Strategies in Size Exclusion Chromatography - American Chemical

---

Chapter 11

Packings for Size Exclusion Chromatography: Preparation and Some Properties

Downloaded by UNIV OF GUELPH LIBRARY on October 8, 2012 | http://pubs.acs.org Publication Date: May 30, 1996 | doi: 10.1021/bk-1996-0635.ch011

D. Horák, J. Hradil, and M. J. Beneš Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského sq. 2, 162 06 Prague 6, Czech Republic

Materials used as packings in size-exclusion chromatography can be divided into organic and inorganic ones. Organic packings include both synthetic and natural products. The preparation of these matrices, the mechanism of porous structure formation, depending on synthesis conditions, as well as the properties of packings, are reviewed.

Since the column is the heart of any chromatographic system, the choice of packing greatly affects the success of any analysis. The basic requirements for the packing material for size-exclusion chromatography (SEC) columns include chemical inert­ ness and minimal adsorption of separated compounds so that the retention may be strictly based on hydrodynamic volume. Further requirements include high porosity which can be either permanent in macroporous resins, or swelling in low-crosslinked polymers. In addition to the chemical properties, the importance of physical pro­ perties such as the particle size must be stressed to achieve an adequate resolution of the packing. The particles should be spherical and uniform in size to minimize mass transfer limitations. Since the column separation efficiency increases with decreasing particle size, the size should be as small as possible, however, with good flow properties. High-speed, high-resolution chromatography also sets special re­ quirements for geometrical and mechanical properties. They play a decisive role in the lifetime of the packing and the stability of the flow-rates through the column. It is therefore desirable that all the factors outlined above should be taken into account when choosing the packing. Generally, packings can be divided into universal and those that have a specific effect or "tailor-made" packings. A universal packing covers a broad molecular-weight distribution of the components of an unknown sam­ ple. Such preliminary assessment allows the subsequent choice of a resin that has the optimum properties for a given separation problem. Chromatographical packings have been already systematically reviewed in many papers and monographs (7-9). The aim of article is to review the synthesis, morphology and other properties of packings which are important for SEC. 0097-6156/96/0635-0190$15.25/0 © 1996 American Chemical Society

In Strategies in Size Exclusion Chromatography; Potschka, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

11. HORAK ET AK

Packings for SEC: Preparation & Some Properties

191

Types of S E C Packings A classification according to the chemical nature of the matrix divides packing materials into several groups: packings based on both synthetic and natural organic polymers and packings based on inorganic materials.

Downloaded by UNIV OF GUELPH LIBRARY on October 8, 2012 | http://pubs.acs.org Publication Date: May 30, 1996 | doi: 10.1021/bk-1996-0635.ch011

Packings Based on Synthetic Organic Polymers A basic contribution to the size-exclusion chromatography (SEC) with synthetic or­ ganic polymer-based packings was made by Moore in 1964 who prepared a series of styrene-divinylbenzene resins by using various précipitants and a suitable concen­ tration of the crosslinking agent (JO). Since then these resins have been extensi­ vely used as packings for SEC (5). Therefore the following discussion is mostly con­ fined to styrene-divinylbenzene copolymers, but the same principles are applicable to other supports, such as methacrylic ester packings which are becoming increasing­ ly popular. Other monomer systems for SEC include acrylamide (77), trimethylolpropane trimethacrylate (72), 4-methylstyrene crosslinked with l,2-bis(p-vinylphenyl)ethane (75), glycidyl methacrylate crosslinked with divinylbenzene (14) or ethylene dimethacrylate (75), etc. A number of packing materials are available on the market: they are based on both hydrophobic and hydrophilic synthetic resins, usually with several pore and particle sizes and suitable for SEC of compounds with molecular weights from below 2 x l 0 up to well over 10 (Table I). Recently, tentacle media, in which freely moving polymer chains are bound to the packing matrix by graft polymerization, have been developed by Merck. Due to the flexibility of polymer chains the tentacle media better fit the molecular structure of the analyzed compound and therefore they show better selectivity, higher capacity and higher separation speed compared with classical packings. Another modern ap­ proach to SEC recently marketed by Supelco uses templated beads. Template poly­ mers were excellently reviewed by Wulff (76). By far the most widely used technique to prepare the synthetic organic polymerbased chromatographical packings remains to be classical suspension polymerization. In a suspension polymerization, monomers (or their solution) are suspended as a dis­ continuous (organic) phase of droplets in a continuous phase and polymerized, typi­ cally by free-radical mechanism, resulting in regular spherical beads. The continuous phase is usually water, as most monomers are water-insoluble. Suspension stabilizers must be used to protect monomer droplets against coalescence. Water-soluble polymers, both natural (natural gums) or synthetic ones (polyvinyl alcohol), polyvinylpyrrolidone), or hydroxypropyl cellulose) are the dispersing agents most generally used. Sometimes surfactants (such as sodium dodecyl sulfate) are added. Inorganic stabilizers, e.g., tricalcium phosphate, magnesium hydroxide, calcium carbonate, bentonite, alumina, are also used. The main parameters which determine the behavior of polymer packings are bead size and mechanical properties, porosity and chemical stability. There are two main classes of polymer packings - gel-type and macroporous re­ sins. 3

7

In Strategies in Size Exclusion Chromatography; Potschka, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

192

STRATEGIES IN SIZE EXCLUSION CHROMATOGRAPHY

Downloaded by UNIV OF GUELPH LIBRARY on October 8, 2012 | http://pubs.acs.org Publication Date: May 30, 1996 | doi: 10.1021/bk-1996-0635.ch011

Table I. Commercial Synthetic Organic Polymer Packings for SEC Product Name

Matrix

Producer (Distributor)

Bio-Beads S-X

ST/DVB

Bio-Rad, U S A

Bio-Gel SEC Hamilton PRP

ST/DVB ST/DVB

Nucleogel GPC

ST/DVB

PLgel

ST/DVB

Bio-Rad Hamilton, Switzerland (Alltech, USA) Macherey-Nagel, Germany Polymer Laboratories, USA (Alltech)

Progel-TSK H

ST/DVB ST/DVB

Supelco, USA Showa Denko, Japan (Alltech, Waters, USA)

ST/DVB ST/DVB DVB

Waters TosoHaas, USA Jordi (Alltech)

Shodex GPC Styragel TSK-Gel H Jordi GPC Bio-Gel Ρ Trisacryl G F Progel-TSK PW T S K - G E L PW Toyopearl HW BiospherGM Fractogel Separon

acrylamide and Ν,Ν'-methy- Bio-Rad lenebisacrylamide N-acryloyl-2-amino-2-hydro- Serva, Germany (Sigma, USA) xymethyl-1,3-propanediol Supelco "ethylene glycol and methacrylate" TosoHaas "ethylene glycol and methacrylate" TosoHaas (Supelco) "ethylene glycol and methacrylate" Labio, Czech Rep. GMA/EDMA Merck, Germany "methacrylate" HEMA/EDMA

Shodex OHpak SB-800HQ " poly hydroxy methacrylate" PL aquagel-OH Asahipak GF-HQ Shodex OHpak Q-800

"polyhydroxyl" poly(vinyl alcohol)

polyvinyl alcohol) Nucleogel G F C (aqua-OH) "hydrophilic"

Tessek, Czech Rep. Showa Denko, Japan (Waters) Polymer Laboratories Showa Denko Showa Denko (Waters) Macherey-Nagel

D V B - divinylbenzene, E D M A - ethylene dimethacrylate, G M A - glycidyl methacry­ late, H E M A - 2-hydroxyethyl methacrylate (ethylene glycol methacrylate), ST styrene

In Strategies in Size Exclusion Chromatography; Potschka, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Downloaded by UNIV OF GUELPH LIBRARY on October 8, 2012 | http://pubs.acs.org Publication Date: May 30, 1996 | doi: 10.1021/bk-1996-0635.ch011

11. HORAK ET AL.

Packings for SEC: Preparation & Some Properties

193

Gel-Type Resins. The gel-type resins are generally low crosslinked (1-5% of crosslinking agent) and appear translucent They do not exhibit any measurable (perma­ nent) porosity in the dry state but swell to varying degrees in organic solvents. They swell much more in a thermodynamically good solvent for the basic uncrosslinked polymer than in a thermodynamically poor solvent. Gel-type resins are often referred to as microporous, because the spaces between the crosslinks occupied by the swell­ ing solvent are considered as small pores. Therefore the gel packings are suitable for SEC of low-molecular-weight compounds. The equilibrium degree of swelling attain­ ed is inversely proportional to the network density of the gel which also determines the working range of the gel; its decrease leads to an increase in the exclusion limit of the gel packing while at the same time reducing the mechanical strength of the particles, which is the limiting factor for the applicability of the packing. The drawback of insufficient resistance against volume changes is overcome in resins with permanent porosity which are used for SEC of high-molecular-weight compounds. Macroporous Resins. The materials referred to as "macroporous" (or "macroreticular") resins, in contrast to the gel-type copolymers, contain a significant non-gel porosity in addition to the gel one, i.e., they are permanently porous in the dry state. Permanent (non-gel) porosity is characterized by the disappearance of transparency of the resin. The higher the porosity, the larger is the opacity. At sufficiently high concentrations of crosslinking agent the non-gel porosity is caused by the addition of an inert diluent to the monomers, which is removed from the final product Any compound which roughly meets the three conditions, can be used as an inert diluent: it must be soluble or miscible with the monomers, does not react during the copolymerization, and at the end of polymerization can be easily removed from the resin obtained. It is obvious that not many compounds fulfil these conditions. According to their properties they cause either only the increase in the gel porosity or non-gel porosity is obtained. The non-gel porosity in reality are channels between agglome­ rates of microspheres of which the resin bead is composed. Mechanism of Porous Structure Formation. The mechanism of the macroporous structure formation was described by Kun and Kunin for the copolymerization of styrene with divinylbenzene in the presence of non-solvating diluent as a process consisting of three stages (17). During the first stage, polymer microgels soluble in the monomer-diluent system are formed. As the monomers are con­ verted into copolymer, the polymer chains become less and less swollen and then become entangled (intramolecularly crosslinked) by further progress of the poly­ merization. At the end of this stage, the nuclei (10-30 nm) are formed. Based on thermodynamic factors, the equation (1) for volume conversion o^. at which phase separation occurs was derived (5) 2

l n ( l - a V3°°) + a v °° + X «x v™) c

c

3

l3

c

+ (v/V )V a v 3

1

c

0O 3

/2 = 0

0)

where v °° is the volume fraction of monomers at the beginning of polymerization, χ is the Flory-Huggins interaction parameter, v/V is the effective degree of 3

1