Rebecca L DeRosa Inamori School of Engineering Alfred University 2008

NTR- 105758

Separating Macromolecules by Size Gel Permeation Chromatography

Laboratory Preparation In order to adequately prepare for this weeks lab you should review the laboratory procedures and safety information. Objectives After in-class presentation, completion of hands-on laboratory experiment and review of the information provided, you should be able to: Identify several laboratory methods for molecular weight analysis of polymers. Confidently discuss the differences between the methods of analysis for polymer molecular weight. Discuss how polymer molecular weight is determined by chromatographic methods. Feel comfortable preparing and working with polymer solutions and using up to date laboratory instrumentation for GPC analysis. Introduction to Gel Permeation Chromatography GPC is one of the most widely used techniques to determine the molecular weight and molecular weight distribution of polymeric materials. Dynamic light scattering and intrinsic viscosity are also used to determine polymer molecular weights, but GPC is the only one that separates the polymers molecules by their size. Using the GPC method, the molecules are separated by introducing a dilute solution of polymer to a column made with a polystyrene gel crosslinked with divinylbenzene. The gel has small pores of specific sizes (i.e. 103 107 diameter). Larger particles pass through the columns first, while the smaller molecules become hung-up in the pores of the column resulting in longer retention times. The retention volume, which is used to characterize the polymer, is calculated from the flow rate, f, ( constant experimental parameter) and the retention time, . The retention volume is the volume of the polymer solution that passed through the column to bring the molecule of one particular size to the detector. The retention volume can be calculated using Equation 1. Equation 1 Vr = f *

The detector is typically a refractometer, which measures the refractive index (n) difference between the pure solvent and the eluting polymer. The change in n is recorded by the computer and printed versus retention time or retention volume. An ultraviolet detector can also be used in the same manner. Polystyrene is a common reference material used to create a calibration curve by plotting the log of the polymer concentration ([M]) of known molecular weight verses the retention volume (Vr): Log[M] vs. Vr. Polymers of unknown molecular weight can be analyzed and their retention volumes compared to the polystyrene calibration curve. A more accurate method is to use what is called a universal calibration curve. Here the log of the polymers hydrodynamic volume [M] is plotted versus the retention volume: Log[M] vs. Vr, where is the intrinsic viscosity of the polymer. By using the hydrodynamic volume, the calibration curve is valid for a wider group of polymers. Using the universal calibration curve, it is necessary to assume that the Mps[]ps is

Rebecca L DeRosa NTR- 105758 Inamori School of Engineering Alfred University 2008 equal to Mx[]x, where PS stands for polystyrene and x for the polymer you will be using. One can extract Equation 2 from the assumption.
+a M 1pmma =

[ ] ps M ps K pmma

Equation 2

The polymer molecular weights (Mn, Mw, Mz and Mv) and molecular weight distribution known as the polydispersity index (PDI) are calculated based on the absolute calibration curve (Log [M] vs. Vr) and universal calibration curve. Polymer molecular weights can be calculated by determining the number of molecules, Ni, at weight Mi, from the curves in the chromatograph. The equations for Mn and Mw are given in Equation 3 and Equation 4 respectively.

Mn =
Equation 3

N M N
i i
i

Mw =
Equation 4

N M N M
i

2 i i

You will use the GPC to help characterize the molecular weight and Mw distribution for poly (methyl methacrylate) or polystyrene. You will use the raw data from the GPC analysis to calculate the different polymer molecular weights. Materials The materials needed for the GPC lab are listed below.

Item
Synthesized or commercially available polystyrene or poly(methyl methacrylate) GPC grade THF Polystyrene standards 5ml syringe Clean vials with labels Milipore syringe filters 500l syringe

Rebecca L DeRosa Inamori School of Engineering Alfred University 2008 Procedure

NTR- 105758

You will be using a GPC to measure the molecular weight of your polymers. Use the following steps to prepare the samples and run the instrument: 1. Prepare five PS standards using a 0.1% solution in HPLC grade THF. a. PS standards should range from 4,000 to 1,800,000 g/mol. b. The instructor may have the standards prepared and run for you before the lab starts. Make sure to gather information from him/her if this is the case. c. Use the following table as a guideline for solution concentrations. Solution Concentrations for GPC Calibration Standards and Unknown Samples Based on Molecular Weight Molecular weight range 0 25K 225K 200K 200K 2MM > 2MM Concentration to use <0.25% <0.1% <0.05% <0.02%

2. Prepare a 0.1% solution of your PMMA unknown using a in HPLC grade THF. Note: Make sure the polymer is completely dissolved. 3. After preparing each sample (and reference) filter the solution and collect the filtered portion in a separate clean and labeled vial. 4. The instructor will assist you with the software setup, sample injection and data collection for the GPC. Reporting You will be required to submit a report based on your work and calculations. This weeks report should include an abstract, introduction and background information, necessary references, experimental section, and results sections. If the instructor wishes, this experimental write up can be combine with one for dilute solution intrinsic viscosity. The following are a set of guidelines for the GPC section of the report. To do for report: 1. Write a concise abstract, introduction, thorough experimental, and detailed results for your report. Your results should include important tables of data, figures, equations for calculations, chemical reactions or schematic drawings that were determined from this laboratory exercise. 2. Record the information about the PS standards, name of the instrument, type of detector used, column sizes (and number), temperature and flow rate. 3. Prepare a relative and a universal calibration curve from the retention time (volume) and known molecular weight standards. Use Log(M) vs Vr and Log([]M) vs Vr for your calibration curves. 4. Make sure to provide a data table used to create your calibration curves.

Rebecca L DeRosa NTR- 105758 Inamori School of Engineering Alfred University 2008 a. The table should have values for M, Vr, [], Log []M, and Log(M). b. You will have to find the Mark Houwink constants for PS for the universal curve. 5. Provide the equations for your calibration curves using up to a 5th order equation. Find the best R2 value. 6. Use the instrument software to calculate the molecular weight averages Mn, Mw, Mv and Mz for your polymer 7. Make sure to report your K and a values and the reference. 8. Provide the polydispersity index from each calibration.

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