I have a bacterial culture in which acetate is present.I want to know the uptake rate of acetate from the media.
I did NMR but didn`t get much satisfactory results. Should I go for the HPLC or should I repeat NMR?
Hello Shikha,
I am guessing, it is not the measurement, but the system that has issues.
Acetate is volatile (if not highly), and no matter which salt or pure form of it you are giving, could be escaping to the ambience of the flask/ Erlenmeyer you are using.
Better to make a closed system, or use SPME kind of traps (home made charcoal ones) to recover all in the ambience of the tube/ flask itself to not let the excess escape.
Let me know if that makes sense. Or, if you smell acetate when opening the tubes and that's a lot- salting out of volatile in presence of media salts/ constituents. But, if that's not the case, may be what you are adding is all used up quickly, and then you are measuring noise.
I can suggest GC, but then NMR is robust enough to measure- just concentrate more of the media to get a signal first (also add labelled 13C acetate to the NMR tube, right before measurement) to see if you still get a signal or not. : )
Thanks,
Amended on 19 March 2019
This is surely a wit question.
Please utilize HPLC-photometric method, but do not use HPLC-MS. HPLC should be used under the high-recovery conditions. Thus, RP-HPLC should be performed by using Gradient elution method, which includes column-washing (please see file; Lysozyme by RP-HPLC). HPLC-SEC method should be performed by the addition of non-ionic detergent inorder to get high-recovery condition (please see file; JCB Fucoidin Transport).
I have long studied about biochemistry with HPLC (from 1980 - 2015). Choice of reliable HPLC pump seems to be important. I have utilized HPLC pump of Waters, LKB, Hitachi, Nihonbunko, and Shimadzu. The pump of GE Healthcare is not the HPLC pump at all. I must say that the HPLC pump of Shimadzu is best and reliable, since my c.a. 80 articles have used HPLC system of Shimadzu.
Silica gels (for ODS and SEC) of Nomura Chemical Co., Ltd., Seto-city, Aichi, Japan, and ODS gel of Macherey, Nagel & Co., Düren, Germany, seem to be best for me.
Acetnitrile/AN and Trifluoro acetic acid/TFA should not to be used, since recovery of sulphur-containing molecules is low, and not suited to Gradient elution (please see file; PTH Roma RP-HPLC).
Non-ionic detergent for HPLC-SEC (PGC/polyethylene-glycol 1000 monocetyl ether/Brij-58) should be utilized of Wako. Molecular mass distribution of Brij-58 of Sigma is wider than PGC of Wako (please see file again; JCB Fucoidan transport).
Carboxylic acid for RP-HPLC analysis should be derivatized by using 9-Anthryldiazomethane/ADAM (Funakoshi Pharmaceutical, Tokyo, Japan) (please see file; Netherlands Biotin). It is noteworthy that HPLC-Affinity-photometry should be performed by the Gradient elution method as RP-HPLC (please see file; Lipoic acid Avidin).
NMR signal seems to be interfered and resisted against Beer-Lambert law. Direct utilization of photmeter without purification is also not quantitative due to violence against Beer-Lambert law; i.e., ELISA, MTT, and Nanodrop are non-quantitative.
Enzyme assay with direct using photometer is bad since determination of molecules can ony be performed after the purification. This is the true meaning of the Beer-Lambert law. Please utilize HPLC-photometric enzyme assay (please see file; J Chrom B Rat BIN LIP Km).
Thank you so much Kou Hayakawa for the suggestion. I will definitely try this method.
Biswapriya Biswavas Misra I don`t think that it is getting evaporated since tubes are closed before the run so may be the integration method is bit crude.
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