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Conventional and Universal Column Calibration

ASTRA supports conventional and universal calibration, allowing chromatographers to build a bridge between their legacy (conventional and universal calibration) data archives, and modern, accurate mass determination through multi-angle light scattering.

The calibration module includes the ability to merge data from multiple calibration standard runs to determine a column profile across the full column separation range. A typical workflow involves collection of data in multiple sample runs (Figure 1), which are then combined (Figure 2) to form the final calibration curve.

Defining Peaks

Figure 1: Define column profile using a series of molecular standards.

Import Peak Data

Figure 2: Import peak definitions from multiple experiments.

The calibration data from multiple runs can be merged to form a complete conventional calibration curve using the signal of a concentration sensitive detector (typically differential refractometer) or universal calibration curve using either the signal from a concentration detector with Mark-Houwink-Sakurada coefficients, or combined signals from concentration and viscometric detectors.

MALS/Universal Calibration Comparison

Figure 3: A conventional column calibration.

MALS/Universal Calibration Comparison

Figure 4: Universal Calibration Curve.

The completed calibration profile can then be used to analyze an unknown sample for average molar mass and molar mass distribution. The calibration results are visible in the column profile, show in in Figure 5, along with fields to hold diagnostic information about the column, such as Plate Count, Asymmetry Factor, Resolution, etc. These fields can be used to track the performance of the column over time.

Conventional Calibration Curve

Figure 5: Column Profile showing coefficients of conventional calibration curve.

Comparison of absolute molar mass derived from MALS and relative molar mass derived from calibration results can then be used to build an archive of comparative data mapping absolute to relative molar mass. Comparison of molar masses obtained by conventional calibration with those from MALS can not only illustrate possible errors generated by conventional size exclusion chromatography, but may also be useful for characterization of branching.

For example, consider the molar mass distributions measured using MALS (PSA_051111_LS) and universal calibration (PSA_051111_UC). The results (Figure 6) are almost identical for this broad polystyrene sample because the analyzed polymer has the same chemical composition as the calibration standards used to determine the column profile.

MALS/Universal Calibration Comparison

Figure 6: Comparison of molar mass versus elution volume plots obtained by MALS, Conventional Calibration, and Universal Calibration.

Contrast the PSA results with those of Alkyd 3,6, shown in Figure 7. The molar masses obtained by conventional calibration (Alkyd_3_6_CC) are far from the correct masses obtained by MALS (Alkyd_3_6_LS). A more accurate result can be obtained using universal calibration (Alkyd_3_6_UC).

MALS/Universal Calibration Comparison

Figure 7: Comparison of molar mass versus elution volume plots obtained by MALS, Conventional Calibration, and Universal Calibration.

While these results certainly show reasonable correlation between universal calibration and MALS analysis for polydisperse samples, things begin to break down when a monodisperse sample is analyzed. Figure 8 shows a comparison of universal calibration and MALS analysis of a 400 KDa polystyrene standard. Note that the universal calibration (due to its strong dependence on elution volume) would lead to the conclusion that the sample is polydisperse, while MALS correctly shows that the sample is monodisperse.

MALS/Universal Calibration Comparison

Figure 8: Comparison of molar mass versus elution volume plots obtained by MALS, and Universal Calibration for a monodisperse sample.

 

   

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Wyatt Technology is the recognized leader in light scattering instrumentation and software for determining the absolute molar mass, size, charge and interactions of macromolecules and nanoparticles in solution.

Wyatt's line of multi-angle static light scattering products couple to size exclusion chromatography (SEC-MALS), field-flow fractionation (FFF-MALS), and stop-flow composition-gradient systems (CG-MALS). Our dynamic light scattering (DLS) products operate in traditional cuvette as well as on-line and automated, high-throughput modes. We also offer unique instruments for electrophoretic light scattering (MP-PALS), differential refractometry, and differential viscosity.



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