Low wavelength and area issues

[vikmurush]

I had a low area count issue on HPLC analysis. I am working at 195nm. Could any of you suggest me my options to increase the area count without changing my concentration. I am injecting 100ul right now on agilent 1100 series. What is the hight injection volume that i can inject on this instrument with out adding any kind of loop? If i change the wavelength is my area going to improve? Help on this issue

[Chris Pohl]

Vikmurush

If I were you, I would first check whether or not you have adequate light arriving at your detector at that wavelength. It's extremely difficult to work at that wavelength because nearly everything absorbs at that wavelength including many contaminants in solvents and water. To check this, increase the wavelength to a more reasonable wavelength, say 250 nm and then zero your detector. When you return to 195, to what level does your baseline rise (assuming your detector doesn't automatically re-zero when you change the wavelength). If your background is above 1 AU than this might be the problem. If it's above 2 AU it's even more likely that this is the cause of the problem. In either case, most likely you would be better off working in a little bit higher wavelength say: 205 as your background will be a lot lower.

[vikmurush]

Thanks a lot Chris. I am working with 30% IPA that is why i am doing at low wavelength. I will try at 198 and 205 to see if i get good area counts and cleaner chromatograpy. If you have any more suggestions that will be great. thanks again

[AA]

If I recall, IPA has a UV cutoff of 210 nm (maybe even slightly greater), using 195 nm would be a poor choice for a solvent containing 30% IPA.

[vikmurush]

AA,

If i go to the higher wavelengths i am getting very low area counts and chrmatography not improved also. Also i am seeing area count reduction throughout the run. Any help will be great

[Chris Pohl]

vikmurush,

Switch to a different solvent. Acetonitrile of good-quality should be usable at the wavelengths of interest (good UV grade acetonitrile has a cut off of 190 nanometers) although it will take a good deal of attention to detail in order to avoid problems with contaminants when working at such wavelengths. In particular, you need to be concerned about trace contaminants in your water and any additives you might be adding to your mobile phase. For example, many buffer ions absorb at this wavelength including acetate, formate and citrate so you need to make sure that you use components suitable for this sort of application.

[vikmurush]

Thanks everybody,

I am currently using acetonitrile:water (45:65) as mobilephase. The sample i am injecting is in 30%IPA. We know that this compound is stable in 30%IPA. Column i am using C18. Instrument HPLC Agilant 1100 series. The problem i am having here is

solvents i am using are HPLC grade (even water)
1. Low area counts
2. Response dropping through out the run for same std

I want to know if there is any way that i could increase my area counts (without changing my loop size) and std response problem. I thought of using sample compartment cool down to solve std response reduction in the run but i have not tried yet. Any suggestion or help will be appreciated.

[bartjoosen]

How do you know that your component is stable in 30% IPA?
Maybe you have checked with UV and measured some degradation products?

I don't know a way to improve your area, but increasing the flow rate also increase peak height, maybe that helps?