Sample pH issues

[magda roth]

How critical is the pH of the sample in HPLC analysis. I am doing lactic acid analysis. Sample prep includes a hydrolysis step with NaOH (high pH), neutralization with H2SO4 conc to lower the pH to get the acid in COOH form. If the pH of the sample gets very low (about 1 or less, is this issue for the column’s life? Mobile phase is water acidified with H2SO4 to pH 2, injection volume 20-50 microliters, flow 1.0 mls/min. Column is Apollo C18 silica from Alltech Grace (supplier says is fine in pH range 1-8). I think I am fine, but would like a “second opinion . As long as we do not charge health care fees!

[tom jupille]

Assuming you're using a "conventional" column (something in the 4 or 4.6 mm id class) that 20-50 microliters gets diluted with mobile phase fairly quickly. If you want to be doubly certain, put in a guard cartridge and let *that* take the hit.

[magda roth]

Assuming you're using a "conventional" column (something in the 4 or 4.6 mm id class) that 20-50 microliters gets diluted with mobile phase fairly quickly. If you want to be doubly certain, put in a guard cartridge and let *that* take the hit.

Thanks Tom, you are the BEST!!!

[mariareese]

Sounds good.. such a nice and helping nature you have. Keep helping .. god bless..

[Consumer Products Guy]

Mag - we validated a similar test procedure for GLP.

We added 10 ml 0.5 N NaOH to saponify all lactic acid esters, then added 10 ml 0.5 N H2SO4 to neutralize remaining NaOH and to drop pH of the sample. We also used dilute H2SO4 as the mobile phase.

By the way, we also used Na lactate as the standard, as lactic acid solutions (such as USP grade) all have a certain amount of internal ester; this is why the USP procedure for lactic acid content uses saponification and back titration.