Acetonitrile buffer compositions

As described for lEC, elution is done by a stepwise or a continuous change of buffer composition. The mildest elution buffer is an aqueous buffer with low ionic strength, e.g., 20 mm Tris-HCl. If it is not successful, desorb with a chaotropic solvent, e.g., 2 M potassium rhodanide (thiocyanate), 2.5 M guanidinium hydrochloride, up to 7 M urea, or with increasing concentrations of methanol or acetonitrile. Especially the use of rhodanide or urea may be accompanied by a chemical modification of amino acid side chains, which disturbs amino acid analysis. 

Z-L-aspartic anhydride (2 mmol) is dissolved in acetonitrile (20 mL). To the solution is added L-phenylalanine methyl ester (2 mmol) under stirring. The reaction continues at 20°C for 2 h. After the reaction, the solution is diluted 10 times with acetonitrile. The composition of the mixture is analyzed by RP HPLC using an ODS column and UV detection at 210 nm. Mobile phase 50% (v/v) acetonitril in 10 mM phosphate buffer, pH 4.15 flow rate 0.5 mL min ... 

Felice et al used a GCE (+0.7 to +0.9 V vs Ag/AgCl) in the analysis of polycyclic aromatic amines, such as 2-aminonaphthalene, 4-aminobiphenyl, and 2-aminoanthracene, in rodent skin samples after topical application of these compounds. The HPLC system used consisted of an ODS-modified silica column with acetonitrile-aq. citrate/perchlorate buffer (7 + 3 or thereabouts) as eluent - the buffer composition and the proportion of acetonitrile were varied in different experiments. A LoD of 0.1 pmol on column could be expected. 

Mixtures of methanol or acetonitrile with either phosphate, TRIS or MES (Table 4.23) buffer solutions are the most frequently used, but many others are being investigated. The higher above 4 the buffer pH is, the greater the EOF generated and the faster the separation. The composition of the mobile phase can have dramatic effects on both the EOF and the selectivity of the separation via the sorption processes with the stationary phase. 

Omura et al.21 used a reverse phase high performance liquTcT cEromatographic column, JASCO PACK SV-02-500, for macrolide antibiotics with methanol, M/15 acetate buffer pH 4.9, and acetonitrile (35 60 5) as solvent. A variable wavelength UV detector using the absorption of the individual compounds gave the required sensitivity. Alterations of buffer pH and the composition ratio of the mobile phase gave selectivity for separation of individual macrolide antibiotics. 

The retention indices, measured on the alkyl aryl ketone scale, of a set of column test compounds (toluene, nitrobenzene, p-cresol, 2-phenyl ethanol, and IV-methylaniline) were used to determine the changes in selectivity of a series of ternary eluents prepared from methanol/0.02M phosphate buffer pH 7 (60 40), acetonitrile/0.02 M phosphate buffer pH 7 (50 50) and tetrahydrofuran/0.02 M phosphate buffer pH 7 (25 65). The analyses were carried out on a Spherisorb ODS reversed-phase column. The selectivity changes were often nonlinear between the binary composition [83]. 

In order to select a carrier solution composition which would provide an overall maximum response for MS detection, two modifiers were selected, acetonitrile and methanol, and two buffers, i.e. ammonium acetate (10 mmol pH 7.5) and ammonium formate (10 mmol L pH 7.5). Biotin and fluorescein-biotin were dissolved in various binding buffer-organic solvent mixtures ranging from 90 10 (v/v) to 50 50 (v/v) at two concentration levels (0.01 ng 1 ng pL ) and 20 pL were injected and analyzed by MS in full-scan and SIM mode. The maximum response was found with 50% methanol, which was about a factor 2x higher than for 10% methanol. Since the proteins can denaturate or protein-ligand complexes can dissociate at relatively low percentages of organic modifier in further experiments only 10% methanol is used in the carrier solution. 

Electrophoresis is carried out with a capillary cartridge containing an extended light path capillary (40 cm x 50 pm i.d.). A buffer solution for column conditioning and electrophoresis should be prepared with the following composition 10 mM potassium dihydrogenphosphate, pH 7,20 mM SDS, 8 mM CD and 20% acetonitrile (v/v). An aliquot of 50 pi of this solution, diluted ten times with distilled and deionised water, should be added to the sample. 

Let s examine Figure 25-26 to see how the systematic procedure works. Step I is to generate chromatogram A by varying the proportions of acetonitrile and aqueous buffer (as in Figure 25-12) to obtain the best separation within the constraint that 0.5 < k < 20. At the best composition, 30 vol% acetonitrile/70% buffer, peaks 4 and 5 are not resolved adequately for quantitative analysis. 

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 ...

In the reversed phase system, buffers are used most often as the mobile phases with small amount of organic modifiers. The use of buffers as the mobile phases has increased the efficiency of the resolution. Ammonium nitrate, triethylammonium acetate (TEAA), and sodium citrate buffers have been used very successfully. A variety of organic modifiers have been used to alter selectivity [2,5,22], Acetonitrile, methanol, ethanol, 2-propanol, and THF have shown good selectivities for various analytes. In the reversed-phase mode, the amount of organic modifiers is typically low, usually of the order of 10-20%. The typical starting composition of the mobile phase is an organic modifier-buffer... 

FIGURE 7 Effect of mobile phase composition on the resolution of enantiomers of different racemates in reversed-phase HPLC on antibiotic CSPs. (a) First ( , O) and second ( , ) enantiomers of 5-methyl-5-phenylhydantoin on a Chirobiotic T column using an acetonitrile-triethylammonium acetate buffer (—) and a methanol-triethylammo-... 

The effect of mobile phase composition, including pH and organic modifiers, was carried out on the chiral resolution of leucine derivatives on the fert-butyl carbamoylated quinine-based CSP [2], The results of these findings are given in Table 2. This table shows that the best resolution was obtained at pH 5, 2mM concentration of buffer, 60% methanol, and 80% acetonitrile concentrations, separately. In another study, the same authors [4] studied the influence of the mobile phase, pH, and temperature on the chiral resolution of leucine derivatives. The effect of temperature on the chiral resolution of leucine derivatives is shown in Figure 2. It is clear from this figure that the chiral... 

To achieve the optimum reversed-phase LC separation, one needs to explore variables such as the analyte chemistry, mobile-phase composition (solvent type, solvent composition, pH, and additives), column composition, column particle size, and column temperature. For pharmaceutical analysis using mass spectrometry, the chemistry of an analyte is rarely changed beyond manipulation of the mobile phase pH, and even there options are limited. Volatile pH modifiers (buffers) are still preferred for LC-MS, and concentrations of these modifiers are kept low. Relatively simply mobile phases consisting of water, acetonitrile, and either formic acid (0.1% v/v), ammonium acetate (1-20 mM), or both have been common. 

Gradient systems let you control flow rate and solvent/buffer changes to improve chromatographic separations. They can be used to sharpen separations and to speed column re-equilibration. A four-solvent gradient system is useful for methods development when equipped with methanol, acetonitrile, ammonium acetate buffer, and formic acid solution. But, many quality control laboratories prefer to use inexpensive isocratic systems that run a constant-composition premixed mobile phase for rapid separations. 

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