Chromatography Forum

Dear members,

I'm trying to develop a method to analyze a mixture of drugs by HPLC-UV. What I don't understand is that when I have a sample with a known concentration of analyte, the response (in absorbance units) is different if I analyze it by HPLC with a UV detector or by using just a UV spectrophotometer. Shoudn't the optical density be the same at equal concentrations regardless of the method used?

All the best,
Mada.

While the absorbtivities will be the same, the optical benches used are not. You will see response differences between a spec and a UV LC detector because of differences in lamps, their relative proximities to gratings, slits and cells...

LC detectors are very compact in an effort to improve sensitivity and reduce instrument size.
UV specs have the luxury of taking 0.5 to 10CM cells, so their designers seldom care if their lamps, gratings, cells and photomultipliers are all as close together as possible. Is you need more sensitivity, use a longer cell!
All LC detectors allow (generally speaking) is a change in cell volume. LC detectors that allow for a change in path length are rare (I think there's one by Thermo).

I think that's pretty well correct, but I'm not 100% certain that this entirely covers it.

Thank you for your answer!

I think I would like to add that the sensitivity of the UV spectrophotometer is higher than the one shown by HPLC-UV. For example, using UV, for a 0.01 mg/mL concentration, I get around 0.2 in absorbance and for the same analyte, I obtain the same absorbance for 1.4 mg/mL concentration using HPLC-UV. I really don't understant what it's happening.

I checked the optical path length of the measuring cell (UV-HPLC) and it is equal with the one from the UV spectrophotometer (1 cm).

These are both static measurements? If so, how do you place the sample in the HPLC detector? Are the instruments single or double beam? How did you zero the machines?

If you are doing an HPLC analysis, the drug is distributed and diluted throughout the peak volume. If you are simply squirting a standard into the HPLC detector, then the absorbance should be reasonably close to the bench spectrophotometer.

In HPLC-UV, I inject a small volume (20 microL) of the standard on a line of the pump, then the volume is carried in and through the column and then it reaches the UV detector. I zero the detector against mobile phase (water/acetonitrile 50/50 v/v).

When using the spectrophotometer, I fill the cuvette with the standard (2 mL) and read the absorbance against the solvent of the sample (tetrahydrofuran). I zero the detector with no cuvettes in the measuring chamber.

Read Mark's post...

a) inject your std directly into the detector by using a syringe with an adapter and check if its similiar

b) approximate with the "mean peak concentration" (don't know if this works, just an idea how I would approximate this)

mass on column: mpeak= Vinj*conc
Peak Volume: Vpeak=Peak width*Flow
mean peak conc: cpeak=mpeak/Vpeak

since this would estimate a rectangular peak use the half peak height for your calculations

How to compare a flowing system to a static one has been discussed some time ago. Let us know if you don´t find that.
Also, from your info I can´t tell whether your spectrometer measurement was correctly performed.

In HPLC-UV, I inject a small volume (20 microL) of the standard on a line of the pump, then the volume is carried in and through the column and then it reaches the UV detector. I zero the detector against mobile phase (water/acetonitrile 50/50 v/v).

When using the spectrophotometer, I fill the cuvette with the standard (2 mL) and read the absorbance against the solvent of the sample (tetrahydrofuran). I zero the detector with no cuvettes in the measuring chamber.

In order to compare the two UV readings the sample has to be dissolved in the same solvent. Zero the detector against the solvent in which the sample is dissolved (THF according to your post). Disconnect the detector, fill the flow cell with THF, zero, fill flow cell with sample, read. This can be easily done with a syringe. Use static readings. Then compare the detector with the spectrometer.

Thank you for your answers!

I'm going to inject my standard directly in the UV-HPLC detector as soon as I can find a syringe with a very small diameter for the needle.

All the best.

Don't use a syringe needle to fill the detector. Use the proper syringe to HPLC fitting adapter. You can get them from from your favorite supplier of HPLC accessories. If you don't have one, try Upchurch http://www.upchurch.com.