Mobile phase scouting

Finally, it is worth emphasizing once again that the C8 reverse phase, with a 3 p particle size, packed in a column 3 cm long and 4.6 mm in diameter is an excellent scouting column. A column of this size can be made to provide very rapid separations and subsequently can be quickly reconditioned to another mobile phase. By using such a column, and employing a gradient from pure water to pure acetonitrile to develop the separation, the complexity of the sample will often be revealed, and from the results an improved phase system can be educed. 

As RP-HPLC is the method of choice for purity analysis, it provides a namral starting point for the selectivity scouting. The approach described here for RP-HPLC is directly applicable to NP, chiral, and other chromatography modes. Figure 7.3 provides examples for stationary and mobile phases providing a starting point for the screening exercise. 

For illustration purposes, we will take the most common case. We will start with a 15-cm long C18 column, 254 nm, and acetonitrile/water in a scouting gradient. Scouting gradients are run much more rapidly than analytical gradients. A mixture of the compounds to be separated is dissolved in 25% acetonitrile in water. A sample is injected into an HPLC equilibrated in the same mobile phase and a 20-min gradient is run to 100% acetonitrile. 

If you do not have a gradient system, I have developed a fast isocratic scouting technique. You select the same column and detector wavelength, but equilibrate the column in 80% acetonitrile in water for our first injection. A strong solvent composition is selected to blow everything off quickly. Look at the peaks if they are resolved, quit. If they are still unresolved, mix the mobile phase with an equal volume of water making 40% acetonitrile, reequilibrate, and shoot again. This time, the peaks should be much farther apart. If not, do another equal dilution with water to 20%, reequilibrate, and reinject the sample. 

A second approach is to use TLC as a scouting technique. A good correlation from TLC to HPLC is generally obtained when mobile phases contain solvents equal to or less polar than ethyl acetate [347], For solvents more polar than ethyl acetate, the correlation is not as useful because the more polar solvent absorbs preferentially to the TLC sorbent [347]. Under these circumstances, compensation for this change in TLC can be made by using a LC solvent mixture less polar than the TLC solvent mixture. Correlations of mobility between TLC and HPLC separations for cephalosporin antibiotics and steroidal hormones have been reported [348-350]. If all else fails a three part series on the basics of separations should be consulted [351-353]. 

A) Determine the optimal pH of the aqueous portion of the mobile phase for gradient scouting studies in Step 5. (paper based evaluation)... 

Mechanistic considerations (e.g., the extensive work published on brush-type phases) or the practitioner s experience might help to select a chiral stationary phase (CSP) for initial work. Scouting for the best CSP/mobile phase combination can be automated by using automated solvent and column switching. More than 100 different CSPs have been reported in the literature to date. Stationary phases for chiral pSFC have been prepared from the chiral pool by modifying small molecules, like amino acids or alkaloids, by the deriva-tization of polymers such as carbohydrates, or by bonding of macrocycles. Also, synthetic selectors such as the brush-type ( Pirkle ) phases, helical poly(meth) acrylates, polysiloxanes and polysiloxane copolymers, and chiral selectors physically coated onto graphite surfaces have been used as stationary phases. 

In reversed-phase and ion-exchange chromatography it is a common procedure to run a gradient scouting run if the conditions for a successful separation are unknown. Such a mn is performed from 10 to 100% B solvent (stronger solvent) with a linear profile. (As already explained, a 100% A mobile phase is often not recommended in reversed-phase chromatography because the alkyl chains are collapsed and equilibration with... 

During initial method development, a set of initial conditions (detector, column, mobile phase) is selected to obtain the first scouting chromatograms of the sample. In most cases, these are based on reversed-phase separations on a Cl 8 column with UV detection. A decision on developing either an iso-cratic or a gradient method should be made at this point. 

Most of the Pirkle phases, and in particular the Whelk-01 phase, are stable to all types of solvent and can be used either in the reversed phase mode, or the normal mode, which again, adds to its universal applicability. In the normal phase mode of development, hexane/IPA would be a good mobile phase from which to start. A mixture, hexane/IPA 80/20 v/v, would be a practical scouting composition to assess the possible level of retention and chiral selectivity. Again,... 

Sample is separated in the first-dimension (Id) column and the fractions obtained are forwarded into the independent second-dimension (2d) column for further separation and characterization. Depending on the methods utilized for the particular separation dimensions, mobile phases in both columns can be either identical or unlike. P 2 delivers the same mobile phase as P 1 in the former case or it is not employed ar all. Results of separation in the Id and the 2d columns are monitored with help of detectors D 1 and D 2, respectively. In the first step, the experimental conditions for the first-dimension separation are optimized. The result of separation is registered with detector D l. Based on results of such scouting experiments, the optimized separation in the Id column is repeated and fractions obtained are forwarded into the 2d column. As discussed below, there are several different options for practical implementation of such transfer. 

The scout gradient process is particularly suited to analyses where a large number of analytes need to be separated. The rapid ramping of the mobile phase up to a very strong mobile phase causes the elution of all compounds in a short period of time. A linear gradient should be used since it is the easiest from which to extract the isocratic conditions. 

C18 layers have been found to have clo,se correlation with HP liquid chromatography reversed-phase columns (Gonnet and Marichy, 1979), and TLC is used widely to scout appropriate mobile phases for HPLC (see Chapter 6). Very polar substances (basic and acidic pharmaceuticals) can be separated on RP layers by ion pair chromatography. 

TLC has been widely used to scout or pilot mobile phases for HPLC (Jork et al., 1981). Because the vast majority of HPLC separations are performed onreversed-... 

Reversed-phase TLC on small commercial C-18 plates (1 x 3 in) is convenient for solvent scouting when one is preparing reversed-phase HPLC conditions for a new retinoid. Mobile phase compositions that give Rf m the range 0.3 to 0.7 on TLC will usually give capacity factors (k ) in the range 1-10 on HPLC. 

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