Mobile phase quality

Guard columns and in-line filters alone will not ensure long life for the analytical column. They are designed to perform a specific function for a finite period of time. They, too, will be short-lived if shortcuts are taken in sample preparation and mobile phase quality control. Use of a guard column does not mean that other good analytical practices can be neglected. Shortcuts mean shorter life for any column. 

All of the components for mobile phase preparation can be purchased from reputable or approved suppliers and any changes in supplier should be checked thoroughly before materials are used. Additional validation of the method may be required when there has been a change in supplier of a critical reagent. All mobile phases should be filtered and degassed prior to use. Further discussion in relation to mobile phase quality is given in Chapter 12. 

It was shown that the effect of the particle size is not significant in HOPC (1). The experiments were conduced using silica gels of the same pore size but with a different average particle size between 15 and 100 /urn. A kinetic effect— enrichment of the mobile phase with high MW components is better at short times before equilibrium is reached—was cited as a possible reason for almost equal quality of separation by large particles. The back-pressure problem was not serious in that range of the particle size. 

Unfortunately, neither the computer nor the potentiometric recorder measures the primary variable, volume of mobile phase, but does measure the secondary variable, time. This places stringent demands on the LC pump as the necessary accurate and proportional relationship between time and volume flow depends on a constant flow rate. Thus, peak area measurements should never be made unless a good quality pump is used to control the mobile phase flow rate. Furthermore, the pump must be a constant flow pump and not a constant pressure pump. 

The effect of the quality of the mobile phase on the operation of the detector being employed is of importance whatever that detector may be. 

A uniform film of analyte, which is required for the production of good quality spectra, can usually be obtained from mobile phases which contain predominantly organic solvents (normal-phase systems). As the percentage of water in the mobile phase increases, however, droplets tend to form on the belt, irrespective of the belt speed. If the belt is not exactly horizontal, and this is often the case, especially after it has been in use for some time, the droplets are likely to roll off the belt and be lost, thus reducing the overall sensitivity of the analysis dramatically. 

Prus and Kowalska [75] dealt with the optimization of separation quality in adsorption TLC with binary mobile phases of alcohol and hydrocarbons. They used the window diagrams to show the relationships between separation selectivity a and the mobile phase eomposition (volume fraction Xj of 2-propanol) that were caleulated on the basis of equations derived using Soezewiriski and Kowalska approaehes for three solute pairs. At the same time, they eompared the efficiency of the three different approaehes for the optimization of separation selectivity in reversed-phase TLC systems, using RP-2 stationary phase and methanol and water as the binary mobile phase. The window diagrams were performed presenting plots of a vs. volume fraetion Xj derived from the retention models of Snyder, Schoen-makers, and Kowalska [76]. 

A very helpful tool for manual application can be the employment of layers with a concentrating zone. The so-called concentrating or preadsorbent zone is a small part of the plate that is covered with an inert but highly porous adsorbent such as diatomaceous earth. Various precoated preparative layers with a preadsorbent zone are commercially available. The effect of the concentrating zone is depicted elsewhere in detail (see Chapter 3, Figure 3.4). In brief, the preadsorbent zone serves as a platform for manual application of any desired performance quality. When development starts, soluble components migrate with the mobile phase front and are... 

One of the most crucial influencing factors in planar chromatography is the vapor space and the interactions involved. The fact that the gas phase is present, in addition to stationary and mobile phases, makes planar chromatography different from other chromatographic techniques. Owing to the characteristic of an open system the stationary, mobile, and vapor phases interact with each other until they all are in equihbrium. This equilibrium is much faster obtained if chamber saturation is employed. This is the reason for differences in separation quality when saturated and unsaturated chambers are used. However, the humidity of the ambient air can also influence the activity of the layer and, thus, separation. Especially during sample application, the equihbrium between layer activity and relative humidity of the... 

Valko et al. [37] developed a fast-gradient RP-HPLC method for the determination of a chromatographic hydrophobicity index (CHI). An octadecylsilane (ODS) column and 50 mM aqueous ammonium acetate (pH 7.4) mobile phase with acetonitrile as an organic modifier (0-100%) were used. The system calibration and quality control were performed periodically by measuring retention for 10 standards unionized at pH 7.4. The CHI could then be used as an independent measure of hydrophobicity. In addition, its correlation with linear free-energy parameters explained some molecular descriptors, including H-bond basicity/ acidity and dipolarity/polarizability. It is noted [27] that there are significant differences between CHI values and octanol-water log D values. 

FTIR in multiply hyphenated systems may be either off-line (with on-line collection of peaks) [666,667] or directly on-line [668,669]. Off-line techniques may be essential for minor components in a mixture, where long analysis times are required for FT-based techniques (NMR, IR), or where careful optimisation of the response is needed. In an early study a prototype configuration comprised SEC, a triple quadrupole mass spectrometer, off-line evaporative FTIR with splitting after UV detection see Scheme 7.12c [667]. Off-line IR spectroscopy (LC Transform ) provides good-quality spectra with no interferences from the mobile phase and the potential for very high sensitivity. Advanced approaches consist of an HPLC system incorporating a UV diode array, FTIR (using an ATR flow-cell to obtain on-flow IR spectra), NMR and ToF-MS. 

The factors affecting Rf include the quality of the stationary and mobile phases, the thickness and activity of the layer, and the amount of sample. Although standards may have the same Rf value as the sample, this does not uniquely identify the compound. For archaeological samples, the best identification achievable is only at a general class level (e.g., triacylglycerols, fatty acids, aromatic, or aliphatic) and not to individual molecular components. 

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. 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

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