Question about PDA Detectors

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8 posts Page 1 of 1
Hello All,

I've recently attached a Prep column to our semi-prep system and was wondering about the responses I've been getting for samples that have been run. Since the flow-rate differs wildly (1 ml/min vs 20 ml/min), I was wondering how this should affect my detection since I have not been getting the usual responses.
On my analytical column, I see responses that frequently blow-out my PDA at specific wavelenghts (254, 320 nm), while the same volume injection on my Prep barely has any response. Is this due to the flow-rate differential between the two methods and, thusly, less-signal because my sample spends less time within the PDA? Or is this an issue that may be related to one of my columns?

Thank you.
A PDA detector is a concentration-sensitive spectrophotometric detector. Its response (the absorbance) is proportional to the analyte concentration in the detector cell. The response is independent of the flow rate. The analyte concentration in the detector cell is proportional to the amount of the analyte (which is proportional to the sample concentration and the injection volume) injected to the column and reciprocal to the dilution of the sample in the column. Preparative columns are wider than analytical columns. The sample dilution is higher in your preparative column. The sample injection volume should be changed proportionally to the column cross-section area (~ diameter squared) when you change the column.

Note that the peak area (the unit is absorbance*time) is reciprocal to the flow rate F since the time needed for the analyte zone to pass through the cell is reciprocal to F. Also, the peak height (the absorbance in the peak maximum) varies to some extent when the flow rate changes because the peak broadening (the analyte zone dispersion) depends on the flow rate (see the Van Deemter equation).
Thanks VMU, that was a wonderful and explanative response.
To sum up what you're saying, due to the column's volume and flow-rate, the sample dilution is much higher on my prep column so while the same amount of material is flowing through, it is significantly diluted due to the significant increase in volume compared to my analytical runs which results in smaller signals on my PDA.
That makes complete sense to me and thank you again for the quick and detailed response.
TS
TylerSmith123 wrote:
To sum up what you're saying, due to the column's volume and flow-rate

To sum up, it's due to the column volume.
The flow rate is scaled with the column diameter to keep nearly the same linear velocity of the eluent in the column.
In addition to the great explanation by VMU, note also that preparative UV cells are usually shorter than analytical cells so sensitivity is reduced at this level too.
Hi Gaetan,

Thank you as well for that explanation! I have a preparative flow-cell, but everything is run through the analytical flow-cell as the prep cell is not installed. My peaks generally go far beyond the limits of the detector, and may even span as much as a minute in length above the upper-limit prior to returning. Both of you have expanded my knowledge of both how to think about detection of my analytes and how that data is processed into the signals I see on my software-- so thank you again!

TS
if you're doing prep flow-rates through an analytical cell, be careful of the back-pressure you're creating at the flow cell. Some are quite delicate, and a high pressure will damage them.
And yes, full-scale-deflection flat top peaks do happen; just don't try to do slope-based peak recognition on a fraction collector if this is happening to you! You know the scenario: you inject a little bit of sample, just to check that the system is working. It all looks good, with a lovely peak, and the fraction collector starting and stopping at the right times. Based on this, you inject all of your precious sample, the fraction collector sees the up-slope of the peak, and swings into action. Three seconds later it sees a completely flat line (because the signal has hit its numerical maximum), the fraction collector declares that the slope is zero, below threshold, stops collecting, and diverts the whole of the rest of the peak into the waste pot. You then have a big dilemma about how clean you think the waste pot was, and whether it's worth putting 4 L of (largely someone else's) waste through the rotary evaporator in the hopes of recovering your sample. Fun, fun fun...
Haha thank you Imh!

I've actually been doing this analysis for quite a while so I have experience in that exact scenario you described (we use the level function instead of the slope function for collection now haha). The really strange thing was figuring out where to input the wavelength I wanted measured for the fraction collector!

It's funny that you brought up the 4L roto-vapping because my lab was doing exactly that to save on storage space from HPLC waste! It was crazy! I could not imagine having to do that and then COLLECTING something out of it.

Back to your first point about the analytical flow-cell with prep flow-rates. Is there anything I should look for or be wary of in the case of analytical flow-cell failures? I can brain-storm a few potential issues with a broken flow-cell, but I don't know how it would manifest on the instrument. Currently, we have a pressure per square inch of ~3200 (this is an old column which is pretty dirty >700 runs and I've got analytical lines mixed in there too haha). Thus far I have had no issues replicating all my standards (run them every month if the column is used), but if you could prepare me in anyway for a future failure then I will be all the more prepared when my lack-luster chromatography backfires!

Thanks again, TS
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