Liquid chromatography buffer

Ion-exchange chromatography involves an electrostatic process which depends on the relative affinities of various types of ions for an immobilised assembly of ions of opposite charge. The stationary phase is an aqueous buffer with a fixed pH or an aqueous mixture of buffers in which the pH is continuously increased or decreased as the separation may require. This form of liquid chromatography can also be performed at high inlet pressures of liquid with increased column performances. 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

While the apparent molecular weight was about 47,000 g/mol or daltons (Da) by mobUify on SDS-PAGE, separate analysis by sedimentation equilibrium measurements and capillary high-performance liquid chromatography (HPLC) in SDS buffer gave values near 23,000 Da. 

Chloroform, sodium chloride, anhydrous sodium sulfate, sulfuric acid (97%), hydrochloric acid (36%), sodium bicarbonate, trifluoroacetic acid, tris(hydro-xymethyl)aminomethane (Tris), special grade Water, high-performance liquid chromatography grade 0.1 M Phosphate buffer solution (pH 7.0)... 

Heat and reflux a 5-g portion of soil sample with 50 mL of methanol-phosphate buffer (pH 7)-water (15 7 28, v/v/v) solvent mixture in a round-bottom flask for 1 h. After cooling, transfer a 10-mL portion of the supernatant to a test-tube and mix with 11 mL of 0.02M H3PO4 solution. Load this solution on to a silica-based SPE cartridge (Analytichem International Clin-Elut 1020) at a flow rate of 1-2 drops per second. Discard this fraction. Elute the analytes with 30 mL of dichloromethane. Concentrate the eluate to dryness with air in a water-bath at a temperature of 40 °C (do not use vacuum). Dissolve the residues in 5mL of HPLC injection solution [900 mL of water - - 50 mL of phosphate buffer (pH 7) 4-50 mL of ACN 4-4 g of TBABr]. Pinal analysis is performed using liquid chromatography/ultraviolet detection (LC/UV) with a three-column switching system. 

Figure 8.43 Separation of enantiomers using complexation chromatography. A, Separation of alkyloxiranes on a 42 m x 0.2S mm I.O. open tubular column coated with 0.06 M Mn(II) bis-3-(pentafluoro-propionyl)-IR-camphorate in OV-ioi at 40 C. B, Separation of D,L-amino acids by reversed-phase liquid chromatography using a mobile phase containing 0.005 M L-histidine methyl ester and 0.0025 M copper sulfate in an ammonium acetate buffer at pH 5.5. A stepwise gradient using increasing amounts of acetonitrile was used for this separation.

Sternson et al. [58] used a high performance liquid chromatographic method for the analysis of miconazole in plasma. Miconazole was extracted from alkalinized plasma with n-heptane-isamyl alcohol (98.5 1.5) and separated by high performance performance liquid chromatography on p-Bondapak Ci8 with ultraviolet detection at 254 nm. The mobile phase was methanol-tetrahydrofuran-acetate buffer (pH 5) (62.5 5 32.5) containing 5 mmol octanesulfonate per liter. The flow rate was 2 mL/min. Recovery was 100%. The relative standard deviation for injection-to-injection reproducibility was 0.4% and that for sample-to-sample variation was 5% at high miconazole concentrations (30 pg/mL) and 1% at low (1 pg/mL) concentrations. The limit of detection was 250 ng/mL. 

High performance liquid chromatography-mass spectrometric methods Nitin et al. [75] developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometric method (LC MS MS) for the simultaneous estimation of bulaquine and its metabolites primaquine in monkey plasma. The mobile phase consisted of acetonitrile ammonium acetate buffer (20 mM, pH 6) (50 50, v/v) at a flow rate of 1 mL/min. The chromatographic separations were achieved on two Spheri cyano columns (5 pm, 30 cm x 4.6 mm), connected in... 

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