Table 6 Troubleshooting advice

2

The refractive index increases even after long equilibration time.

High denaturant concentrations can cause detergent precipitation.

Dilute sample to lower denaturant concentration and perform experiment at this concentration.

Denaturant precipitates.

2B

No plateau is reached.

Loop volume is too small; hence, the applied volume does not completely fill the measurement chamber.

Install larger sample loop.

2B

Change between plateaus is not pronounced enough for reliable discrimination.

Flow rate of syringe pump is too high.

Reduce flow rate to (0.1–0.2) mL/min.

2B

Baselines are unstable.

Disconnection of syringe causes pressure changes and injection of air bubbles.

Wait for a few minutes until the baseline is stable again.

5, 21, 38, 50

RI baseline is unstable.

Too much gas dissolved in solvent.

Make sure your buffer is degassed before using it in triple-detection SEC.

The flow rate has changed.

Use the same flow rate during system equilibration and measurement to allow the baseline to stabilise.

19–21, 50

Measurement cannot be started.

Sample tray is not placed correctly. Connection between software and LS or RI detector is lost.

Remove tray and make sure it is put back in place correctly. Close ASTRA, restart the detectors, and subsequently restart ASTRA.

21, 50

No data acquisition in ASTRA.

Method in ChemStation was started before the sample set in ASTRA was started.

Stop method in ChemStation, check that enough sample is left, and start ASTRA data acquisition before restarting the method run in ChemStation.

28

Reference detector is not the one with the broadest signal.

Wrong reference detector chosen. Inappropriate peak selection.

Make sure to choose the detector with the broadest signal. This is normally the last detector in line and should be the RI detector. Make sure you set the peak boundaries from halfway up the peak to the point where all detector signals have returned to baseline.

55

Baseline cuts peaks to be analysed.

High denaturant or salt concentrations cause baseline instabilities.

Set baselines manually for each detector individually, such that the flanks of peaks of interest essentially reach baseline level without being cut or shifted upwards.

59

Systematic deviation from linearity of one of the LS detectors.

Detectors are not normalised correctly.

Check detector normalisation values. If necessary, repeat normalisation. If the sample analysed reveals a unimodal particle size distribution, normalisation can be done using the actual measurement according to the procedure described in step 33|.

59

Data points cannot be fitted with a linear fit.

Scattering particles are large (i.e., >50 nm), and, thus, the Zimm plot is significantly curved.

Select a different fit model in the peak section.

59

Molar mass plot is bent upwards or downwards within the analysed region (smiley or anti-smiley effect).

Band-broadening correction is incorrect.

Check settings for instrument and mixing terms; if necessary, repeat band-broadening correction for current solvent system.

60

The results obtained are far from expected or reasonable values.

Incomplete separation of different species.

Install a different SEC column that is able to separate the species of interest. Try manual, more complex analysis algorithms that are able to distinguish contributions from different species (see Results and Discussion).

60, 63

Analysis is not possible. Molar masses are displayed as N/A in the final report.

Baseline correction, peak selection, or constants needed for analysis were not adapted to the system being analysed.

Check if baseline settings and peak selection are correct and if the saved constants correspond to the system you are analysing.