I chanced upon a HPLC protocol that uses acetonitrile, Potassium phosphate and tetrabutylammonium bromide as the solvent for reverse phase chromatography. I believe potassium phosphate is most probably present to balance the charges of proteins but am not particularly sure about the use of tetrabutylammonium bromide. I heard that it serves as a counterion but is this not the same as that of potassium phosphate? Also, may I know when are these compounds required and are there any other counterions that can be used instead?
Thank you all in advance.
Generally speaking there is no good reason to use an ion pair reagent for retention of proteins on a reversed phase column. Furthermore, it's not a good idea to use a cationic ion pair reagent on a C-18 silica reversed phase column unless you plan to dedicate the column to that application since it's quite difficult to remove the cationic reagent quantitatively. Finally, it's generally a bad idea to use a bromide salt with a UV detector since bromide absorbs in the UV. You would be a lot better off using tetrabutylammonium phosphate assuming you have a good rationale in spite of the issues noted above.
Tetrabutylammonium bromide serves as an ion-pair reagent. It complexes charged molecules enabling us to do, for example, a nucleotide separation with a C18 column, e.g. ATP analysis.
It is used inl reverse-phase ion pair-high performance liquid chromatography for separation of heparin, heparan sulfate and low molecular weight-heparins disaccharides and oligosaccharides. Tetrabutylammonium bisulfate as ion-pairing reagent. Both heparin/heparan sulfate disaccharides are generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit. It is resistant to enzymatic action. This used in HPLC method to separate and quantify uncommon disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation.
Ravi, may I know what's the reason behind the increased sensitivity as compared to ion exchange? Dogan, I suppose it can bind differentially to different states of nucleotides? Ie ATP and ADP? How about single inorganic phosphate?
Ion pairing agents like TBA+ in RP-HPLC generally are used to sharpen peaks and improve separation efficiency, which will affect signal-to-noise and LOD/LOQ, as well as peak capacity. The potassium phosphate is added to control pH; in RP-HPLC of many compounds, it is very important to choose a pH with the pKa's of the compounds to be separated in mind, to manage co-migration. Your choice of ion pairing agent and/or buffer will depend entirely on what kind of analytes you are trying to separate. Can you give more information about your analytes?
Hi Abigail, I'm analyzing the relative abundance of GTP, GDP and GMP. The use of TBA bromide has been suggested in some papers but I wonder if alternatives can be used. Thanks!
Hi Adam, I haven't done any method development on nucleotides myself, but since you are trying to separate small anionic compounds, its makes sense that you would need a cationic ion paring agent like TBA+. Is there a reason you don't want to use TBA+? Everything I've encountered is some form of quaternary ammonium ion. There are many variants of this available commercially. The phosphate buffer won't replace the ion pairing reagent, but it can be used to control the charge state on the three nucleotide phosphates and tune selectivity. If you want for some reason to eliminate the ion pairing reagent, you are probably in for some serious method development. Good luck!
Ion-exchange chromatography systems have previously been utilized in HPLC analysis of ionic samples. Recently, reversed phase partition chromatography using ion-pair reagents has been developed and utilized. The ionic samples form an ion-pair with ion-pair reagents in the mobile phase to become electrically neutral. The increase in hydrophobic character of the ion-pair results in a greater affinity for the reverse stationary phase and leads to sample resolution.
UV and fluorescence detectors are widely used. Therefore ion-pair reagents must lack UV absorption and fluorescence themselves to obtain highly sensitive detection of samples. The UV absorption of sodium alkanesulfonates and quaternary ammonium salts is minimal so that these reagents can be used for reliable HPLC analysis. On the other hand, when a sample lacks sufficient UV absorption or fluorescence, the use of sodium 9,10-dimethoxyanthracene-2-sulfonate allows for high-sensitivity detection as a fluorimetric ion-pair reagent.
Recently, use of LC-MS in which mass spectrometry is incorporated in HPLC as a detector has become widespread. Sodium alkanesulfonates, a general ion-pair reagent, being non-volatile crystals pose a problem in that they contaminate the interface.
TBA Bromide in this case is used as a cationic ion-pairing reagent to improve retention of organic analytes that need to be separated in HPLC using C18 Silica columns. Other cationic counterions like cetylpyridinium bromide may be used if your analyte is anionic in character.
Generally speaking there is no good reason to use an ion pair reagent for retention of proteins on a reversed phase column. Furthermore, it's not a good idea to use a cationic ion pair reagent on a C-18 silica reversed phase column unless you plan to dedicate the column to that application since it's quite difficult to remove the cationic reagent quantitatively. Finally, it's generally a bad idea to use a bromide salt with a UV detector since bromide absorbs in the UV. You would be a lot better off using tetrabutylammonium phosphate assuming you have a good rationale in spite of the issues noted above.
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