Rapid Elution Gradients

Gradient elution techniques are the most versatile and popular techniques for solving the general elution problem in liquid chromatography. The advantages of gradient elution are enhanced peak resolution, faster analysis times, and better detectability. The major disadvantage is that the compositions of the stationary and mobile phases change during the course of the separation and column regeneration is needed before the next analysis. In order to perform gradient elution in MLC, the concentration of micelles and/or organic modifier may be increased during the course of the separation. In a micellar concentration gradient, re-equilibration time at the end of a gradient run is not necessary, and in an organic modifier gradient the re-equilibration time is very short.

Micellar gradients Rapid micellar elution gradients can be performed in MLC because re-equilibration time for the column is not necessary. This is because the amount of surfactant adsorbed on the stationary phase remains practically constant after reaching equilibrium when the concentration of surfactant in the mobile phase is above the c.m.c. Accordingly, micellar elution gradients are compatible with electrochemical detection. Figure 5 shows the separation of eight organic compounds using a micellar gradient and electrochemical detection.

Organic modifier gradients Organic solvent gradients in MLC require short re-equilibration times at the end of the gradient mainly due to the small range of organic modifier concentration used in MLC in order to maintain micelle integrity. In this case, the change in the concentration of organic modifier is not sufficient to change the concentration of adsorbed surfactant monomer in the stationary phase. In MLC, t

50 nA

32 16 0

Time (min)

Figure 5 Micellar gradient elution separation with electrochemical detection at # 1.2 V. Flow rate: 1.0 mL min-1. Mobile phase A: 0.05 mol L"1 SDS/3% 1-propanol, pH 2.5 with phosphate buffer, sodium perchlorate added to balance conductivity with solvent B. Mobile phase B: 0.112 mol L"1 SDS/3% 1-pro-panol, pH 2.5 with phosphate buffer. Gradient program A to B in 12 min. Key: 1, hydroquinone; 2, resorcinol; 3, catechol; 4, phenol; 5, p-nitrophenol; 6, o-nitrophenol; 7, p-chlorophenol; 8, p-bro-mophenol. Column: Altex Ultrasphere C18 (15 cm x 4.6 mm i.d.). (Reproduced with permission from Dorsey JG, Khaledi MG, Landy JS and Lin JL (1984) Gradient elution micellar liquid chromatography. Journal of Chromatography 316: 183-191, copyright Elsevier Science Publishers B.V.)

organic modifier gradients are useful since although a limited range of organic modifier may be used, the solvent strength can be compensated with a concurrent micelle concentration gradient. Figure 6 shows the separation of a mixture of amino acids and peptides in MLC under isocratic and gradient conditions.

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