Chromatography Forum

what is the difference between analyzing parameters of UV-spectrophotometer and HPLC UV detector....
if in both techniques we are using same detector then why there is difference in sensitivity?????

I'm no expert, but I can offer some ideas...

LC detectors are built to measure absorbance, allowing very small volumes of sample to be used. To do this, they have anywhere from <1mm up to 5cm effective cell lengths while being quite small in all other dimensions. Their signals are also highly amplified, optics highly focused etc. etc. etc. This is all allowable without loss in precision as the position of everything is more tightly controlled than in a typical UV spec.

UV specs use a variety of flow cells that are (compared to a LC) loosely held in place and can have path lengths of (typically) 5mm-10cm. This means that the optical bench must be much more flexible, and therefore, have a smaller dynamic range.

shantaram, are you talking about using a conventional spectrometer with a flow cell in HPLC?

To elaborate a bit on the previous response:

Although we think of both spectrophotometers and HPLC UV detectors as measuring absorbance, this is incorrect. Both systems measure transmittance (the fraction of the incident light that is transmitted by the flow cell). They calculate absorbance (the negative log of transmittance). This may sound like a distinction without a difference, but it's important because the process involves measuring (potentially small) differences in large values. That means that maximizing signal/noise ratio at the photodetector is critical. To oversimplify a bit, photodetector noise is roughly proportional to the square root of the light intensity (signal). Therefore, the more light you can get to the photodetector, the better your s/n ratio and the better your sensitivity.

For a given light source and flow cell path length, two things limit the amount of light available: the cross-sectional area of the cell ( a bigger cell can capture more light), and the slit width of the monochromator (this controls the bandpass; a wider range of wavelengths will contain more light).

In HPLC, minimizing extra-column volume is critical, so an HPLC detector will be designed with a very narrow cell (typically, 1 mm diameter) but because spectral resolution is not very important, the slit width or bandpass values are typically large. As DR pointed out, the very narrow flow cell requires precise alignment with the optical bench.

In a spectrophotometer, the sample cell can have a very large cross-section, but spectral resolution is very important. A spectrophotometer will thus typically be designed with a large cell and a narrow bandpass.

If I were less long-winded, I'd simply say that spectrophotometers are designed around spectral constraints, while HPLC detectors are designed around spatial constraints.

To ... the more light you can get to the photodetector, the better your s/n ratio and the better your sensitivity...
two things limit the amount of light available: the cross-sectional area of the cell ( a bigger cell can capture more light), and the slit width of the monochromator... spectrophotometers are designed around spectral constraints, while HPLC detectors are designed around spatial constraints.

Tom, thanks for the "long winded" answer - us newbies depend on these plainly worded explainations!
I think my last lab used the spec to sub for lack of scanning ability on our HPLC, we could scan for wavelength on the spec to get us in the right neighborhood for the HPLC... Probably not a best practice but it was better than just guessing...

Jade, there is nothing wrong with that if one has enough stuff to see it in the conventional spectrometer. I tried this the other way around once. To get a better spectrum I collected the HPLC peak, put the liquid in the spectrometer and was shock-surprised that nothing could be seen. It turned out that all the spectrometers I ever used have a sensitivity of about 1/100 of that of my detectors.

Using a lab spectrophotometer to determine the absorbance maximum for a UV detector is a good idea, as long as you have enough material to do this. Many LC UV detectors can scan, but you have to do an infusion or stop the flow to do this, you can't scan fast enough to get the entire spectrum during the time it takes for the peak to elute. That's one reason why some people buy photodiode array detectors, they can collect the full spectrum to determine the absorbance maximum, then for routine operation you can monitor only that wavelength. It is similar to the difference in scan and SIM in mass spectrometry.

Linear used? to make a detector which scans the spectrum in less than a second with 1 nm resolution. I have one in the lab. A peak which was too short for this was never encountered. Never did a stop flow with that one. Sensitivity is higher than in most, if not all, array detectors.