Mobile phase optimum mixture

The mixture of acetonitrile/water (1 1, v/v) was selected as most effective mobile phase. The optimum conditions for chromatography were the velocity of mobile phase utilization - 0,6 ml/min, the wave length in spectrophotometric detector - 254 nm. The linear dependence of the height of peack in chromathography from the TM concentration was observed in the range of 1-12.0 p.g/mL. 

If the mixture to be separated contains fairly polar materials, the silica may need to be deactivated by a more polar solvent such as ethyl acetate, propanol or even methanol. As already discussed, polar solutes are avidly adsorbed by silica gel and thus the optimum concentration is likely to be low, e.g. l-4%v/v and consequently, a little difficult to control in a reproducible manner. Ethyl acetate is the most useful moderator as it is significantly less polar than propanol or methanol and thus, more controllable, but unfortunately adsorbs in the UV range and can only be used in the mobile phase at concentrations up to about 5%v/v. Above this concentration the mobile phase may be opaque to the detector and thus, the solutes will not be discernible against the background adsorption of the mobile phase. If a detector such as the refractive index detector is employed then there is no restriction on the concentration of the moderator. Propanol and methanol are transparent in the UV so their presence does not effect the performance of a UV detector. However, their polarity is much greater than that of ethyl acetate and thus, the adjustment of the optimum moderator concentration is more difficult and not easy to reproduce accurately. For more polar mixtures it is better to explore the possibility of a reverse phase (which will be discussed shortly) than attempt to utilize silica gel out of the range of solutes for which it is appropriate. 

The newcomer to chromatography, faced with a hitherto unknown sample, would do well to start with a C8 silica based reverse phase and an acetonitrile water mixture as a mobile phase and carry out a gradient elution from 100% water to 100% acetonitrile. From the results, the nature and the complexity of the sample can be evaluated and a more optimum phase system can be inferred. 

The peak capacity is not pertinent as the separation was developed by a solvent program. The expected efficiency of the column when operated at the optimum velocity would be about 5,500 theoretical plates. This is not a particularly high efficiency and so the separation depended heavily on the phases selected and the gradient employed. The separation was achieved by a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. Initially the tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase. 

Mobile phases are of a greater variety than the restricted number of stationary phases. Many solvents and their mixtures are used as a mobile phase. The possibility of slight modification of solvent proportions in a mixmre permits the increase of mobile phase number and, thus, different results in the component separation of the analyzed sample. That is why the optimum mobile phase selection becomes one of the basic operations for the success of the analysis. 

Pelander et al. [81] developed a computer program for optimization of the mobile phase composition in TLC. They used the desirability function technique combined with the PRISMA model to enhance the quahty of TLC separation. They apphed the statistical models for prediction of retardation and band broadening at different mobile phase compositions they obtained using the PRISMA method the optimum mobile phase mixtures and a good separation for cyanobacterial hepatotoxins on a normal phase TLC plate and for phenolic compound on reversed-phase layers. 

Experimental values of H, obtained from equation (4.46) and plotted against the rate of flow of mobile phase for a given solute and set of conditions, produce a hyperbolic curve showing an optimum flow rate for maximum efficiency (Figure 4.15). The position of the maximum varies with the solute, and a family of curves can be derived for the components of a mixture. The most efficient flow rate for a particular sample is, therefore, a matter of compromise. The equation also indicates that the highest... 

Adequate resolution of the components of a mixture in the shortest possible time is nearly always a principal goal. Establishing the optimum conditions by trial and error is inefficient and relies heavily on the expertise of the analyst. The development of computer-controlled HPLC systems has enabled systematic automated optimization techniques, based on statistical experimental design and mathematical resolution functions, to be exploited. The basic choices of column (stationary phase) and detector are made first followed by an investigation of the mobile phase composition and possibly other parameters. This can be done manually but computer-controlled optimization has the advantage of releasing the analyst for other... 

The Snyder-solvent-selectivity triangle concept can be combined with a mixture-design statistical technique to define the optimum mobile-phase composition for a particular separation A feature of this mixture-design... 

In this experiment, a mixture of methyl, propyl, and butyl paraben (structures shown in Figure 13.14) in methanol solvent will be separated by reverse phase HPLC. Mobile phase compositions of varying polarities will first be tested to see which one gives the optimum resolution of this mixture, and following this, a standard curve for methyl paraben will be constructed and its concentration in this solution determined. 

Such solvent systems continued to be used even though the lack of solubility of triacyl-glycerols with carbon numbers greater than 46 in this mobile phase has been noted. The solvent gradients that would be required for optimum separations of complex triacylglycerol mixtures are not compatible with RI detection. Therefore, ultraviolet detectors have also been used, but the range of mobile phases is limited, since TGs absorb only in the far-UV range. 

A liquid-liquid system can be created by coating a particulate matter with a thin layer of a liquid phase, similar to the way packed columns are used in GLC. To maintain such an LLC column, the stationary phase should be insoluble in the mobile phase, just as GLC phases need to be involatile at the temperature of operation. Unfortunately, insolubility is an absolute demand that can at best be approximated in practice. The solubility of the stationary phase in the mobile phase becomes even more critical once some flexibility is desired with regard to the choice of the mobile phase. For example, mixtures of several pure solvents are usually required in order to adapt the eluotropic strength (polarity) of the mobile phase such that the capacity factors fall in the optimum range. 

In RPLC retention varies exponentially with the composition of the mobile phase, i.e. approximately straight lines are obtained in a plot of In k vs.

retention behaviour of each individual solute, then the optimum conditions (LSS gradient, see section 5.4) correspond to a linear gradient (figure 6.2b). Linear gradients will indeed be optimal when acetonitrile-water mixtures are used as the mobile... 

The predictive method of Jandera et al. [628] requires knowledge of the isocratic retention data of all solute components in binary and (preferably) ternary mobile phase mixtures. Once these data are available, the method may be very helpful in obtaining an adequate (but not an optimum) separation with a ternary gradient. Unfortunately, the data required for the application of this predictive method are almost never available, and hence a large number of experiments need to be performed before any predictions can take place. When this is the case the method is of very little practical use. 

The optimum process for this binary separation would be to have fixed positions for the introduction of mobile phase and feed, and fixed collection points for the two components of the mixture whilst having the ability to move the stationary phase upwards. In practice it is impossible to engineer a system where the column bed moves, but it is possible to simulate the movement. Such a system is shown schematically in Figure 1.6 where four columns are set in sequence with four multi-port valves between the columns. 

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