Chromatography Forum

How to determine if a column is dead?

What is delay volume?

Why did I get negative peaks for pressure reading? is that because of Bubbles?

by the way.
Is MWD the same as VWD or the same as DAD?

Thanks a lot!

1) The preferred way to determine if your column is dead is to periodically run a test solution (available from the column manufacturer) and look at the peak shape and retention time. When peak shape has degraded then the column is "dead".

2) Delay volume (also known as dead volume) is the amount of liquid (mobile phase) contained in the column. This can be determined by looking at the retention time of a compound that is not retained by the column and dividing by the flow rate.

3) How much does your pressure fluctuate? A little fluctuation is normal. A large fluctuation could be caused by bubbles in the pump head, bad check valves, seals, or pistons. First purge the pump to get rid of bubbles. If that doesn't help, take out the check valves and clean them. If that doesn't help replace check valves. If that doesn't help replace seals. If that doesn't help replace pistons.

4) I think MWD is multiple wavelength detector. This one can do multiple wavelengths at one time similar to DAD. VWD is variable wavelength detector which can only do one wavelength at a time (although that wavelength can be changed).

Sassman,

If you divide the retention time (min) by the flow rate (mL/min) you’ll end up with a pretty unusual unit

Best Regards

Thank you guys.

t0*f=dead volume, right?

Yeah, I meant multiply by the flow rate. Units have to cancel.

t0*f=dead volume, that is correct.

Delay volume is most often the gradient delay volume, not the column dead volume. The gradient delay volume is the volume between the point where the two solvent are mixed and the column inlet.

make sure you degas your mobile phases(s) even if you have a degasser on your instrument. Most service personnel will tell you the same thing.

If you make up a 50ug/ml solution of uricil and inject this into your column it will come through with the buffer front. knowing the pump speed and the retention time for the uricil this will give you the volume from the sample loop to the detector.

In the same vein as Uwe Neue referenced ...

You can also run 100% water and 0.3% acetone in water.

1. Start at 100% water and hold for three or so min.
2. Slowly, over about 5 min ramp up to 100% 0.3% water.
3. Take the midpoint of that ramp line and drop a tangent down to the x-axis (time).
4. Subtract the time of the midpoint from the time you held 100% water (3min); we will call this T50/50.
5. Take the gradient time (Tg, or 5 min) and divide by two and subtract this number from T50/50 (essentially you will have T50/50 - Tg/2). Call this result Td.
6. Td multiplied by the flow rate will sive you the system dwell volume.

I generally use a flow of about 3 mL/min do to this, and use a zero dead volume union for the "column". I have also found it more beneficial to know the system delay than the column dwell...

I haven't tried the technique, or thought out how it provides the delay volume, but...

Step 3. Is that really a tangent?.

Please keep having fun,

Bruce Hamilton

Nope, it is not a tangent. It should have been drop a line down from the midpoint that is perpendicular to the x-axis.

Thanks for catching that one.