Can anybody HPLC protocol for lactic acid isolation from bacterial culture?
Certainly! Here's a basic HPLC protocol for lactic acid isolation from a bacterial culture:
Materials:
HPLC system equipped with a UV detector
C18 reversed-phase HPLC column
Mobile phase: aqueous solution of 0.1% H3PO4 (pH 2.5)
Sample of bacterial culture containing lactic acid
Procedure:
Centrifuge the bacterial culture at 10,000 x g for 10 minutes to pellet the cells. Remove the supernatant and filter it through a 0.22 μm syringe filter to remove any remaining bacterial cells or debris.
Inject a small volume of the filtered culture supernatant onto the HPLC column. Use a sample loop appropriate for the desired injection volume.
Set the flow rate to 1 mL/min and use the mobile phase to elute the lactic acid. The retention time for lactic acid is typically between 5 and 10 minutes, depending on the specific conditions of your HPLC system.
Monitor the eluent with the UV detector set to 210 nm. Lactic acid has a strong absorbance at this wavelength, which allows for sensitive detection.
Collect the eluate containing lactic acid in a clean, dry vial.
To quantify the amount of lactic acid in the sample, compare the peak area or peak height of the lactic acid peak to a calibration curve generated from known concentrations of lactic acid standards.
Note that this protocol is intended as a starting point and may need to be optimized for your specific bacterial culture and HPLC system. Additionally, appropriate safety precautions should be taken when working with bacterial cultures and HPLC systems.
From some reference sources verify before applying
For lactic acid quantitation I would think 1st of Elissa or a Boehringer-Ingelheim kit (we used them for cheese analysis).
Hello, before injection of a small volume into HPLC just add 4N sodium acetate 1/10 volume and freeze the sample to precipitate the proteins, centrifuge the sample at 10000g, and then inject supernatant.
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