Column selectivity, HPLC

Scheme 4.5 illustrates HPLCphase selection. Column manufacturers may have an applications database from which they can recommend a column and a method. Specific methods have been established for quite a large number of analytes, such as additives (e.g. antioxidants). Column selection and column technology have been reviewed [549]. Contrary to GC, and with the exception of SEC, selectivity in HPLC is determined not by the column alone but also by the mobile phase. There is therefore no one-for-one assignment between an analytical problem and the best column for this problem. 

Detector selectivity is much more important in LC than in GC since, in general, separations must be performed with a much smaller number of theoretical plates, and for complex mixtures both column separation and detector discrimination may be equally significant in obtaining an acceptable result. Sensitivity is important for trace analysis and for compatibility with the small sizes and miniaturised detector volumes associated with microcolumns in LC. The introduction of small bore packed columns in HPLC with reduced peak volume places an even greater strain on LC detector design. It is generally desirable to have a nondestructive detector this allows coupling several detectors in series (dual... 

D acquarica, I., Gasparrini, F., Giannoli, B., Badaloni, E., Galletti, B., Giorgi, F., Tinti, M.O., Vigevani, A. (2004). Enantio- and chemo-selective HPLC separations hy chiral-achiral tandem-columns approach the combination of CHIROBIOTIC TAG and SCX for the analysis of propionyl carnitine and related impurities. J. Chromatogr. A 1061, 167-173. 

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

The determination of endogenous compounds and drugs in biological matrices has always presented a formidable challenge as one has to consider various factors before attempting to develop a suitable HPLC assay. These include the physicochemical properties of the compound such as the pKa value, solubility, volatility, particular functional groups (e.g., possessing chromophores, fluorophores, or electroactive characteristics), potential metabolites, and the required sensitivity and specificity. All these aspects will determine the type of extraction processes, analytical column selection, and suitable detector systems to be used as part of the HPLC apparatus. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Column selection high performance liquid chromatography Electrical safety in the laboratory Flammable and explosive Instrumentation of HPLC (slide show and discussion)... 

SOURCE C. S. Young and R. J. Weigand, An Efficient Approach to Column Selection in HPLC Method Development," LCGC 2002, 20, 464. 

Figure 25-29 Separation of six compounds on (a) phenyl- and (b) Cle-silica columns with 3-p.m particle size using 35 65 (vol/vol) acelonitrile/0.2% aqueous trifluoroacetic acid. Column size 7 x 53 mm flow rate = 2.5 mL/min. [From C. S. Young and R. J. Weigand, "An Efficient Approach to Column Selection In HPLC Method Development." LCGC 2002,20.464. Courtesy Alltech Associates.]...

Like buying computer software, the first step is to decide exactly what you will be using the HPLC for today and possibly in the future. I m not talking about specific separations at this point those decisions will be used to control column selection, which we will discuss in a moment. What I m really looking for is an overall philosophy of use. 

Selecting a column for an HPLC separation is a matter of asking yourself a series of questions (Fig. 5.4). You first must determine how much material you wish to separate in a single injection (preparative vx. semipreparative vs. analytical). The next question involves the separation mode to be employed (size exclusion vx. ion exchange vx partition). Finally, there is the question of solubility controlling solvent and column selection in all modes. 

Orlando and Bonato [73] presented a practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma. Ci8 solid-phase extraction cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol-hexane (70 30) as the mobile phase, at a flow-rate of 0.5 ml/min. The detection was carried out at 302 nm. The method is linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%. 

If the separation factor is unity, the peaks coincide, and no separation has occurred. If the separation factor is 1.3, the column selectively retards one component 30% more than the other. The larger the a value, the easier the HPLC separation is to achieve however, an a value of 1.1-1.4 is typically desired. As we show later in this chapter, because resolution is influenced by three factors, separations can be attained for a values smaller than 1.1. In some modes of modern HPLC, meaningful resolution can be achieved with a values as low as 1.05, which means that the column retards the second component only 5% more than it retards the first component. 

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