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Absorbance Correction

Multi-angle light scattering (MALS) analysis is based on the intensity of scattered light as measured by an array of photodiodes. If the correct mass of a sample is to be measured, we must account for known variations in the intensity of the laser source so that we do not incorrectly attribute such changes to the sample being analyzed.

ASTRA® has the ability to account for variations in the laser intensity due to power fluctuations by dividing the measured signal by the laser monitor. ASTRA has now been extended to account for changes in measured intensity due to absorbance by the sample. This is accomplished by applying an additional correction to the measured intensity based on changes in the forward laser monitor signal.

Data Collection Requirements

To apply the Forward Monitor Correction to a sample, the forward monitor signal must be measured at the time of data collection. The DAWN® and the miniDAWN® measure the forward monitor signal with every collection.

Analysis

The standard laser monitor correction works well when there is no interaction of the signal with solvent or sample. This is typically not the case with the forward monitor correction, because the presence of sample directly affects the measured intensities. If the collection is stopped during a solvent peak, or when the baseline is otherwise changing, the measurement of average monitor value is inaccurate. Therefore, the forward monitor correction requires the user to specify a peak range for the pure solvent. The correction uses this 'pure solvent' peak to compute the forward monitor average. An example is shown in Figure 1.

Defining Peaks

Figure 1: Specify sample and pure solvent ranges for analysis.

Results

The forward monitor correction adjusts the measured light intensity based on the change in the forward monitor, in proportion to the square root of the ratio between the forward monitor signal and the average forward monitor signal. Consequently, small fluctuations in forward monitor signal are expected to yield small changes in the measured molar mass. The following examples illustrate these effects.

Example 1: Absorbing Sample

Consider an absorbing sample, shown in Figure 2, where the forward laser monitor (measured on aux channel 2) has an average value of ~3.05 V, but drops to ~2.35 V in the presence of the absorbing sample (a drop of ~23%). Consequently, the expected change in molar mass should be around that value. A comparison of the sample with and without the forward monitor correction is shown in Figure 3 and Table 1.


Table 1: Absorbing Sample
    Sample Sample with Correction    Change
Mn   2.157e+5 (0.3%) 2.598e+5 (0.3%) 20%
Mw   2.166e+5 (0.3%) 2.615e+5 (0.3%) 21%
Mz   2.174e+5 (0.6%) 2.629e+5 (0.7%) 21%
Mw/Mn   1.004 (0.4%) 1.007 (0.5%) -
Mz/Mn   1.008 (0.6%) 1.012 (0.8%) -

Defining Peaks

Figure 2: Comparison of 90° Detector and Forward Monitor signal.

Defining Peaks

Figure 3: Comparison of sample with and without the Forward Monitor correction.

Example 2: Less Absorbing Sample

In contrast, consider a minimally absorbing sample, where the forward laser monitor (measured on aux channel 2) has an average value of ~3.05 V, but drops to ~3.01 V in the presence of an absorbing sample (Figure 4). The change in voltage is ~1.2%, therefore the expected correction in molar mass should be around this amount. A comparison of the sample with and without absorbance correction is shown in Figure 4 and Table 2.



Table 2: Less Absorbing Sample
    Sample Sample with Correction    Change
Mn   1.411e+5 (0.6%) 1.413e+5 (1.0%) 0.1%
Mw   1.416e+5 (0.6%) 1.418e+5 (1.0%) 0.1%
Mz   1.420e+5 (1.0%) 1.423e+5 (2.0%) 0.2%
Mw/Mn   1.003 (0.9%) 1.004 (1.0%) -
Mz/Mn   1.006 (1.0%) 1.007 (2.0%) -
Defining Peaks

Figure 4: Comparison of 90° Detector and Forward Monitor signal.

Defining Peaks

Figure 5: Comparison of sample with and without the Forward Monitor correction.

 

   

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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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