Peak from water at 210 nm

[carnie]

I'm using a C8 (4.6 x 250) Inertsil column, the mobile phase is 0.23% perchloric acid/acetonitrile (65:35) premixed, isocratic. The column temperature is 25C. injection volume 100 microliters, detection 210 nm. I'm hoping to use this method for determination of related substances however I'm getting a rather broad peak from water (the diluent) at about 10 minutes which will mask some of the related substances. I've changed HPLC, column, water and the peak is always there. Is this typical at 210 nm? Or is there something strange going on?

[Bruce Hamilton]

100ul is quite a large injection. and I'm assuming about 1ml/min flow?.

With a 100ul injection, I'd be worried that some of the sample wasn't soluble in the mobile phase if the sample concentration is high..

What happens if mobile phase is injected, does the peak disappear?.
What happens if 10ul of water is injected, does the peak change?.
What happens if you inject 65/35 H2O: CH3CN without perchloric?>

If it's linked to the injection, I'd look to change that - add acetonitrile and perchloric, or reduce injection volume.

Please keep having fun,

Bruce Hamilton

[carnie]

Thank you for your ideas. Here are the results:

What happens if mobile phase is injected, does the peak disappear?: No, same
What happens if 10ul of water is injected, does the peak change?: Peak is still there only smaller (roughly proportional)
What happens if you inject 65/35 H2O: CH3CN without perchloric?: Peak remains but shape is tailing .

I've calculated that I can afford to decrease the injection volume to about 60 microlitres and still achieve the required sensitivity%. I can't easily change the solvent due to the preceeding procedures. I can't change the wavelength. Any other parameters that you think may be worth looking at please let me know.
Thanks again

[DR]

Try making your own MP & diluent with polished water that has been further filtered through an Empore extraction disk (search my posts featuring "Empore" - I've posted directions etc. at least once). It may help.

[carnie]

I didn't have the filters you mentioned but tried purging the water with helium before injection and the peak has disappeared.
Thank you all for your excellent advice.