gpc初级读本

gpc初级读本 GPC - Gel Permeation Chromatography GPC Introduction •Why is GPC important? •How GPC works •GPC systems Gel permeation chromatography (GPC) is one of the most powerful and versatile analytical techniques available for understanding and predicting polymer performance. It is the most convenient technique for characterizing the complete molecular weight distribution of a polymer. Waters commercially pioneered GPC in 1963. Since then, Waters has continued to develop and explore new GPC applications and improve the instrumentation that makes GPC so powerful. Why is GPC important? GPC can determine several important parameters. These include number average molecular weight, weight average molecular weight, Z weight average molecular weight, and the most fundamental characteristic of a polymer its molecular weight distribution. These values are important, since they affect many of the characteristic physical properties of a polymer. Subtle batch-to-batch differences in these measurable values can cause significant differences in the end-use properties of a polymer. Some of these properties include: Tensile strengthAdhesive strenth Elastomer relaxation time松弛时时Cure time固化时时 Brittleness脆性Elastic modules时性模量 Flex life时性Melt viscosity融融粘度 Impact strength冲时强度Hardness ToughnessSoftening temperature DrawabilityTear Strength Adhesive tackStress-crack resistance Coefficient of friction Materials characterization Understanding the make up of a polymer is particularly important due to the variety of resins available for the same purpose, the high cost of specialty resins or compounds, and the value added to the polymer during manufacturing. For example, the cost of a resin used in a printed circuit board is very low, but the cost of the finished board is very high. Poor quality resin can result in an unacceptable finished circuit board. Where a polymer's end-use application requires precision performance or endurance under harsh conditions, the need for polymer characterization is particularly acute. Because GPC fulfills these needs better than any other single technique, it has become an extremely valuable tool for materials characterization in the polymer industry. Telling good from bad 抗拉强度Two samples of the same polymer resin can have identical tensile strengths and melt viscosities, and yet differ markedly in their ability to be fabricated into usable, durable products. These differences can be attributed to subtle, yet significant variations in the molecular weight distributions of the two resin samples. Such differences, if undetected, can cause serious product defects. Though they are subtle, differences such as those shown in the molecular-weight distributions to the left, could cause marked variations in the performance of the polymer. In addition to providing the molecular weight distribution, GPC also separates a complex polymeric compound into its component parts - polymer, oligomer, monomer, and additives. How GPC works GPC separates molecules in solution by their "effective size in solution." To prepare a sample for GPC analysis the resin is first dissolved in an appropriate solvent.Inside the gel permeation chromatograph, the dissolved resin is injected into a continually flowing stream of solvent (mobile phase). The mobile phase flows through millions of 渗透highly porous, rigid particles (stationary phase) tightly packed together in a column. The pore sizes of these particles are controlled and available in a range of sizes.Cross sectional view of porous particle The width of the individual peaks reflects the distribution of the size of molecules for a given resin and its components. The distribution curve is also known as the molecular weight distribution (MWD) curve. Taken together the peaks reflect the MWD of a sample. The broader the MWD, the broader the peaks become and vice versa. The higher the average molecular weight, the further along the molecular weight axis the curve shifts and vice versa. You can see then how easily the MWD profiles of two resins can be compared. If the MWD profile of an incoming resin doesn't match that of the control resin (i.e. one that is known to process well) closely enough, the incoming resin can be modified or process conditions can be changed to make sure the resin processes properly. If the differences between the control resin and the incoming resin are too severe, the incoming resin can be returned to the supplier as unacceptable. The Size Separation Mechanism Molecules of various sizes elute from the column at different rates. The column retains low molecular weight material (small black dots) longer than the high molecular weight material (large black dots). The time it takes for a specific fraction to elute is called its "retention time". GPC Systems In designing instrumentation for GPC, a variety of requirements must be satisfied. Injectors are needed to introduce the polymer solution into the flowing system. Pumps deliver the sample and solvent through the columns and system. Detectors monitor and record the separation. Data acquisition accessories control the test automatically, record the results, and calculate the molecular weight averages. The gel permeation chromatograph contains a number of different components that work together to provide optimum system performance with minimum effort. Schematic of a basic gel permeation chromatograph. Schematic of a basic gel permeation chromatograph This diagram illustrates how the sample is injected into the mobile phase and the path the sample takes to the detector. 1. Pump Pumps the polymer in solution through the system. Different polymers produce solutions of different viscosities. To compare data from one analysis to the next, the pump must deliver the same flow rates independent of viscosity differences. In addition, some detectors are very sensitive to the solvent flow rate precision. Such constant flow must be a critical feature of the instrument.2. Injector Introduces the polymer solution into the mobile phase. The injector must be capable of small volume injections (for molecular weight determinations) and large volume injections (if fraction collecting is desirable). The injector should not disturb the continuous mobile phase flow. It should also be capable of automatic multiple sample injection when the sample volume is large. 3. Column Set Efficiently separates sample components from one another. High efficiency columns give maximum separating capability and rapid analyses. Every column must provide reproducible information over extended periods for both analytical and fraction collecting purposes. 4. Detector 洗脱Monitors the separation and responds to components as they elute from the column. 无时的Detectors must be nondestructiveto eluting components if they are to be collected for further analysis. In addition, the detectors must be sensitive and have a wide linear range in order to respond to both trace amounts and large quantities of material if necessary. 折射差示折光Since all compounds refract light, the differential refractometer (RI) is referred to as a "universal" detector. As a result it is the most widely used detector to monitor molecular weight distribution. The refractive index of polymers is constant above approximately 1000 MW. Therefore, the detector response is directly proportional to concentration. Beside information about molecular weight averages and distribution obtained with RI, the use of UV absorbance detectors may provide information about composition, while on-line 散射 light scatteringdetectors and viscometers provide information about polymer structure. 5. Automatic data processing equipment Automatically calculates, records, and report numerical values for Mz, Mw, Mv, Mn, and MWD. Data systems can also provide complete control of GPC systems so that large numbers of samples can be run unattended and raw data can be automatically processed. Today's GPC software offerings need to be able to provide special calculations for multi-detection 修正processing, band broadening correction, special calibration routines and polymer branching determination, just to name a few. GPC/SEC Separations - Theory and System Considerations •Introduction to size separation •Monomers, oligomers, polymers and molecular weight distributions •Molecular weight averages, Mn, Mw, Mz, Mz+1 Configuring a GPC System: •Solvent management •Sample management •Column selection •Detector options •Data handling Calibration of the GPC System •Relative, Narrow Standard Calibration •Broad Standard Calibration •Universal Calibration Performing a GPC Analysis •Solvent Selection •Solvent Selection Guide for Room Temp. Organic Soluble Polymers 高温•Solvent Selection Guide for Elevated Temperature Organic Soluble Polymers •Discussion of Organic Solvents for GPC 甲基丙时酸时 •Solvent Selection Guide for Water Soluble Polymers with Methacrylate 填充物Gel Column Packings •Concentration •Preparing the Sample GPC/SEC Separations - Theory and System Considerations Introduction to size separation: Gel Permeation Chromatography, (GPC), also known as Size Exclusion Chromatography, (SEC) is really the easiest to understand of all 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 ;,TCBat 140? C. Once the sample has been suitably dissolved, it is introduced via an injection mechanism 时时onto a set of columns which act as a molecular filtration system. The columns are 交时二乙时基苯packed with a crosslinked gel, (styrene/divinylbenzene copolymer for organic applications, for example), 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. 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 decades ago was on chewing 口香糖gum. Chewing gum is really synthetic rubber, plus additives such as flavors, stabilizers, etc. Here is a representation of the original GPC chromatogram,separated on several columns of various pore sizes connected in series. The polymer (rubber in this case) elutes first because it is the largest molecule, followed by the "additives" in decreasing order of size. 增塑时This could just as well be a chromatogram of PVC with a mixture of plasticizers, 抗时氧antioxidants and UV stabilizers. Monomers, Oligomers, Polymers and Molecular Weight Distributions Monomers have a single molecular weight, and are said to be monodisperse. Examples would be ethylene, styrene, vinyl chloride, etc. After monomers, we have dimers, trimers, tetramers, pentamers, etc., which are called oligomers. As we get to higher molecular weights, the group is called polymers. Polymers have a 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 聚时polymer, such as a 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, 聚时时时力 (such as for polyolefins). Controlling the kineticsof 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. Here we show an an overlay of two molecular weight distributions of a polymer (in this case polystyrene): Molecular Weight Averages, M, M, M, M.nwzz+1 Once we obtain the molecular weight distribution of the polymer sample, we need a way to quantify it. We assign molecular weight averages across this distribution by simply doing statistics. There is a height, (H, also represented as concentration, C) a retention time, ii and a molecular weight, (M), attributed to each slice. The molecular weight is obtained i from a calibration curve (see next section). We next perform a summation to obtain the various molecular weight averages that describe the polymer molecular weight distribution. The PD shown is the ratio of the weight average and number average molecular weights and is called the polydispersity, or sometimes simply, the dispersity of the polymer. This summation is just a simple way to obtain these four molecular weight statistical moments and describe the molecular weight distribution. There are other techniques to obtain these molecular weight averages: •Number average, Mn, may be obtained by membrane osmometry, or end group 滴定analysis, (titration, NMR, etc.) 光散射•Weight Average, Mw, may be obtained by light scattering •Z Average, Mz, and Z + 1 Average, Mz + 1, may be obtained by ultracentrifugation超速心法离 Once we have calibrated our GPC system, we can obtain all of these averages with a single injection. 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. The system (shown above) consists of a pump, an injector of some sort, either manual or automated, the column set, the detector(s), and some sort of data handling device. In 脱气装置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 viscosites. We will now discuss the system in more detail. 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 Alliance system, 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 保留时时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 the 精度 precisionof your molecular weight average measurements immensely over some of the low flow precision traditional pumps that are still being used. With the solvent manager that is used with the Alliance system, the flow precision is remarkably under 0.075% without any flow rate correction! Some pumps in the marketplace claim similar flow precision but with software flow rate correction. Be wary of pumps in the marketplace that list a specification of 0.3% (and worse) if you are putting a GPC system together. 梯度 The Alliance system solvent manager also provides exceptional gradientand 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 Alliance system allows you to do both high performance GPC and gradient analysis with a single system. Sample Management The next step in configuring our system is to decide how we wish to introduce the standards and samples for the separation. The least expensive way to do this is with a manual injector. You manually fill a loop (known volume) and open a valve to let the solution flow onto the column set with the eluent stream. This is fine if you happen to be running just a few samples now and then. However, if you are running several samples each day, it may be better to consider an autosampler. The autosampler used most today for room temperature GPC analysis is the 2707 Autosampler. This all-electric autosampler will allow you to set up a full tray of samples to run unattended for as long as the analyses take. The injection volume accuracy and 非常卓越的reproducibility is unsurpassed, which is critical for molecular weight sensitive detector mass measurements, (such as with a viscometer or light scattering detector), where the exact mass loading must be known. Another option for an autosampler is the Alliance system. There are five different carousels, each holding up to 24 samples (120 total sample capacity). 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 "Linear" columns, which are also called 混合子交时器离"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 气孔 column with a single pore size. If the blending of poresis 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 will 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): Molecular weight Pore size range 100 - 1000 50 A 250 - 2500 100 A 1,000 - 18,000 500 A 3 5,000 - 40,000 10A 4 10,000 - 200,000 10A 5 50,000 - 1,000,000 10A 6 200,000 - > 5,000,000 10A 7 500,000 - ~20,000,000 10A ~1,000 - 10,000,000 Mixed Bed - High ~100 - 100,000 Mixed Bed - Low 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 分辨率not for dissolution purposes, but to increase the resolution of the separation, 渗透enhance the permeation process, and in some cases, to decrease the viscosity of 反时 the solvent (DMF, for example), and reduce backpressureacross the column bank. 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 洗液脱硅时to the eluent. In some cases, the d RI for the sample and eluent (siliconesand 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 2414 refractometer (and previous Model 2410 and 410) have been the industry standard for many years. Another detector that is used often for GPC is the UV detector. Obviously, we need to have 时色时some chromophorepresent that will absorb in the UV to get a signal. The UV detector is excellent for styrenic type polymers, (polystyrene, styrene/isoprene, styrene/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 as the eluent composition changes. Waters 2489 UV Detector provides excellent sensitivity, linearity, and overall outstanding performance for GPC/HPLC analysis of UV absorbing polymers and additives. 光时二管时列极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 provides a way to obtain not only the intrinsic 特性粘度viscosity [h] of the polymer, but also the "absolute" molecular weight and estimation of long chain branching. The RI detector is our concentration detector, (C), and 时力 the viscometer provides us with [h](C). Using the two signals in tandemwill provide us with the intrinsic viscosity at each slice across the elution profile of the polymer. We can then use Benoit's 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 cell (on- line in this case) that contains the sample solution. The incident beam will be scattered by the polymer particles that are in solution. Depending on the design of light scattering detector (small angle or multi-angle), the weight average molecular weight, Mw, can be measured accurately with or without the radius of gyration result of polymer in solution. 串时In both cases of the viscometer and the light scattering detector in tandem with the RI, 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 common detectors used today for GPC analysis are the RI, UV/PDA, Viscometer and Light Scattering. Data Handling Once we have configured the main hardware portion of our system, we must now consider the software options for control of this system and processing the data. With today's very powerful computers, calibration and molecular weight distribution calculations may be done in seconds. Empower Software may be used for both conventional GPC (RI only) data reduction, as well as for RI/Viscometry detection. Empower 2 supports many calibration procedures, including Relative Calibration, Cumulative Matching and Hamielec Broad Standard Calibration, and Universal Calibration. Zero through fifth order curve fits, along with a unique Bounded Calibration, and a splines fit, are all supported. See the Empower link for more detailed information. Calibration of the GPC System In order to assign a molecular weight to each retention time slice for the eluted 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 well-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, (M/M = 1). wn The closest we can come to achieving this is to use polymer standards that are polymerized specifically for this purpose, such as the anionically 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 on-line viscometer) may also be used. We will discuss each of these in some detail: 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 ～;,聚戊二时异poly(methylmethacrylates)PMMA, polyisoprenes, polybutadienes 聚丁二时, poly(THF), but certainly polystyrene is the major narrow standard used for 水时乙时氧organic GPC analysis. In the case of aqueous GPC, poly(ethylene oxides) are 乙时乙二醇the most widely used, along with poly(ethylene glycols) for low molecular 支时淀粉丙糖 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. 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 供时商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 膜渗透时超alternative methods, (membrane osmometry, light scattering, ultracentrifugation 速心离, 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 Hamielec in 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 时量控制 molecular weight values from the result of the search routine). However, for the QClab 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. There are a few other narrow standards available for organic GPC, such as poly(methylmethacrylates), polyisoprenes, polybutadienes, poly(THF), but certainly polystyrene is the major narrow standard used for organic GPC analysis. In the case of aqueous GPC, poly(ethylene oxides) are the most widely used, along with poly(ethylene 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. 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 [η] and molecular weight M is plotted vs. retention. 流力时体学体The [η]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 无时卷曲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 α), and the intercept is called log K. The resulting equation, known as the Mark-Houwink equation, is: a[η] = KM 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 ([η]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 i 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, M. i This Universal Calibration concept has wide applicability, especially for random coil type polymers, which represents the majority of polymers being analyzed today. Other polymer 棒状球体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 branched a polymer 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 polyethylenes 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 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' = [η ]/[η] One can do further calculations to determine the branching brlin 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 •Determinationof branching Performing a GPC Analysis The most important criteria in preparing to do a GPC analysis is finding a suitable solvent to dissolve the polymer. This sounds trivial enough, but remember that GPC is a separation technique based on the size of the polymer in solution. Polymer chains will open up to a certain relaxed conformation in solution, and the solvent chosen will determine what this size will be. Many polymers are soluble at room temperature in various solvents, but in some cases, (especially for highly crystalline polymers), high temperature is required for dissolution. Another important aspect for GPC sample preparation is the concentration chosen. If the mass loading of the sample onto the column set is too high, there may be concentration or viscosity effects, which will give rise to incorrect elution volumes. Another consideration is whether or not to filter the polymer solution. We will discuss some of these sample preparation considerations. Solvent Selection Guide for Room Temp. Aqueous Soluble Polymers Polymer Class Eluent Polyethylene oxide 聚时乙时氧Neutral0.10M NaNO3 Polyethylene glycol聚乙时氧Neutral0.10M NaNO3 Polysaccharides, Pullulans Neutral0.10M NaNO3 Dextrans 葡萄聚糖Neutral0.10M NaNO3 Celluloses 时时素(water soluble)Neutral0.10M NaNO3 Polyvinyl alcohol 聚乙时醇Neutral0.10M NaNO3 Polyacrylamide 聚丙时时胺Neutral0.10M NaNO3 Polyvinyl pyrrolidone Neutral hydrophobic80:20 0.10M NaNO/Acetonitrile乙时3 Polyacrylic acid Anionic时子离Anionic0.10M NaNO3 Polyalginic acid/alginates Anionic0.10M NaNO3 Hyaluronic acid Anionic0.10M NaNO3 Carrageenan Anionic0.10M NaNO3 Polystyrene sulfonate Anionic hydrophobic80:20 0.10M NaNO/Acetonitrile 3 Lignin sulfonate Anionic hydrophobic80:20 0.10M NaNO/Acetonitrile 3 DEAE Dextran Cationic0.80M NaNO3 Polyvinylamine Cationic0.80M NaNO3 Polyepiamine Cationic0.10% TEA n-Acetylglucosamine Cationic0.10M TEA/ 1% Acetic Acid Polyethyleneimine Cationic, hydrophobic0.50M Sodium Acetate/ 0.50M Acetic Acid Poly(n-methyl-2-vinyl pyridinium) I Cationic, hydrophobic0.50M Sodium Acetate乙酸时/0.5M Acetic Acid醋酸 salt Lysozyme Cationic, hydrophobic0.50M Acetic Acid/0.30M Sodium sulfate Chitosan Cationic, hydrophobic0.50M Acetic Acid/0.30M Sodium sulfate Polylysine Cationic, hydrophobic5% Ammonium Biphosphate/ 3% Acetonitrile (pH = 4.0) PeptidesCationic, hydrophobic0.10% TFA/ 40% Collagen/Gelatin Amphoteric80:20 0.10M NaNO/Acetonitrile3 Note that in many cases where sodium nitrate is shown, many workers have used acetate, sulfate, sodium chloride, etc. 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 0.45um (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 0.20um 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. 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 时时法时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 假定 everything is assumedto be prepared at room temperature. Remember that if viscometry or light scattering analysis is being performed, the exact mass injected needs 密度校正 to be determined. This will require density correctionsif the analysis is being done at elevated temperature. These concentrations shown are to be used assuming a maximum of 100ul injection volume per column. Molecular Weight Range Concentration Range (weight per volume) w/v MW > 1,000,000 0.007- 0.02% 500K - 1,000,0000.02 - 0.07% 100K - 500K0.07 - 0.10% 50K - 100K0.10 - 0.13% 10K - 50K0.13 - 0.16% <10K0.16 - 0.20% Preparing the Sample Now that we have successfully dissolved the standards and samples in our chosen solvent, and have installed our GPC 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 filter the sample solution prior to injection. Generally, as in the case of the solvent filtration discussed previously, we would choose a 0.45 um membrane fluorocarbon filter. In some cases, where there is very fine particulate material (such as carbon black, titanium dioxide, silica, or other fillers), a 0.20 um 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. 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/min so the analysis time for a three column set would be ~45 min. 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 in Empower Software, or we may choose to go in to each raw data file and manually integrate each sample. Columns and Column Selection: Which columns should I use and why? Column selection is critical to ensure that one obtains the correct molecular weight distribution of the polymer of interest. Initial factors to consider: Solubility Water soluble: ?•Ultrahydrogel column line Organic soluble: ?分辨率 •Styragel HR for high resolutionwork •StyragelHT for high temperature work •StyragelHMW for ultra high molecular weight polymers What solvent should I buy my columns packed in and why? HR, HT, and HMW columns are packed in either: •THF •Toluene •DMF Specialty columns packed in methanol specifically for analysis at room temperature with 六丙醇氟异HFIP (hexafluoroisopropanol) are available. If you are using a solvent other than these four for your application, there are a couple of rules of thumb to think about. If you are doing a "room temperature" application in a solvent such as chloroform or methylene chloride, convert over from THF. If you plan on doing high temperature work in TCB, ODCB时二时苯甲苯, for example, convert over from toluene at ~85 - 90? C. If you are going to use 极性二甲基乙时胺a solvent that is very polar, such as DMAC (Dimethylacetamide) or NMP (n- methylpyrollidone), convert over from DMF. I currently have columns in solvent "A" and wish to change to solvent "B"? Generally, one can switch directly from one solvent to another at 0.1 - 0.2 ml/minute (see your column care and use manual) if the two solvents are miscible. If the solvents are not 中性miscible, an intermediate solvent (which both solvents are miscible in) will have to be used. In which order should I place the columns and why? Generally, it does not matter what order the columns are placed in. The order will not affect the molecular weight distribution calculations of the eluting polymer. It is a good idea, however, to always place the 100 A or 50 A columns at the end of the set, as the styrene/divinylbenzene gel in these columns tend to be softer and less durable. What flow rate should I use in my GPC column? It is recommended not to exceed 1.0 mL/min for the 7.8 mm i.d. analytical columns. The 最佳 "optimum"resolution for these columns is approximately 0.70 to 0.80 mL/min. The optimum flow rate for the 4.6 mm i.d. narrow bore columns is 0.3 to 0.35 mL/min. See your care and use manual for more details. Should I gradually increase flow and temperature when starting up columns? 强制的 It is mandatoryto ramp up the flow rate for analytical GPC columns, particularly the HR series. Sudden increase in flow (and subsequently pressure) will certainly damage the columns. Temperature ramping is not as critical. Generally, we ramp the flowrate from 0.0 to 1.0 mL/min over a 60 second interval, and the temperature from ambient to 150 ?C (as and example) over several hours. How do I choose the pore size range of my columns? The range of pore sizes is chosen by determining the approximate molecular weight range of the sample of interest. If one knows that the molecular weight range is low for example, 3(such as an epoxy resin), than a column set of 10, 500, 100, and perhaps a 50 A column 3would be used. If a medium molecular weight PVC is the sample of interest, then a 10, 4510, and 10 set would be used. Choosing individual pore sizes targeted at the molecular weight range of the polymer provides the highest resolution. If the molecular weight range is not known, or is very broad, it is a good idea to use mixed bed, (i.e. "linear', or "extended range") columns which provide a mixture of pore sizes. What is resolution and how much do I need? In GPC analysis, resolution means range of molecular weight separated in an incremental volume of elution. We would like to maximize this whenever possible. The easiest way to maximize this is to add more columns (and therefore analysis time, unfortunately). Another way is to use smaller particle size, (~ 5u ) which will increase efficiency. The trade-off here is column durability and lifetime. In separations where oligomers, additives, multi-modal distributions, are present, resolution may be important. If the sample is a high density polyethylene with a broad distribution, resolution may not be as important. Waters manufactures columns in the high resolution range (HR series) which are 5 u, the HT series, ~10 u (good for high temperature work and multiple solvent changeovers), and the HMW series, which have 20 u particles. These are good for very high molecular weight samples where shearing is a problem and resolution is not as critical. Calibration: What is a "narrow" standard? What is a "broad" standard? •"Narrow" standards are those where the polydispersity is less than ~1.10. The polydispersity is defined as the ratio of the weight average molecular weight, (M) to the w number average molecular weight (M). n •"Broad" standards have polydispersities greater than 1.10 and are usually the same polymer as the sample to be analyzed. If I use "narrow" standards can I inject more than one standard at a time? In conventional GPC with RI detection it is certainly acceptable to inject a mixture of standards, as long as there is sufficient resolution among the eluted standards. We would suggest a maximum of three. With advanced detection such as viscometry, where the area under the curve for the standard needs to be known accurately, one standard at a time should be injected. What standard(s) should I use for my polymer? For most people, a narrow standard "relative" calibration is fine. In this case, polystyrene standards are the usual choice for organic GPC, but PMMA's, polyisoprenes, polybutadienes and polyTHF narrow standards may be used. For aqueous GPC, narrow polyethylene oxides, polyethylene glycols and pullulans (polysaccharides) are available. If the user needs the "true" molecular weight (relative to the calibrant not being good enough), the broad standard (or reference) with the same chemical nature as the samples may be used. How reliable are broad standards? In most cases, broad standards that are commercially available have been well characterized by techniques that provide a reasonable Mw, Mn, Mz, etc. There is a certain amount of trust in purchasing these standards that the values reported are accurate and obtained with excellent precision. After all, your calibration curve is based on these values. One can also send out a representative sample that is typical of samples run in the 时助laboratory for analysis by these ancillary techniques. Many contract labs and universities can perform these analyses and provide you with Mn, Mw, Mz, etc. on your sample that you wish to use as a broad standard. Can I use k and alpha values from the Polymer Handbook? The concept of Universal Calibration, developing a calibration based on log hydrodynamic volume instead of log molecular weight allows one to obtain "absolute" molecular weights for unknowns. A plot of log [intrinsic viscosity] vs. log [molecular weight] results in what is referred to as the "Mark-Houwink" or "viscosity law" plot. The slope of this curve is alpha, 截距 and the interceptdetermines k, (the Mark-Houwink constants). In the absence of an in-line viscometer detector, the Mark-Houwink constants may be employed, provided that they are well known for not only the narrow standards to develop the universal calibration, but also the unknown. The values found in the Polymer Handbook must be for the correct polymer of interest, in the correct molecular weight range, in the solvent being used and at the temperature of operation. Sample Preparation: How do I prepare my mobile phase? In most cases, the only step required to prepare the mobile phase is filtration. The solvent 醋酸时时时素 should be filtered through a 0.45m(micron) fluorocarbon filter, (acetatetype for aqueous GPC). What additives are important and when should I use them? In certain cases, some mobile phase additive is required. For example, 0.05M Lithium 二甲基乙时 胺甲基时时吡咯Bromide is added to polar solvents such as DMF, DMACand NMP. 聚脂胺These polar solvents are used to analyze polar polymers such as polyurethanes or 聚时时胺偶作用 极虚polyimides, and there is a dipole interactionthat occurs, causing artificial 假shoulders to appear on the high molecular weight end of the distribution. This interaction is eliminated with the addition of the salt. In high temperature analysis of 聚时时大时 polyolefins, approximatelyone gram per 4 liters of an antioxidant (most any hindered phenol will do) needs to be added to the mobile phase (TCB, for example). This will help to reduce oxidation of the sample as it sits in the sample carousel at high temperature prior to injection. How do I prepare my samples? (i.e. temperature, time, and mixing) The main question one needs to ask before trying to do GPC analysis is: What is my sample soluble in? Waters began as a company doing GPC, and we have developed a long list of solvents and temperatures for almost every polymer that has ever been run by GPC. The amount of time the sample takes to dissolve (whether room temperature or elevated 时晶temperature) usually depends on two things: the molecular weight and the crystallinity度. The higher the molecular weight and the more crystalline the polymer, the longer it takes to dissolve. Usually, two to three hours with slight stirring will dissolve the sample. In some cases, (for example, ultra high molecular weight polyethylene, for example) 超 声several hours are required. High speed mixing, ultrasonicdissolution, and microwave 微波 降解 dissolution should be avoided, unless carried out without any degradationto the polymer. What concentration and injection volume should I use? As a rule of thumb, a polymer with a peak molecular weight of 100,000 should be prepared in the solvent at a concentration of ~0.10 - 0.12%, (weight/volume). This represents approximately 1 to 1.2 mg. of sample (or standard) per ml. of solvent. As you go up in molecular weight, the concentration should decrease accordingly. A high molecular weight polymer (such as ~3,000,000 weight average) should be analyzed at a 时时脂 氧concentration of < 0.02% (w/v). On the other hand, an epoxyresin with a molecular weight under 1,000 can be run at a concentration of 0.20%. At these concentrations, the maximum injection volume per 7.8 x 30 mm column should not exceed 100 μl. Detectors: What advantages does having a viscometer and/or light scattering detector on-line give me? As polymer characterization chemists wish to obtain more information about their specific samples, more people are leaning towards "advanced detection" techniques. Having a viscometer on-line with the refractive index detector gives you three main advantages over having just an RI alone: •"Absolute" molecular weight via universal calibration •Intrinsic viscosity of the polymer across the distribution •Branching information as related to long chain branching. The light scattering detector will allow you to obtain: "Absolute" weight average molecular weight (Mw) without establishing a calibration curve radius of gyration of the polymer branching information as for the viscometer. Applications for Room Temperature GPC The room temperature GPC applications for organic soluble polymers were all performed ?on the Alliance System. In most cases, the Styragel HR columns were used for the analysis. Because of the unique design of the solvent manager in the Alliance system, the flow rate precision is better than 0.075%, and the flow is virtually pulseless. Pulseless flow is extremely important for people doing light scattering, as a pump that pulses will loosen the column "fines" and cause the spikes in the chromatogram. Calibration The first step in any GPC analysis is to calibrate the system. Below, you see a polystyrene narrow standard calibration curve that was obtained on Alliance? using THF as the eluent. The molecular range covered is ~250 to 3M. The column set consisted of 2 HR 5E's (mixed bed) and a single HR2 (500A). The columns were heated to 40 ?C in the column heater, thand the flow rate was 1.0 mlL/min. The calibration curve is a 5 order fit. The curve looks excellent, but there is also something very interesting to note. There are three injections of each standard shown on the curve, (3 injections each from three different vials). So the total number of points on the curve is 39! (If you look hard enough you may be able to see some evidence of a very small amount of scatter for a couple of the standards). The retention time reproducibility of the narrow standards is less than 0.04%, a result of the superior flow delivery of the Alliance system. We sometimes need to do our GPC analysis in solvents other than THF. Below is a narrow standard calibration curve using poly(methyl methacrylate) standards, with dimethylformamide being used as the eluent. We prefer to use PMMA's rather than polystyrene when working with DMF, as low 不一致 molecular weight polystyrene standards tend to have inconsistentretention times, eluting later than expected. The polystyrene oligomer standards, (molecular weight under ~700, for example), may show retention beyond the total volume, V. The PMMA narrow T standards do not exhibit this tendency, and are preferred for work in DMF. The same column set that was used for the polystyrene calibration was used here, (2 HR 5E's plus a single HR2). The only difference is that these columns were packed in DMF. Lithium bromide, at a concentration of 0.05M, was added to the DMF. This is to prevent any polar interaction between sample and eluent, as most samples run in DMF tend to be very polar. As for the polystyrene curve, there are three injections for each standard, so, in this case, there are 36 calibration points on the curve. There seems to be even less scatter on this curve than the polystyrene curve. The columns were heated to 80? C to reduce the viscosity of the DMF. Running Very Dilute Solutions A well characterized broad polystyrene standard, Dow 1683, was run on the Alliance system with the dRI detector and THF as the eluent. The concentration was 0.15%, and a 300ul injection was made. The broad standard was injected again, (also 300ul) but this time at a 0.015% concentration (10 times less). You can see the comparisons in the figures below. Note that the signal for the 0.15% concentration is ~15mV. With the 信比噪baseline noise being 14 uV, we have a S/N of >1000:1. The signal for the 0.015% injection is only 1.5mV, but we still are able to attain a S/N of >100:1, and is easily integrated. The smoothness of flow, along with the integrated degasser, allows us to run 稀时的 very dilutepolymer solutions, and still get the S/N needed for reproducible GPC work. This is very important when we need to run very low concentrations of high molecular weight samples. We can now run extremely low concentrations and still get the correct results without sacrifice of S/N. 时性 体ElastomerAnalysis Determining the molecular weight distribution of elastomers (both natural and synthetic) is a very important analytical technique used to correlate with physical properties. Elastomer formulations may be very complicated, with blends of polymers being used, as well as 硫化时antioxidants, plasticizers, vulcanizers, accelerators, and a variety of fillers (carbon black, titanium dioxide, silica, etc.). The entire formulation may consist of only 50% (or even less) of the elastomer. These formulations are used extensively in the automotive and aerospace industries for everything from tires to O-ring seals. As is always the case in GPC analysis, the first thing we must do is calibrate our system, so here we show athird-order 聚丁二时 calibration curve using polybutadienenarrow standards as the calibrants. 聚戊二时 异There are also polyisoprenenarrow standards available. Once again, two HR 5E’s and a single HR2 were used for the column bank, maintained at 75 ?C. In the case of 甲 苯elastomers, tolueneis usually the solvent choice. THF may be used in many cases, but toluene tends to do a better job at dissolving some elastomers such as natural rubber, (cis – 1,4 polyisoprene). The dRI detector was used with the Alliance system. We chose polybutadiene narrow standards as they are similar in structure to most of the elastomers we looked at. Note the outstanding reproducibility for the applications that show the multiple distribution overlays. Below are a few additional elastomer applications of interest: Polycarbonate The GPC analysis is pretty straightforward, using either THF or methylene chloride as the eluent. We decided to see how good the precision was for the Alliance system by doing something a little different. We had a series of polycarbonates that we ran GPC analysis on Alliance system in Milford, and had the same samples run on an Alliance system at a Waters site outside the U.S., with even a slightly different column set. Shown below are the amazing agreement obtained between the two lab sites. Aqueous Samples Aqueous GPC analysis brings a whole new set of challenges to the polymer characterization 高效chemist. Most conventional, high performance packings for aqueous GPC analysis are prepared from hydrophilic methacrylate gels, with residual carboxylate groups, giving the column chemistry an overall anionic charge. When doing GPC analysis on water soluble polymers, one must be cognizant of the fact that there could be a charge interaction between the sample and the packing material, unless certain steps are taken. Theoretically, if the polymer is neutral, you could do the analysis in pure water. If there is any anionic charge to the polymer, it will be excluded by the column and elute at the void volume if pure water is used as the eluent. On the other hand, if the polymer has an overall cationic charge, (and you use pure water as the eluent), the sample will stick to the column and never elute. A lot of these ionic problems can be overcome quite easily with the addition of an electrolyte, such as 0.10M NaNO. Even for neutral samples, it is a good 3 idea to use 0.10M NaNO as the eluent. Some of the problems that need to be overcome 3 with the correct eluent (see solvent selection guide for water soluble polymers) are as follows: 1.Intramolecular Electrostatic Interactions – Polyelectrolyte expands due to the charges on the molecule itself. 2.Ion Exclusion – Sample polyelectrolyte and packing material have the same charge (both anionic, for example). 3.Ion Inclusion – Polyelectrolyte charge is opposite to that of the packing; sample will stick to the column, (cationic sample, for example) and not elute. pH may need to be adjusted. 4.Ion exchange – This phenomenon may occur as in the case of ion inclusion, where the packing and sample have opposite charges. An ion exchange reaction occurs, causing the sample to elute late or not at all. 5.Hydrophobic Interactions – The non-ionic portion of the polyelectrolyte sample interacts with the non-polar sites of the packing material. This problem can easily be remedied by adding 20% of an organic modifier (acetonitrile, for example) to the eluent.There are occasionally other interactions that can occur, such as association effects and memory effects, but the 5 problems shown above are the ones most encountered. The aqueous solvent selection guide shown previously will help you to choose the correct eluent for you particular application. We have chromatograms for nearly all of the applications shown in the guide. Just let us know if you need any help with your particular samples. Three different water soluble polymers were run on the Alliance system, with refractive index detection. The three polymers analyzed (shown below) were Hydroxyethyl cellulose, Pectin, and Polyalginic Acid. Note the excellent reproducibility of the multiple molecular weight distribution overlays. In all cases a three column Ultrahydrogel set (2 Linear plus a 120) was used. Note that the eluent was 0.10M sodium nitrate, an excellent choice for neutral and anionic hydrophilic polymers. Narrow polyethylene oxide standards were used to develop the calibration curve, so the molecular weight averages shown are based on PEO’s. 聚时Nylon and Polyester The GPC analysis of nylons and polyesters have historically been very difficult to do, with 肩甲 酚m-cresolat 100? C being the solvent choice for many years. There have been a variety of other solvent choices that workers have tried, but one that works quite well is hexafluoroisopropanol, (HFIP). HFIP has an advantage over m-cresol in that nylons and polyesters dissolve at room temperature. One disadvantage is cost: HFIP costs approximately $1,000 per liter. This is the reason we have investigated the GPC analysis of these two popular polymers using HFIP with solvent efficient columns, which are 4.6mm i.d. These are 30cm long columns, but the more narrow i.d. (as compared to conventional 7.8mm columns) allows for a large savings in solvent use (and disposal costs). The flow rate is usually ~0.35 ml/minute, which will give approximately the same eluent linear velocity as 1.0 ml/minute with the 7.8 x 300mm columns we usually use. These solvent efficient HFIP columns are specially packed in methanol for conversion directly to HFIP at 0.05 ml/minute. For our analysis of nylons and polyesters, 0.05M sodium trifluoroacetic acid was added to the HFIP, to prevent any polar interactions. Nylons in particular will exhibit tailing on the low molecular weight end if the salt is not added to the HFIP. Once again, the Alliance GPC system with dRI detector was used for the analysis. Because of the low system volume (low dispersity) of the Alliance system, excellent resolution may still be obtained with the 4.6mm columns. We used a column designation of HR2, HR3, and HR4, which represents 34 high resolution columns in the 500, 10, and 10angstrom range. The RI and the columns were maintained at 30 ?C and the injection volume was only 25 µl for the narrow PMMA standards and samples. Polystyrene does not dissolve in HFIP, so the narrow poly(methyl methacrylate) standards are used, and they work very well. Here we show a third-order calibration curve for the PMMA standards in HFIP (triplicate injections of each standard). The extraordinary retention time reproducibility of the standards is obvious from the curve. The first set of samples run was Poly(ethylene terephthalate), (PET) and Poly(butyleneterephthalate), (PBT) shown here. Also shown below is an overlay of 5 molecular weight distributions of Nylon 6/6. A Nylon 6/6 broad standard was used for the calibration, so the molecular weights shown are "accurate" for the nylon sample. The last work shown in HFIP is for two medical plastic grade Polyether/amide copolymers, used to make catheters. The two samples did not have the same physical properties nor the same "processability", yet FTIR, thermal analysis, rheological measurements, melt flow index, etc., showed no discernible differences between the two samples. If you look at the two molecular weight distributions individually (shown here as 5 overlays), they indeed do look quite identical to eachother. However, if you look at the overlays of the 5 MWD’s for each here, you can easily see some differences between the two. The reproducibility of the Alliance system gives you the confidence to say that these small MWD differences are indeed real, and not due to injection-to-injection variability. 混时时时Gradient Analysis of Polymer Blends, Copolymers, and 蒸时光散射时时器 Additives with ELSDand PDA Detection •Introduction •Experimental •Results & Discussion •Summary Abstract In recent years there has been increased interest in using gradient HPLC techniques, such as Gradient Polymer Elution Chromatography (GPEC), with polymers for determining the compositional drift of copolymers, the composition of polymer blends, or for the analysis of polymer additives. Depending upon the gradient conditions and columns selected for analysis, separations may be obtained dependent on molecular weight or based upon precipitation, or adsorption mechanisms. The use of an Evaporative Light Scattering Detector (ELSD) allows one to perform solvent gradients with a universal mass detector and observe both UV absorbing and non-UV absorbing polymer samples without baseline disturbances from the solvent gradient. The addition of a Photodiode Array Detector (PDA) allows for compositional analysis across the molecular weight distribution of many copolymers, can be useful for the identification of components in a polymer blend, and also is invaluable for the quantitation of polymer additives and other small molecules in traditional reverse phase separations. This section demonstrates the advantages of gradient analysis of polymers as compared to results obtainable with Gel Permeation Chromatography. The instrumentation used to carry out this work is described and examples of this technique for the analysis of polymer blends are shown. The effects of column functionality and solvent composition on the separation of polystyrene standards and samples is described and the best conditions observed are used to analyze various copolymers for monomer composition. Finally, the traditional use of gradient separations with the same instrumentation for the analysis of several types of polymer additives is also shown. Introduction The most common chromatographic method for the analysis of polymers is Gel Permeation Chromatography (GPC) where the separation is based upon the size of the polymer sample in solution, or the hydrodynamic volume of the polymer solution.Figure 1 shows the chromatograms obtained using GPC for a polystyrene sample, polystyrene-acrylonitrile copolymer (25% acrylonitrile) and a polystyrene-butadiene rubber (50% styrene) analyzed separately.Even though the samples are of different molecular weight, the hydrodynamic volumes are similar enough that the polymer peaks are observed at nearly the same retention time.The chromatograms obtained for the GPC analysis of a blend of approximately the same concentration of each of the polystyrene, polystyrene-acrylonitrile, and the polystyrene-butadiene samples are also shown in Figure 1. This chromatogram shows no separation of the three different polymers and demonstrates the impracticality of GPC for the analysis of most polymer blends. However, when this same polymer blend is analyzed in a gradient mode, the three components can easily be baseline resolved as demonstrated in Figure 2 which shows the overlay of two replicate injections of the polymer blend run on a prototype divinylbenzene-vinylpyrolidone column with a gradient from 100% Acetonitrile (ACN) to 100% Tetrahydrofuran (THF) over 20 minutes. Using this technique, the samples are dissolved in THF and then injected into the chromatographic system running 100% ACN. The polymers in the blend are insoluble in acetonitrile and precipitate onto the column. As the gradient proceeds, the polymers in the blend are redissolved according to their solubilities and are eluted from the column as well resolved peaks. This mechanism is similar to Gradient Polymer Elution Chromatography (GPEC). Other gradient methods for the analysis of polymers have been described in the literature which are performed under conditions where the polymers remain in solution and are separated by an adsorption mechanism, but these are generally for polar polymers that are soluble in alcohols or ketones run on bare silica columns and are not discussed here. Experimental All gradient work was carried out using the following system configuration unless otherwise noted. System:Waters Alliance 2690 Separations Module with column heater at 30 ºC Detector 1:Waters 996 Photodiode Array Detector Detector 2: Alltech Model 500 ELSD with LTA Adapter (Drift Tube at 40º C, 1.75 Liters/min Nitrogen) Data System: Waters Millennium 32 Chromatography Manager Column:As listed in Figures, 30 ºC Flow Rate:1mL/min Samples:10 - 25 µl injections of 0.2 - 0.5% samples Gradient:Linear gradient, conditions and mobile phases as listed in Figures. The most commonly used detector for GPC is the Refractive Index (RI) detector; however, the sensitivity of the RI to changes in mobile phase composition makes it unacceptable as a detector for Gradient Polymer Analysis. Figure 3 shows the chromatograms obtained for the 25 µl injection of a 0.5% solution of a styrene-acrylonitrile copolymer (25% Acrylonitrile) run on a prototype DVB/Vinylpyrolidone column with a gradient from 100% ACN to 100% THF in 20 minutes using a refractive index detector (RI), a photodiode array detector (PDA), and an evaporative light scattering detector (ELSD). As soon as the mobile phase change from the gradient reaches the RI detector (~2.5 minutes) the RI signal goes offscale, completely overloading the detector. The chromatogram obtained from the PDA detector at 260nm (or any UV detector) demonstrates that UV detection is much better suited for gradient analysis than RI detection. The chromatogram does show baseline drift with the change in mobile phase but there is still good sensitivity for the polymer sample and the drift can easily be eliminated by baseline subtracting a blank gradient run. The third chromatogram in Figure 3, obtained using an ELSD, demonstrates the superior performance of the ELSD for gradient applications. The detector is essentially insensitive to the changes in the mobile phase composition since the solvents are evaporated prior to detection. This, combined with the excellent sensitivity for polymer samples, makes the ELSD the detector of choice for gradient analysis of polymers. By combining a PDA with the ELSD, one can detect and quantitate unknowns with the ELSD and use the PDA to determine peak purity, for the identification of unknowns through library matching, and for compositional analysis of copolymers. Using this system, a wide variety of different types of polymers, polymer blends and copolymers can be analyzed. Figure 4 shows an overlay of chromatograms obtained for many types of polymers run on a Nova-PakC18 Column with a 30 minute gradient from 100% ACN to 100% THF including polyvinylchloride, polymethylmethacrylate, polystyrene, polystyrene-butadiene block copolymer, polydimethylsiloxane, polystyrene-isoprene block copolymer, and butyl rubber. When using this technique for the analysis of polymer blends or copolymers, it is necessary that the separation be independent of molecular weight so that the polymers are separated only by composition. Unfortunately, since this is primarily a precipitation/redissolution mechanism, some molecular weight dependence is inevitable, but it can be minimized through the judicious selection of columns, mobile phases, and gradient conditions. Figure 5 shows an overlay of chromatograms obtained from a series of narrow polystyrene standards run on a SymmetryShield C8 Column (3.9 mm x 15 cm) with a gradient form 100% ACN to 100% THF in 10 min. The standards from 43,900 to 2,890,000 MW elute in a band from approximately 9 to 9.5 minutes. The lower MW standards elute earlier, with many of the oligomers well resolved. These lower molecular weight standards are soluble or nearly soluble (9100 MW) in the starting conditions of the gradient (100% ACN) and are therefore separated by the traditional reverse phase mechanism. Figure 6 shows an overlay of the chromatograms obtained for the same standards run under identical conditions on a prototype DVB/vinylpyrolidone column (3.9 mm x 15 cm). A similar pattern is observed with the 43,900 to 2,890,000 standards eluting in a slightly narrower band. The separation of the lower molecular weight standards is somewhat different; however, this is not surprising due to the different reverse phase characteristics of the two columns. By changing to a Nova-PakC18 Column (3.9 mm x 30 cm) and using a 30 min gradient, the chromatograms shown in Figure 7 were obtained. Using these conditions, the molecular weight dependence for the 43,900 MW and higher polystyrene standards is nearly eliminated. As expected the lower MW standards that are soluble in ACN are eluted earlier in the chromatogram, however, the low MW oligomers are being split into three peaks, indicating that they are being separated by their differing end groups. The choice of mobile phase used as the non-solvent can have significant effects on the separations obtained from gradient analysis of polymers. Figure 8 shows an overlay of chromatograms obtained for the same standards run on a C18 Column (3.9 mm x 15 cm) with a linear gradient from 100% Methanol Nova-Pak (MeOH) to 100% THF in 30 min. These results show a clear dependence on molecular weight from the well-resolved oligomers early in the chromatograms to the 8 million MW standard. This is undesirable for the purposes of copolymer or polymer blend analysis, as it would be difficult to determine whether differences in retention time were due to compositional differences or MW differences. This non-solvent effect can also be seen when analyzing broad MW polymer samples. Figure 9 shows the chromatograms obtained for NBS706 broad polystyrene standard run C18 Column (3.9 mm x 15 cm) with a 30 min gradient using first ACN and on a Nova-Pak then MeOH as the non-solvent with THF as the solvent for both injections. When using ACN as the non-solvent, a more desirable sharp peak is obtained whereas when MeOH is used as the non-solvent, a very broad peak is obtained. Our work has shown that for THF soluble polymers, the best separations were observed with the 100% ACN to 100% THF gradient. These conditions give a rugged method that can be used for a wide variety of polymer blends and copolymers. Gradient analysis is a powerful tool for evaluating copolymer materials. A series of random styrene-butadiene rubbers (SBR) were run using this 100% ACN to 100% THF gradient on a prototype DVB/Vinylpyrolidone column (3.9 mm x 15 cm) in 20 min. Five different SBRs with composition ranging from 50% styrene to 5.2% styrene were injected along with a narrow polystyrene standard (355K MW) and a narrow polybutadiene standard (330K MW). An overlay of the resulting chromatograms is shown in Figure 10. The different SBRs are easily separated by their relative amounts of styrene and butadiene. These SBRs were previously analyzed by traditional GPC to be sure that the molecular weights were high enough that molecular weight dependence would be insignificant, and the molecular weights were all found to be approximately 200,000 to 300,000 by relative calibration with polystyrene. Using the gradient results, a calibration curve was constructed to determine % styrene vs retention time and is shown in Figure 11. The plot exhibits a good correlation between % styrene and retention time so that this method could be used to determine the approximate composition of an unknown SBR. The UV data from the PDA could also be used to crosscheck the results from the ELSD.In a similar manner, Figure 12 shows the chromatograms obtained for a series of block styrene-butadiene copolymers with a similar separation as the random SBRs. The data is plotted in Figure 13 showing a calibration curve similar to the one obtained for the random SBRs. Using this gradient method, species with only slight differences in structure can easily be separated. Figure 14 shows an overlay of individual injections of polymethylmethacrylate, polymethylmethacrylate, poly-n-butylmethacrylate, poly-n-hexylmethacrylate, and poly- laurelmethacrylate run on a Nova-PakC18 Column (3.9 mm x 15 cm) with a gradient of 100% ACN to 100% THF in 30 minutes. The chromatograms show excellent separation between each component in the homologous series of methacrylates and could easily be resolved with a faster gradient. The chromatogram in Figure 15 shows the separation of the same methacrylates injected as a mixture and run under identical conditions demonstrating an identical separation when the components are run in a mixture. This same method using identical conditions also has utility for analyzing lower molecular weight compounds. Figure 16 shows an overlay of chromatograms for two low molecular weight waxes. The two waxes are well separated and slight differences between the oligomer ratios can be observed. Low molecular weight polymer additives can be analyzed with this method by the traditional reverse phase mechanism. Many types of polymer additives will be shown using the following conditions that were chosen to be compatible with a mass spectrometer:System:Waters Alliance 2690 Separations Module with column heater at 30º C Detector 1:Waters 996 Photodiode Array Detector Detector 2:Alltech Model 500 ELSD with LTA Adapter (Drift Tube at 40º C, 1.75 Liters/min Nitrogen) Data System:Waters Millennium32 Chromatography Manager Column:Symmetry C8, 2.1mm x 15cm, 30º C Flow Rate:0.29 mL/min Gradient: Linear Ternary Gradient, 30 mins; 70/10/20 to 1/79/20 HO/ACN/THF2 Figure 17 shows the separation of Tinuvin 440, Tinuvin 900, and Tinuvin 328 that are UV stabilizers commonly used in polyolefin resins. Even though these compounds are difficult to extract from polyolefin resins with good recovery, once extracted they can be analyzed easily with good sensitivity using this method. Several different types of phthalate plasticizers are separated in Figure 18. Phthalates, which are commonly used as plasticizers in PVC resin, have come under scrutiny recently as possible carcinogens. Phthalates, particularly diethylhexylphthalate (DEHP), are used routinely in medical devices such as catheters and IV bags and in children’s toys possibly exposing patients and children to high levels of this suspected carcinogen. This method is a simple means for analyzing these phthalate compounds. Figure 19 shows the chromatograms for the slip agents oleamide and erucamide and the antistat stearic acid. These compounds, which have very little UV absorbance, exhibit poor sensitivity with UV detection but can easily be detected with the Evaporative Light Scattering Detector. Figure 20 shows the separation of Irganox 1076 and Irgafos 168 that are two antioxidants commonly used in polyolefins and other polymers. Irganox 1076 is a hindered amine and Irgafos 168 is a phosphite ester that degrades easily. The chromatogram shows two peaks for Irgafos 168. The second peak is the main Irgafos 168 peak while the first peak is actually the oxidized Irgafos 168 impurity that was present in the sample. This method is not meant to be an optimized method but only a general method for use with a wide variety of additives. Figure 21 shows 12 overlays of a separation of 10 common antioxidants run using a modified version of the approved ASTM method for the analysis of additives in polyolefins. The column, mobile phases, flowrate, and gradient conditions were optimized to obtain the shortest analysis time and maximum sensitivity allowing for the analysis of these 10 antioxidants in less than 10 minutes. The method utilizes both a mobile phase gradient and a flow rate gradient resulting in an extremely reproducible and sensitive method. The analytes were detected with a PDA at 230 nm which besides giving excellent sensitivity, also allows for peak identification using the library matching capabilities of the photodiode array detector. The instrument and conditions used to carry out this separation are shown in Figure 22. Summary The use of gradient methods for the analysis of polymers allows for separations that are essentially independent of molecular weight. Individual polymers in blends having the same molecular weight distribution can easily be separated and copolymers can be separated by their monomer ratios. Using the same instrumentation, mostcommon polymer additives may also be analyzed. The Evaporative Light Scattering Detector is a universal detector which is unaffected by changes in mobile phase gradient composition and the Photodiode Array Detector allows for positive identification of many compounds and compositional analysis of copolymers. These gradient methods are highly reproducible techniques and are extremely well suited for deformulation applications. Additives Analysis using Gradient HPLC People involved in polymer characterization by chromatographic techniques do not exclusively use GPC to analyze their samples. Many times we need to use liquid chromatographic techniques by adsorption or partition chromatography to get the information we need. Conventional reverse phase and, at times, normal phase separation techniques are used to quantitate polymer additives, as an example. Obtaining the molecular weight distribution of your polymer sample may be just one part of the characterization process. What about the additives that are formulated into the polymer to offer stabilization or processing enhancement? They can be even more important than the polymer itself. We need to think about using the correct UV stabilizers and antioxidants for protection against degradation, plasticizers to improve flexibility, antistats for polyolefins, flame retardants, accelerators to enhance the crosslinking (or curing) process, and so forth. We have done an extensive amount of work with polymer additives, and you can find some of our published work detailed in the Journal of Liquid Chromatography, volume 14 #3, (1991) and volume 16, #7, (1993). How do we analyze polymer additives? First, we need to think of what we are trying to accomplish. Do we need to know if the correct amounts of each additive are present in the formulation? Are we trying to "deformulate" a competitive material? Do we need to extract the additive package out from the polymer matrix? Chances are, the answers to these questions will be "Yes". GPC analysis is not the best way to separate, identify and quantitate the levels of additives present. Most of the additives are quite close to each other in size and molecular weight, so we need to use HPLC to separate them. A simple gradient technique, with optional flow programming, works very well in getting many different types of additives separated in a short run time. A gradient analysis consists of varying the eluent, or mobile phase composition, usually from a "weak" solvent to a "strong" solvent over a period of time. This composition variation is usually done in a linear fashion for additive analysis. Since we are varying the eluent composition throughout the chromatographic run, the refractive index detector can not be used. Most of the polymer additives we deal with have some chromophore that absorbs ultraviolet light, so a UV detector is used primarily. If there are no chromophores present, an evaporative light scattering detector may be used. We can also change the flow rate throughout the run, usually increasing flow to get the later eluters to come out more quickly. The column usually chosen for additive analysis is either an octadecylsilane (C18) or Octylsilane (C8) column, ~15 cm in length. An example of a reverse phase gradient (with flow program) separation of a series of common antioxidants and UV stabilizers 9 overlay of 12 injections is shown here. The gradient conditions are quite simple: 70% acetonitrile/30% water initially, then proceed to 100% acetonitrile in a linear fashion after just 5 min. There is also a flow program, from 2.0 mL/min initially to 6 min, then ramping to 3.0 mL/min in just 12 sec. The table of data shows the remarkable reproducibility results (retention time and area RSD's) for each additive. This is further testimony to the incredible flow and sample delivery reproducibility of the Alliance system. The UV detection was carried out at 230 nm. The PDA detector looks at all wavelengths (that you choose to view) simultaneously, which allows you to obtain the UV spectra for each additive. This spectrum may then be stored in a library and compared to a stored library of known additive standards. The only drawback to the library search is that a large majority of antioxidants are hindered phenols, which all have very similar spectra. In this case, you will have to rely only on the retention time for identification purposes. Another alternative is to add a mass spectrometer detector to the system. This will provide an electron impact spectrum which is library searchable.