Choice of stationary phase

The suitability of a stationary phase for a particular application depends on the selectivity and the degree to which polar compounds are retarded relative to what their retardation would be on a completely non-polar stationary phase. Since retention time is a function of temperature, flow-rate, stationary phase type and loading or film thickness it cannot be used to relate the retention characteristics of one column to another. Various retention index methods have been described such as evaluating the partition and separation properties of solute-stationary phase systems. Kovats (1958) devised a system of indexing chromatographic retention properties of a stationary phase with respect to the retention characteristics of n-alkanes, alkanes being used as reference materials since they are non-polar, chemically inert and soluble in most common stationary phases [8-10]. The retention index (RI) for the n-alkanes is defined as 

Alternatively, the RI for an individual compound, RIc, may be calculated using the following equation  

Rohrschneider and McReynold extended the RI system to predict a PI for various stationary phases measured at a column temperature of 120°C with a 20% (w/w) loading, to minimise retention contributions from the diatomite support [11,12]. A set of five reference compounds were selected to reflect a range of polar characteristics and functional groups benzene, X butanol, Y 2-pentanone, Z nitromethane, [/ and pyridine, S. Squalane, 2, 6, 10, 15, 19, 23-hexamethyltetracosane (C30H62), is used as the reference stationary phase as it is a readily available completely non-polar, non-volatile liquid, bp = 176 at 0.05 mm. The values of X, Y, Z, U and S represent the relative affinities of the reference compounds for the stationary phase, calculated as the differences, ARI, between the RI of the reference on a chosen stationary phase compared to the RI on squalane. The polarity index, PI, is the mean of the RI values (Table 5.2). 

Although PI gives an indication of the polar character of a stationary phase it is necessary to consider the individual values for each reference compound to obtain a true picture of the selectivity of the stationary phase. Note that 

A method to select the appropriate stationary phase for analysis of a sample mixture is to consider the polar characteristics of the analytes and select a stationary phase of similar polarity. An analyte with similar polar character to the stationary phase will be well retained, the principle of like attracts like applies, and useful retention is then likely to occur leading to adequate selectivity and separation of the analytes primarily on the basis of volatility. Conversely, if the solute is immiscible with the stationary phase then little or no retention difference will be obtained. Further useful indication of retention characteristics may be obtained by analysing a sample on a non-polar and polar column, for example, a dual column GC fitted with Apiezon/OVlOl and Carbowax 20M columns temperature programmed 50-220°C at 10°Cmin with a final hold of 10 min. The chromatogram will indicate the polarity of stationary phase required for the components and the analysis can be repeated with columns of differing polarity, e.g. OV17, OV1701. Tables of RIs for various classes of compounds have been published, mainly for squalane, the reference non-polar stationary phase and Apiezon L, with RI values for Carbowax 20M as a reference polar stationary phase [10]. 

The type of column chosen for a particular separation depends on the compound and the aim of analysis. Pharmaceutical companies may have a preferred list of columns that have good demonstrated performance in regard to pH/temperature stability. These columns that have been selected by a specific laboratory are known to be stable within predefined pH and temperature regions in which method development/column screening are employed. A good understanding of the chemical stability of the stationary phases is needed, and some examples are shown in Section 8.10. 

Screening columns from each of the following various column classes should provide for the desired chromatographic selectivity, even for the most challenging separations (1-3) C8 or C18 stable at pH 2, pH 2-8, and pH 8-11 (4) phenyl (5) pentafluorphenyl (6) polar embedded and stationary phases that could be run in 100% aqueous. A certain number of columns in each of the six column classes and subclasses could be chosen as standard columns that the chromatographers choose as a first choice for performing method development. These standard columns could be chosen based on some 

For more hydrophobic compounds, a stationary phase that has a lower surface area should be used. For very polar compounds that cannot be retained on traditional CIS phases, less hydrophobic columns such as C4 and polar embedded stationary phases could be used. However, all this is also dependent on the pH of the analysis since some columns are not stable at low pH ( 2) and higher pH ( 7) for extended periods of time. This should be taken into careful consideration when defining a column(s) during the development of a method. 

Moreover, the effect of pH on a particular compound s retention needs to be determined first before exploring the retentivity and selectivity of different columns. The strategy and choice of the optimal pH for analysis was discussed in Chapter 4 and is further reinforced in the case studies within this chapter. After the optimal pH is chosen for the separation and the gradient has been optimized on a particular column and the optimal selectivity still has not been achieved between critical pairs, then a column screening can be performed. For method column screening, generally columns with 10-cm or 5-cm X 3.0-mm i.d. could be used that are packed with 3-pm particles. Implementation of a column switcher that can use six different types of stationary phases such as two types of C18 from different vendors, phenyl, two polar embedded, and pentafluorphenyl is suggested. 

Moreover, once a particular column or columns that have provided the best selectivity are chosen, an automated method optimization may be performed. This would include employment of an integrated HPLC method development system such as AMDS/Drylab such that the gradient slope/temperature 

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The Diacel columns can be used for the separation of a wide variety of compounds, including aromatic hydrocarbons having hydroxyl groups, carbonyls and sulfoxides, barbiturates, and P-blockers (35,36). There are presendy nine different cellulose derivative-based columns produced by Diacel Chemical Industries. The different columns each demonstrate unique selectivities so that a choice of stationary phases is available to accomplish a separation. 

Excellent selectivity (wide choice of stationary phases)... 

The choice of stationary phase and its degree of activity is determined by the nature of the sample. If sample components are adsorbed too strongly, they may be difficult to elute or chemical changes may occur. Weakly polar solutes should be separated on highly active adsorbents otherwise they may elute rapidly with little or no resolution. Strongly polar solutes are better separated on adsorbents of low activity. Silica gel can be prepared with a... 

Until the introduction of capillary columns, it was not possible to separate all the amino acids found in proteins on one column. The choice of stationary phase will depend upon the types of derivatives that have been prepared and in some situations it still may be preferable to use two different columns simultaneously. 

Coupling a screening system with an analytical fraction collector can be helpful when very small amounts (submilligrams to single-digit milligrams) are required. It often also provides the first opportunity to isolate enriched samples for further use in the method development, for example, as retention time markers. The choice of stationary phases in the method development system can be based on... 

Over 100 stationary phases of various types have been described in the literature for packed columns, which are slowly being abandoned. However, for bonded phase capillary columns the choice of stationary phase is limited because the generation of the film at the surface of the column requires a different principle than impregnation. Generally, two families of compounds are used to modify the polarity polysiloxanes and polyethylene (silicones) glycols. Very special phases such as cyclodextrins can be used for enantiomeric separations. Stationary phases can be used between a minimum temperature under which equilibrium is too slow to occur and a maximum temperature above which degradation of the polymer occurs. The maximum temperature depends on the film thickness and the nature of the polymer. 

A successful separation depends on the right choice of stationary phase, which is characterised by several surface parameters. 

The strong points of this technique are its absence of interaction with the stationary phase, rapid dilution and the potential to recover all of the analytes. Because the technique permits the separation of nominal masses ranging from 200 to 107 Da, its main applications are in the analyses of synthetic and natural polymers. The choice of stationary phase for a given separation is made by examination of the calibration curve of various columns. The column of choice is that which provides a linear range over the masses of the compounds found in the sample. The calibration has to be conducted with the same type of polymers because macromolecules can have various forms ranging from pellet-like to thread-like. The data presented in Table 7.1 show the domains of application of the three gels shown in Fig. 7.3, depending on the standards that are used. 

The high efficiency of open tubular columns makes the choice of stationary phases less complex than with packed columns. A separation that is incomplete on a packed column would have better resolution using an open tubular column. The separation of the xylene isomers on a packed column using 7,8-benzoquinoline requires 20 min and 14,600 theoretical plates whereas the same separation can be performed on an open tubular column in less time but using squalane and utilizing 68,000 theoretical plates. 

Another technique is supercritical fluid chromatography (SFC), which is a chromatographic technique that in many ways is a hybrid of GC and HPLC. It is recognized as a valuable technique for the analysis of thermolabile compounds, which would not be amenable to analysis by GC or HPLC. Few applications have been reported for SFC in the field of OCP and OPP determination (16). The advantages reported for SFC are versatility in separation (by the addition of modifier or the choice of stationary phase) and detection (with LC or GC detectors). However, SFC is a little-used technique because it still presents a wide range of instrumental problems (14-16). 

The choice of stationary phase will be influenced by the polar character of the components of the mixture. In general, mixtures with components of high polarity separate better on chromatography when the more polar stationary phases are used. The chromatographic separation of a mixture is judged to be successful... 

Fast screening is often accomplished by TLC, as indicated in Figure 12.2. The choice of stationary phase can be based on sample solubility and polarity according to Figure 12.3. Silica gel is commonly used for a test run, and homemade silica gel layers on microscope slides (see Chapter 10) provide fast and inexpensive screening. A polar mobile phase is used First, followed by less polar ones and mixtures, depending on the chromatographic results. 

Heron, S. and Tchapla, A. (1994) Choice of stationary phases for separation of mixed triglycerides by liquid phase chromatography. Analusis, 22, 114-126. 

Choice of Stationary Phases Over seven hundred substances have been used as stationary phases. The choice of stationary phase for a particular separation is often a matter of serendipity, depending on a combination of experience, prejudice, intuition, and the desire to use an existing system if it will do the job. 

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