Choosing the Stationary Phase

The type and amount of stationary phase influence the retention of compounds in the column. The effect of various variables upon retention time (Ir) or retention volume ( Vr) of a compound can be found from Equation 2.4. Vr = Ir F, where F is the flow rate in milliliter per minute  

If a nonpolar stationary phase is used, the sample components will have little or no interaction vith the stationary phase (y°° is close to 1) and separation is obtained according to the boiling point (Bp) of the components. 

If a more polar stationary phase is used, the separation is obtained according to both polarity (interaction) and boiling point. This means that it is possible to separate compounds with the same Bp, but with different functional groups. 

The liquid used as stationary phase must be thermally stable and give minimal bleeding (loss of stationary phase because of evaporation). 

The silicones have good thermal stability, and stationary phases with a wide variety of R and R, and hence polarity, are easily made (Table 2.5). 

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These few remarks should suffice to demonstrate the importance of the precise knowledge of the various layer materials and the precise documentation of their use. Such differences should also be taken into account when choosing the stationary phase so that the impression is not later produced that phase A is better or worse than phase B. 

It is often easier to elute drugs from silica than from either cellulose or alumina, and only bonded stationary phases should be used in reversed-phase systems or else the organic stationary phase will also be eluted. These points should be considered when choosing the stationary phase. 

In choosing the stationary phase, the following two major factors are to be considered —... 

The extent to which two solutes are separated depends exclusively on the relative magnitudes of their individual distribution coefficients (K(a)) and (K(b)) and the amount of stationary phase with which they can interact, (V(a)) and (V(b)). Consequently, for them to be separated, either their distribution coefficients must differ (choose appropriate phase systems), the amount of stationary phase with which they interact must differ (choose a stationary phase with exclusion properties), or a subtle combination of both. 

The analyst must remember that solubility of a polymer in the chosen eluant is a necessary, but not sufficient, requirement for ideal GPC separations. Once injected on the column, the polymer has a choice of partitioning onto the stationary phase or remaining in the solvent. It is imperative that the analyst choose solvent and column conditions such that the ideal, nonadsorptive, GPC mechanism can occur. 

LC is a limiting technique that occurs when the column outlet pressure is near ambient and we choose well-behaved liquids as our mobile phases. Our only means of adjusting solute retention (after selecting the stationary phase and the... 

In lc there are other sorption mechanisms that can cause separation, depending on whether we choose to use a liquid or a solid as the stationary phase, or what kind of solid we use. Liquid-liquid chromatography (11c) uses a liquid stationary phase coated onto a finely divided inert solid support. Separation here is due to differences in the partition coefficients of solutes between the stationary liquid and the liquid mobile phase. In normal phase 11c the stationary phase is relatively polar and the mobile phase relatively non-polar, whilst... 

The scouting exercise is comprised of a screening of the stationary phases with aqueous gradients and organic modifiers such as acetonitrile, methanol, tetrahy-drofuran, and others. For the reasons discussed in the Isolation section, the use of additives and buffers is avoided or minimized. Having identified a suitable chromatographic system (e.g. as in Figure 7.4), the practitioner may choose to further... 

If separation according to functionality cannot be achieved because of a small difference between the interaction energies of the functional group and the chain groups, one can try to improve the efficiency of the separation system, but it is necessary to choose another stationary phase which is more selective with respect to functional groups. 

CHO, —OC(O)H, and -0C(0)CH3 may also be obtained. The value of AHgvp or a Particular functional group is a measure of the strength of interaction between the solute and the stationary phase. Thus, knowledge of AHevp for a mixture, of solutes can help in choosing the liquid phase to obtain optimum separation. 

Choosing the mobile and the stationary phase of roughly the same polarity (i.e. Sm x Ss). While this has the desired effect on retention, the remedy is equally effective for all solutes, independent of the value of 5, and therefore it creates a very non-selective phase system (see the discussion on selectivity below). 

Before proceeding with a discussion on the individual modes of separation it is worth reviewing a brief glossary of terms in association with the stationary phase properties. A simple strategy for selection of the stationary phase will naturally follow, in order to help the reader to choose a supplier wisely. 

Let us look at the benzene-cyclohexane separation more closely as we summarize how GC works. The boiling points of benzene and cyclohexane are nearly the same, 80.1 and 81.4°C respectively. Any GC separation will have to depend on differences in the intermolecular interactions between the stationary phase and these two analytes, both of which are nonpolar hydrocarbons. What differences could be exploited with GC Benzene has a -n-electron cloud, which should make it more susceptible to induction effects and perhaps dispersion attractions (Chapter 3). Therefore we should choose a stationary liquid phase that would accentuate this difference—a polar one also, using the like-dissolves-like rule we might choose an aromatic compound that would interact more with benzene than with cyclohexane. One possible liquid phase that meets these criteria is dinonylphthalate, and it has been used to separate benzene and cyclohexane. The relative retention has been found to be 1.6, which represents a very good separation.1... 

Chapter 3 ended by noting that an alternative way of estimating the polarity of a stationary phase was to use probes. Basically, the extent of interaction between the stationary phase and a given sample will be reflected in the adjusted retention time (or partition ratio or retention index, /) of the solute. Thus, by choosing as solute probes those chemicals that are thought to have particularly strong selective interactions, one can get a measure of the relative magnitude of that interaction from its retention index. 

There are a variety of ways to approach the important task of choosing a stationary phase (a column) for a GC analysis. Chapter 12 elaborates on the searching of the literature and logical decision-making processes based on the information that is at hand. The procedures outlined here are more concerned with the fundamentals of matching a liquid phase to a separation problem, drawing on theoretical principles as much as possible. 

Applications. SEC is the easiest LC technique to understand and apply. Choosing the stationary and mobile phases is relatively easy as just described, and the separation achieved on the selected system is predictable. 

Intermolecular forces were discussed in Chapter 3 and extended to GC stationary phases in Chapter 8. Rohrschneider, followed by McReynolds, investigated the nature of GC stationary phases by using a few common chemicals as probes. Their retention on a given liquid reflected the extent of their interaction with the stationary phase. By choosing probes with selective interactions, they could determine a set of numbers that characterize the liquids under study. 

In everyday method development practice, it is important to ensure the separation of target compounds, matrix components, and other impurities. The elution of the analyte at the void volume means that it did not interact with the stationary phase and thus could not be separated from other components that do not interact with the surface either. To ensure the analyte interaction with the stationary phase, it is usually recommended to choose chromatographic conditions when any component of interest elutes with at least 1.5 void volume values or even greater. The error of the void volume determination for these purposes could be 20% or even greater (insofar as these void volume values are not used for any calculations but just to estimate where the least retained analye elutes). The use of uracil, thiourea, or allantoin as analytical void volume markers is most common in practical analytical work. 

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