UV-1084 HPLC problem

[creature]

good evening to everyone,

I use HPLC to determine UV-1084 in PE
My method uses:
mobile phase: isopropanol/tetrahydrofuran 80/20:93% and 0.2%HAC: 7%
column: KROMASIL 100 C18 5μm 33x4.6mm
perkin elmer series 200 LC pump
785 A UV/VIS detector
column: brand new
lamp: new

problem: no peaks, even when standards made of 100% nickel compound powder(UV-1084) are injected
When i inject isopropanol i take its peak so detector is ok?
Any ideas?
thanks in advance
Philip

[creature]

anyone?

[tom jupille]

When i inject isopropanol i take its peak so detector is ok?

Not necessarily. In fact, since you have IPA in your mobile phase, I would expect injecting pure IPA would give only a small refractive index anomaly peak at t0.

What wavelength are you using?

Also, have you run this method successfully in the past? If so, is anything different? Do you have an old column you can swap in temporarily?

[creature]

Now it works, baseline is smooth, but the peak of the compound comes out at the same time with the peaks of the IPA and the THF while in the past with the same method mobile fase peaks apeared at about 1min and compound peak at 5min. how can i make the peak of the compound come later?
wavelength is 278. I have two new identical columns, did swap from one to the other, same results.
thanks for responding.

[tom jupille]

My guess is that you are essentially doing HILIC, partitioning between a water-rich layer on the surface of the silica and a relatively water-poor mobile phase. That mixture of IPA/THF complicates matters, and it's difficult so sort out what's going on from a distance.

If you can contact the developers of the method they may be able to give you some insight into what's going on and how to increase retention. My first inclination would be to decrease the aqueous content (the water/acetic acid portion) in small increments (maybe a percent or so at a time), making sure you allow a long equilibration time between changes.

[carls]

This additive is very hydrophobic.

Decrease the IPA/THF in 5% increments until you get the desired retention.

If this method has worked in the past without modification try remaking the IPA/THF (and verify the proportions specified in the method)