Gradient separations organic acids

With a modified gradient program, starting with a lower initial chloride concentration and a more shallow gradient, some organic acids such as lactic add, pyruvic acid, formic acid, acetic add, malonic add, hippuric add, fumaric add, citric acid, and maleic acid can also be determined in serum. The respective example in Eig. 9-209 is a first attempt, and shows that more time has to be invested to optimize the separation. 

RPC with mobile phases containing relatively high concentrations of salts (0.1-0.5M) is occasionally employed for separations of organic acids or bases [67-69], Eqnation 5.7 can often describe the effect of the concentration of organic solvents on the retention in these phase systems, so that Eqnations 5.8 and 5.9 can be principally used for calculation of the retention data in the LC with organic solvent gradients. 

Offline precolumn derivatization is the most common alternative in this respect it involves separating the esters obtained from the organic acids by reversed-phase chromatography, which amply surpasses solvophobic chromatography (i.e., the use of undissociated acids as such) and allows gradient elution techniques to be applied, thanks to the wider lipophilicity range covered by the derivatized compounds. 

Figure 1 shows the LC/MS thermospray total ion chromatogram of 0.25 p.g standard mixture of the six sulfonylurea herbicides. Gradient HPLC conditions were used to separate the six compounds in less than 25 minutes total run time. The mobile phase composition was kept isocratic at 30% acetonitrile/.05M formic acid for the first 15 minutes to separate the four herbicides HARMONY, ALLY, OUST and GLEAN. A gradient from 30% acetonitrile to 60% in 10 minutes was then used to elute EXPRESS and CLASSIC. An acidified mobile phase is used with sulfonylureas to keep them in the undissociated form which is retained on the HPLC column (3). Organic acids are recommended for use with LC/MS to prevent the formation of deposits in the mass spectrometer source and to prevent clogging of the thermospray interface probe tip. In this work we used formic acid. 

Figure 14.4 Separation of organic acids (as anions) and inorganic anions in wine (reproduced with permission of Dionex). Conditions column, 25cm x 4mm i.d. and precolumn stationary phase, lonPac AS11-HC mobile phase, 1.5 ml min nonlinear gradient from 1 mM to 60mM NaOH and from 0 to 20% methanol temperature, 30°C conductivity detector after packed suppressor. Peaks 1 = lactate 2 = acetate 3 = formate 4 = pyruvate 5 = galacturonate 6 = chloride 7 = nitrate 8 = succinate 9 = malate 10 —tartrate 11—fumarate 12 —sulfate 13 = oxalate 14 = phosphate 15 —citrate 16 = isocitrate 17 = c/s-aconitate 18 = frans-aconitate.

Consider the case of a pair of weak organic acids with Kd values of 3.0 and 3.5, whose pK values are 4.2 and 4.8, respectively. What are the capacity factors of these acids using an aqueous eluent at pH 1 At pH 4.4 At pH 7.0 How many plates would be needed to separate the acid which elutes first before 0.5% of the second elutes at each of these pH values Is a gradient elution scheme feasible here ... 

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