Chromatography Forum

Our lab has recently acquired the Microsolv HPLC qualification kit which requires a stop flow wavelength scan with a "wavelength calibration solution". I have an Agilent 1100 DAD with the newest version of Chemstation installed. I am supposed to program in 5 NIST wavelengths then scan over ±5 nm.
has anyone ever run this method with the Chemstation/DAD? Is there a way to program it automatically? I have been trying for hours to figure out how to determine the absorbance values over this range and am coming up with nothing. Do I have to do this manually? How then do I document my values?

*What do the directions say ?

You might want to speak to the company that sold you the kit for more instruction. I am not a big fan of these "qualification kits" as you really should develop an instrument qualification and validation procedure according to a set of SOP's and Policies that you generate for your own system. You can sometime use the "kits" as templates to develop your own procedures from, but you should invest in some help (outside or inside your lab) in this area if it is unfamiliar to you.

Stopped flow scanning is something we do when you only have a single wavelength detector (VWD) with the scanning option (as all of HP/Agilent's VWD's have). This technique is used because the user does not have a real scanning detector such as a DAD to monitor several wavelengths. The VWD's can be configured to scan a sample (e.g. 200 to 500 nm) in the flow cell once the pump flow is set to zero and the sample is trapped inside.

If you have a DAD (or even a VWD), then you should be able to run the built-in wavelength calibration test which places a holmium oxide glass slide in the light path. This provides and compares stored wavelength values against those measured per the old NIST 2034 guidelines (usually five of the wavelengths are used). You can also make up your own holmium oxide solution (4% HO in 10% perchloric acid) or use other common standards such as caffeine for calibration comparison.