Mobile phase preparation mixing components

Mixing errors manual mobile phase preparation. These tend to occur as a result of operator error during the dispensing and mixing of the individual components. The final concentration of each component within the mixture can vary depending on the way in which it is prepared. 

Mixing errors automated mobile phase preparation. Problems encountered with an automated system can still be the result of analyst error. For example, the wrong component may have been dispensed into the HPLC solvent reservoir. Where there is an apparent issue in relation to the retention time of a compound, the mobile phase should always be checked for accuracy of preparation. Proportioning valve errors can also cause a problem with automated systems. When the valve ceases to dispense the correct amount of either solvent, the retention time of the compounds of interest will vary. 

In this case, the TLC system most commonly employed uses silica gel plates and a mobile phase of ethyl acetate/methanol/25% ammonia (85 10 5, by volume). The plates are prepared and the chromatogram developed in the standard way. After development, the plate is removed from the mobile phase, the solvent front marked, and the plate dried. Visualization of barbiturates is best achieved by the use of a mercuric chloride-diphenylcarbazone reagent. The latter is prepared as two component solutions, i.e. (i) 0.1 g of diphenylcarbazone in 50 ml of methanol, and (ii) 0.1 g of mercuric chloride in 50 ml of ethanol. These solutions should be freshly prepared and mixed just before use. The presence of barbiturates will give rise to blue-violet spots on a pink background when using this reagent system. 

The separation selectivity can be significantly affected as a result of different pH shift of different buffers even at the same organic composition. For example, if two buffers are prepared at the same pH, one using an acidic buffer such as phosphate and another using a basic buffer such as ammonia, both at WpH 8, the separation of a mixture of ionizable components could be different. This could be attributed to the different mobile-phase pH after the aqueous is mixed with the organic. Espinosa et al. [64] analyzed A,A-dimethyl-... 

Method transfer from one laboratory to another one (from development to routine, from manufacturer to customer and so on) can be difficult because HPLC separations are influenced by many parameters. At the new place the resolution of a critical peak parr can be worse than required or the whole chromatogram looks different. In order to prevent such surprises, whenever possible, it is necessary to describe every detail of the method column dimensions, stationary phase (maybe even the batch number), preparation of the mobile phase (the order the individual components are mixed can be critical), temperature, volume flow rate, extra-column volumes of the instrument, the dwell volume in the case of gradient separations (see Section 4.3) as well as detection and integration parameters. It can be useful to designate alternative stationary phases, i.e. materials which are located close to each other in representations such as Figure 10.9. The true temperature in a column oven must be verified because it can differ from the requested one Method transfer also includes the detailed description of sampling, storage and sample preparation. 

Mobile phases should be as simple as possible and prepared from pure grades of solvent. Mixtures composed of more than four components should be avoided because of problems associated with reproducible preparation. Mobile-phase proportions are designated in parts by volume so that the sum is usually 100. Quantitative techniques should be used to prepare the mobile phase and the constituents should be mixed thoroughly before use. Multisolvent mobile phases should be discarded after use. 

To prepare 1,000 mL of a mobile phase containing 70% methanol and 30% water, measure out 700 mL methanol and 300 mL water in separate measuring cylinders. Mix both components together mix thoroughly and filter and degas before use. 

Exceptions to the never filter rule occur when mobile phases contain MPMs (particularly salts) added to the solvent. When this is the case, the solvent containing ffie salts should be prepared and filtered independently and then mixed with the other high-purity solvent components. For example, consider a 50/50 v/v methanol/water (0.05 M acetate buffer at pH 4). Here the aqueous acetate buffer is prepared from glacial acetic acid and sodium acetate, filtered, and then added in equal volumes to methanol. 

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