Add an additional column to further resolve the ghost peak from the sample peak of interest.

Gradually change the flow rates (by no greater than 0.1 mL/min2) to prevent large pressure changes from shocking the column.

These columns do not have ghost peak issues when new or well maintained. Wyatt offers SEC columns optimized for light scattering measurements which minimize shedding particles, have low baseline noise, and can be ready to run samples in a short amount of time.

Select a column with minimum shedding.

For details see TN1012 Using the Baseline Subtraction Procedure in ASTRA.

If the ghost or system peak is reproducible in multiple blank injections, you can subtract the light scattering baselines of a blank injection from the sample injection using ASTRA’s Baseline Subtraction procedure.

Please note that the pressure during load is only shown on the front panel and typically not part of the data recorded in the HPLC data file. Use an HPLC system with minimum pressure change during the load and inject steps.To eliminate or prevent these ghost peaks: If the ghost peak is seen even with a blank injection, then it is due to the system and is a true ghost peak. For details see TN1011 Baseline Determination for Imperfect Chromatography. If the peak from the large particles in the sample bleeds over into other sample peaks, you can adjust the baselines of the sample peak of interest to exclude the contribution from the larger particles. Light scattering is a great tool for characterizing these large aggregate peaks as even without a concentration signal available to determine molar mass, the size (RMS radius) and number density can still be determined. If the early peak is due to the sample, then it is not actually a ghost peak, but a real sample peak that needs to be considered in the analysis. The ghost peak is most likely coming from column shedding during injection, and to a lesser degree, coming from your HPLC system (filters, frits, injectors, tubing, etc.) or mobile phase. Otherwise, if the peak at the total elution time can be seen in both the sample and blank injection chromatograms with similar intensities, it is an indication of system or ghost peak. Dynamic & Electrophoretic Light Scattering.

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