The Column - June 2008 - (Page 19) June 2008 www.thecolumn.eu.com The Column Tips & Tricks: GPC/SEC molecule (total penetration volume). The measured elution volume is not only related to the molar mass of the molecule (size) but also to its chemical nature. How are GPC/SEC calibration curves created? There are several ways and methods to construct a GPC/SEC calibration curve. Table 1 shows an overview of the different techniques including their advantages and disadvantages. All the techniques described in Table 1 are based on the use of molar mass calibration standards. The most popular method for calibration is the method that uses molar mass standards with a narrow MMD. Here the elution volumes of the molar mass standards are measured and plotted against the logarithm of the molar mass. GPC/SEC guidelines recommend using at least three molar mass standards per molar mass decade.4 Although the concentration is not needed to obtain accurate and precise GPC/SEC results it is important for samples with a narrow MMD (e.g., molar mass standards) to be in the recommended concentration range.5 Figure 1 also shows the recommended concentration range, that depends on the molar mass of the calibration standard. After measuring the elution volumes and plotting them versus the logarithm of the molar masses (in general the molar mass at the peak maximum),2 a fit function that describes the shape of the calibration curve, has to be chosen. Unfortunately there is no general optimum fit function that is recommended for all column/column combinations, but users have to select a proper fit function based on multiple criteria. As discussed before, the fit function will not be linear. GPC/SEC fit functions are based on polynomial functions with a degree of 3 (cubic) or higher (4–7). There are also special dedicated fit functions available that are based on polynomial Table 1: GPC/SEC calibration methods and their advantages/disadvantages. Method Calibration with molar mass calibration standards with narrow MMD Advantages • easy and straight forward • precise method3 • accurate method3 for chemical and structural identical samples and calibration standards • accuracy is based on the average deviation of all calibrants • easy and accurate method • ideal for internal referencing of analytical conditions • calibration is precise Disadvantages • results only accurate for chemical and structural identical substances • standards with narrow MMD are not available for all polymer types Broad calibration with well characterized standard with broad MMD • calibration only accurate for chemical and structural identical substances • only a limited number of standards commercially available • a single broad standard covers only a limited molar mass range • only a limited number of standards commercially available • accuracy is limited by quality of the standard • high and low molecular weight region are less accurate • accuracy depends on the accuracy of the Mark-Houwink coefficients Integral calibration/Cumulative calibration • easy and accurate method • calibration is precise • calibration accurate for chemical and structural identical substances • easy and accurate method • matching calibration curves for all different polymer types can be constructed from one calibration curve • easy and accurate method • one calibration curve valid for all types of samples Universal calibration I: Calibration with molar mass calibration standards with narrow distribution and Mark-Houwink coefficients Universal calibration II: Calibration with molar mass calibration standards with narrow distribution with additional on-line viscometer detection • increased experimental complexity • additional experimental error from concentration dependence, band broadening and detector delay 19 http://www.thecolumn.eu.com
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