The Mobile Phase

Unlike GC, the mobile phase in HPLC can play a major role in the separation process. There are no major restrictions apart from those imposed by the stability of the stationary phase (or column itself) to solvent or pH and the requirements of mutual solubility of the compo- 

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Rf, Rg, Rf A measure of retention of solute in chromatography defined as the ratio of the distance travelled by the solute to the distance travelled by the mobile phase. 

The average linear velocity u of the mobile phase in terms of the column length L and the average linear velocity of eluent (which is measured by the transit time of a nonretained solute) is... 

When the mobile phase is a gas, a compressibility factor j must be applied to the adjusted retention volume to give the net retention volume ... 

Now t[i is a minimum when k = 2, that is, when = 3t . There is little increase in analysis time when k lies between 1 and 10. A twofold increase in the mobile-phase velocity roughly halves the analysis time (actually it is the ratio Wu which influences the analysis time). The ratio Wu can be obtained from the experimental plate height/velocity graph. 

In these expressions, dp is the particle diameter of the stationary phase that constitutes one plate height. D is the diffusion coefficient of the solute in the mobile phase. 

Assumed reduced parameters h -linear velocity of the mobile phase. 

Chromatographic separations are accomplished by continuously passing one sample-free phase, called a mobile phase, over a second sample-free phase that remains fixed, or stationary. The sample is injected, or placed, into the mobile phase. As it moves with the mobile phase, the sample s components partition themselves between the mobile and stationary phases. Those components whose distribution ratio favors the stationary phase require a longer time to pass through the system. Given sufficient time, and sufficient stationary and mobile phase, solutes with similar distribution ratios can be separated. 

Analytical separations may be classified in three ways by the physical state of the mobile phase and stationary phase by the method of contact between the mobile phase and stationary phase or by the chemical or physical mechanism responsible for separating the sample s constituents. The mobile phase is usually a liquid or a gas, and the stationary phase, when present, is a solid or a liquid film coated on a solid surface. Chromatographic techniques are often named by listing the type of mobile phase, followed by the type of stationary phase. Thus, in gas-liquid chromatography the mobile phase is a gas and the stationary phase is a liquid. If only one phase is indicated, as in gas chromatography, it is assumed to be the mobile phase. 

A chromatographic peak may be characterized in many ways, two of which are shown in Figure 12.7. The retention time, is the elapsed time from the introduction of the solute to the peak maximum. The retention time also can be measured indirectly as the volume of mobile phase eluting between the solute s introduction and the appearance of the solute s peak maximum. This is known as the retention volume, Vr. Dividing the retention volume by the mobile phase s flow rate, u, gives the retention time. 

Besides the solute peak, Figure 12.7 also shows a small peak eluted soon after the sample is injected into the mobile phase. This peak results from solutes that move through the column at the same rate as the mobile phase. Since these solutes do not interact with the stationary phase, they are considered nonretained. The time or volume of mobile phase required to elute nonretained components is called the column s void time, or void volume. 

From equation 12.1 it is clear that resolution may be improved either by increasing Afr or by decreasing wa or w-q (Figure 12.9). We can increase Afr by enhancing the interaction of the solutes with the column or by increasing the column s selectivity for one of the solutes. Peak width is a kinetic effect associated with the solute s movement within and between the mobile phase and stationary phase. The effect is governed by several factors that are collectively called column efficiency. Each of these factors is considered in more detail in the following sections. 

The distribution of a solute, S, between the mobile phase and stationary phase can be represented by an equilibrium reaction... 

A solute s capacity factor can be determined from a chromatogram by measuring the column s void time, f, and the solute s retention time, (see Figure 12.7). The mobile phase s average linear velocity, m, is equal to the length of the column, L, divided by the time required to elute a nonretained solute. 

The solute can only move through the column when it is in the mobile phase. Its average linear velocity, therefore, is simply the product of the mobile phase s average linear velocity and the fraction of solute present in the mobile phase. 

Nonideal asymmetrical chromatographic bands showing (a) fronting and (b) tailing. Also depicted are the corresponding sorption isotherms showing the relationship between the concentration of solute in the stationary phase as a function of its concentration in the mobile phase. 

The process of changing the mobile phase s solvent strength to enhance the separation of both early and late eluting solutes. 

To determine how the height of a theoretical plate can be decreased, it is necessary to understand the experimental factors contributing to the broadening of a solute s chromatographic band. Several theoretical treatments of band broadening have been proposed. We will consider one approach in which the height of a theoretical plate is determined by four contributions multiple paths, longitudinal diffusion, mass transfer in the stationary phase, and mass transfer in the mobile phase. 

One contribution to band broadening due to the time required for a solute to move from the mobile phase or the stationary phase to the interface between the two phases. 

An equation showing the effect of the mobile phase s flow rate on the height of a theoretical plate. 

To increase the number of theoretical plates without increasing the length of the column, it is necessary to decrease one or more of the terms in equation 12.27 or equation 12.28. The easiest way to accomplish this is by adjusting the velocity of the mobile phase. At a low mobile-phase velocity, column efficiency is limited by longitudinal diffusion, whereas at higher velocities efficiency is limited by the two mass transfer terms. As shown in Figure 12.15 (which is interpreted in terms of equation 12.28), the optimum mobile-phase velocity corresponds to a minimum in a plot of H as a function of u. 

One of the most important advances in column construction has been the development of open tubular, or capillary columns that contain no packing material (dp = 0). Instead, the interior wall of a capillary column is coated with a thin film of the stationary phase. The absence of packing material means that the mobile phase... 

The most common mobile phases for GC are He, Ar, and N2, which have the advantage of being chemically inert toward both the sample and the stationary phase. The choice of which carrier gas to use is often determined by the instrument s detector. With packed columns the mobile-phase velocity is usually within the range of 25-150 mF/min, whereas flow rates for capillary columns are 1-25 mF/min. Actual flow rates are determined with a flow meter placed at the column outlet. 

A chromatographic technique in which the mobile phase is a gas and the stationary phase is a liquid coated either on a solid packing material or on the column s walls. 

The most widely used particulate support is diatomaceous earth, which is composed of the silica skeletons of diatoms. These particles are quite porous, with surface areas of 0.5-7.5 m /g, which provides ample contact between the mobile phase and stationary phase. When hydrolyzed, the surface of a diatomaceous earth contains silanol groups (-SiOH), providing active sites that absorb solute molecules in gas-solid chromatography. 

Thermal Conductivity Detector One of the earliest gas chromatography detectors, which is still widely used, is based on the mobile phase s thermal conductivity (Figure 12.21). As the mobile phase exits the column, it passes over a tungsten-rhenium wire filament. The filament s electrical resistance depends on its temperature, which, in turn, depends on the thermal conductivity of the mobile phase. Because of its high thermal conductivity, helium is the mobile phase of choice when using a thermal conductivity detector (TCD). 

When a solute elutes from the column, the thermal conductivity of the mobile phase decreases and the temperature of the wire filament, and thus its resistance, increases. A reference cell, through which only the mobile phase passes, corrects for any time-dependent variations in flow rate, pressure, or electrical power, ah of which may lead to a change in the filament s resistance. 

A chromatographic technique in which the mobile phase is a liquid. 

In liquid-liquid chromatography the stationary phase is a liquid film coated on a packing material consisting of 3-10 pm porous silica particles. The stationary phase may be partially soluble in the mobile phase, causing it to bleed from the column... 

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

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