Acetonitrile for HPLC

Dissolve the residue in an appropriate volume of acetonitrile for HPLC analysis. 

DNPH is often susceptible to formaldehyde or acetone contamination. It should, therefore, be crystallized with acetonitrile to remove any impurities. Repeated crystallization may further be performed to achieve the desired level of purity for DNPH. A 100-mL aliquot of aqueous sample is buffered with a citrate buffer and pH adjusted to 3 0.1 with HC1 or NaOH. The acidified sample is then treated with DNPH reagent and heated at 40°C for an hour under gentle swirling. The DNPH derivatives of aldehydes and ketones formed according to the above reaction are extracted with methylene chloride using liquid-liquid extraction. The extract is then solvent exchanged to acetonitrile for HPLC determination. 

The DNPH derivative may alternatively be extracted by solid-phase extraction on a sorbent cartridge, conditioned with 10 mL dilute 1M citrate buffer (1 25 dilution) and 10 mL saturated NaCl solution. The extract loaded on the cartridge, and the derivative eluted with acetonitrile for HPLC analysis. 

Alternatively, DNPH coated sihca gel or florisil adsorbent may be used instead of impinger solution derivative eluted with acetonitrile for HPLC determination. 

Dinitrophenyl hydrazine (DNPH) was purchased from Wako Pure Chemical Inc. (Osaka Japan). Silica gel TLC plate used was Silica gel 60 TLC aluminum No. 5553 sheet Merck, Germany. Acetonitrile for HPLC grade was purchased from Nacalai Tesque Inc. (Tokyo Japan). All other chemicals were of the best grade available from commercial source. DNPH reagent prepared was 0.2% DNPH in 2% HCl-methanol. 

After extracting fluthiacet-methyl from the soil extract with n-hexane, pass the residual aqueous layer through a dual cartridge of Sep-Pak Plus NH2 and Sep-Pak Plus C18 to adsorb the free form of fluthiacet-methyl on Sep-Pak Plus Gig. Remove the Sep-Pak Plus C18, wash it with 0.5% acetic acid and acetonitrile-water-acetic acid (20 80 0.5, v/v/v), elute with acetonitrile-water-acetic acid (50 50 0.5, v/v/v) and quantify the free form by HPLC. The operating conditions for HPLC are the same as those for fluthiacet-methyl, except that the mobile phase is acetonitrile-water-acetic acid (50 50 0.5, v/v/v) (retention time 8.8 min). 

Second cleanup Transfer the above carbon tetrachloride solution into a glass column packed with 7 g of silica gel saturated in carbon tetrachloride. Rinse the column, first with 2 mL of carbon tetrachloride and then with 35 mL of hexane-ethyl acetate (17 3, v/v). Elute benfuracarb with 30 mL of the same hexane-ethyl acetate solution. Concentrate the eluate to near dryness by rotary evaporation and prepare the GC/HPLC-ready sample solution by dissolving the residue either in benzene for plant material or in acetonitrile for water and soil. 

Methanol, ethanol, acetonitrile, benzene, trifluoroacetic anhydride, triethylamine, distilled water reagent grade (Wako Pure Chemical Industries, Ltd, Japan) Methanol, distilled water specially prepared reagent for HPLC (Wako Pure Chemical Industries, Ltd, Japan)... 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

C for 1 h. A 100 pL portion of the solution was injected onto a column (15 cmx 3.2 mm) of LiChroscob RP-18 (7 pm) for HPLC at room temperature, using acetonitrile-0.033 M phosphate buffer of pH 8.2 (1 2) containing 0.05% of ethyle-nediamine as the mobile phase (eluted at 1 mL/min). Fluorimetric detection involved excitation at 338 nm and measurement at 540 nm (or with a 430 nm cutoff filter). For 50 300 ng of drug injected on to the column, the coefficient of variation was 7-8%. The method permits a simple determination of (z>)-penicilla-mine in serum at therapeutic levels. 

A six-port valve was used in both manual and semi-automated SPME interfaces and PEEK tubing used to connect the HPLC system to the SPME probe. A Cohesive HTLC 2300 with dual pumps along with a Sciex API 3000 mass spectrometer was used for LC/MS/MS and a Symmetry Shield RP-18 (5 ji, 50 x 2.1 mm) for HPLC. A quaternary pump with flow switching was used for desorption chamber flushing along with MS make-up flow and a binary pump for LC/MS/MS. Acetoni-trile/0.1% acetic acid in water (90 10, solvent B) and 10 90 acetonitrile/0.1% aqueous acetic acid (solvent A) were used, with 10% B for 0.5 min ramped to 90% B in 2 min and held at this concentration for 1.5 min before returning to 10% B for 1 min at a flow rate of 0.5 mL/min. 

Fig. 2.145. Semipreparative HPLC separation of the subunits from P. cruentum B-PE. The elution profile monitored at 226 nm is shown. The identity of each peak was detemined from the visible absorption spectra, the known distribution and content of PEB (phycoeryhtrobihn) and PUB (phu-courobihn) and SDA-PAGe. Dotted line symbolizes the gradient in percentage of acetonitrile (for quantitative details see text). The order of subunit elution is y. a, p. The elution profile shows a partial resolution of at least three j- species. Reprinted with permission from R. Bermejo et al. [317].

HPLC has been shown as an effective method in the fractionation and preparation of AHLs for structural analysis. Preparation of AHL-containing samples for HPLC analysis requires their extraction with organic solvents such as dichloromethane or ethyl acetate [37]. Usually, C8 reverse-phase columns are employed and samples eluted with either gradient or isocratic mobile phases, e.g. acetonitrile-water. Fractions are analysed for the presence of AHLs using the biosensors described in the previous section. AHLs from active fractions can then be identified using more powerful techniques (see following sections). 

Differences between acetonitrile and methanol are in price, transparency, toxicity, and viscosity methanol for HPLC is approximately by a factor of 3 cheaper than acetonitrile of identical purity ... 

The HPLC-FTIR technique has recently been used to identify six catechins and two methyl-xanthines present in green tea extracts." " A reversed-phase separation of the compounds was performed on a C-18 column equilibrated at 30°C using an isocratic mobile phase of acetonitrile-0.1% formic acid (15 85), prior to introduction to the deposition interface linked to the FTIR detector. The solvent was evaporated at 130°C and spectra were collected every 6 sec during the run. Two distinct designs for HPLC-FTIR interfaces have been developed flow cells and solvent elimination systems. Flow cell systems acquired spectra of the eluent in the solvent matrix through IR transparent, nonhydroscopic windows. The... 

Reagents. Organic solvents for HPLC separations—methylene chloride, methanol, isopropyl alcohol, hexane, and acetonitrile—were obtained as HPLC grade from Fisher Scientific. Type I water for HPLC and for the preparation of other aqueous solutions was purified as described previously (7). All HPLC solvents were filtered through a 0.45-/zm Millipore membrane filter (Millipore Corporation) and degassed... 

HPLC and Isolation of Mutagenic Fractions. Analytical and semipreparative reverse-phase HPLC separations were performed by using a water-to-acetonitrile linear gradient (J2). Separations were carried out on a Hewlett Packard Model 10084 B equipped with an automatic sampling device, a solvent programmer, a variable absorbance detector, and an automatically steered fraction collector. The instrument was fitted with a 3.9-mm X 30-cm prepacked analytical column of 10-/zm silica particles bonded with octadecylsilane (Bondapack-Cis) for analytical scale. For semipreparative scale separations, the HPLC was fitted with a 7.8-mm X 30-cm prepacked column packed with 10-/xm silica particles bonded with octadecylsilane. Samples for HPLC were injected at volumes of 20 /xL (flow rate 1 mL/min) and 80 /zL (flow rate 4 mL/min), and the absorption was measured at 254 nm. Fractions... 

Free fatty acids are separable by GC by the inclusion of phosphoric acid in the packing so, for HPLC analysis, the phosphoric acid or other equivalent strong acid is included in the mobile phase. On a SUPELCOSIL LC 18 column, a model mixture of free fatty acids was separated with a mobile phase containing tetrahydrofuran, acetonitrile, water, and phosphoric acid (6 64 30 0.1) at pH 2 (Fig. 1) (15). Oleic and elaidic acids, palmitoleic and palmitelaidic acids, and linoleic and linoelaidic acids were well separated, but margarine fatty acids presented a difficult problem. Ultraviolet detection of 220 nm was used to prepare this chromatogram. 

The mobile phases usually used for HPLC separation of fatty acid derivatives are acetonitrile-water, methanol-water, and acetonitrile-methanol-water. Elution with methanol-water mobile phase only failed to resolve linolenic (18 3) and myristic (14 0) acids. 

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