Mobile phase

The mobile phase is the interstitial space in the packed column. The mass balance for a solute in the mobile phase can be obtained from the continuity equa- 

From a mass spectrometry perspective, the pump must be pulse free, i.e. it must deliver the mobile phase at a constant flow rate. Pulsing of the flow causes the total ion current (TIC) trace (see Chapter 3) - the primary piece of information used for spectral analysis - to show increases in signal intensity when analytes are not being eluted and this makes interpretation more difficult. 

In contrast to gas chromatography, in which a number of different types of injector are available and the selection of which is often crucial to the success (or otherwise) of the analysis, a single type of injector is used almost exclusively in HPLC. 

From a quantitative perspective, the way in which the injector functions is crucial to the precision and accuracy which may be obtained and therefore these two parameters are of paramount importance. 

Loops are not calibrated accurately and a loop of nominally 20. il is unlikely to have this exact volume. This will not affect either the precision of measurement and, as long as the same loop is used for obtaining the quantitative calibration and for determining the unknowns , the accuracy of measurement. 

From a mass spectrometry perspective, the injector is of little concern other than the fact that any bubbles introduced into the injector may interrupt the liquid flow, so resulting in an unstable response from the mass spectrometer. 

Not all antibiotics are retained and separated well on the same type of analytical columns. Columns of different retention mechanism and/or mobile-phase additives are required for improved chromatographic performance, which is discussed further in Section 6.3.3. 

Several chromatographic parameters or characteristics are important when choosing a column or selecting an LC elution profile for residue method development and validation. These characteristics include the following  

Retention factor (k) or capacity factor, which is a measure of the relative speed of an analyte through a column and is calculated as  

Resolution Rs), which describes the degree of separation between two analytes, is based on the distance between two peaks and their dispersion. Resolution is calculated as a ratio of the distance between peaks to their width. 

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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 column is swept continuously by a carrier gas such as helium, hydrogen, nitrogen or argon. The sample is injected into the head of the column where it is vaporized and picked up by the carrier gas. In packed columns, the injected volume is on the order of a microliter, whereas in a capillary column a flow divider (split) is installed at the head of the column and only a tiny fraction of the volume injected, about one per cent, is carried into the column. The different components migrate through the length of the column by a continuous succession of equilibria between the stationary and mobile phases. The components are held up by their attraction for the stationary phase and their vaporization temperatures. 

The distance d corresponds to the movement of solute and mobile phase from the starting (sample spotting) line. Subscript r represents an ion-exchange resin phase. Two immiscible liquid phases might be represented similarly using subscripts 1 and 2. ... 

Concentration of solute in mobile phase Cm Diffusion coefficient, liquid film Dt... 

Retention Behavior. On a chromatogram the distance on the time axis from the point of sample injection to the peak of an eluted component is called the uncorrected retention time The corresponding retention volume is the product of retention time and flow rate, expressed as volume of mobile phase per unit time ... 

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 ... 

The relative retention is dependent on (1) the nature of the stationary and mobile phases and (2) the column operating temperature. 

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. 

The volume of mobile phase needed to move a solute from its point of injection to the detector (Vr). 

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... 

Note that this equation is identical to that describing the extraction of a solute in a liquid-liquid extraction (equation 7.25 in Chapter 7). Since the volumes of the stationary and mobile phase may not be known, equation 12.4 is simplified by dividing both the numerator and denominator by V thus... 

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. 

In their original theoretical model of chromatography, Martin and Synge treated the chromatographic column as though it consists of discrete sections at which partitioning of the solute between the stationary and mobile phases occurs. They called each section a theoretical plate and defined column efficiency in terms of the number of theoretical plates, N, or the height of a theoretical plate, H where... 

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. 

Use of column selectivity to improve chromatographic resolution showing (a) the variation in retention time with mobile phase pH, and (b) the resulting change in alpha with mobile phase pH. 

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