The Column - February 2009 - (Page 17)

February 2009 www.thecolumn.eu.com The Column Tips and Tricks: GPC/SEC Tips & Tricks: GPC/SEC Do’s and Don’ts in GPC/SEC Light Scattering Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany. Light scattering (LS) is one of the few absolute, theoretically founded methods for the determination of molar mass and molecular size of polymers and biopolymers. Light-scattering instruments became commercially available in the late 1950s. LS detectors for GPC/SEC appeared in the mid 1970s. Since then they became an important tool to investigate macromolecules. Expectations are high, when the method is implemented in the laboratory. However, even an absolute method is not suitable for all types of macromolecules, requires knowledge of evaluation parameters and thorough set-up measurements. the sample needs to be weighed in accurately using an analytical balance. • For method 2 the refractive index increment (dn/dc) of the sample and the concentration detector calibration factor must be known. The calibration factor can be measured using a reference substance (e.g., poly(styrene) or pullulan) with accurately known dn/dc and known concentration(s). (Method 1 needs to be applied for the reference substance to determine the detector response.) If the detector calibration factor is determined precisely, method 2 produces more accurate results. Errors resulting from deviating injection volume, adsorption on the column or non-soluble sample parts are not affecting the slice concentration (and, therefore, the molar mass). It is nevertheless recommended to weigh in the sample precisely because then it is possible to quantify the sample recovery. This helps to identify systematic errors or malfunctioning system components early. Weigh in Your Samples Precisely While GPC/SEC does not require the accurate knowledge of the mass in a chromatographic slice, GPC/SEC light scattering does. To calculate the molar mass from the scattering intensity the sample concentration (injected mass) in the chromatographic slice must be known.1 An error in the slice concentration results in an identical error for the molar mass determination. The slice concentration can be measured with a concentration detector (e.g., an RI, UV detector). Two different methods can be used: • Method 1 requires the accurate knowledge of the bulk sample concentration. The slice concentration is calculated from the batch concentration assuming that 100% of the sample elutes from the column and that the injection system works properly. For this method Author: Daniela Held E-mail: Dheld@polymer.de Be Aware of The dn/dc The dn/dc of the sample must be known to calculate the molar mass from the scattering intensity. The dn/dc is the change, dn, of the solution’s refractive index n with the molecular concentration change, dc. The dn/dc of a sample depends (amongst others) on the chemical nature of the sample, the composition, the solvent, the temperature and the wavelength of the light source used. It also depends on the molar mass but can be assumed to be constant above approx. 10 000 Da.2 Any error in the dn/dc results John Rensten/Getty Images 17 http://www.thecolumn.eu.com

Table of Contents for the Digital Edition of The Column - February 2009

The Column - February 2009
Contents
Rapid Sample Preparation Technique for the Extraction of Dioxins and Furans from Animal Tissue and Animal Feed Using Pressurized Fluid Extraction
News
Incognito
Market Trends and Analysis
Tips and Tricks: GPC/SEC
Multi-residue Pesticide Analysis in Foods Using Modified QuEChERS Extraction and Ion Trap GC–MSn Analysis
Quantification of Protein Phosphorylation Using Chip-based LC–MS
A New Perspective on the Challenges of Mass Spectrometry
Q&A

The Column - February 2009