Mobile phase preparation degassing

Mobile phase Prepare a filtered and degassed mixture of methanol and 1-penta-nesulfonate sodium solution (60 40). Make adjustments if necessary (see system suitability under Chromatography <621 >). 

Mobile Phase Prepare a filtered and degassed 87 13 v/v mixture of 0.025 M phosphoric acid (previously adjusted to a pH of 3.0 0.1 with triethylamine) and acetonitrile. 

Mobile Phase Prepare a filtered and degassed 65 35 mixture of acetonitrile water. 

Mobile Phase Prepare 0.005 N sulfuric acid that has been suitably filtered and degassed. 

Mobile Phase Prepare a filtered, degassed solution of 0.01 N sulfuric acid in water. 

All of the components for mobile phase preparation can be purchased from reputable or approved suppliers and any changes in supplier should be checked thoroughly before materials are used. Additional validation of the method may be required when there has been a change in supplier of a critical reagent. All mobile phases should be filtered and degassed prior to use. Further discussion in relation to mobile phase quality is given in Chapter 12. 

Although the method is simple and straightforward, there are a few important points to consider. First, since 200 pL are being injected on to a 2-mm diameter HPLC column, compatible solvents must be injected on to the column and the amount of acetonitrile used in the prepared samples should be exactly as directed. Second, the temperature of the column and the use of a degassing system for the mobile phase are critical components required to guarantee reproducible chromatography. In addition, the standards should be stored in a refrigerator when not in use. 

Left, Charlie Focht of the Nebraska State Agriculture Laboratory prepares the mobile phase for an atrazine assay. Note that the vacuum flask is positioned in an ultrasonic cleaner bath. Simultaneous vacuum filtration and sonication provide a more efficient means for degassing. Right, Charlie adjusts the flow rate setting on the HPLC pump. 

Using a graduated cylinder, measure 300 ml deionized water into a vacuum flask. Add 700 ml HPLC-grade acetonitrile to the flask. Mix solution with stir bar and magnetic stir plate. Degas the mobile phase daily in an ultrasonic bath under vacuum or using an in-line degasser. Prepare fresh for each analytical run. 

This compound is determined using HPLC analysis. A Diluent reagent is prepared as a 9 1 mixture of water and methanol. The Mobile Phase is prepared as a filtered and degassed solution by dissolving 5.6 g of monobasic potassium phosphate in 820 mL of water in a 1-liter volumetric flask, adjusting with phosphoric acid to a pH of 4.3, diluting with methanol to volume, and mixing. Adjustments in the composition may be made if required by the System Suitability requirements. 

According to the USP 26 and the Indonesian Pharmacopoeia, the assay for mefenamic acid is performed by a liquid chromatography method. A buffer solution is prepared as a 50 mM solution of monobasic ammonium phosphate, adjusted with 3 M ammonium hydroxide to a pH of 5.0. The system uses a filtered and degassed mixture of acetonitrile, buffer solution, and tetrahydrofuran (23 20 7) as the mobile phase. The liquid chromatograph is equipped with a 254 nm detector and a 4.6 mm x 25 cm column that contains packing LI. The flow rate is about 1 mL/min. The column efficiency is not less than 8200 theoretical plates, the tailing factor for the analyte peak is not more than 1.6, and the relative standard deviation for replicate injections is not more than 1.0% [1, 4]. 

Common HPLC problems are caused by component malfunctions (pump, degasser, injector, detector, data system, column), and faulty preparation of the mobile phase or sample preparation. Problems can be categorized into several areas ... 

Degassing your mobile phase solvent-this is an important step and the best approach is to prepare the solvent composition (e.g. 50 50 v/v methanol water, for isocratic RPHPLC) and then filterthrough a 0.22 porosity filter using a Buchner flask arrangement (p. 28). 

This procedure guarantees a reproducible preparation of your mobile phase and avoids problems associated with volume contraction. Always prepare a sufficient amount of mobile phase (approx. 1 1) and degas it. Degassing is possible with helium or the built-in degasser (see Tip No. 5). If you use buffers, you should filter them through a membrane. The eluent container should be well covered in order to avoid dirt contamination. 

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