Reversed-phase liquid chromatography required method performance

Analyses to quantitatively relate the log k values to a combination of molecular interaction energy values, solvent effects, and/or electron localization energy values (calculated as HOMO or LUMO) in reversed-phase liquid chromatography, have been performed. The practical application of this method requires a limited number of standard compounds for column calibration. 

As a consequence of the previous considerations Kieber et al. [75] have developed an enzymic method to quantify formic acid in non-saline water samples at sub-micromolar concentrations. The method is based on the oxidation of formate by formate dehydrogenase with corresponding reduction of /3-nicotinamide adenine dinucleotide (j6-NAD+) to reduced -NAD+(/3-NADH) jS-NADH is quantified by reversed-phase high performance liquid chromatography with fluorimetric detection. An important feature of this method is that the enzymic reaction occurs directly in aqueous media, even seawater, and does not require sample pre-treatment other than simple filtration. The reaction proceeds at room temperature at a slightly alkaline pH (7.5-8.5), and is specific for formate with a detection limit of 0.5 im (SIN = 4) for a 200 xl injection. The precision of the method was 4.6% relative standard deviation (n = 6) for a 0.6 xM standard addition of formate to Sargasso seawater. Average re-... 

In the past decade, we have benefited from major improvements in the sensitivity and efficiency of both chromatography and electrophoresis. High-performance liquid chromatography (HPLC) has become firmly ensconced as a powerful method for protein analysis. Ironically, reversed-phase chromatography, which employs denaturing conditions, has found a particularly wide application, at least when only analytical information is sought. This is the case in most routine analytical work required for many biotechnological applications. 

The stable nonvolatile intermediate phenylthiocar-bamoyl derivatives are formed in basic media and can be analyzed directly by reverse-phase high-performance liquid chromatography (RP-HPLC). Their cyclization into hydantoins requires acid catalysis. This mode of derivatization is a very important supplement to the Edman s method of N-terminated sequencing of polypeptides. Before GC analysis, any hydantoins can be converted into N-trifluoroacetyl or enol-O-trimethylsilyl derivatives, which increases the selectivity of their determination in complex matrices. 

Reversed-phase high performance liquid chromatography (HPLC) is the preferred method of quantifying domoic acid for both regulatory and research applications. Detection can be accomplished using either UV absorbance or fluorescence, although the latter requires derivatization of the molecule but also confers greater sensitivity (Table 20.1). 

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