RSD failing HPLC

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

3 posts Page 1 of 1
Hi All,
I have been having ongoing issues with a high RSD value for 5 consecutive injections from the same HPLC vial. Getting RSD between 1.9-2.5% (Spec <2.0%)
The mobile phase is a mixture of dioxan and water (575:400)
Run time is 40mins
Flow rate is 0.7ml/min
Injection volume 5uL
So far we have tried to eliminate the column, system, mobile phase.

The mobile phase has been re-made multiple occasions with different degassing methods/ stirring mobile phase while on system
A new column was primed and used with similar results.
The barrel of the injector was changed out on system.
A different system was used which had recently been used for other analysis with no issues.
The analysis has been carried out many times in the past with RSD of less than 1%.
Any help would be greatly appreciated.
I there is more than one measurable peak in your chromatogram, try calculating the area ratio for two peaks. If the RSD of the ratio is substantially better than that of the individual peaks, that suggests that the major contributions to error occur before the peaks have separated, so look at things like injector problems. If the RDS of the ratio is substantially worse than that of the individual peaks, that suggests that the major contributions to error are uncorrelated between the peaks, so look at thinks like peak shape, integration settings, baseline noise, etc.

In any case, since the method works OK on another system, are there visible differences in the chromatogram (as above: signal/noise ratio, peak shape as they affect integration settings).
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
My boss has decades of experience running dioxan / water methods and mentioned that proper degassing of this difficult mixture is critical to reliable results. Please use either a chemically compatible inline vacuum degasser (never assume that any vacuum degasser is compatible. Check first.) OR use continuous high-purity Helium sparging to degass the two liquids. Sonication and vacuum filtering of mobile phase are of no use here as they only temporarily remove gas from the liquid resulting in poor RSD. You must insure that the HPLC pump is fully primed, all channels are operating perfectly and the liquid is fully mixed before it hits the column. Double check your detection settings (esp, the Bandwidth and never use any software based "reference" wavelengths). 100% perfect operation of the pump and flow should be verified through the column (monitor back pressure and detection baseline for stability). Do not start an analysis until the system is fully stabilized.
3 posts Page 1 of 1

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