High performance liquid chromatography sample injection

FIGURE 24.6 FIA manifold for online dialysis coupled with HPLC. AS acceptor stream C carrier DC dialysis cell DM dialysis membrane DS donor stream HPLC high-performance liquid chromatography IV injection valve MCI and MC2 mixing coils PI and P2 pumps S sample W waste. 

High performance liquid chromatography (HPLC) is an excellent technique for sample preseparation prior to GC injection since the separation efficiency is high, analysis time is short, and method development is easy. An LC-GC system could be fully automated and the selectivity characteristics of both the mobile and stationary... 

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. 

Procedure Flavonoids are then further purified with 2 ml of methanolic HC1 (2 N), followed by centrifugation (2 min, 15 600 g), hydrolyzation of 150 il of suspension in an autoclave (15 min, 120 C). A reverse osmosis-Millipore UF Plus water purification system is used in high performance liquid chromatography (HPLC) with an autosampler. After injections of 5 pg of samples, the mobile phases flow at a rate of 1 ml/minute with isocratic elution in a column at 30 C. 

As a consequence of the previous considerations Kieber et al. [75] have developed an enzymic method to quantify formic acid in non-saline water samples at sub-micromolar concentrations. The method is based on the oxidation of formate by formate dehydrogenase with corresponding reduction of /3-nicotinamide adenine dinucleotide (j6-NAD+) to reduced -NAD+(/3-NADH) jS-NADH is quantified by reversed-phase high performance liquid chromatography with fluorimetric detection. An important feature of this method is that the enzymic reaction occurs directly in aqueous media, even seawater, and does not require sample pre-treatment other than simple filtration. The reaction proceeds at room temperature at a slightly alkaline pH (7.5-8.5), and is specific for formate with a detection limit of 0.5 im (SIN = 4) for a 200 xl injection. The precision of the method was 4.6% relative standard deviation (n = 6) for a 0.6 xM standard addition of formate to Sargasso seawater. Average re-... 

Jemal M., Xia Y., and Whigan D.B., 1998. The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples. Rapid Commun Mass Spectrom 12 1389. 

Figure 7.5 Schematic diagram of a high performance liquid chromatography (HPLC) system. The solvent(s) are pumped through the system, and the sample injected just before the column where separation occurs. Detection is often by UV/visible spectrophotometry at a fixed wavelength.

High Performance Liquid Chromatography (HPLC) (Chapter 30) gives an elaborate discussion of theoretical aspects. Instrumentation encompasses the various important components e.g., solvent reservoir and degassing system pressure, flow and temperature pumps and sample injection system ... 

Assay of the reaction mixture. A 50 /iL sample was removed from the reaction and the dichloromethane component was evaporated under nitrogen for 20 s. The sample was then resuspended in 600 /iL isopropanol and assayed by chiral high-performance liquid chromatography. A 250 mm x 4.6 mm Chiralpak AD-H column was used with an eluant of 85 15 heptane/ethanol, a flow rate of 3 mL min a temperature of 10 °C, a detection wavelength of 245 nm and a sample injection volume of 2 fL. 

In the separation of biomolecules, sample preparation almost always involves the use of one or more pretreatment techniques. With high-performance liquid chromatography (HPLC), no one sample preparation technique can be appHed to all biological samples. Several techiques may be used to prepare the sample for injection. For example, complex samples require some form of preffactionation before analysis, samples that are too dilute for detection require concentration before analysis, samples in an inappropriate or incompatible solvent require buffer exchange before analysis, and samples that contain particulates require filtration before injection into the analytical instrument. 

Jemal, M., Huang, M., Jiang, X., Mao, Y., and Powell, M. L. (1999a). Direct injection versus liquid-liquid extraction for plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry. Rapid Commun. Mass Spectrom. 13 2125-2132. 

High, K.A., Azani, R., Fazekas, A.F., Chee, Z.A. and Blais, J.-S. (1992) Thermospray-microatomizer interface for the determination of trace cadmium and cadmium-metallothioneins in biological samples with flow injection- and high-performance liquid chromatography-atomic absorption spectrometry. Anal. Chem., 64, 3197-3201. 

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