a non-linear function:

4 = Q(t) (polynomial, exponential, etc.)

Flow-rate gradients are characterized by Q = dqldt, and are constant (Q = a) for linear functions and variable [Q = <D(t)] for other types of function.

One of the main assets of these flow-gradient patterns is that they can be used to establish concen- tration-gradients along the flow system. With this principle, various applications can be implemented in a rapid, easy and automatic way, as shown below.

Description of the technique

The earliest reference to the flow-rate gradient technique involved a hydrostatic head [2], and the creation of gradients along a single-channel mani-

0 1992 Elsevier Science Publishers B.V. All rights reserved

polymer coatings as stationary phases in high-performance liquid chromatography

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