High performance liquid chromatography transfer volume

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

Particulate sorbents are available almost exclusively in the shape of micrometersized beads. These beads are packed in columns and represent currently the most common stationary phases for high-performance liquid chromatography (HPLC). Despite their immense popularity, slow diffusional mass transfer of macromolecular solutes into the stagnant pool of the mobile phase present in the pores of the separation medium and the large void volume between the packed particles are considered to be major problems in the HPLC of macromolecules, frequently impairing their rapid and efficient separation [1]. 

The injector is an interface that achieves reproducible and accurate transfer of a sample volume between the system operator and the chromatographic column in a front (band) as narrow as possible. Usually, for analysis by high-performance liquid chromatography (HPLC), samples are prepared in a liquid state (as a solution in an appropriate solvent or mixture of solvents, obtained as a result of sample preparation of solid or liquid samples). For practical reasons (avoiding column overloading), even for analytes in a liquid state under ambient conditions, dilution in appropriate solvents is more often used. 

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