Eluent or mobile phase

Proteins contain a variety of functional groups and interact with the stationary phase at a number of simultaneous sites on the protein molecule, each more or less affected by change in eluent, or mobile-phase, pH or ionic strength. The equilibrium constant for the dissociation of the adsorption complex thus contains a product of many eluent-sensitive concentration terms, and the equilibrium position is very sensitive to elution conditions. Under the elution conditions, some proteins in a mixture may be tightly bound by the stationary phase (t), oo) while others are unretained (t R 0). Differential migration (Section 19.2.1) is replaced by extreme retention values. 

Carrier electrolyte or buffer Eluent or mobile phase... 

Chapter 4 deals with the selection of a chromatographic system, i.e. the optimal combination of stationary phase, eluent or mobile phase for a given separation task. These key issues focus not only on solely scientific questions, but also take into account economy, speed, time pressure, hardware requirements, automation and legal aspects towards documentation, safety and others. Obviously, such rules of thumb may not cover all possible scenarios, but they may be useful in avoiding pitfalls. 

An HPLC system, shown schematically in Figure 2.1, consists of a solvent reservoir, which contains the eluent or mobile phse a pump, often called a solvent delivery system an injector through which the sample is introduced into the system without a drop in pressure or change in flow rate the analytical column, which is usually stainless steel and contains the solid packing or stationary phase and a suitable detector to monitor the eluent. 

Mobile Phases An organic solvent (called co-solvent or modifier) is very often used in SFC to increase the solubility of the crude mixture in the eluent and fine-tune the eluent strength. Mobile phases on SFC use from 0 to 40% of modifier. Fresh modifier is pumped in the system vith a high pressure pump. The modifier is collected at the liquid outlet of the separators. 

Eluent The solvent or mobile phase used in SPE or chromatography. 

In reeent years, tire use of elevated temperatures has been reeognised as a potential variable in method development. Witlr inereased temperature, aqueous-organie mobile phases separations ean improve, viseosity deereases and diffusion inereases so baek pressures are redueed. At higher temperatures (usually with superheated water > 100 °C under modest pressures) water alone ean be used as the mobile phase and eair provide unique separation opportunities. The absenee of an organie solvent enables the use in HPLC of alternative deteetors sueh as FID or on-line LC-NMR using deuterium oxide as the eluent. 

The influence of NH., and CO, on the chromatographic behaviour of benzoic acid and its derivatives (o-, m-, p-hydroxybenzoic, nitrobenzoic, aminobenzoic, chlorobenzoic acids) was studied. The work was carried out by means of upgoing TLC on Sorbfil plates. Isopropanol- and ethyl acetate-containing water-organic eluents were used as mobile phases in the absence or presence of gaseous modifiers in the MP. The novel modification of TLC has been found to separate benzoic acids with different values of their dissociation constants more effectively than water-organic mobile phases. 

If the mobile phase is a liquid, and can be considered incompressible, then the volume of the mobile phase eluted from the column, between the injection and the peak maximum, can be easily obtained from the product of the flow rate and the retention time. For more precise measurements, the volume of eluent can be directly measured volumetrically by means of a burette or other suitable volume measuring vessel that is placed at the end of the column. If the mobile phase is compressible, however, the volume of mobile phase that passes through the column, measured at the exit, will no longer represent the true retention volume, as the volume flow will increase continuously along the column as the pressure falls. This problem was solved by James and Martin [3], who derived a correction factor that allowed the actual retention volume to be calculated from the retention volume measured at the column outlet at atmospheric pressure, and a function of the inlet/outlet pressure ratio. This correction factor can be derived as follows. 

More commonly, a fraction, based on chemical type, molecular weight or volatility, is heart-cut from the eluent of the primary column and introduced into a secondary column for more detailed analysis. If the same mobile phase is used in both dimensions, fractions may be diverted by means of pressure changes-an approach first used in 1968 in GC-GC by Deans (35), and applied by Davies et al. in SFC-SFC (36). If the mobile phases are different, valves are employed, and special... 

Traditionally, LC and GC are used as separate steps in the sample analysis sequence, with collection in between, and then followed by transfer. A major limitation of off-line LC-GC is that only a small aliquot of the LC fraction is injected into the GC p. (e.g. 1 - 2 p.1 from 1 ml). Therefore, increasing attention is now given to the on-line combination of LC and GC. This involves the transfer of large volumes of eluent into capillary GC. In order to achieve this, the so-called on-column interface (retention gap) or a programmed temperature vaporizor (PTV) in front of the GC column are used. Nearly all on-line LC-GC applications involve normal-phase (NP) LC, because the introduction of relatively large volumes of apolar, relatively volatile mobile phases into the GC unit is easier than for aqueous solvents. On-line LC-GC does not only increase the sensitivity but also saves time and improves precision. 

An alternative way of eliminating water in the RPLC eluent is to introduce an SPE trapping column after the LC column (88, 99). After a post-column addition of water (to prevent breakthrough of the less retained compounds), the fraction that elutes from the RPLC column is trapped on to a short-column which is usually packed with polymeric sorbent. This system can use mobile phases containing salts, buffers or ion-pair reagents which can not be introduced directly into the GC unit. This system has been successfully applied, for example, to the analysis of polycyclic aromatic hydrocarbons (PAHs) in water samples (99). 

In this technique, the development distance is increased linearly in 10- or 20-mm steps (with evaporation of the mobile phase from the plates after each step) by using the same solvent or a series of solvents for modified IMD technique. In gradient IMD, the eluent strength is rednced stepwise. 

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