Potential mobile phases

Electrolysis of mobile phase constituents will cause a continuous detector response (background current) resulting in a chromatographic baseline level that differs from the electrical detector zero-response level. The difference, baseline- offset, is an important analysis parameter, because baseline fluctuations (noise, drift) due to fluctuations in electrolysis conditions (potential, mobile phase flow rate, temperature) are proportional to baseline offset. See Figure 2-5 for an example of the influence of flow pulsation at different baseline offset... 

As noted in this data, water acetonitrile seemed to offer greatest promise as a potential mobile phase due to the greater observed sensitivity. However, the... 

Very recently, the separation of polar analytes has also been performed by using pure water under subcritical conditions. Subcritical water has several unique characteristics. For example, the dielectric constant, surface tension, and viscosity of water are dramatically decreased by raising the water temperature while a moderate pressure is applied to keep water in the liquid state. At 200 -250°C, the values of these physical properties are similar to those of pure methanol or acetonitrile at ambient conditions. Therefore, subcritical water may be a potential mobile phase for polar analytes. SFC mobile phases other than CO2 are reviewed separately in this encyclopedia. 

The vapour pressure of volatile samples has a great influence on retention behaviour, this being one of the analogues with GC. Compounds that are potential mobile phases are listed in Table 23.1. The first two are obviously unsuitable for temperature-sensitive analytes. By far, carbon dioxide is used most frequently. Since it is very nonpolar a common B solvent is methanol. 

TABLE 23.1 Potential mobile phases for supercritical HPLC... 

In an excellent article by Bell et al. [356], the retention of 12 carotenoids (zeaxanthin, lutein, echinenone, / -cryptoxanthin, and a-, 9-cis-a-, 15-cis-a-, 9 -cis-a-, 13-cir-a-, ] -, 3-cis-P-, and 15-c/j- -caiotene) was studied with respect to temperature (277 K to 323 K) on C,g, C30, and C34 columns (A = 450 nm). Methanol was used as a mobile phase on the C g column and 95/5 methanol/methyl r-butyl ether on the C30 and C34 columns. The authors noted that acetonitrile was a potential mobile phase modifier but that high acetonitrile levels ofien led to decreases in recoveries. The use of dichloromethane was discouraged since residual HCI due to natural solvent degradation was implicated in poor recoveries as well. The latter is supported by the fact that low molecular weight alkenes are ofien used as preservatives in dichloromethane. A series of van t Hoff plots (essentially Infc vs. 1 /T) were presented where C]g phase showed near linearity and C30 and C34 phases exhibited nonlinear relationships for most carotenoids. 

The use of an amperometric detector is emphasized in this experiment. Hydrodynamic voltammetry (see Chapter 11) is first performed to identify a potential for the oxidation of 4-aminophenol without an appreciable background current due to the oxidation of the mobile phase. The separation is then carried out using a Cjg column and a mobile phase of 50% v/v pH 5, 20 mM acetate buffer with 0.02 M MgCl2, and 50% v/v methanol. The analysis is easily extended to a mixture of 4-aminophenol, ascorbic acid, and catechol, and to the use of a UV detector. 

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. 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Mobile phases with some solvating potential, such as CO2 or ammonia, are necessary in SGC. Even though this technique is performed with ambient outlet pressure, solutes can be separated at lower temperatures than in GC because the average pressure on the column is high enough that solvation occurs. Obviously, solute retention is not constant in the column, and the local values of retention factors increase for all solutes as they near the column outlet. 

In the introduction to this chapter, MD-PC was defined as a procedure in which substances to be separated were subjected to at least two separation steps with different mechanisms of retention (5). Discussion of the basic potential modes of operation showed that because of the versatility which resulted from being able to combine mobile phases of different composition, more than two development steps can easily be realized by the use of "D techniques. 

Returning now to the subject of the chapter, in addition to appropriate retentive characteristics, a potential stationary phase must have other key physical characteristics before it can be considered suitable for use in LC. It is extremely important that the stationary phase is completely insoluble (or virtually so) in all solvents that are likely to be used as a mobile phase. Furthermore, it must be insensitive to changes in pH and be capable of assuming the range of interactive characteristics that are necessary for the retention of all types of solutes. In addition, the material must be available as solid particles a few microns in diameter, so that it can be packed into a column and at the same time be mechanically strong enough to sustain bed pressures of 6,000 p.s.i. or more. It is clear that the need for versatile interactive characteristics, virtually universal solvent insolubility together with other critical physical characteristics severely restricts the choice of materials suitable for LC stationary phases. 

In effect, the composition of the mobile phase, and thus the selectivity of the chromatographic system, has been changed. As mentioned in the text, dynamic FAB operates effectively with lower concentrations of matrix than static FAB and although its effect may be minimal it should always be considered. Post-column addition of matrix overcomes potential problems of this nature. 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

LCEC is a special case of hydrodynamic chronoamperometry (measuring current as a function of time at a fixed electrode potential in a flowing or stirred solution). In order to fully understand the operation of electrochemical detectors, it is necessary to also appreciate hydrodynamic voltammetry. Hydrodynamic voltammetry, from which amperometry is derived, is a steady-state technique in which the electrode potential is scanned while the solution is stirred and the current is plotted as a function of the potential. Idealized hydrodynamic voltammograms (HDVs) for the case of electrolyte solution (mobile phase) alone and with an oxidizable species added are shown in Fig. 9. The HDV of a compound begins at a potential where the compound is not electroactive and therefore no faradaic current occurs, goes through a region... 

The metabolic and pharmacokinetic profile of sucralose (this is a novel intense sweetener with a potency about 600 times that of sucrose) in human volunteers was studied by Roberts and coworkers [82]. Part of this study was realized using PLC in the following chromatographic system in which the stationary phase was silica gel and the mobile phase was ethyl acetate-methanol-water-concentrated ammonia (60 20 10 2, v/v). Separated substances were scraped off separately, suspended in methanol, and analyzed by filtration, scintillation counting, or enzymatic assay. It was shown that the characteristics of sucralose include poor absorption, rapid elimination, limited conjugative metabolism of the fraction absorbed, and lack of bio-accumulative potential. 

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