PST 522E Synthesis and Characterization of Macromolecules

CHAPTER 10
GEL PERMEATION CHRORAMATOGRAPHY (GPC)

1. THEORETICAL PART 1.1. Introduction to size separations Gel Permeation Chromatography, (GPC), also known as Size Exclusion Chromatography, (SEC) is really the easiest to understand of ail the liquid chromatographic techniques. The separation is based strictly on the size of the sample in solution, and there should be no interaction with the column packing, (adsorption, partition, etc.), as you have with conventional HPLC. The mode of separation is not based on molecular weight but on the size of the material being analyzed (usually a polymer) in solution. In other words, to do GPC correctly, the sample must be dissolved in a suitable solvent. The concentration of the sample in solution depends on the molecular weight, but a concentration of 0.10% (w/v) for a polymer of molecular weight -100,000, is typical. (See more in the Sample Prep. Section below): At times, the sample solution must be heated to dissolve the sample. For example, some polyolefins need temperatures greater than 120 C to dissolve, and are typically run in 1,2,4 trichlorobenzene at 140 C. Once the sample has been suitably dissolved, it is introduced via an injection mechanism onto a set of columns which act as a molecular filtration system. The columns are packed with a crosslinked gel, (styrene/divinylbenzene copolymer for organic applications), which contain surface pores. These pores can vary from small to quite large, and act as the molecular filters mentioned above. The larger size molecules will not fit into the smaller pores. Conversely, the smaller molecules will fit into most of the pores, and be retained longer.

Figure 1: The larger molecules will elute first according to BOCOF's law (Big Ones Come Out First) One of the first GPC demonstrations performed by Waters -30 years ago was on chewing gum. Chewing gum is really synthetic rubber, plus additives such as flavors, stabilizers, etc.

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Figure 2: GPC Chromatogram Above a representation of the original GPC chromatogram, separated on several columns of various pore sized connected in series. The polymer (rubber in this case) elutes first as it is the largest molecule, followed by the additives in decreasing order of size. This could just as well be a chromatogram of PVC with a mixture of plasticizers, antioxidants and UV stabilizers. 1.2. Monomers, Oligomers, Polymers and Molecular Weight Distributions Monomers have a single molecular weight, and are said to be mono disperse. Examples would be ethylene, styrene, vinyl chloride, etc. After monomers, we have dimmers, trimers, tetramers, pentamers, etc., which are called oligomers. As we get to higher molecular weights, the group is called polymers. Polymers have distribution of chain lengths, and, therefore molecular weights. Depending on how the polymerization was carried out, this distribution can be narrow, or quite broad. As an example, a condensation, or step-growth, such as polyester, (polyethyleneterephthalate), will have a fairly narrow distribution of molecular weights. On the other hand, a free radical polymerization may produce a polymer with a very broad distribution of chain lengths and molecular weights, (such as for polyolefins). Controlling the kinetics of the polymerization is extremely important in obtaining a desired molecular weight distribution. That is why GPC is such an important technique to the polymer chemist.

Figure 3: Molecular Weight Distribution (MWD)

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Chapter 10 Gel Permeation Chromatography

In the below, we show an overlay of 2 molecules weight distributions of a polymer (in this case polystyrene). Molecular Weight Averages, Mn, Mw, Mz, Mz+1

Figure 4: Calculation of MW Averages There are other techniques to obtain these molecular weight averages: Number average, Mn, may be obtained by membrane osmometry, or end group analysis, (titration, NMR, etc.) Weight Average, Mw, may be obtained by light scattering Z Average, Mz, and Z+1 Average, Mz+1 may be obtained by ultracentrifugation

1.3. Configuring a GPC System Now that we have an understanding as to what molecular weight averages are all about, we are ready to put a system together.

Figure 5: GPC System The system (shown above) consists of a pump, an injector of some sort, either manual or automated, the detector(s), the column set, and some sort of data handling device. In addition, it is a good idea to use a degasser, particularly when using THF with a refractive index detector. The columns are almost always heated to some elevated temperature, even for room temperature soluble applications to insure low pressure drop and uniform viscosities. We will now discuss the system in more detail.

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1.4. Solvent Management The pumps that are being used today with Waters GPC systems are really sophisticated fluid handling devices. In the case of the fluidics system being used in the 2690 module, it is really a solvent manager. The single most important thing to consider in choosing a fluidics module for GPC analysis is the flow precision. The calibration of the system is a plot of retention time (or volume) vs. the log of the molecular weight. Any minor flow fluctuation will result in a potentially large error in molecular weight. It is to your advantage to use the most precise fluid-handling device you can. This will improve precision traditional pumps that are still being used. In the case of the Waters new 515 fluid handling unit, the flow precision is better than 0.10%. With the solvent manager that is used with the 2690 module, the flow precision is remarkably under 0.075%! This translates to incredible precision of your molecular weight measurements often below 1% RSD. Be wary of pumps in the marketplace that list a specification of 0.3% (and worse) if you are putting a GPC system together. The 515 fluid handling device is ideal for isocratic (single solvent) GPC separations. It is a low-cost unit that provides exceptional flow precision for GPC analysis. You may put two 515s together for gradient (two-solvent) capability, if HPLC analysis of polymer additives, epoxy or phenolic resins is desired. A step up in performance is the solvent manager and a sample manager (autosampler) in one convenient, integrated package. The solvent manager may be used as an isocratic fluid-handling device, or up to a four solvent gradient device. The system represents a major improvement over the traditional gear-driven pump. The two heads are independently controlled by a microprocessor, each with its own transducer. There is even a compression step that eliminates any air bubbles, which would cause problems with a refractive index detector. The result is extremely smooth, precise flow, resulting in molecular weight average precisions regularly less than 1%. The 2690 solvent manager also provides exceptional gradient and flow program performance. Many polymer characterization chemists are realizing how important it is to analyze the additive package, in addition to determining the molecular weight distribution of the polymer, in many cases, the additive package has as much to do with the successful application of a finished product as the polymer used to fabricate the product. Any errors in the compounding (incorrect antioxidant, or incorrect level of plasticizer, for example) of the additives into the main formulation may result in unacceptable physical properties and performance. In order to successfully characterize the additive package, a reverse- phase gradient HPLC analysis is performed. In addition to polymer additives, epoxy and phenolic resins are regularly analyzed by both GPC (to examine the oligomer distribution) and gradient HPLC (to characterize the isomer and impurities). The 2690 module allows you to do both high performance GPC and gradient analysis with a single system. For analysis of polymers soluble only at high temperature Waters provides the Alliance GPC2000 and GPCV2000 integrated systems (pump, injector, columns and detectors all integrated in one housing) The systems are also ideal for use at room temperature. The fluid ics system used in the Alliance GPC2000 and GPCV2000 provides flow rate precision of better than 0.075%. It can be heated up to 50 C to reduce viscosity of certain solvents.

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1.5. Column Selection Once we have found a suitable solvent to dissolve the polymer, and prepared our narrow standards and samples at the correct concentration, we are ready to start our analysis. We have chosen the correct column set to do the analysis (or have we?), so we are ready to go. However, let's review the procedure to choose the correct column set. Many people like to use what used to be called "Linear4' columns, which are now called "Extended Range" or "Mixed Bed" columns. These columns are blends of different pore sizes, the idea being to cover a broader molecular weight range than a column with a single pore size. If the blending of pores is done carefully enough, the column calibration curve may indeed be linear. The drawback in using these mixed bed columns is that you will have less resolution over a finite molecular range than if you used individual pore size columns. For example, if you were running a series of epoxy or phenolic resins, say with a molecular weight range of a few hundred to five thousand, what column set would you use? The first consideration is to have enough pore volume in the column set to obtain the correct separation, i.e. the correct distribution profile of the polymer. One column is certainly not enough, and two may still not be enough. One should use at least three columns in series to guarantee that we have enough pore volume to ensure a successful separation. Now, what columns wilt we use to analyze our epoxy or phenolic resin? Should we use a "mixed bed" column set, with a mixture of pore sizes? Or should we use a series of individual pore size columns to really target the molecular weight range of interest? The following table lists the molecular weight range of separation for individual pore size columns of styrene/divinylbenzene packings, based on polystyrene chain length exclusion limits (in Angstroms): Table 1: Column Sets Molecular Weight Range 100 - 1000 250 2500 1000 18000 5000 40000 10000 200000 50000 1000000 200000 - >5000000 500000 - ~20000000 ~1000 10000000 ~100 - 100000 Pore Size (Angstroms) 50 A 100 A 500 A 103 A 104 A 105 A 108 A 107 A Mixed Bed High Mixed Bed - Low

For the above case of the epoxy and phenolic analysis, a column set of 50A, 100A, 500A would be ideal. With this column set, we have maximized our molecular weight separation power by using a column set specific for that range. Another example would be polycarbonate, with a weight average molecular weight of 28,000, and a number average of-12,000. A four column set of a 100A, 500A, 103A, and 104A would be ideal for this analysis.

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Again, we have targeted Individual pore size columns at the specific molecular weight range of our polycarbonate sample. On the other extreme of molecular weight, suppose we had a sample of UHMWPE (ultra high molecular weight polyethylene)? Should we choose a series of mixed bed columns, or can we target our column set to the specific molecular weight range of the polyethylene? Lets assume that the sample has a weight average molecular weight of 4,000,000, and a number average of 300,000. Z Average for this polymer may be >10,000,000. What column set should we use? If we look at our table, a set of 10, 106, and a 107 may be the best way to go. Unfortunately, we do not always have the luxury of knowing what the real molecular weight is for our samples. Many times we are analyzing new samples, or competitive materials, and we have no idea what the molecular weight range is. In these cases, it is a good idea to put three mixed bed columns together, or maybe two mixed beds with a single 500A column. The reason we may want to consider putting a low pore size column like a 500A would be to provide some pore volume at the low molecular weight end, where the low molecular weight tail of the sample may be unresolved from the "impurity" peaks we always see at the end of the chromatogram. So, two mid-to-high molecular weight range columns in series with a single 500A column would make a very good "scouting" column set, where we could get a good linear calibration range over several orders of magnitude in molecular weight This column set would be ideal where we have very little idea as to what the molecular weight of the sample is, or if the molecular weight range of the polymer is very broad, (i.e. the sample has a very broad polydispersity). The column selection table shown above is for organic soluble polymers and styrene/divinylbenzene packings. The table below shows the effective molecular weight range of separation for water soluble polymers, based on polyethylene oxide exclusion limits and the methacrylate gel columns used for aqueous GPC: Table 2: Effective molecular weight range of separation for water soluble polymers Molecular Weight Range 50 - 1500 500 10000 1000 30000 5000 400000 50000 4000000 200000 - ~10000000 2000 - ~10000000 Pore Size (Angstroms) 100 A 500 A 103 A 104 A 105 A 108 A Mixed Bed

Just one more word about columns. If you looked through the GPC solvent guide, you noticed that a typical operating range of temperatures were shown. In GPC analysis, we almost always heat the columns to some elevated temperature as shown in the solvent guide, (even for room temperature applications). The purpose of heating the columns is not for dissolution purposes, but to increase the resolution of the separation, enhance the permeation process, and in some cases, to decrease the viscosity of the solvent (DMF, for example), and reduce backpressure across the column bank.

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1.6. Detector Options The most widely used detector today for GPC analysis is the differential refractometer. It is a concentration sensitive detector that simply measures the difference in refractive index (dRI) between the eluent in the reference side, and the sample + eluent in the sample side. It is a "universal" detector (unlike an UV detector, for example) in that you will get a response for any polymer that has a significant difference in refractive index as compared to the eluent In some cases, the d Ri for the sample and eluent (silicones and THF, for example) is very small, resulting in a poor signal. In that case, we need to find another eluent that will dissolve the polymer and provide a significant d RI. The Waters 2410 refractometer (and previous Model 410) have been the industry standard for the last 11 years. More 410 Rl's are being used for GPC today than all other RI detectors combined. Another detector that is used often for GPC is the UV detector. Obviously, we need to have some chromophore present that will absorb in the UV to get a signal. The UV detector is excellent for styrenic type polymers, (polystyrene, styrene/Isoprene, stynene/butadiene, ABS, etc.), epoxies, phenolics, polycarbonates, polyurethanes, and aromatic polyesters, for example. If gradient analysis is being performed, (solvent composition being changed throughout the run) the UV detector must be used, since the RI detector would continue to drift ps the eluent composition changes. Waters 2487 UV detector provides excellent sensitivity, linearity, and overall outstanding performance for GPC/HPLC analysis of UV absorbing polymers and additives. We can also use a photodiode array (PDA) detector, which is a step up from the UV, and is a powerful, information-rich detector. An array of photodiodes is used in this detector, where we can look at a wide variety of wavelengths instantaneously. For example, we could set the PDA to look at a wavelength range from 190 to 800 nanometers (nm), instead of looking at just one or two wavelengths as for most UV detectors. Now we can look at the actual UV spectra for the polymer sample (or additives). This allows us to determine something about the chemical composition distribution. We can determine if an SBR (styrene/butadiene rubber) is a block or random copolymer, for example. We can create spectral libraries, which we can compare our unknown samples with. This can be done for polymers or with polymer additives. We can now try to identify which additives are present in compounded, finished materials. The PDA can be used to help deformulate competitive compounds as well. As polymer characterization chemists strive to learn as much as they can about their samples, other detection options are considered. As we move into the world of "advanced" detection for GPC analysis, we begin to consider the molecular weight sensitive detectors such as viscometry and light scattering. The viscometer detector will be discussed in some detail in the calibration section that follows. Essentially, putting a viscometer detector in line with the refractometer, (as with the Alliance GPC/GPCV2000 systems), provides a way to obtain not only the intrinsic viscosity [n] of the polymer, but also the "absolute" molecular weight and estimation of long chain branching. The RI detector is our concentration detector, (C), and the viscometer provides us with [n](C). Using the two signals in tandem will provide us with the intrinsic viscosity at each slice across the elution profile of the polymer. We can then use Benoifs Universal Calibration concepts discussed in the next section to obtain the absolute molecular weight of the polymer sample. The light scattering detector, coupled with the refractometer, is another powerful mode of advanced detection for GPC analysis. Essentially, a laser beam is focused into a ceil (on-line

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in this case) that contains the sample solution. The incident beam will be scattered by the polymer particles that are in solution. The intensity of scattered light is proportional to the size of the scattering particles. We can look at the intensity of scattered light at various angles and determine the radius of the sample, and subsequently, the weight average molecular weight, Mw, very accurately. The light scattering detector gives us (M)C across the elution profile, slice by slice, for the polymer. An extrapolation is done to zero angle to determine the weight average molecular weight. In both the cases of the viscometer and light scattering detector in tandem with the Rl, we obtain a lot of very useful information. Using a triple detector approach provides very meaningful data, as long as the user is able to interpret it all. For a more detailed discussion on multi- detector data reduction, please refer to our reference section. There are other techniques for advanced detection of polymers and additives, such as Mass Spec, but the major detectors used today for GPC analysis are the Rl, UV/PDA, Viscometer and Light Scattering. 1.6. Data Handling 1.6.1. Calibration of the GPC System In order to assign a molecular weight to each retention time slice for the eiuted polymer, we must calibrate our system, or more specifically, the column set There are several ways to do this, but the easiest is to use a relative calibration based on a set of weil-characterized polymer standards with as narrow a molecular weight distribution as possible. Ideally, we would like to use a set of standards that are monodisperse, i.e., a single molecular weight, with the weight and number average ratio (dispersity) being equal to one, (Mw / Mn = 1). The closest we can come to achieving this is to use polymer standards that are polymerized specifically for this purpose, such as the antonically polymerized polystyrene narrow standards Standards cover a very broad molecular weight range, from monomer to molecular weights >10,000,000, with a dispersity of <1.10. For a calibration standard to be really considered narrow, and acceptable for use in GPC calibration, the dispersity should indeed be <1.10. There are also ways to do a broad standard calibration, and Benoit's Universal Calibration procedure (with or without an online viscometer) may also be used. We will discuss each of these in some detail: 1.6.2. Relative, Narrow, Standard Calibration We call the conventional narrow standard calibration technique a relative calibration because the molecular weight averages obtained are relative to the calibrant For example, if one were running polyethylene as a sample, and calibrated the column set with polystyrene narrow standards, the molecular weights obtained after integration would be based on polystyrene, and incorrect for polyethylene. This is fine for many people, however, who are simply comparing molecular weights obtained for an unknown against a set of "acceptable" values. Whether these molecular weight values are really "absolute" for their polymer of interest is unimportant; just as long as these values obtained are In the acceptable range There are a few other narrow standards available for organic GPC, such as polymethylmethacrylates), polyisoprenes, polybutadienes, poly(THF), but certainly polystyrene is the major narrow standard used for organic GPC analysis. In the case of

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aqueous GPC, poly(ethylene oxides) are the most widely used, along with poly(ethy!ene glycols) for low molecular weight, and the pullulans, which are polysaccharides based on triose structures. After running the series of narrow standards, a polynomial fit is then performed, (usually third or fifth order), and the resulting log M vs. retention time (or volume) calibration curve is plotted. 1.6.3. Broad Standard Calibration One can also calibrate the GPC column set using a broad standard that is the same polymer being run as the unknown. Broad standard can be purchased from a variety of different vendors, and the standard should be well characterized, i.e. the number, weight, Z and possibly viscosity average molecular weights have been determined by alternative methods, (membrane osmometry, light scattering, ultracentrifugation, for example). An alternative would be to use an actual "sample" of material (that is present in a significant quantity), where the molecular weight averages have been determined by these other techniques. The advantage to this is being able to use a polymer that has the same structure as the unknown samples being analyzed day in and day out. The molecular weight averages that are known are entered into the software, and the broad standard is chromatographed in the usual manner, under the same conditions that the unknowns will be chromatographed. The software does a Simplex search routine, fitting the chromatographed broad standard shape to the given molecular weight averages. The resulting calibration curve will consist of the data points for each average. If only a number and weight average are provided, the resulting calibration curve will consist of these two points plus the peak molecular weight, or a three point calibration curve. This broad standard is based on work done by Hamlelecin 1969. It is recommended that two broad standards of different molecular weights be used, to increase the molecular weight range of the calibration curve. Even with using two broad standards with two known molecular weight averages, only a six-point calibration curve is obtained, (using the peak molecular weight values from the result of the search routine). However, for the QC lab running the same polymer every day, in the same molecular weight range as the broad standards, this calibration works very nicely, and provides absolute molecular weight weights 1.6.4. Universal Calibration The concept of Universal Calibration was introduced by Benoit et al. in 1967. Instead of plotting the log molecular weight of a series of narrow standards vs. retention, the log of the product of the intrinsic viscosity [n] and molecular weight M is plotted vs. retention. The [n]M product is related to the hydrodynamic volume. Benoit found that plotting a series of hydrodynamic volume values for a variety of narrow standards resulted in a singular calibration curve. In other words, all of the points fit the same curve. Once this "Universal" calibration has been established, any random coil polymer can be run in the appropriate solvent, and the molecular weight determined based on the Universal curve. Benoit used a glass capillary viscometer to measure the viscosities of the narrow standards and samples. After establishing the Universal curve, we can also plot the log of the intrinsic viscosity vs. the log of the molecular weight for the narrow standards. This plot is called the viscosity law plot, or, the Mark-Houwink plot. The slope of this plot is alpha, (sometimes called a), and the intercept is called log K. The resulting equation, known as the Mark-Houwink equation, is:

[n] = K M a
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Figure 6: Universal Calibration curve and viscosity law plot Above, a typical Universal Calibration curve and viscosity law plot for a series of polystyrene standards. The Polymer Handbook contains many K and alpha values for a variety of different polymer/solvent combinations. One can input these empirical constants into many of the commercial GPC software packages available today, and obtain "absolute or accurate molecular weights for many polymers. One must be sure that the values in the handbook are accurate for the polymer to be analyzed, or errors will occur. Today, we can use an on-line viscometer detector, along with the differential refractive index (dRI) detector, to directly obtain the molecular weight of each slice. The dRI is the concentration (C) detector, and the viscometer detector gives us the product of intrinsic viscosity and concentration ([n]C). Dividing the viscometer signal by the dRI signal gives us the intrinsic viscosity [n <] of each slice across the polymer peak. We now know both the intrinsic viscosity and, of course, the retention time (or volume) of each slice, so we can go back to the Universal Calibration curve and obtain the molecular weight of each slice, Mi. This Universal Calibration concept has wide applicability, especially for random coil type polymers, which represents the majority of polymers being analyzed today. Other polymer conformations, such as rods, spheres, or globular shaped (such as proteins) may not behave the Universal concepts. There can be no interaction of the polymer and the eluent or column packing material for Universal Calibration to work. Another advantage to using Universal Calibration and on-line viscometry/dRI detection is the ability to determine how a polymer branched is, relative to a known linear polymer standard. This technique is quite sensitive to long chain branching (as opposed to short chain branching), and is important to help predict how a certain polymer will process, or what the final physical properties will be, in comparison to the linear counterpart. As an example, one can run a linear polyethylene broad polymer, (such as "NBS 1475", or any other known linear polyethylene), with the resulting Mark-Houwink values being determined from the experiment The resulting Mark-Houwink plot (or viscosity law plot) will be linear, with a constant slope, (alpha will be constant across the molecular weight distribution). The K and alpha values can then be input into the software, and any subsequent unknown polyethylene can be analyzed, with the viscosity law plot being compared to that of the known linear polyethylene. If the unknown exhibits any long chain branching, the viscosity/molecular weight relationship is not linear; i.e. the viscosity will not increase linearly with molecular weight The greater this deviation from linearity, the greater

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the level of long chain branching. An accurate alpha can be obtained for a branched polymer only at low molecular weights, where there is no long chain branching, and the slope is constant Once the polymer is at a molecular weight where there is long chain branching, alpha is continuously changing, (may even approach zero), and becomes meaningless. A simple ratio of the viscosity law plot of the branched polymer to the linear polymer gives us the branching index, (g'), where: g' = [n]br/[n]lin One can do further calculations to determine the branching frequency, what type of branch is present, etc. It is obvious that adding a viscometer detector on-line with a refractive index detector can provide much more information about your polymer, specifically: "Absolute" or accurate molecular weights for your polymer via Universal Calibration Calculation of the intrinsic viscosity of your polymer, Determination of branching

1.7. Solvent Selection Tetrahydrofuran, (THF), for example, is the most common organic solvent used at room temperature. THF is great for PVC, polycarbonate, polystyrene, poly(methyl methacrylate),(and other acrylic compounds), and many others. Toluene works very nicely for elastomers, such as polybutadiene, polylsoprene, SBR, etc. Hexafluoroisopropanol (HFIP) will dissolve most nylons, as well as PET (poty(ethyteneterephthalate)and PBT (poly(butyleneterephthalate)) at room temperature, polyolefins are typically run at high temperature (-135 -145 C) in 1,2,4 trichlorobenzene. For more information on solvents, see the tables below: Table 3: Solvent Selection Guide for Room Temp. Organic Soluble Polymers Polymer Solvent Temp.

Acrylonitrile/Methylmethacrylate Cellulose Acetate Cellulose Acetate/Butyrate Cellulose Acetate/Propionate Cellulose Triacetate Diallyl Phthalate Ethyl Cellulose Epoxy Resins Phenolic Resins Polyglycolic Acid

THF THF THF THF THF THF THF THF THF THF

40 40 40 40 40 40 40 40 40 40

Polyester Alkyd Resins

THF

40

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Table 3 (continued): Solvent Selection Guide for Room Temp. Organic Soluble Polymers Polymethylmethacrylate and other Acrylics and Acrylates Polypropyleneglycol Polystyrene Polystyrene/Acrylonitrile, (SAN) (Low ACN) THF THF THF THF THF 40 40 40 40 40

Polysulfone Polyurethane (Some) Polyvinylacetate Polyvinylbutyral Polyvinylchloride Polyvinylchloride/Acetate Polyvinylidenechloride Polyvinyformal Thermosetting Polyesters Rosin Acids

THF THF THF THF THF THF THF THF THF THF

40 40 40 40 40 40 40 40 40 40

Polybutadiene Polychloroprene (Neoprene) Polydimethylsiloxane (Silicone Oils and Rubber) Polyisobutytene (Butyl Rubber) Polyisoprene, Natural Rubber Chlorinated Rubber Styrerre/butadiene Rubber (SBR) Styrene/isoprene (SI)

Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene

75 75 75 75 75 75 75 75

Acrylonitrile/Butadiene Styrene (ABS) Acrylic/Styrene/Acrylonitrile (ASA) Acrylic/Butadiene/Acfylonitriie (ABA) ABS/Polycarbonate Carboxy methylcellulose Polyacrylonitrile

DMF* with 0.05M LIBr DMF* with 0.05M LIBr DMF* with 0.05M LIBr DMF* with 0.05M LIBr DMF* with 0.05M LIBr DMF* with 0.05M LIBr

85 85 85 85 85 85

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Table 3 (continued): Solvent Selection Guide for Room Temp. Organic Soluble Polymers

Polybutadiene/Acrylonitrile Polystyrene/Acrylonitrile (SAN)

DMF* with 0.05M LIBr DMF* with 0.05M LIBr

85 85

Polyurethane (Most) *!n most cases, DMAC may be substituted for DMF, again with the 0.05M LiBr

DMF* with 0.05M LIBr

85

Melamine/Formaldehyde

HFIP with 0.05M NATFA (Trifluoroacetic acid, sodium salt)

35

Nylon/ Polyamides (all nylons plus most other Polyamides)

HFIP with 0.05M NATFA

35

Polyethylene terephthalate (PET) Polybutylene terephthalate (PBT)

HFIP with 0.05M NATFA HFIP with 0.05M NATFA

35 35

Table 4: Solvent Selection Guide for Elevated Temperature Organic Soluble Polymers Polymer Chorinated Polyethylene Ethylene/Propylene Diene Monomer (EPDM) Solvent TCB* TCB* Column/Injector Temp. 135-145 135

Polyethylene Polyethylene/Ethyl acrylate PolyethyleneA/inyl acetate (EVA)

TCB* TCB* TCB*

135-145 135-145 135

Polyethylene/Methacrylic acid Polyphenylene Oxide Poly-4-Methyl pentene Polypropylene Ultra High Molecular Weight PE (UHMWPE) *In most cases, ODCB may be substituted for TCB

TCB* TCB* TCB* TCB* TCB*

135-145 135 135 135-145 145

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Table 4 (continued): Solvent Selection Guide for Elevated Temperature Organic Soluble Polymers

Polyamide-imide Polyether-imide Polyether Sulfone Polyimide Polyvinylidene Fluoride Polyacetals Polyetherketone (PEK) Polyetheretherketone (PEEK) Starch Cellulose

NMP with 0.05M LiBr NMP with 0.05M LiBr NMP with 0.05M LiBr NMP with 0.05M LiBr NMP with 0.05M LiBr NMP with 0.05M LiBr 1:1 TCB/Phenol 1:1 TCB/Phenol DMSO DMF with 6M LiCI

100 100 100 100 100 145 145 145 50-100 85

1.7.1. Discussion of Organic Solvents for GPC Here is a little more detail on the organic solvents mentioned above: Table 5: Organic Solvent Selection Guide Solvent/Full Name Boiling Point (C) Comments

THF - Tetrahydrofuran Toluene DMF - N,N Dimethylformamide DMAC - N,N Dimethylacetamide HFIP -1,1,1,3,3,3-Hexafiuoro-2-propanol TCB -1,2,4 Trichlorobenzene

66 111 153 166 58 213

Highly Flammable, Peroxides may form Highly Flammable Strong Irritant/Toxic (0.05M UBr added to minimize polar effects) Same as DMF Very Expensive, Corrosive Vapors harmful Toxic; 1.0 gram/4 liters of an antioxidant should be added Somewhat toxic, also needs antioxidant

ODCB - orthodichlorobenzene

173

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Table 5 (continued): Organic Solvent Selection Guide NMP - n-methyl pyrrolldone CHCI3 - Chloroform CH2Cl2 - Methylene Chloride DMSO - Dimethyl sulfoxide 202 61 40 189 Strong Irritant/Toxic (0.05M UBr added as for DMF) Known carcinogen Toxic, possible carcinogen Non-toxic

There are several other solvents and solvent blends that are occasionally used, such as decalin, (for polyolefins), xylene, (for amorphous polypropylene), m-cresol and trifluoroethanol (for certain nylons and polyesters), etc. We have attempted to show the solvents used most often as eluents in GPC analysis. Table 6 : Solvent Selection Guide for Water Soluble Polymers with Methacrylate Gel Column Packings Polymer Class Eluent

Polyethylene oxide Polyethylene glycol Polysaccharides, Pullulans Dextrans Celluloses (water soluble) Polyvinyl alcohol Polyacrylamide Polyvinyl pyrrolidone Polyacrylic acid Antonie Polyalginic acid/alginates Hyaluronic acid Carrageenan Polystyrene sulfonate Lignin sulfonate DEAE Dextran Polyvinylamine Polyepiamine

Neutral Neutral Neutral Neutral Neutral Neutral Neutral Neutral hydrophobic Anionic Anionic Anionic Anionic Anionic hydrophobic Anionic hydrophobic Cationic Cationic | Cationic

0.10M NaNO3 0.10M NaNO3 0.10M NaNO3 0.10M NaNO3 0.10M NaNO3 0.10M NaNO3 0.10M NaNO3 80:20 0.10M NaNO3/Acetonitrile 0.10M NaN03 0.10M NaN03 0.10M NaN03 0.10M NaN03 80:20 0.10M NaNO3/Acetonitrile 80:20 0.10M NaNO3/Acetonitrile 0.80M NaNO3 0.80M NaNO3 0.10% TEA

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Chapter 10 Gel Permeation Chromatography

Table 6 (continued): Solvent Selection Guide for Water Soluble Polymers with Methacrylate Gel Column Packings
n

-Acetylglucosamine

Cationic

0.10M TEA/1 % Acetic Acid

Polyethyleneimine

Cationic, hydrophobic 0.50M Sodium Acetate/ 0.50M Acetic Acid

Poly(n-methyl-2-vinyi pyridinium) : Cationic, hydrophobic 0.50M Sodium Acetate/0.5M Acetic Acid salt Lysozyme Chitosan Polylysine Peptides Collagen/Gelatin Cationic, hydrophobic 0.50M Acetic Acid/0.30M Sodium sulfate Cationic, hydrophobic 0.50M Acetic Acid/0.30M Sodium sulfate Cationic, hydrophobic 5% Ammonium Biphosphate/ 3% Acetonitrile (pH = 4.0)

Cationic, hydrophobic 0.10% TFA/ 40% Amphoteric 80:20 0.10M NaNO3/Acetonitrile

Note that in the many cases where sodium nitrate is shown, many workers have used acetate, sulfate, sodium chloride, eta We recommend sodium nitrate, which has shown to minimize ionic interferences very consistently for neutral and anionic compounds. The reason for these various eluents is because of the overall anionic charge of the packing material. The methacrylate based gel packing for aqueous GPC has an overall anionic charge, which can cause ion exclusion for anionic samples and ion adsorption for cationic samples if run in water alone. One should always filter the eluent under vacuum before use in the chromatographic system. With the organic solvents, a fluorocarbon filter is generally used. The filter pore membrane size is generally 0.45m (micron). For aqueous GPC (filtration of the water), an acetate type of membrane filter is used. If one is preparing to do a light scattering analysis, it may be a good idea to filter the eluent though a 0.20m filter Some organic solvents such as DMF are very viscous and do not wet the surface of the fluorocarbon filter very well. A good tip is to wet the filter surface initially with methanol, then quickly start the DMF filtration. You would then discard this small volume of methanol/DMF mixture, then start the DMF filtration before the filter dries out. 1.8. Concentration Once we have chosen the proper solvent for the analysis, the next step is to prepare the narrow standard and sample solutions. We need to be careful to use enough concentration to be able to get an acceptable signal-to-noise, but at no risk of overloading the column and risking concentration effects. The table below is a general "rule of thumb" to be used as a guide as to what concentration should be prepared. These concentrations are in percent, where 1.0 mg/mL is 0.10%. No correction is made for temperature, so everything is assumed to be prepared at room temperature. Remember that if viscometry or light scattering analysis is being performed, the exact mass injected needs to be determined. This will require density corrections if the analysis is being done at elevated temperature. These concentrations shown are to be used assuming a maximum of 100ul injection volume per column.

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Chapter 10 Gel Permeation Chromatography

Table 7: Molecular Weight Range Molecular Weight Range

MW> 1,000,000

Concentration Range (weight per volume) w/v 0.007 - 0.02% 0.02 - 0.07% 0.07 - 0.10% 0.10 - 0.13% 0.13 - 0.16% 0.16 - 0.20%

500K -1,000,000 100K -500K 50K -100K 10K - 50K <10K 1.9. Preparing the Sample

Now that we have successfully dissolved the standards and samples in our chosen solvent, and have installed our GPG columns, we are ready to start making injections. The next choice we have to make is whether or not we should filter the sample solution. In nearly all cases, we should fitter the sample solution prior to injection. Generally, as In the case of the solvent filtration discussed previously, we would choose a 0.45m membrane fluorocarbon filter. In some cases, where there is very fine particulate material (such as carbon black, titanium dioxide, silica, or other fillers), a 0.20m filter may be used. Obviously, when we start to use very fine filter sizes, polymer shear may become a concern. Filtering a high molecular weight polymer through a 0.20m filter would certainly cause some shear degradation One may have to choose not to filter the sample at all, and hope there is no pressure increase due to plugging of the system in-line filter or column frit. If the analysis is to be run at room temperature, the filtration is very easy. If the application is to be done at high temperature, then it Is best to let the system automatically filter the sample solution for you at the operation temperature, as done by the GPC2000 and GPCV2000 systems. Otherwise, you would be filtering the solutions in a hood with gloves and a heated syringe barrel. Now we can start making injections of the standards and samples. As mentioned previously, we will inject a maximum of 100ul per column, at the concentrations shown in the table. Our run time will be approximately 15 minutes per column at a flow rate of 1.0 ml/minute, so the analysis time for a three column set would be -45 minutes. Once the sample set has been run, it is time for the data handling system to process the results according to the integration method we designated and furnish a completed report This can be done automatically in a "Run and Report" mode, or we may choose to go in to each raw data file and manually integrate each sample.

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