Temperature and baseline with calibration problem

[LabProARW]

I have a couple of Shimadzu HPLC Prominence UV models. NOTE: This question also applies to my 1990s model Hitachi HPLC. I have a lab where the temperature sometimes varies 10 deg.F within a hour. I can see this reflected on my baseline. I am doing reverse phase chromatography and my column is in a temperature controlled oven. My calibration gets hosed up by this temperature variance. *So my question is what is it about temperature which throws a UV HPLC off so bad... especially considering I have a column oven? Is it the electronics, the viscosity of eluent, the dissolved gases in eluents, other??

Thank you for all concrete and knowledgeable answers.

[tom jupille]

Most conventional UV detectors have a certain amount of sensitivity to refractive index. We wish they didn't, but absent something like a total-internal-reflectance type cell, it's difficult to get a perfectly collimated light beam through the cell, so at least some of the light will enter the cell at an angle. If the RI of cell contents changes, then some of that off-angle light will not make it to the photodetector. As an aside, that's one of the reasons we often see the "mid-gradient hump" when running gradients.

Your column may be in an oven, but if the detector cell and (more important) the transfer line are at ambient temperature, then the RI of the mobile phase may change enough to cause a problem.

A trick I've used in the past (originally suggested by John Dolan) is to slit a piece of plastic tubing lengthwise and slip it over the transfer line to provide at least some insulation. And on one occasion, I resorted to cutting slits for the tubing in a large cardboard box and then putting the box over the detector (ugly and crude, but it decreased the baseline noise measurably).

[pwm]

Yes have heard too of insulating the capillary from column compartment to detector cell.

Which part of the calibration is getting hosed in what way?

Ive seen some people add a little back pressure to flow cell outlets using pressure regulator, or just short length of smaller ID tube. Key word little, you dont want to rupture the cell here. I believe this helps with dissolved gas problems, and less potential of out gassing in the flow cell. I have not tried myself, or have ever needed, but the theory does have some logic to it I suppose.