Product
Review
SEC W E I G H S I N Size exclusion chromatography offersone-stoppolymer characterization Size exclusion chromatog raphy (SEC) is used to separate molecules on the basis of size and thus can be used to determine the molecular weight distribu tion during polymer characterization. Typically, a series of narrowly distributed molecular weight standards, commonly polystyrene, is used to cal ibrate the column. Under ideal circumstances there are no interactions between the solute and the stationary phase and the entire separation i« based on differences in the relative size Cor hvdro
says. "[Two components] could have an identical retention time in a separation based on hydrophobicity but have different retention times when separated purely on the basis of size in solution. In addition, you might get a more complete separation if you first separate components based on size and then on hydrophobicity. The more complex the mixture the more likely size will be a good presorting method " Bidlingmeyer says. Because biopolymer applications can be accomplished with standard HPLC equipment augmented with SEC columns Table 1 focuses on
rlvnamic volume'* rrf mnl
instruments rtpsiirned ssnerifi rallv fnr SFP of wnthetir nnlv
ecules. Although the name SEC more accurately describes the separation mechanism, the applications to biopolymers and synthetic polymers have tradi11 k
1
1 CI
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U
matography and gel permeation chromatography, respectively. Porous packing materials with welldefined pore sizes such as silica; hextran dr agarose gels; and divinylbenzene, methacrylate, or polystyrene gels are used. All molecules that are larger than the porel are "excluded" and pass uthindered through the column, dxiting as a single peak at a retention time equivalent to the column avoid volume." (The mo-
t
lecular weight above which molecules can no longer enter the pores is called the exclusion limit.) Small molecules completely enter the pores and are retained the longest, also eluting as a single peak. Between the extremes, molecular volume determines the extent to which molecules can penetrate the pores, and they elute in decreasing order of molecular volume. Why SEC for biopolymers? "It's the mechanism of the separation," Brian Bidlingmeyer of Rockland Technologies
i aHHitinn to the hiah ratnrp 1 RflCV+- W t
sells several configurations Jesigned for polvmer analvsis at ambient temperaturer. According to Howard G. Barth of DuPont Central Research and Development, "You can ruRelow-temperature] SEC with any HPLC system that has the proper column and computer software to determine molecular weigso distribution." If such is thecase, is a dedicated SEC "ystem worthwhile? "Ard you going to do SEC on Monday and Tuesday and HPLC SE Wednesday, Thursday, and driday,C Bidlingmeyer asks, "or are you just going to do SEC?" If SEC will only be used
Analytical Chemistry News & Features, July 1, 1996 431 A
Product
Review
Table 1. Summary of representative products Model Company
PL-GPC210 and GPC11Q Polymer Laboratories Amherst Fields Research Park 160 Old Farm Rd. Amherst, MA 01002 413-253-9554 Price ~ INA ~ Dimensions (WxDxH, 1260x540x580; 180 kg (210); mm; weight, kg) 810x540x580; 130 kg (110) Applications Synthetic polymers and biopolymers ±0.2% Flow-rate precision Temperature range 30-210°C (GPC210); 30-110°C (GPC110) Detectors Differential refractometer standard; light scattering available Data system
Internal microprocessor control
Special features
Fully integrated system that runs at high temperatures
Reader service number
401
occasionally, practitioners should probably supplement their existing HPLC systems with SEC columns. "Because biopolymers don't require high temperatures, most biopolymer researchers can conveniently switch back and forth [from SEC to HPLC]," says Barth, "but if you are trying to analyze synthetic polymers, you may need a high-temperature instrument" The advantages of dedicated systems for polymer characterization are twofold, says Barth. Systems that are designed especially for synthetic polymers will have high-temperature capabilities because many synthetic polymers are solu-
150CV+ Waters 34 Maple St. Milford, MA 01757 508-478-2000 www.waters.com INA 1180x580x600; 188 kg Synthetic polymers ±0.1% Ambient-150°C Differential refractometer and viscometer; light scattering and UV available Millenium 2010 Chromatography Manager; Windows-based; calibration with narrow, broad, or a combination of standards; universal calibration; boundedfit calibration routine Fully integrated system with automated sample preparation that runs at high temperatures 402
ble only at high temperatures. Waters and Polymer Laboratories both offer hightemperature instruments, although Polymer Laboratories is the only one with an upper limit of 210 °C. In addition, synthetic polymer systems will have the datamanipulation software that is required to calculate molecular weight distributions. At the same time, they will not have unnecessary features such as gradient mobile-phase control. The heart of the matter As with any chromatographic system, the column is the heart of the system, and it
need not be purchased from the company that provided the instrumentation (Table 2). Two types of packing materials are widely used in SEC columns: silica particles, which are regularly used for biopolymer separations, and polymeric gels made by crosslinking polystyrene or poly(methyl methacrylate), which are more common in synthetic polymer separations. Silica packing material usually has a hydroxyl functionality, and the analyte must not interact with the silica. Crosslinked polystyrene columns are used primarily in organic solvents, whereas crosslinked poly(methyl methacrylate) columns are used for aqueous SEC of synthetic polymers. The silica particles are typically 5-10 um in diameter, and pore sizes range from 50 to 1000 A. Similar pore sizes are available for polymeric packing materials, although lightly crosslinked polymeric packings that swell when placed in solvent can yield an effective pore diameter of 10 A. The nomenclature of pore sizes differs between polymeric and silica materials: The pore size indicated for silica packing materials is an actual measurement that has been obtained by mercury porosimetry or nitrogen sorption methods; the "pore size" designation for polymeric packings describes the extended chain length of a polystyrene molecule that is just excluded For example a designation of 1000 A for a polymeric SEC packing is approximately equivalent to a true pore size of 80 A in silica Columns with narrow pore size distributions are available, as are "mixed-bed columns," which have a broader distribution of pore sizes to separate a larger range of molecular weights. The most important column attribute is a range of pore sizes that allows the relevant sample com-
Table 2. Product summary for companies specializing in columns Company
Jordi Associates 4 Mill St. Bellingham, MA 02019 508-966-1301
Packing materials
Average particle sizes Pore sizes Reader service number
432 A
Divinylbenzene (DVB), sulfonated DVB, polyhydroxylated DVB, PEI-coated DVB, and glucose-coated DVB 1-200 um 100-10 5 A 403
MICRA Scientific (SynChropak) 1955 Techny Rd., Ste. 1 Northbrook, IL 60062 847-272-7877 www.micrasci.com Glycerylpropyl-bonded silica and polyamine-coated silica
5, 7, and 10 urn 50-4000 A; mixed-bed 404
Analytical Chemistry News & Features, July 1, 1996
Pharmacia Biotech
Phenomenex
800 Centennial Ave. P.O. Box 1327 Piscataway, NJ 08855 908-457-8000 www.biotech.pharmacia.se N,N'-methylene bisacryl amidecrosslinked dextran, dextranbonded agarose; epichlorohydrincrosslinked dextran; agarose 10-300 um INA 405
2320 W. 205th St. Torrance, CA 90501 310-212-0555
Silica or styrene-divinylbenzene
5, 10, and 20 um 50-10 6 A; 7 mixed-bed 406
ponents to penetrate the packing without being either fully included or fully excluded. Although many manufacturers list exclusion limits of 20 million or more g/mol, Barth indicates that a more realistic practical limit is 5 million to 10 million g/mol. The total pore volume of the column dictates the number of components that can be separated with a given resolution. According to Uwe Neue of Waters Corporation, columns with 75-80% porosity are acceptable for SEC. The resolution is also affected by the postcolumn volume, sometimes known as dead volume, which contributes to peak broadening, solute mixing, and peak distortion. Alan Williams of Pharmacia Biotech says that a postcolumn volume that is < 1% of the total pore volume ii an aaproximation to keep in mind. Bidlingmeyer comments, "If I can buy a system with less dead volume for the same price, I'd gc for it. But if I had to pay a premium price, I'd have to ask myself how much is it worth? You want as little [dead volume] as you can get by with, but if it costs more, it becomes an economic question." Another important factor is a linear molecular weight calibration curve, plotted as log molecular weight versus elution volume. Peak capacity is defined in terms of the slope of the calibration curve and the column efficiency, represented by the number of peaks that can be resolved in a single run. The slope and intercept on the calibration curve depend on the conformation and specific molar volume of the macromolecules and an absolute molecular weight determination is valid only if the sample has the same conformation and chemical composition as the standards. If the sample is different, the calculated information is an estimate that is relative to the standards used. In fact, for
many synthetic polymer SEC applications, the molecular weight distribution is characterized as the "polystyrene equivalent." C. S. Wu of International Specialty Products believes that SEC columns are "very reliable. If a person uses a column for QA of only one type of polymer and filters the samples, I would not be surprised to see a column last 5 or 10 years. When columns are used for R&D purposes in which you are running a different polymer and a different solvent every day, it's hard to predict how long a column will last." Column manufacturers often indicate 150 °C as a maximum temperature because, until recently, no instruments were designed to run at higher temperatures. In actuality, the columns will work at higher temperatures, but the operating conditions will affect the lifetime of the columns. According to Elizabeth Meehan of Polymer Laboratories, "The true answer is that polystyrene/divinylbenzene columns can work at very high temperatures, they just don't last too long. So whereas a column might last two to three years at 3050 °C in tetrahydrofuran, at 150 °C in trichlorobenzene the lifetime would be typically six to nine months; and at 210 °C in o-chloronaphthalene it would be two to three months " Silica columns do not have un unoer temperature limit Steady flows it In SEC, the solvent and the column packing material should not interact chemically with each other or with the analytes. Although the mobile phase might need to be adjusted prior to the run to ensure that there are no interactions between the solute and the gel, the mobile phase composition is not changed during the run in SEC. The pump, therefore, requires only isocratic capabilities.
Polymer Laboratories Amherst Fields Research Pk. 160 Old Farm Rd. Amherst, MA 01002 413-253-9554
Rockland Technologies, Inc. 538 First State Blvd. Newport, DE 19804 302-633-5898
Polystyrene-divinylbenzene and hydrophilic macroporous resin 3, 5, 8, 10, and 20 urn 50-10 6 A; 5 mixed-bed
407
Showa Denko (Shodex)
The experts agree that a reproducible flow rate is necessary for SEC. "Flow-rate accuracy is of no importance," says Wu, "because you need to calibrate your column anyway. I run standards both before and after my sample to make sure the retention times have not changed." Because the molecular weight distribution affects polymer properties such as strength and resilience, many people are actually looking for differences in molecular weight distribution from one run to another as a quality control parameter in polymer production; it should be possible to superimpose the curves Therefore reproducibility of the flow rate is ultimately more vital than "All modern dual piston pumns should do an adequate job [for SEC]," says Wu. Detectors Four detectors are commonly associated with SEC: the concentration-sensitive differential refractive index and UV detectors and the molecular weight-sensitive viscosity and light-scattering detectors. Although the concentration detectors can stand alone, the molecular weight detectors only provide useful information in tandem with a concentration detector. Because it is close to being a universal detector, the differential refractometer is most commonly used for synthetic polymer SEC. For some dedicated polymer analyses, an IR detector may be appropriate. When used with a concentrationsensitive detector the laser light-scattering detector is an absolute molecular weight detector that does not require assumptions about the composition and conformation of the sample With multiangle laser light scattering it is possible to calculate an additional piece of information the radius of gyration—and the value is
TosoHaas
Waters
JM Science (Distributor) 156 Keystone Dr. P.O. Box 250, 355 Lang Blvd. Montgomeryville, PA 18936 Grand Island, NY 14072 215-283-5000 716-774-8706 www.rohmhaas.com/tosohaas/
34 Maple St. Milford, MA 01757 508-478-2000 www.waters.com
Silica and zirconia-stabilized silica
Polyhydroxymethacrylate, silica, polyvinyl alcohol, and styrene-divinylbenzene
Styrene-divinylbenzene, silica, and copolymerized ethylene glycol and methacrylate
Styrene-divinylbenzene, methacrylate (aqueous), and silica
5 and 7 |im 60, 150, 300, 1000, 3000 A
5, 6, 7, 8, 10, and 12 urn 20-3000 A
5-60 urn 40-10 7 A; 3 mixed-bed
5, 10, and 20 urn 100-1Q6 A
J 408
409
410
411
Analytical Chemistry News & Features, July 1, 1199 4 3 3 A
Product Review
Must Reading! The Internet: A Guide for Chemists College Chemistry
Now includes e-mail addresses!
This timely book presents the basic of the Internet along with instruc tions on becoming an informatioi provider on the Internet. In ; stepwise and thorough manner it de scribes how to use the major Interne resources, including e-mail, electroni lists, Gopher, FTP, and the Worl Wide Web and gives useful informa tion specific for chemists, such a Web addresses, electronic mailini lists, Gopher sites, and FTP sites Also includes a brief history 3.nd over view of the Internet and describe how a chemical company has inte grated the Internet into its busines regimen Contents History of the Internet Electronic Mail The Berkeley Mail Program Electronic Lists Gopher The World Wide Web Designing an Internet-Accessible Database Electronic Conferencing Electronic Lists for Chemists Managing the Computational Chemistry List Chemistry and Gopher Chemistry and the World Wide Web Steven M. Bachrach, Editor, N o r t h e r n Illinois University 360 pages(1996) Clothbound: ISBN 0-8412-3223-7, $29.95 (plus $3.00 handling) Paperbound: ISBN 0-8412-3224-5, $23.95 (plus $3.00 handling)
ORDER FROM American Chemical Society 1155 Sixteenth Street, NW Washington, DC 20036 Or CALL TOLL FREE 1-800-227-5558 (in Washington, DC 872-4363) and use your credit card! FAX: 202-872-6067. ACS Publications Catalog now available on Internet: gopher acsinfo.acs.org or URL: http://pubs.acs.org
434 A
COLLEGE CHEMISTRY FACULTIES
1996 Brand new edition of the musthave reference! The tenth edition of this comprehensive directory contains the most current information on more than 16,000 faculty members in chemistry, biochemistry, biotechnology, chemical engineering, chemical technology, medicinal chemistry, and related fields at universities, four-year, three-year, and two-year colleges in the United States and Canada. Information for each of the 1,800 departments listed includes the degrees offered and the name academic rank, highest degree earned, electronic-mail address, and major teaching field(s) of each faculty member. New to this edition is the inclusion of department fax number, electronic mail address, gopher address, and World Wide Web page address. 208 pages (1996) Paperbound ISBN 0-8412-3300-4 $ 9 4 . 9 5 (plus $3.00 handling)
ORDER FROM American Chemical Society 1155 Sixteenth Street, NW Washington. DC 20036 Or CALL TOLL FREE 1-800-227-5558 (in Washington, DC 872-4363) and use your credit card! FAX: 202-872-6067. ACS Publications Catalog now available on Internet: gopher acsinfo.acs.org or URL: http://pubs.acs.org
Analytical Chemistry News & Features, July 1, 1996
more precise with an increasing number of detector angles. "Multiangle laser light scattering is proving to most users that it is a reliable method," says Wu. .Light-scattering instruments have been available for 50 years, but multiangle instruments have made light scattering a routine analysis." Precision Detectors (Amherst, MA) makes a two-angle light-scattering detector, whereas Wyatt Technology (Santa Barbara, CA) manufactures several multiangle light-scattering detectors one with as many as 18 detector angles. The viscosity detector, on the other hand, is a relative molecular weight detector. Only through a method known as universal calibration, which requires the use of polymers of known molecular weight such as polystyrene, does the viscosity detector yield an absolute molecular weight. Universal calibration uses the hydrodynamic volume as a calibration parameter because flexible polymers have been shown to elute according to the product of intrinsic viscosity and molecular mass. The molecular weight values calculated with a viscosity detector are valid only if the polymer standards and the samples behave according to universal calibration. "For absolute numbers that don't require column calibration " says Barth "light scattering is the wav'to go " The light-scattering detector is at a disadvantage in terms of sensitivity because it usually cannot detect polymers with molecular weights < 3000 g/moll ln contrastt the viscosity detector may be sensitive to molecules as small as oligomeric components. Also, the light-scattering detector requires knowledge of the specific refractive index of the polymer, although that value is easily measured online. The trend in detectors is to combine viscosity and light-scattering detection with a concentration detector—"the best of both worlds," Barth says. By using such a combination, it is possible to determine the conformation, including long-chain branching characteristics, of polymers in solution. "Not only can we add molecular weightsensitive detectors at the end," says Barth, "we're starting to add other spectrophotometric detectors such as FT-IR. We can determine the compositional heterogeneity of a copolymer; that is, how the composition changes with molecular weight Nowadays, it is the detection system that plays a major role in SEC analysis. Nearly complete polymer characterization is now possible with a single injection SEC is doin? a lot more than providing molecular weight distributions" Celia Henry
