Chromatography Forum

Is it acceptable to use a USB spectrophotometer with a flow cell (12.5mm) as an HPLC detector?

Thank you!

If you can get your UV/Vis spectrophotometer to continuously read and collect data give it a try.

Our very first HPLC (mid-1970s) actually used a standard UV-vis with a small flow cell as its detector !

I suspect that you will find that the relatively huge volume of that cell will render the UV/VIS useless for LC work. Think about how long it should take to elute one of your peaks and your flow rate and how long it would take to fill the cell...

As Consumer Products Guy noted, a fair number of labs got this approach to work in the 1970's and early 80's. It requires a capillary flow cell (not a regular cuvette, like DR seems to be thinking of) that is aligned correctly to intercept the light beam of a conventional spectrophotometer. Some manufacturers (e.g., LKB) sold kits for the purpose. I might note that Waters' Z-cell for the detector in its CapLC is a refined version of this approach.

I suspect that you will find that the relatively huge volume of that cell will render the UV/VIS useless for LC work. Think about how long it should take to elute one of your peaks and your flow rate and how long it would take to fill the cell...

Cell volumes are available as low as .018ml, but I don't know how this compares to an ordinary detector.

Software might be an issue too, my spectro won't scan long enough!

A regular HPLC absorbance detector has a cell volume of about 8-10 µl for use at a flow rate of 1 ml/min. Lower flow rates necessitate smaller cell volumes so you don't lose your peak height to diffusion in the extracolumn volume. For microbore and narrow-bore columns, with flow rates of 50- and 200-µl/min, cell volumes of 0.5 and 1-2 µl are common.

Concerning your apprehension over the scanning rate: If you want to get a complete absorption spectrum of a peak on the fly, then the usual procedure is to shut off the flow when the peak enters the flow cell. This is not easy to do. If you just want to monitor absorbance at a single wavelength, then you sample the light beam on a timed basis using an A-to-D converter.