Acetonitrile or Methanol

The molar ratio of fluorine to toluene spans the range from 0.40 to 1.00 [38] hence under-stochiometric fluorine contents were employed. The concentration of toluene in the solvent was 1.1 mol 1 . As liquid batch volume always 20 ml was applied. Acetonitrile or methanol was taken as solvent for the aromatic compound. 

At least four chromatographic standards prepared at concentrations equivalent to 50-70% of the limit of quantitation (LOQ) up to the maximum levels of analytes expected in the samples should be prepared and analyzed concurrently with the samples. In LC/MS/MS analysis, the first injection should be that of a standard or reagent blank and should be discarded. Then, the lowest standard should be injected, followed by two to four blanks, control samples, fortifications or investigation samples, followed by another chromatographic standard. This sequence is then repeated until all the samples have been injected. The last injection should be that of a standard. In order to permit unattended analysis of a normal analysis set, we recommend that samples and standards be made up in aqueous solutions of ammonium acetate (ca 5 mM) with up to 25% of an organic modifier such as acetonitrile or methanol if needed. In addition, use of a chilled autosampler maintained at 4 °C provides additional prevention of degradation during analysis. 

Typical protein precipitation procedures use one volume of plasma plus three to six volumes of acetonitrile or methanol (or a mixture) with the internal standard at an appropriate concentration for the assay. Poison et al.102 reported that protein precipitation using acetonitrile eliminates at least 95% of the proteins after filtration or centrifugation, the supernatant can often be directly injected into the HPLC/MS/MS system. Usually this step is performed using 96-well plates that are ideal for semi-automation of sample preparation. Briem et al.103 reported on a robotic sample preparation system for plasma based on a protein precipitation step and a robotic liquid handling system that increased throughput by a factor of four compared to a manual system. 

Protein precipitation is used routinely in bioanalytical laboratories in the pharmaceutical industry. Plasma is mixed with an excess (3 to 5 times) of organic solvent (typically acetonitrile or methanol) or an acid such as formic acid. The proteins precipitate out of solution, the sample is centrifuged, and the supernatant is analyzed. While this method is relatively fast and simple, the extract contains salts and lipids that can interfere with subsequent analyses. Often, a second technique such as SPE is used for further cleanup. Table 2.4 exhibits various samples that... 

Reversed-phase separations currently dominate in CEC. As a result, the vast majority of the mobile phases are mixtures of water and an organic solvent, typically acetonitrile or methanol. In addition to the modulation of the retention, the mobile phase in CEC also conducts electricity and must contain mobile ions. This is achieved by using aqueous mixtures of salts instead of pure water. The discussion in Sect. 2 of this chapter indicated that the electro osmotic flow is created by ionized functionalities. The extent of ionization of these functionalities that directly affects the flow rate depends on the pH value of the mobile phase. Therefore, the mobile phase must be buffered to a pH that is desired to achieve the optimal flow velocity. Obviously there are at least three parameters of the mobile phase that have to be controlled (i) percentage of the organic solvent, (ii) the ionic strength of the aqueous component, and (iii) its pH value. 

Mobile phase—a liquid that carries the sample through the column. Typical mobile phases in reversed phase LC are a mixture of water with acetonitrile or methanol. 

In summary, the use of RPLC is ideal for pharmaceutical analyses because of the broad range of commercially available stationary phases because the most common RPLC mobile phases (buffers with acetonitrile or methanol) have low UV cut-off wavelengths, which facilitate high sensitivity detection for quantitation of low-level impurities and because selectivity can readily be controlled via mobile phase optimization. Additionally, the samples generated for selectivity screening (as detailed above) are typically aqueous based. In subsequent phases of pharmaceutical development, aqueous-based sample solvents are ideal for sample preparation and are, under limited constraints, compatible with MS detection required to identify impurities and degradation products. 

Store the columns in acetonitrile or methanol or in a mixture of water and organic solvents. Cap them with closed fittings to prevent the columns from drying out. 

Acetonitrile shows in mixtnres with water, a better solnbility for salts. It is therefore recommended in ion-pair chromatography [52], Basic analytes also show better peak shapes in acetonitrile-buffer mixtures than with methanol. The proper selection, whether acetonitrile or methanol, should be used as the organic component in the mixtnre with a bnffer, however, the type of RP column used classical RP or a shielded RP column is also important. For demonstration with basic analytes, a standard mixture of anti depressives is used. Iso-eluotropic mixtures of methanol and acetonitrile are used. For standardization, the concentration of buffer components are also be kept constant. The analyte structures and the eluent mixtures are summarized in Table 2.2. As selectivity is worse in acidic eluents, a pH value of 7 has been used. Two phases... 

According to some recent literature, typical conditions for biogenic amine determination are precolumn derivatization with Dnsl-Cl followed by separation on C8 or C18 column with gradient elution with mobile phases consisting of water or phosphate buffer and acetonitrile (or methanol) or postcolumn derivatization with OPA and gradient elution with mixtures of sodium acetate buffer and methanol (or acetonitrile). In the latter case, a counterion (such as hexanesulfonic or octansulfonic acid) is usually added in the mobile phase. 

Since the 1970s numerous HPLC methods using lEC, RP and ion-pair chromatography have been proposed. In the last years, RP chromatography has become the most used method, thanks to its simplicity, sensitivity, and compatibility with different detection techniques. The stationary phases usually used are C18 or phenyl-bonded silica-based phases. More recently, alternative stationary phases, such as polar-embedded, polar endcapped, and perfluorinated phases, have been successfully tested for folate analysis [577]. The mobile phase is usually a mixture of phosphate or acetate buffer and acetonitrile or methanol. 

The most common method is RP-HPLC. Microbore-HPLC [613] and narrow bore columns [614] are applied with good results in increasing sensitivity. A recent comparison between NP and RP microbore columns confirms the better suitability of RP for this purpose [615], The mobile phase is usually composed of acetonitrile or methanol. 

The view-cell reactor is made of titanium and has two sapphire windows, a gas inlet valve and an outlet valve, as shown in Figure 3. The view cell is interfaced with a pressure transducer, a thermocouple, and a pressure relief valve. The pressure and temperature are computer-monitored during the reaction. 0.6 ml of 50 wt% H2O2/H2O (10.41 mmoles), 0.20ml of pyridine (2.47 mmoles), or some other base, was dissolved in 5 ml of acetonitrile or methanol, and was added to the reactor. 2.2 ml of supercritical CO2 was charged after lOOmg of propylene (2.38 mmoles) had been added to the reactor. The reactor was heated with a band heater at 40 - 70°C for 3, 6, 12, and 24 hr reaction periods. Following a batch conversion experiment, the amounts of products formed were determined by GC and GC/MS. 

Vacuum Filtering of Mobile Phase. Vacuum filtering of the mobile phase should be avoided in a procedure that is very sensitive to the level of the organic in the mobile phase. Vacuum suction will evaporate the volatile organic portion during filtration (e.g., acetonitrile or methanol), and may lead to variation of the chromatography. 

Thus, simple deproteinization of plasma with trichloroacetic acid, perchloric acid, phosphoric acid, or acetonitrile, followed by centrifugation and direct injection of the supernatants, yielded low recoveries of malachite green and leuco-malachite green, probably due to insufficient debinding of the analytes (495). Acidification or alkalinization of plasma and subsequent extraction with ethyl acetate also resulted in poor recoveries. In contrast, protein denaturation with a mixture of either acetonitrile or methanol and citric acid could substantially improve Ute recovery of the analytes, possibly due to the pairing-ion function of Ure citrate ions. 

Electrospray ionization (ESI) was first employed more than 20 years ago, but it is fairly recently that it became a routine technique for the soft ionization of a wide range of polar analytes, including biomolecules. For this technique, the analyte is usually dissolved in a mixture of an organic solvent (most commonly acetonitrile or methanol) and water with a pH modifier [e.g. formic (methanoic) or acetic (ethanoic) acid for positive ion mode]. The presence of the pH modifier ensures that ionization takes place in the solution state. This is the only common case where ionization occurs before ion vaporization the exact mechanism of the vaporization (Figure 5.6) is still not clearly understood in ESI. 

Apart from pure benzene and pure polar solvents, either acetonitrile or methanol, we have considered xp = 0.2 and xp = 0.7 molar fractions of the polar solvent. Systems ranging from 256 (pure benzene) to 512 (pure polar solvents) molecules were used. From well equilibrated (1 ns) simulations with the coumarin in the ground state So, one to two hundred equally distant configurations were selected. In these configurations the coumarin state was switched to the Si state and the solvent was let to relax in a series of 10 ps long NVE simulations. The solvent response was monitored using the normalized time-dependent stokes-shift function ... 

Whereas in acetonitrile the number of double bonds seems to be more important for solubility, in methanol the chain length seems more important. Utilization of the different properties of these two solvents offers some possibility for the separation of fatty acids whose differences in chain length and degree of unsaturation may make them difficult to separate with the use of either acetonitrile or methanol alone. 

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