Method development isocratic separations

Lewis, J. A., Snyder, L. R., and Dolan, J. W., Initial experiments in high-performance liquid chromatographic method development. II. Recommended approach and conditions for isocratic separation, ]. Chromatogr. A, 721,15,1996. 

Many industrial laboratories conducting significant amounts of additive analyses have developed a universal HPLC method which may be used to separate most of the additives of interest. Thomas [417] has reported a method that can separate over 20 common primary and secondary stabilisers. Verdurmen et al. [197] employ a gradient ranging from 60 % acetonitrile/40 % water to 100% acetonitrile subsequently, all components are eluted off the column in isocratic mode. Irganox 1063 is used as a suitable internal standard since this compound is not frequently encountered in commercial polymers, elutes without overlap to other additives and shows good UV absorbency. In order... 

Some LC/MS users adhere to isocratic separation because of the myths around gradient elution (it is complex to develop and transfer between instruments and laboratories, it is inherently slower than isocratic methods because of re-equilibration, and other reasons summarized by Carr and Schelling6). A researcher may have a very good reason to use an isocratic method, for example, for a well defined mixture containing only a few compounds. The isocratic method would certainly not be useful in an open access LC/MS system processing varying samples from injection to injection. 

The approach to method development is similar to the one described for HPLC and can be characterized as a rapid stationary phase screen using column and solvent switching with gradient elution followed by development of an isocratic preparative method. SFC has been successfully applied to the analytical and preparative separation of achiral and chiral compounds. 

Hupka et al. [29] developed a method for the determination of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in the blood of heroin victims. The method is based on immunoaffinity SPE, RP-HPLC isocratic separation (mobile phase 90% lOmmol KH2PO4, 2mmol 1-heptanesulfonic acid, adjusted to pH 2.5 with H3PO4 and 10% acetonitrile flow rate 1.5 mL/min), and laser-induced native fluorescence detection. 

Method development is vastly simplified by computer simulations using commercial software. With input from a small number of real experiments, a program can predict the effects of solvent composition and temperature in isocratic or gradient separations. You can select optimum conditions in minutes with the computer instead of days in the lab. Of course, you must verify the prediction by a real experiment. Commercial software saves huge expenses in method development in industrial laboratories. 

Figure 25-28 Isocratic method development for HPLC, using solvent composition, % B. and temperature, T, as independent variables. % B and T are each varied between selected low and high values. From the appearance of chromatograms resulting from conditions A-D, we can select intermediate conditions to improve the separation.

Steps in method development (1) determine the goal of the analysis, (2) select a method of sample preparation, (3) choose a detector, and (4) use a systematic procedure to select solvent for isocratic or gradient elution. Aqueous acetonitrile, methanol, and tetrahydrofuran are customary solvents for reversed-phase separations. A separation can be optimized by varying several solvents or by using one solvent and temperature as the principal variables. If further resolution is required, flow rate can be decreased and you can use a longer column with smaller particle size. Criteria for a successful separation are 0.5 < < 20, resolution >2.0, operating... 

It is sometimes not possible to develop an isocratic separation for complex mixtures of compounds. Binary gradient methods development starts with a... 

Gradient systems let you control flow rate and solvent/buffer changes to improve chromatographic separations. They can be used to sharpen separations and to speed column re-equilibration. A four-solvent gradient system is useful for methods development when equipped with methanol, acetonitrile, ammonium acetate buffer, and formic acid solution. But, many quality control laboratories prefer to use inexpensive isocratic systems that run a constant-composition premixed mobile phase for rapid separations. 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

Effect of pH on the Retention/Selectivity of the Isomers. The first step in method development is to understand the effect of pH on the separation characteristics of the method. The pKa values of the ortho and the para isomers was estimated by ACD (Advanced Chemistry Development software) to be 9.0 and 9.5, respectively. Obviously the best pH to carry out the separation would be at pH that is less than 2 units lower than the analyte that has the lowest pKa. This would be at pH values less than 7.0. However, to illustrate the effect of pH on the separation selectivity of the isomers, a controlled pH study at isocratic conditions was conducted. 

In this context it should further be pointed out that a commercial method development computer software package (DRYLAB) has been successfully applied to optimize enantiomeric separations of a multi-component amino acid sample utilizing isocratic and gradient elution techniques 389. ... 

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