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Column Bleed?
Posted: Mon Feb 02, 2009 3:29 am
by wan
I'm not experienced with HPLC and was flushing my system (Agilent 1100) to get a baseline when I saw this.
Red: Pressure, Blue: VWD 254nm
Column: BDS C18
Mobile phase: 50:50 Methanol:Water
Flow rate: 0.8 mL/min
Is that due to column bleed?
Posted: Mon Feb 02, 2009 11:34 pm
by tom jupille
Since the four negative dips in pressure are closely followed by four baseline excursions, it looks more likely that you had momentary losses of prime (outgassing from the solvents?). Difficult to tell more without being able to read the y-axis units. Any "bleed" from the column would be negligible, and very un-likely to show up at 254 nm.
Posted: Tue Feb 03, 2009 7:03 am
by wan
I degassed the solvent for 15 minutes prior to use, so I'm not too sure what else can cause that.
Today, I experience the same thing:
Red: Pressure, Blue: VWD 254nm
Column: BDS C18
Mobile phase: 50:50 Methanol:Water
Flow rate: 0.5 mL/min
What else can cause such observations?
Posted: Wed Feb 04, 2009 3:47 pm
by tom jupille
The only thing that can cause what you are seeing is a temporary "loss of prime". Most likely possibility is an air bubble, as discussed (more on that below). Another possibility is a sticking or malfunctioning check valve.
Re degassing, some questions:
1. Are you pre-mixing your mobile phase, or are you mixing on-line?
2. If you are mixing on-line, which version of the 1100 do you have? the low-pressure mixer (a single high-pressure pump with a proportioning valve), or the high-pressure mixer (two high-pressure pumps)?
3. How, exactly, did you degas?