Mobile-Phase Preparation

The practices of isocratic and gradient sorptive chromatography are very different. Isocratic chromatography tends to be very sensitive to the details of mobile phase preparation, temperature, pump speed, and sample composition. Gradient chromatography is usually more tolerant of small variations in these factors but may be extremely sensitive to column history, equilibration time, and gradient preparation. 

Mobile phase Prepare a filtered and degassed mixture of methanol and 1-penta-nesulfonate sodium solution (60 40). Make adjustments if necessary (see system suitability under Chromatography <621 >). 

FIGURE I Retention times of two analytes using various mobile preparations vs. pump blend. Note that procedures A and D are almost equivalent while column B or C (using volumetric flasks) yields retention times which are either too high or too low depending on whether methanol or water is filled first.This phenomenon is caused by the negative volume of mixing. Procedure A is recommended for all mobile phase preparation. 

Mobile Phase Preparation. Distilled water and 6M urea were filtered under vacuum using a 0.22ym membrane filter (Type GS, Millipore). 

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

Polyelectrolyte contraction can be followed by determining the Kj as a function of mobile phase ionic strength (104). In practice, however, the mobile phase ionic strength must be sufficiently high to ensure that the chain is in a contracted state. In this way, small changes in ionic strength, which may be inadvertently introduced during mobile phase preparation, will not affect the elution behavior of the sample. Also, if the ionic... 

Mobile Phase - Prepare a filtered and de-gassed mixture of alcohol-free chloroform, n-hexane, and tetrahydrofuran (about 70 30 1 by volume). The ratio of components and the flow rate may be varied to meet system suitability requirements. 

Determine final chromatographic conditions Evaluate alternate column lots. Based on this information and considering the instrumentation and personnel who will be using the method, set final chromatographic conditions, including mobile phase preparation. 

Mobile Phase Prepare a 80 17 3 mixture of 0.01 M phosphoric acid, acetonitrile, and methanol. Adjustments in the composition may be made if necessary... 

Mobile Phase Prepare a filtered and degassed 87 13 v/v mixture of 0.025 M phosphoric acid (previously adjusted to a pH of 3.0 0.1 with triethylamine) and acetonitrile. 

In the basic system the mobile phase is contained in a reservoir. This reservoir may or may not be pressurized (to approximately 3-6 psi). The mobile phase should be prefiltered before being placed into this reservoir, yet a filter is usually placed in the reservoir to be a sinker to hold the inlet at the bottom of the reservoir and to remove any accidental contamination of coarse particulate matter (20-30 /um) before it reaches the solvent delivery system (the pump). Since this is a low-pressure line, Teflon tubing (1 in. OD or larger) is usually used to ensure that sufficient volume of mobile phase reaches the solvent delivery system. Addition considerations for mobile phase preparation are given in Chapter 6. 

If a single solvent will not accomplish the separation, try a mixture of two solvents of different types (e.g., on silica, try mixing a hydrocarbon and a halogenated hydrocarbon for fairly nonpolar solutes, or a hydrocarbon and an ether for moderately polar compounds). This simplifies mobile phase preparation and column equilibration. 

Mobile Phase Prepare a filtered and degassed 65 35 mixture of acetonitrile water. 

Mobile Phase Prepare 0.005 N sulfuric acid that has been suitably filtered and degassed. 

Mobile Phase Prepare a filtered, degassed solution of 0.01 N sulfuric acid in water. 

Mobile Phase Prepare a 0.04 M potassium hydrogen phthalate solution by transferring 16.4 g of potassium hydrogen phthalate into a 2-L volumetric flask, diluting to volume with water, and mixing. Filter the solution through a 0.45- im pore-size filter (Millipore, or equivalent). 

Mobile Phase Prepare a 60 40 mixture of Solution A and Solution B, filter, and degas. Make adjustments to the Mobile Phase, if necessary, to obtain baseline separation of thiamine hydrochloride and methyl benzoate. 

Figure 4-38. Elution of a mixture of ionizable components on a XTerra MS CIS (Waters) column with a 60% ACN mobile phase prepared from wpH = 8.0. (A) Phosphate buffer. (B) NH4 /NH3 buffer. Compounds are (1) 2-nitrophenol, (2) 2,4,6-trrmethyIpyridine, (3) 3-bromphenoI, and (4) NN dimethylbenzylamine. (From reference 64, with permission.)...

In both MEKC methods reported, the mobile phase consisted of borate buffer containing surfactant and acetonitrile. It was foimd that mobile phase prepared at pH 9.75 gave better resolution compared to other conditions, and increasing pH also increased the migration time of ezetimibe. An analyte concentration of 25 mM was chosen due to its lower current, and the sharp peaks observed [41]. In addition, for the analysis of ezetimibe in combination with another drug such as simvastatin, increasing the borate concentration increased both resolution and migration times. 

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