Mobile-phase adjustment

Regarding the type of organic modifier, there has not been any particular reason for selecting methanol or acetonitrile in the preparation of the mobile phase. Adjusting the mobile phase at a pH value lower than 3.0, the ionization of the carboxylate moiety is suppressed and increased retention of the analytes can be achieved. Under these conditions, however, problems may appear due to the high sensitivity of some penicillins at these pH values. 

A procedure for the analysis of fruit for residues of postharvest preservatives and ethoxyquin was described by Gieger (132). Residues were extracted with dichloromethane via ultrasonic treatment. The extracts were analyzed on a C,8 5-/zm column, with methanol/0.01 M ammonium acetate (61 39) as a mobile phase adjusted to pH 7.5-7.7. Recovery for ethoxyquin was 65-78%, with a detection limit of approximately 0.05 mg/kg. 

An example of two ion exchange separations are shown in Figure 5-32. The first separation is of phthalic acid, terephthalic acid and isophthalic acid using a 10-mM boric acid mobile phase adjusted to pH 9.2 using sodium hydroxide. The other separation in Figure 5-32 is of caffeine, w-acetyl-p-aminophenyl, aspirin, and salicylamide. In order for all of these compounds to be eluted in a reasonable time, the 10-mM boric acid solution also contains 20 mM sodium nitrate for ionic strength adjustment. 

The product, 7-hydroxycoumarin, was separated on a Nov-Pak C8 column (3.9 mm X 150 mm). The mobile phase, adjusted to pH 3.5, was a 35 65 mixture of methanol and 1% acetic acid. The flow rate was 1 mL/min. The postcolumn eluate was mixed with 1.0JV NaOH pumped at a flow rate of 0.5 mL/min. Complete mixing occurred via a T-joint followed by 1.5 m of Teflon tubing. The fluorometer was set to use excitation and emission wavelengths of 368 and 456 nm, respectively. 

Wrong pH value of mobile phase. Adjust pH. For basic compounds lower pH usually provides more symmetric peaks. 

Sample preparation 1 mL Serum -i- 50 pL 4 pg/mL nisoldipine in mobile phase, adjust to pH 9 with 1 M KOH, add 0.5 g KCl, add 3 mL diethyl ether, shake gently (Fisher Roto-Rack) for 10 min, centrifuge at 1000 rpm for 15 min. Separate and retain ether layer, extract aqueous layer as before. Evaporate tdl ether layers to dryness under diy nitrogen... 

Figure 5-20. Retention factor as a function of concentration of perchlorate anion on basic analyte. A pH of mobile phase adjusted with perchloric acid,...

B 50 mM sodium phosphate buffer with pH adjustment using perchloric acid, C pH of mobile phase adjusted with perchloric acid and NaClO salt then added. 

For the optimization of nano-HPLC separations of GSL extracts, reverse-phase (RP) HPLG with a Cjg column and amidophase under different mobile phase adjustments in both isocratic and gradient elution mode can be considered for neutral and acidic GSLs. Adequate conditions for separation criteria, considering either the ceramide or the carbohydrate moiety, were elucidated using different solvent mixtures. As the matrix solvent exerts an influence on ionization efficiency, its composition must be adapted accordingly so as to achieve ionization of both the major and minor components present in the fractions. 

Sample solvent incompatible with mobile phase. —> Adjust solvent. 

Minocycline and four production impurities (e.g., 4-epiminocycline, 6-deoxy-6-demethyltetracycline, 7-didemethylminocycline, 7-monodemethylminocycline) were baseline resolved on a Cg column (A = 280 nm) using a 20/55/20/5 DMF/water (0.2 M ammonium oxalate)/water (0.1 M EDTA)/water mobile phase adjusted to pH 7.0 with tetrabutylammonium hydroxide [1369]. The author noted that with DMF levels of more than 25% in the mobile phase, precipitation of the mobile phase EDTA occurred. In these solvents a decrease in the concentration of the EDTA to 0.01 M did not prevent a precipitate from forming. Elution was complete in 40 min. 

In the mixture design statistical approach, first the appropriate solvent system is chosen, and the composition of each mobile phase adjusted. Then the seven experiments are carried out and the values measured. These values are then substituted into the following equation, in order to establish the values of the constants ... 

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