Chromatography Forum

I have two HPLC and i just want to know why the area count of same solution is higher in UV then PDA detector

I have two HPLC and i just want to know why the area count of same solution is higher in UV then PDA detector

They have different sensitivities.

thankd dblux,
but still my answer is not known. i want to know why this phenomenon happen. both are working on lambert beer law's . both have same lamps or is due to any other physical or electronics related issue.

with regards
chandresh

Do you have the same flow cell size in both?

Another hint - check spectral bandwidth of both detectors.

If there is any signal conversion going on (analog to digital) then you possibly have a different factor in each instrument.

You may also check all your system settings. For instance with PerkinElmer instruments and TotalChrom, if you change the sample loading syringe size you have to change it in both the software and on the instrument. If you don't, you have a situation where it may be treating a 200uL syringe as though it were 100uL and thus injecting only half as much per injection.

I also agree with checking flow cell size and bandwidth, those would have been my first two suggestions.

As paulw indicated the most likely explanation is your data settings. The area is the sum of the voltage measurements made. So if you have one system reading data at 10 Hz (i.e., 10 times per second) and another one reading data at 20 Hz, the second system will report twice the area, even for exactly the same detector settings.

In addition, as has been suggested, the slit width (or bandpass on a PDA) may be different so that a detector with a wider bandpass may be sampling proportionally more "off-maximum" signal.

All of this relates to the reasons why a calibration plot is good only on one instrument run by one operator on one day!

As paulw indicated the most likely explanation is your data settings. The area is the sum of the voltage measurements made. So if you have one system reading data at 10 Hz (i.e., 10 times per second) and another one reading data at 20 Hz, the second system will report twice the area, even for exactly the same detector settings.

???

Tom, peak area is the sum of (voltage measurements multiplied by time of duration). Do you remember those tall rectangles - bars one by one, resembling integration time ? When you increase sampling rate by 2 you parallely decrease "rectangle" width by 2 (on time axis) and area stays unchanged. We simplify here and bear in mind that low frequency sampling leads to loosing apexes.
In other words reading time from your wristwatch more frequently won't advance time at all.

in somewhere at chromatography forum on internet i read that there is a photomultiplier tube used in UV/Vis detectors which is may be one of the region for higher sensitivity as compared to DAD.

I was going to mention this exact point - that different photomultiplier tubes can yield at least slightly different responses, other things being equal (which in a PDA vs. UV, even from the same vendor, is rarely if ever true).

In addition to DR's comment, what about the fact that the physical optics in a PDA compared to a standard VWD are completely different.

Let's see what can cause the differences:
1) different optics
2) different detector....PMT vs Diodes
3) different flow cells
4) different aquisition rates
5) different slit widths pre / post flow cells
6) different lamp life
7) different a/d conversions
and should I go on and on.....

They are different detectors.......

Let's see what can cause the differences:
1) different optics
2) different detector....PMT vs Diodes
3) different flow cells
4) different aquisition rates
5) different slit widths pre / post flow cells
6) different lamp life
7) different a/d conversions
and should I go on and on.....

They are different detectors.......

PMTs often have a gain setting where most PDAs don't if I recall correctly. It might be possible to adjust the gain of a PMT to match the signal response of a PDA if you really need similar signals, but otherwise, yea, they are just two different detectors with difference responses to the same analyte.