Classical HPLC Buffers

Which buffers are preferred for an application depends primarily on the choice of detector. For this reason, we distinguish between classical HPLC buffers, which are used with UV detection, and MS-compatible buffers, which are highly volatile. We discuss first the classical buffers. 

In LC/MS, the typical buffer concentration used today is around 10 mM. This results in a reduced control over retention compared to classical HPLC. At the same time, the specificity of the mass spectrometer compensates for the sloppiness... 

Solvents and additives Purity of solvents Number, position, and width of solvent signals Additives such as acids, bases or buffers should not give rise to additional signals in the NMR spectrum Use of NMR-pure solvents Use of solvents that show only one signal, such as acetonitrile, acetone, and water Use deuterated solvents (capillary techniques) or at least replace water with DjO for classical HPLC-NMR Use deuterated or perhalogenated analogues of additives... 

High-performance liquid chromatography (HPLC) is a well-established separation technique it is able to solve numerous analytical problems and there is the possibility of acting on the mobile phases with appropriate additives to improve the quality of the peak. Of course, any additive must be compatible with the MS detector nonvolatile buffer or eluent additives cannot be used strong acids such as trifhioroacetic acid (TFA) may cause significant signal suppression in positive ionization. Different stationary phases are used as an alternative to the classical C18 Phenyl, HILIC, fluorinated, etc. 

The classical measurement of LogP is the shake flask method [17]. A known amount of drug is dissolved in a flask containing both octanol phase and aqueous buffer at controlled pH to ensure the existence of only nonionic form (at least two units from the drug pA) ). The flask is shaken to equilibrate the sample between two phases. There must be no undissolved substance present in both phases. After the system reaches its equilibrium, which is time- and temperature-dependent, the concentration of drug is analyzed by HPLC in both phases. Partitioning coefficient is calculated as... 

The modulator (in NPLC, a strong, polar solvent, or in IXC, a buffer) or the organic modifier (in RPLC, methanol, acetonitrile, or THE) may affect the retention of the components of the sample in different possible ways. In the most classical case, such as in ion-exchange chromatography or in normal phase HPLC, the... 

Many approaches to determine thermodynamic solubility in drug discovery have focused on miniaturizing the classic shake-flask method with solid samples manually weighed and dispensed into 96-well plates or 96-vial arrays [32-34]. The 96-well format enables the use of liquid handling robots to improve throughput. As with the standard shake-flask method, aqueous buffer is added and the solution is agitated for a minimum of 24 h prior to plate filtration or centrifugation to remove the supernatant, which is then analyzed by HPLC-UV or direct UV. 

The classical procedure for reversed-phase HPLC of oligonucleotides involves chromatography on octadecylsilyl columns in ammonium or triethylammonium acetate buffer with an acetonitrile gradient (11). 

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