When running peptide standard a stable baseline was observed but my samples are giving a very significant baseline drift (wavy)?
To be precise protein samples were digested in 6N HCl and were then neutralised with NaOH followed by freeze drying. 100 mg of freeze dried samples were then dissolved in MQ water to be analysed by HPLC for peptide formation, but it started giving unstable baseline. Also the same column and mobile phase gave stable baseline when peptide standard from sigma aldrich was analysed before the samples.
@ Farooq, thankyou for your reply. Actually baseline is not periodic but actually it is just like a wave going all through the negative part of the baseline. The same system is giving a nice baseline and chromatogram with standard so there should not be any problem with the bubbles. I will check the pH of the mobile phase and sample and see if it works. How much difference in pH(mobile phase and sample) is acceptable so that it gives a stable baseline??
Swati,
This is a great question... I get asked this every once in awhile, and there are ten (10) possible causes for baseline drift:
*Column temperature fluctuation (whether from changes in room temperature, or a faulty column oven)
*non-homogenous mobile phase
*contaminant (or air) buildup in the detector cell
*plugged outlet line (post detector)
*mobile phase mixing problem (or change in flow rate)
*slow column equilibration
*mobile phase contaminated, deteriorated, or prepared with low quality materials
*strongly retained materials in sample
*mobile phase recycled but detector not adjusted (when using a mobile phase recycler)
*Detector (when using UV) is not set at absorbance maximum but at slope of curve
Given your analytes, I believe in all likliehood it is the 'strongly retained materials', in which case a good column cleaning procedure should work well for you.
Is this helpful? If you would like additional information on this, and other troubleshooting issues, you can always go here:
https://phenomenex.blob.core.windows.net/documents/a8cb7bc3-eb65-4244-8910-faeb1818df8c.pdf
Paul
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