Premixed mobile phases

Traditionally, most pharmaceutical assays are isocratic analysis employing the same mobile phase throughout the elution of the sample. Isocratic analyses are particnlarly common in quality control applications since they nse simpler HPLC eqnipment and premixed mobile phases. Notable disadvantages of isocratic analysis are limited peak capacity (the maximnm nnmber of peaks that can be accommodated in the chromatogram), and problems with samples containing analytes of diverse polarities. Also, late eluters (such as dimers) are particularly difficult to quantitate in isocratic analysis due to excessive band broadening with long retention times. 

Use premixed mobile phases for isocratic analysis to reduce mobile phase variations. 

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. 

Salt precipitation (especially in reversed-phase chromatography with high concentration of organic solvent in mobile phase) Ensure mobile phase compatihility with buffer concentration decrease ionic strength and water-organic solvent ratio premix mobile phase. 

Better peak widths and peak shapes are usually obtained if the sample is introduced onto the column as part of a premixed mobile phase. Placing the injection valve in the modifier line instead of the premixed mobile phase line allows the user to slowly add the solute to the mobile phase at a greatly reduced local modifier concentration. Such an approach requires that the solute be strongly retained at the modifier concentration flowing through the column, so that the peak can be focused. 

Lukulay, P.H. McGuffin, V.L. Comparison of solvent modulation with premixed mobile phases for the separation of corticosteroids by liquid chromatography. J.Liq.Chromatogr, 1995, 18, 4039-4062... 

For acrylate polymers with higher levels of carboxylic acids, THF can be modified by the addition of acids such as acetic, phosphoric, or trifluoroacetic. Levels as high as 10% acetic acid are considered acceptable by most manufacturers for their styrene/DVB columns. If such a modified mobile phase is used, it may need to be premixed rather than generated using a dynamic mixing HPLC pump because on-line mixing often leads to much noisier baselines, particularly when using a refractive index detector. 

There are two types of gradient programmer. In the first type, the solvent mixing occurs at high pressure and in the second the solvents are premixed at low pressure and then passed to the pump. The high pressure programmer is the simplest but most expensive as it requires a pump for each solvent supply. There can be any number of solvents involved in a mobile phase program, however, the majority of LC analyses usually require only two solvents but up to four solvents can... 

The catalytic reaction was carried out at 270°C and 101.3 kPa in a stainless steel tubular fixed-bed reactor. The premixed reaction solution, with a molar ratio catechol. methanol water of 1 1 6, was fed into the reactor using a micro-feed pump. To change the residence time in the reactor, the catechol molar inlet flow (Fio) and the catalyst mass (met) were varied in the range 10 < Fio <10 mol-h and 2-10 < met < 310 kg. The products were condensed at the reactor outlet and collected for analysis. The products distribution was determined quantitatively by HPLC (column Nucleosil 5Ci8, flow rate, 1 ml-min, operating pressure, 18 MPa, mobile phase, CH3CN H2O =1 9 molar ratio). 

Beker and Lovrec (119) developed the method for the determination of BHT in poultry premix. BHT was extracted from the sample with methanol, extract filtered, and injected onto HIBAR LiChrosorb RP-18 column. As a mobile phase, 5% water in methanol with a flow rate of 1 ml/min was used, and eluent was detected at 280 nm. The recovery varied from 98.7 to 101.8%, with a coefficient of variation from 1.58 to 3.05%. 

BHT Poultry premix extraction into methanol, filtration, and dilution with mobile phase HIBAR LiChrosorb RP-18, 25-cm X 4-mmTD column... 

The mobile phase was composed of a buffer and acetonitrile in the ratio of 68 32, vol/vol. The pH of the mobile phase was adjusted to 7.5 with orthophosphoric acid. The buffer used in the mobile phase consisted of 10 mM disodium hydrogen phosphate and 10 mM SDS in double-distilled water. The mobile phase was premixed and filtered through a 0.45-pm nylon filter and degassed. 

For HPLC-MS/MS assays, the mobile phase is an important consideration. For reversed-phase HPLC systems (the most common), mobile phase A is water plus one or more modifiers while mobile phase B is usually either acetonitrile or methanol with one or more modifiers. Modifiers have to be volatile for HPLC-MS/MS assays. Typical modifiers are acetic acid, formic acid, and ammonium acetate. Formic acid is so popular that it is now available as a premixed HPLC solvent (0.1% in water or acetonitrile). Most other modifiers are not recommended. Triethylamine (TEA) and trifluoroacetic acid (TFA) are a problem as they are known to cause ion-suppression problems. For some special HPLC-MS/MS assays that need ion-pairing reagents, hexylamine and heptafluorobutyric acid have been found to be successful [51-53], Gao et al. [54] recently evaluated a series of ion-pairing reagents in terms of their suitability for an HPLC-MS/MS assay. 

Mobile phase Hexane l,2-dichloroethane EtOH/trifluoroacetic acid 58 35 7 (EtOH/ trifluoroacetic acid was premixed 20 1.)... 

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