Chromatogram Troubleshooting
Jake Morrow
Created on July 20, 2021
Chromatogram Check Points
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Transcript
Baseline: Indicated by the green trace, this should be flat, however not completely flat. There will always be some noise on the baseline when the pumps switch from A to B and back to A, as is evident from the bumps present. This green trace displays the system noise being cancelled out from the final result, and should not cause any alarm.
Pressure: It is paramount that the pressure remains steady throughout the run in order to achieve optimal analysis conditions. Pressure is measured in units of Bar. Pressure fluctuations typically do not exceed 1- 2 bar when system pressure is 10-25 Bar, and typically do not exceed 2-3 Bar when system pressure is >25 bar. Within-run Δp of less than 20% will not be flagged with a Code 3 Peak Analysis & Results Code and in the case of the Premier should be considered normal as long as QC values are within range.
Peak Shape: The peaks should be sharp and resolute. The top of the first peak should not be much wider than the second hash mark in the chromatography window. The second peak should return to baseline and should not shoulder in any way, which would indicate full and efficient elution.
Valley: The valley should always return to baseline. Sometimes it is difficult for elevated HbA1c samples to achieve a full return to the baseline, but it should be very close. Elevated baselines are indicators of columns with high injection counts.
Tail: The second peak should return to baseline and should have plenty of time to allow the system to re- equilibrate and elute all biological material off of the column before the next injection so as to avoid carry- over.
Retention Times: The retention times should remain consistent for each column. There may be minor differences from column-to-column, but these will be ≈1% fluctuations at most.
Area counts: The system area count default settings will provide results from 250,000-2M. Ideal total peak area counts are typically from 800,000-1,000,000.