Reversed-phase chromatography tailing

In reversed-phase chromatography, tailing may show up or increase as the column ages. Tliis is due to a partial and slow hydrolysis of the stationary phase. This process exposes additional silanols, which cause or increase peak tailing. This is a natural part of the column aging process. If the column lifetime is too short as a result of this effect, you may want to incorporate amine modifiers into the mobile phase, even if they are not needed with a brand-new column. 

While the technique of ionic suppression (or ionization control) is only effective with weakly ionic species, ion-pair chromatography has been developed for strongly ionic species and again utilizes reverse-phase chromatography. If the pH of the solvent is such that the solute molecules are in the ionized state and if an ion (the counter-ion) with an opposite charge to the test ion is incorporated in the solvent, the two ions will associate on the basis of their opposite charges. If the counter-ion has a non-polar chain or tail, the ion-pair so produced will show significant affinity for the non-polar stationary phase. 

The influence of various structural and physicochemical parameters of the stationary and mobile phases on the tailing of a cationic dye in reversed-phase chromatography has been studied in detail. Measurements were performed in a C8 reversed-phase column (80 X 4.6 mm). The isocratic mobile phase was ACN-0.01 M aqueous HC1 (90 10, v/v). Analyses were carried out at 20°C and the flow rate was 1-5 ml/min. The concentration of the cationic dye, l,l -didodecyl-3,3,3, 3 -tetramethylindocarbocyanine perchlorate (Dil) in the model solutions varied between 0.9-309 pM. The dependence of the chromatographic profile of the dye on the injected concentration is illustrated in Fig. 3.112. Calculations and mathematical modelling indicated that the peak tailing of the dye can be... 

M.J. Wirth, E.A. Smith and S.R. Anthony, Measurement and simulation of tailing zones of a cationic dye in analytical-scale reversed-phase chromatography. J. Chromatogr.A, 1034 (2004) 69-75. 

E.A. Smith and M.J. Wirth, pH dependence of tailing in reversed-phase chromatography of a cationic dye measurement of the strong adsorption site surface density. J. Chromatogr.A, 1060 (2004) 127-134. 

The origin of tailing peaks of basic compounds in reversed-phase chromatography has been an issue in the chromatographic literature for many years. Erom the foregoing discussion, it follows... 

Olieman et al40 found that reversed-phase ion-pair chromatography was the most suitable technique to analyze a series of morphinan derivatives. Normal reversed-phase chromatography on octadecyl columns, as well as straight-phase systems gave too much tailing -particularly for alkaloids with a high retention time (Table 7.4). 

Molecules which are highly polar give rise to problems of long retention times and peak tailing in adsorption chromatography and the solution to this problem lies in the use of reversed-phase chromatography, which employs a non-polar stationary phase in conjunction with a polar mobile phase the polar molecules now have little affinity for the hydrophobic support and are eluted relatively quickly by a polar eluent such as a methanol/water mixture. 

You would like to separate polar solutes with reversed-phase chromatography. Let us assume the compounds are basic and you would like to test several columns for suitability. Of course, you do everything just right and choose good columns with an endcapped stationary phase. Despite your careful choice, you obtain under identical chromatographic conditions (mobile phase composition, pH, temperature, etc.) reasonable results with one column, whereas the use of a second column, endcapped as the first, results in tailing peaks. Why ... 

In Tip No. 44, we started to deal with the most difficult problem in reversed-phase chromatography, the separation of polar and ionic organic solutes. In this Tip, the subject is further elaborated. At least if you obtain tailing peaks and have excluded all other possibilities (see Tip No. 44) you know you deal with ionic solutes, where bases are more annoying than acids. The conditions suitable for the separation of ionic solutes are listed below in Table 45-1. Stationary phases and mobile phases listed side by side in the Table do not necessarily have to be used together it is possible to succeed with one or the other. 

A. flos-aquae strains they obtained an excellent linearity in their calibration curve in the range, 20-100 ng (on-column) (Harada et al., 1989). Wong and Hindin studied the applicability of LC-UV for the isolation, identification, and quantification of AN. Both normal phase and reversed phase chromatography were examined, but both phases showed poor chromatograms with broad peaks with extensive band tailing (Wong and Hindin, 1982). 

The special phases with an embedded polar functional group are designed for reversed-phase chromatography. The polar function shields the silica surface, preventing the interaction of analytes with the acidic silanols on the silica surface. For many samples, they exhibit a significant difference in selectivity compared to simple hydrocarbon bonded phases. Also, the tailing of basic analytes is reduced. 

The solvent systems most often used in reverse-phase chromatography are mixtures of either acetonitrile or methanol and water. Addition of a salt (usually ammonium acetate) is required to eliminate tailing of retinoids containing a free carboxylic acid moiety. The amount of salt needed varies, depending on the column being used. As little as 0.2% could be utilized for a Spherisorb ODS... 

Amlnoalkyl and cyanoalkyl (nitrile) bonded phases (the alkyl group is usually propyl) are moderately polar. The former is particularly useful in separating mixtures of sugars and other carbohydrates (Fig. 2(b)), whilst the latter is used as a substitute for unmodified silica, giving more reproducible retention factors and less tailing, especially with basic solutes. Both normal-phase and reversed-phase chromatography is possible by appropriate choice of eluents. 

The effect on chromatography is to complicate the separation greatly. If we consider a reverse-phase separation, the first thing we notice is an almost irreversible binding of protein to the column. Even after protein removal, we find polar peaks, which overload the early part of the chromatogram and tail into the compounds of interest. The components that are more nonpolar than our compounds of interest adhere to the column and must be washed off before the next injection. To ensure polar elution before our target compounds and nonpolar removal afterwards, we are almost forced to run solvent gradients. 

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