Mobile-phase changing

D. Tong and K. D. Battle, Band broadening during mobile phase change in unified cliromtography (GC-SEC) , 7. Microcolumn Sep. 5 237-243 (1993). 

The ideal reference electrode has a stable and reproducible potential versus the mobile phase, that does not vary with mobile phase changes. 

When the pH of the mobile phase changes linearly in time the pH change can be... 

The refractive index detector, considered to be almost universal, is often used in series with a UV detector in the isocratic mode to provide a supplementary chromatogram. This detector, which is not highly sensitive, has to be temperature controlled, as does the column (0.1 °C). The baseline of the chromatogram has to be set to an intermediate position because it can lead to either positive or negative signals (Fig. 3.18). The detector can only be used in the isocratic mode because in gradient elution the composition of the mobile phase changes with time, as does the refractive index. Compensation, which is easily obtained in the case of a mobile phase of constant composition, is difficult to carry out when the composition at the end of the column differs from that at the inlet. 

Step elution is similar to gradient elution in that the composition of the mobile phase changes during the separation process. However, in step elution the change is not continuous. It involves a sudden change in the composition of mobile phase followed by a period where the mobile phase is held constant. This procedure results in a mobile phase profile that resembles a series of steps. 

While ionic strengths as low as 1 mM have been used with the cell illustrated in Figure 1, most LCEC experiments are carried out with a minimum of 0.05 M buffer salts in the mobile phase. Postcolumn mobile phase changes (pH, ionic strength, solvent content) and post-column reactions (redox cross reactions, derivatiza-tions, enzyme catalyzed reactions) can expand the utility of electrochemical as well as other detectors. These subjects have recently been treated in some detail (9). Suffice it to say that direct detection, without post-column chemistry, is always preferable (more reliable, more sensitive, less expensive). 

When used in the normal-phase mode, bonded-phase packings are stable to hydrolysis, have a shorter equilibration time to mobile phase changes than unbonded silica, and do not require water deactivation or humidified mobile... 

Another factor to consider with gradients is that, as the composition of the mobile phase changes, mobile-phase viscosity and refractive index change. 

A series of instruments for rotation planar chromatography have been described. These are based on the work of Hopf [61 ], more than 50 years ago, who introduced an apparatus in which the mobile pha.se was propagated by centrifugal forces. Subsequently methods have been developed by a number of researchers to control the mobile phase movement (for review, see [62 ). The separation can be performed in various types of chambers, such as in a normal chamber, a microchamber, or an ultramicrochamber. The separation takes place during constant rotation and the flow rate of the mobile phase changes throughout, i.e. the flow rate is inversely proportional to the square distance from the centre of the supply. 

High viscosity of mobile phase Change mobile phase, or increase column temperature. Change to lower viscosity solvent. 

Highly UV absorbing mobile phase. Change detection wavelength taking into account the UV cutoff of mobile phase solvents. 

The selection of an appropriate mobile phase is another aspect of concern. Most often the commonly used LC mobile phase is not compatible with thermospray LC/MS, for instance owing to the use of non-volatile buffers can be left out or replaced by volatile ones. In other cases the buffers are present for retention time reproducibility, which mostly is not very important for identification. In other applications however a correspondence between UV or fluorescence peaks and MS identification is obligatory, which makes mobile phase changes unattractive. In this respect it is often overlooked that LC-UV and LC/MS give different responses as a result of different detection principles. For not too complex samples a UV photo-diode array detector can be used to link up the chromatographic peaks obtained under different mobile phase conditions. To cut short, despite many successes also many potential problems are met in LC/MS to which tailor-made creative solutions are needed. 

Protein is not eluted Decrease salt concentration if it is high increase if it is low . Change salt to one that decreases the surface tension. Add amine or another silanophile to the mobile phase. Change pH away from the isoelectric point of the protein. Change stationary phase to one with longer ligate and/or greater chain density, i.e. increase phase ratio. 

The last direct method often used for determination of the colloidal properties of crude oils is gel permeation chromatography. The principles of this method were described in section 2.1.2. Normally, this method is used for analyzing the molecular weight distribution of substances. However, it is possible to use it to analyze colloidal properties as well if an appropriate solvent is used as the mobile phase. This solvent must not change the native disperse particles. Almost all the solvents that can be used in this analysis as a mobile phase change the size of native particles. This is why this analysis is usually used for estimating the particle size in the sample solution. 

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