Preparation of the mobile phase

This section describes some of the problems that can occur with the mobile phase in hplc. Many of these problems arise because of the presence of impurities, additives, dust or other particulate material, or dissolved air. It is always best to try to prevent these problems by a little attention to detail and the use of simple good housekeeping procedures. Although it is always tempting to try to save time and expense by the neglect of such matters, if you do this you will store up trouble for yourself in the long term. 

A microparticulate hplc column is a very efficient filter, and if the mobile phase contains any particulate matter, or acquires it from the pump and/or the injection valve due to wear, it will collect at the top of the column. If this happens, the pressure drop across the column for a given flow will gradually increase, and the column may eventually become completely blocked. To prevent this happening, the mobile phase should always be filtered before use, preferably through a 0.5 p,m porosity filter, and guard and scavenger columns should be used as a matter of routine (see Section 5.3.2). 

Many reagent grade solvents contain levels of impurities that make them unsuitable for long term use in hplc. Sometimes the impurities are added deliberately as antioxidants, stabilisers, or for denaturing. Wherever possible, hplc grade solvents should be used to prepare mobile phases, or alternatively the solvents should be adequately purified before use. 

Distilled or deionised water contains small amounts of organic impurities which can cause problems in long term use with bonded phase columns in the reverse phase mode. The non-polar stationary phase will collect these organics, which can alter the nature of the stationary phase or sometimes produce spurious peaks (Fig. 4.3c is an example of this). Water purification can be done by distillation from permanganate, by passage of the water through bonded phase columns, or by means of commercial systems, eg the Milli- 

Impurities in other solvents may affect chromatographic behaviour, or detection, or both. Chlorinated solvents such as di- or trichloromethane are stabilised against oxidative breakdown by the addition of small amounts of methanol or ethanol. 

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Why are methanol or acetonitrile preferred to other water miscible solvents for the preparation of mobile phases for reverse phase chromatography ... 

The chromatography method, including the manufacturer and model for the LC system and autoinjector, if applicable, should be provided. Ideally, the exact same equipment that was used for method development should be used to validate the method. After the method has been validated, other types of equivalent equipment may be used but, since factors such as system dead volume and carryover may impact the method, additional validation experiments may be required to support these changes. (Section 10.2.10). Similar to what was described above for the preparation of solutions and reagents, a description of all chemicals and solvents used for the preparation of mobile phases should be provided along with examples of how these are prepared and stored when... 

For preparative separation, the mobile phase can be selected by performing preliminary analytieal TLC experiments. In PLC, the chromatographic chamber has to be saturated within 2 h beeause the development of preparative plates is much slower than the analytical development. In the analytical preassay during the selection of mobile phase composition, the chromatographic chamber must be hned with a sheet of filter paper to obtain a saturated atmosphere with mobile phase vapor. Then, the optimized analytical mobile phase can be transferred imchanged to preparative separations in the saturated developing chamber. 

Antidrcular development is very rarely applied in planar chromatographic practice for preparative separation. This mode of separation was introduced by Kaiser [40]. Studer and Traitler adapted the antidrcular U-chamber from CAMAG to preparative separations on 20 x20 cm plates [22], as mentioned earher. The sample mixture was spotted at the circumference of the plate, and the mobile phase was moved from the circumference to the center of the plate. The bands obtained are elongated, but their diameter measured perpendicular to the direction of mobile phase movement... 

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. 

Developing an isolation approach is an activity that is frequently overlooked or addressed as an afterthought. However, solubility and stability data may dictate the development of a chromatographic method that requires the elaboration of the isolation, that is, it is more complicated than a simple evaporation of the mobile phase. The development of the chromatographic process should be linked to and interactively codeveloped with the isolation. Ideally, the isolated impurity sample should not contain other compounds or artifacts, such as solvents, mobile-phase additives or particulate matter from the preparative chromatography, as they may interfere with the structure elucidation effort or adversely affect the stability of the impurity during the isolation process. Therefore, it is preferable to avoid or minimize the use of mobile-phase additives. However, should this prove to be impossible, the additive used should be easy to remove. The judicious choice of mobile phase in the HPLC process increases the ability to recover the compound of interest without or with minimum degradation. The most common... 

Mobile phases were prepared by adding the appropriate quantities of ion-pairing reagent solutions and phosphoric acid to distilled water with subsequent titration to pH 7.0 (+.05) with ammonium hydroxide. In the case of mobile phase B, pH adjustment was carried out before addition of methanol. The final buffer concentrations (1.5 mM in A and 5.0 mM in B) are expressed as... 

Elution is one of the critical step for successful separation. Sample application in affinity chromatography is performed usually by injection or application in the presence of mobile phase which is prepared in appropriate pH, ionic strength and solvent composition for solute-ligand binding. This solvent is referred as application buffer [8]. In the presence of application buffer, compounds which are complementary to the affinity ligand will bind while the other solutes in the sample will tend to pass through the column as nonretained compounds. After... 

By adjusting the composition of the mobile phase, a wide variety of solutes of differing polarity can be separated easily. Even closely related molecules can be resolved on efficient HPLC columns. Mobile phases may need to be complex—depending on the nature of the sample components—and it may be necessary to use several solvents and additives such as acids, bases, salts, or surfactants. The stationary phase can be changed to aid in achieving a separation, but this necessitates owning an assortment of packed LC columns, which is expensive. In addition, the diversity of mobile phases that can be prepared is far greater than the variety of commercially available stationary phases. 

If a is not satisfactory, try a binary mixture in which the two solvents are very different in individual eluent strength, with one solvent predominant (>80%) (e.g., on silica, try a small percentage of an alcohol in a hydrocarbon). Accurate preparation of mobile phases and column equilibration... 

One considerable disadvantage of coated polysaccharide type CSPs, however, is the high solubility of the SO in many organic solvents, e.g. chloroform, ethylacetate, and tetrahydrofuran, restricting the choice of mobile phases that can be used. Accordingly, inflexibility in the optimization of separations and enantioselectivity is a considerable drawback this counts in particular for preparative separations, where often the solubility of the SAs in the mobile phase is limited and thus loadability and finally the productivity rate is reduced. 

The possibility of zonal sample dosage in equilibrium conditions (after the front of mobile-phase and continuous-chromatogram development, which is provided by a horizontal sandwich chamber) was utilized by Glowniak et al. [3] in preparative chromatography of simple coumarins and furano-coumarins found in Archangelica fruits, performed with a short-bed continuous development (SB-CD) technique. 

The advantages of CLC are consequences of its miniaturization [9]. Due to its miniaturized size, it requires much less stationary phase than does ordinary HPLC and, as a consequence, more expensive phases can be used to prepare the columns. This includes chiral phases, experimental new materials, expensive biocompounds, and so forth. In the same way, the amount of mobile phase is very small, thus leading to a savings in buying, storing, and discarding the solvent, allowing... 

Preparation of Mobile Phase. In preparing the mobile phase, dissolved gases in the solvent must be removed or suppressed. The solvent must also be free of particulate matter. When the mobile phase is composed of two or more solvents, the solvents must be adequately mixed. 

These phases have been prepared by polymerization of N, N -diaUyl derivatives of tartardiantide and grafted onto silica gel. As these phases are crosslinked and bonded to sihca gel, they are insoluble in organic solvents and there is no limitation regarding the choice of mobile phase. Normal mode, reversed-phase and supercritical fluid conditions have been applied. They show good mechanical stability, but the relatively high content of achiral sihca gel in these CSPs reduces their loading capacity. A few preparative separations have been reported on these two CSPs (Kromasil) [52-54], but so far they have not been widely used for preparative apphcations. 

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