Acetonitrile blending with buffers

Semisolid samples such as muscle and liver tissues can be homogenized by blending with water or an appropriate aqueous solution such as a buffer in a mechanical or an ultrasonic device to expose the residue to the extraction solvent. Fatty tissue samples are sometimes subjected to heating at 40 or 60 C until fat becomes liquid, prior to extraction of the analytes with hexane (433) or acetonitrile (434), respectively. An alternative pretreatment approach is the enzymatic digestion of the tissue by means of proteolytic enzymes such as subtilisin A (429, 435-437). 

FigurB 25-26 Application of the method development triangle to the separation of seven aromatic compounds by HPLC. Column 0.46 x 25 cm Hypersil ODS (C)e on 5-(j.m silica) at ambient temperature ( 22°C). Elution rate was 1.0 mL/min with the following solvents (A) 30 vol% acetonitrile/70 vol% buffer (B) 40% methanol/60% buffer (C) 32% tetrahydrofuran/68% buffer. The aqueous buffer contained 25 mM KH2P04 plus 0.1 g/L NaN3 adjusted to pH 3.5 with HCI. Points D, E, and F are midway between the vertices (D) 15% acetonitrile/20% methanol/65% buffer (E) 15% acetonitrile/16% tetrahydrofuran/69% buffer (F) 20% methanol/16% tetrahydrofuran/64% buffer. Point G at the center of the triangle is an equal blend of A, B, and C with the composition 10% acetonitrile/13% methanol/11% tetrahydro-furan/66% buffer. The negative dip in C between peaks 3 and 1 is associated with the solvent front. Peak identities were tracked with a photodiode array ultraviolet spectrophotometer (1) benzyl alcohol (2) phenol (3) 3, 4 -dimethoxyacetophenone (4) m-dinitrobenzene (5) p-dinitrobenzene ...

The most popular phase is octadecyl (C18 or ODS), and most separations are reversed-phase, the mobile phase being a blend of methanol or acetonitrile with water or an aqueous buffer. For weakly acidic or basic solutes, the role of pH is crucial because the ionized or protonated forms have a much lower affinity for the ODS than the corresponding neutral species, and are therefore eluted more quickly. The dissociation of weak acids and the protonation of weak bases are shown by the following equations... 

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