product review
The SEC Need for Speed A field that has seen incremental improvements now faces a high-throughput revolution. Laura DeFrancesco with contributions from Cheryl M. Harris
T
his summer in New Hampshire, the Gordon Research Conferences will hold for the first time talks on combinatorial and high-throughput materials science. And researchers in the field know that highthroughput materials science would not flourish at its current rate without highthroughput size-exclusion chromatography (SEC). For Eric Amis, chief of the Polymer Division at the National Institute of Standards and Technology (NIST) based in Gaithersburg, Md., high throughput opens doors to new possibilities in materials science. “Being able to explore a broad range of different polymers and analyze them automatically with a high-throughput approach will help people expand the range of materials that they look at,” says Amis. “That’s the most compelling reason to use high throughput. You can afford intellectually to broaden your horizons.” Advances in SEC have also opened doors for companies to sell their latest SEC instrumentation. The separations technique can be applied to biopolymers as gel filtration chromatography and to synthetic polymers as gel permeation chromatography (GPC). When biopolymer mixtures are really complex, SEC can be ideal for presorting because it separates molecules in solution on the basis of size. Some company representatives say the pharmaceutical industry is catching on to the advantages of using SEC in the lab for biopolymers. But it is primarily in the polymer laboratory where SEC has been a mainstay for decades. Molecular weight and molecular weight distribution are important char-
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product review
Table 1. Selected SEC instruments. features
PL-GPC210 and GPC110
Alliance GPC2000 series system s Alliance GPC/HPLC system s
Com pany
Polymer Laboratories Amherst Fields Research Park 160 Old Farm Rd. Amherst, MA 01001 413-253-9554
Waters 334 Maple St. Milford, MA 01757 508-478-2000
Waters 334 Maple St. Milford, MA 01757 508-478-2000
URL
www.polymerlabs.com
www.waters.com
www.waters.com
Dim ensions (w ⳰ d ⳰ h) mm
GP210: 1260 ⫻ 540 ⫻ 588 GPC110: 810 ⫻ 540 ⫻ 580
937 ⫻ 610 ⫻ 560
580 ⫻ 600 ⫻ 570
Synthetic polymers and biopolymers
Synthetic polymers and biopolymers
Synthetic polymers, biopolymers, and polymer additives
±0.07%
±0.075%
±0.075%
GPC210: 30–220 ºC GPC110: 30–120 ºC
Ambient to 180 ºC
Ambient
Differential refractometer standard; light scattering, viscometer, and FTIR available
Differential refractometer standard; viscometer optional, and light scattering available
Differential refractometer, multichannel UV–vis, photodiode array, fluorescence, and conductivity
Internal microprocessor control
Millennium 2010 Chromatography Manager
Millennium 2010 Chromatography Manager
Fully integrated system
Fully integrated system with auto- Semi-integrated system for GPC matic sample mixing and filtration and quaternary solvent HPLC
M odel
Applications
Flow rate precision Tem perature range Detectors
Data system
Special
acteristics of polymers, which are inherently polydisperse, and SEC has been widely used to determine these parameters. While there have been some improvements in the technology—better performing beads, faster columns— these improvements have been incremental, according to Howard Barth of Dupont Experimental Station in Wilmington, Del. “[SEC is] a mature technique. It’s been around for many years, and there’s really, in my opinion, not much in terms of a quantum leap or revolutionary changes,” he says. But as the materials industry embraces high-speed approaches for synthesizing and screening new products, SEC will have to adapt to keep pace. Analytical Chemistry explores SEC and its effect on the big business of synthetic polymers, as well as its steady climb into the pharmaceutical industry. Table 1 lists representative companies that sell complete SEC systems for synthetic polymers. Biopolymer applications can be performed with standard HPLC equipment enhanced with SEC columns. Table 2 lists examples of companies that sell selected SEC
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parts, such as columns and detectors. Interested readers should contact the individual companies for full details.
Bead basics For synthetic polymers, SEC—or GPC, as it’s commonly referred to for synthetic polymers—is conceptually simple. When polydisperse material passes through a column of porous beads, the movement of small molecules is retarded in the pores, while larger ones passes through the column unimpeded. This results in the separation of the various species according to their size. While SEC may sound simple, manufacturers of SEC columns and their customers face some challenges. For example, separation by size alone requires that the polymer not interact with the column material, otherwise the method becomes more like affinity chromatography. However, different polymers are soluble in different solvents, running the gamut from organic to aqueous. Hence, it takes a range of different packing materials to satisfy the needs of polymer chemists, and suppliers of SEC columns have to
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constantly invent new approaches for working with new polymers and solvent combinations. Another problem with SEC is that the separation is strictly time-dependent— species are characterized by the time it takes for them to exit the column. Any change in flow rate between runs, however slight, can affect reproducibility. Furthermore, determining molecular weight from elution profiles requires calibrating the column with molecular weight standards, which may or may not share the hydrodynamic properties of the material of interest. Here again, any change in the column flow rate means the column must be recalibrated. Most users don’t care about absolute molecular weight, according to John McConville, vice president of sales at Polymer Laboratories in Amherst, Mass. In many cases, SEC is used strictly as a relative technique. For those who do need absolute molecular weights, detectors that measure it, such as light scattering detectors, can be added on. For different polymer/solvent combinations, manufacturers have developed
product review
Table 2. Selected SEC columns. Com pany
Agilent Technologies 395 Page Mill Rd. Palo Alto, CA 94303 800-227-9770
Jordi Associates 4 Mill St. Bellingham, MA 02019 508-966-1301
Eprogen, Inc. (MICRA) 8205 S. Cass Ave. Ste. 106 Darien IL 60561 630-963-1481
Phenomenex 2320 W. 205th St. Torrence, CA 90501 310-212-0555
Polymer Laboratories Amherst Fields Research Park 160 Old Farm Rd. Amherst, MA 01002 413-253-9554
URL
www.agilent.com/chem
www.jordiassoc.com
www.eprogen.com
www.phenomenex.com
www.polymerlabs.com
Product
Zorbax PSM/PSM S
JordiGel DVB SynChropak GPC JordiGel Glucose-DVB SynChropak CATSEC JordiGel Hydroxylated-DVB JordiGel Sulfonated DVB JordiGel Wax DVB
PhenoGel
PLGel, PL Aquagel, PLHTS, PLHFIP, PLOLigoPore, PL Mixed LS
Packing m aterials
Silica, silanized, and unsilanized
DVB, glucose DVB, hydrox- Glycerol-bonded silica and Styrene DVB ylated DVB, sulfonated DVB, polyamine-bonded silica wax DVB
PV/DVB, some proprietary materials
Particle sizes (µm )
5, 8
5
5, 7, 10
3, 5, 6, 8, 10 15, 20
102–105
60, 100, 300, 500, 1000, 4000 50–106 and mixed beds
Proteins, peptides, water- Typical polymers, isoprene Organic, aqueous, high soluble anionic and neutral units, plant pigments, speed, polar solvent, polymers triglycerides oligomer, GPC with lightscattering detector
Pore sizes (Å) 60, 300, 1000 Applications
Polymers in organic and aqueous eluents
Organic, neutral polar, and dextran; negatively charged polar; positively charged polar
Special features
Withstands up to 3000 psi
Available in different High temperatures and pressures, available in bulk dimensions
beads with various coatings. For organic solvents, most suppliers use some form of polystyrene heavily cross-linked with divinylbenzene (DVB) to prevent the bead from being dissolved by the solvent. An exception to this strategy is the organic beads sold by Jordi Associates, which use pure, cross-linked DVB. The advantage here, according to President Howard Jordi, is that shrinkage in organic solvents is less when the styrene is taken out of the bead. The company has added polar surface chemistries to the bead, which it says further reduces shrinkage. Jordi also offers a glucose-bonded DVB bead that he says runs in water, hexane, and freon. But there is a trade-off. With derivatized beads, glucose becomes part of the pore structure, in essence clogging the pores. “So, if you want the ultimate resolution, you’re better off running standard DVB with the pores wide open,” says Jordi. “The degree of resolution isn’t as good with bonded columns as with the nonbonded.” Styrene and DVB beads are inherently hydrophobic and hence not suitable for aqueous environments. Thus, there
5, 10, 20
Stable to 140 ºC; ultratemp to 205 ºC; mixed bed, and different dimensions
are almost as many solutions to handling aqueous conditions as there are manufacturers. Some switch to an entirely different synthetic bead—polyacrylic, for example. Others use nonsynthetic beads, such as silica, whereas some derivatize DVB beads with hydrophilic species. “Aqueous SEC is a witch’s brew,” says McConville. “Getting material that meets the requirements—right particle size for efficiency and right porosity for discrimination and right surface for avoiding interactions—is really an art. [The wizard] Merlin would be perfectly at home here. It’s both artful and magical.”
Smaller, faster SEC As the materials industry is under increasing pressure to produce better products at lower cost in less time, SEC is facing new challenges. “The whole idea is: Can we get things done quicker with fewer people?” says Rick Nielson, a product manager at Waters Corporation. This means providing faster SEC columns. There are different ways of getting faster GPC: One can increase the flow
50–106 and mixed beds
Operating temperatures up to 220 °C; different dimensions
rate, use shorter columns, or do both. But changing these parameters can affect the resolution and the quality of the data. According to President Michael Gray of Polymer Standards Service, “It’s all about pore volume.” His company has approached this problem by developing new materials with greater pore volumes, which they combine with shorter, fatter columns to bring run times down to a few minutes. In industry, standard columns are typically 7.5- to 8.0-mm i.d. and 300 mm long, says Gray. A typical SEC column using an organic mobile phase normally has an analysis time of about 15 min; for water-based separations, it’s 15–30 min, he says. His company has developed column technology that has reduced analysis times to about 3 min for organic-phase separations and about 6 min for water-based separations. Symyx Technologies, Inc., based in Santa Clara, Calif., is another example of a company pushing SEC further. It is also developing very high-throughput technologies for generating hundreds to thousands of new materials, as well as the in-
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product review
Table 2. Selected SEC columns (continued). Com pany
Polymer Standards Service 10111 Colesville Rd. #123 Silver Spring, MD 20901 301-681-9624
Showa Denko 13-9, Shiba Daimon 1-Chome, Minato-ku, Tokyo 105-8518 Japan +81-3-5470-3235
Tosoh Biosep 156 Keystone Dr. Montgomeryville, PA 18936 800-366-4875
Waters 30 Maple St. Milford, MA 01757 508-478-2000
URL
www.polymer.de
www.sdk.co.jp/index_e.htm
www.tosohbiosep.com
www.waters.com
Product
SDV, Polefin, HighSpeed, GRAM, MCX, Suprema, HEMA, PFG, Novema
Asaipak GF, Shodex
TSK-Gel SW, PW, HW TSK-Gel H
Styragel, ultrahydrogel
Packing m aterials
Polystyrene/DVB, sulfonated SDV, Vinyl alcohol polymer, porous or based on application S/DVB
Silica- or polymer-based
Styrene DVB, methacrylate
Particle sizes (µm )
3, 5, 8, 10, 20
5, 6, 7, 9
5, 10, 13
5, 6, 10, 20
60–1000
10–250
50–107; 120–2000
Aqueous and nonaqueous
Aqueous and nonaqueous
Pore sizes (Å) 50–108 and mixed beds Applications
High temperature, high speed, polar Aqueous and nonaqueous solvent, anionic neutral, water soluble, hexafluoroisopropanol analysis
Ambient to 180 ºC
Special features
strumentation for analyzing them. The company has developed the Rapid GPC module, which automatically analyzes polymer samples in a rapid, serial method with data quality similar to that obtained from traditional GPC. These rapid analysis techniques can also be done simultaneously using a bank of GPC columns running in parallel. Symyx company representatives are discreet about their column technology, but they say they’ve reduced 60- to 90-min separations to one-tenth of that time. The general approaches, they say, involve using “high-ratio” (shorter and fatter) columns, overlapping injection, and automated preparation and injection. With conventional GPC, which typically runs at 1 mL/min, increasing the flow rate 2- to 3-fold wreaks havoc with the distribution data and retention times because of the time-dependent nature of the separation. But fast GPC can still be used to indicate the trend in polymer reaction. “You can monitor the trend,” says McConville. “You can see that [the product] is changing its molecular weight upward. You can then use the technique in a different way.” Software can also be used to deal with resolution loss in fast SEC. According to Nielson, “Band spreading becomes more of a problem as you try to get things done faster.” Waters offers software that report-
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edly corrects band spreading and gives accurate size average, weight average, and z average molecular weight in 7 min.
SEC gets versatile MALDI TOF technology, the darling of proteomics and genomics, is garnering the attention of the polymer industry as well. This MS technique can measure chemical composition quickly and directly. However, MS is still problematic with polymers that have molecular weight distributions greater than a polydispersity of 1.2, or that differ in polymerization by more than 20%. Analysts may be forced to use SEC as a way to prefractionate polymers before running them on a mass spectrometer. This, according to Barth, may be the most effective use of this technique. “Rather than looking at SEC as the method of doing molecular weight distribution, it’s really combining techniques,” he says. “SEC may become more of a preparation technique, to clean up the sample for MS or NMR or other spectroscopic techniques.” SEC for biopolymers is a growing area in the pharmaceutical industry, says Gray, “but it has not been as established in that marketplace until fairly recently.” His “gut reaction” is that SEC is being used more for synthetic polymers; but in the past three or five years, column technology has caught up. That, coupled with
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better commercial detectors that characterize biological samples easily, has made the pharmaceutical industry take notice of using SEC in the laboratory. Advances have made SEC technology grow so much that companies are quick to jump on them and patent them. Symyx has been issued numerous patents related to their rapid GPC technology, according to McConville. “Name it, it’s covered— instrumentation, concept, speed, detectors, actual operation,” he says. This has led some to question whether Symyx’s extensive number of patents will be a barrier to the continued development of highthroughput GPC. But according to Amis, in materials, there is little sign of that happening, and the GPC field is flourishing. Such growth in materials science with the help of high-throughput GPC can only mean good things to come for scientists like Amis, who believe that applying combinatorial approaches to polymer synthesis and analysis is truly revolutionary. “To the extent that we can develop simple and elegant tools that let people extend their investigations and give them ways to think differently, that’s a huge advance,” he says. Laura DeFrancesco is a freelance writer based in Pasadena, Calif. Cheryl M. Harris is an associate editor with Analytical Chemistry.
