Optimization of Separation Conditions

3 Optimization of Mobile/Stationary-Phase Composition, Including Temperature It should be taken into account that the highest enantioselectivity is observed at the lowest degree of nonchiral interactions, that is, at the level of a nearly non-retained first enantiomer. Moreover, enantioselectivity increases with lower temperature according to Equation 3.16. 

This effect on resolution may be counterbalanced by increased viscosity, leading to lower efficiency of the system. Therefore, fine tuning of mobile-phase composition and temperature should be carefully taken into account for production-scale systems as some economic benefits have to be considered against a higher complexity of the separation system, for example, in terms of controlling temperature and small amounts of modifier. 

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The qualitative analysis of retention behaviour in liquid chromatography has now become possible. Quantitative retention-prediction is, however, still difficult the prediction of retention time and the optimization of separation conditions based on physicochemical properties have not yet been completely successful. One reason is the lack of an ideal stationary phase material. The stationary phase material has to be stable as part of an instrument, and this is very difficult to achieve in normal-phase liquid chromatography because the moisture in organic solvents ages the silica gel. 

J. E. Madden, M. J. Shaw, G. W. Dicinoski, and P. R. Haddad, Simulation and Optimization of Retention in Ion Chromatography Using Virtual Column 2 Software, Anal. Chem. 2002, 74, 6023 P. R. Haddad, M. J. Shaw, J. E. Madden, and G. W. Dicinoski, Computer-Based Undergraduate Exercise Using Internet-Accessible Simulation Software for the Study of Retention Behavior and Optimization of Separation Conditions in Ion Chromatography, J. Chem. Ed. 2004, 81, 1293 http //www.virtualcolumn.com. 

Optimization of Separation Conditions Determination of Racemate Solubility... 

Chemometries has played two major roles in MEKC for analysis of the data collected from the separation and detection of analytes, and for efficient optimization of the separation conditions. Regarding data analysis, chemometrics can allow deconvolution of poorly resolved peaks (15,16) and quantification of the corresponding analytes. Chemometrics can also be employed for multivariate calibration (17), characterization of complex samples, and to study peak purity. Sentellas and Saurina have recently reviewed the role of chemometrics applied to data analysis in CE (18). For MEKC in particular, chemometrics has been used more widely as a tool for optimization of separation conditions. The focus of this chapter is to exemplify the utility of chemometric methods for the optimization of separation conditions in MEKC. 

One of the current trends in separation science is the development of comprehensive or multidimensional separation systems, in which CE and CE-MS are also achieving relative importance. Chemometric approaches like the ones described in this chapter will surely be of great help for the optimization of these more complicated separation systems. Current trends toward miniaturization in separation science are also well known. Ultrafast separations, extremely low sample requirements, and automation of the arrangement are some of these goals. Chemometrics will surely provide an interesting and challenging approach for the optimization of separation conditions in these miniaturized systems, including microchips for years to come. 

Optimization of separation conditions using chemometric approaches 231... 

OPTIMIZATION OF SEPARATION CONDITIONS USING CHEMOMETRIC APPROACHES... 

In practice, separation of enantiomers by the use of chiral stationary phases is not free from problems. Chiral stationary phases are difficult to prepare reproducibly, are sometimes of lower chromatographic efficiency than expected, and optimization of separation conditions is restricted by the fixed nature of the chiral centres. Chiral mobile phases are free from many of these problems, optimization of the separation is more convenient, and conventional reversed-phase columns may be used. Thus N-(2, 4-dinitrophenyl)-L-alanine-n-dodecyl ester has been used as a non-ionic chiral mobile phase additive for the resolution of 1-azahexahelicenes by reversed-phase chromatography. The resolution obtained was found to be a function of the mobile phase polarity and the concentration of chiral additive used. 

Figvire 4. Flow-chart for the optimization of separation conditions in reversed-phase LC. Reproduced with permission from Ref. 9. Copyright 1984, Elsevier. 

Rozylo, J., and Janicka, M. (1991b). Thermodynamic description of liquid-solid chromatography process in the optimization of separation conditions of organic compound mixture. J. Liq. Chromatogr. 14 3197-3212. 

Felhofer, J., Hanrahan, G., and Garcia, C.D. (2009) Multivariate versus univariate optimization of separation conditions in micellar electrokinetic chromatography. Talanta, , 1172-1178. 

Computer programs enable the user to use well-established relationships between separation, retention, and chromatographic conditions to allow the prediction of results of different experiments and optimization of separation conditions, working mainly with a PC rather than with an HPLC system. In this case, a user enters some information such as run data and conditions, and works with the software interactively. 

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