Abstract
Efficiency in HPLC can be enhanced by increasing the column length and/or decreasing the particle size. The use of high temperature in HPLC has emerged as a valuable tool to overcome the increase in column backpressure when using small packing particles, as it allows for reduction in mobile phase viscosity. In this study, high plate count was obtained by coupling sub-2 mu m columns at elevated temperature to reduce the viscosity of the mobile phase, thus reducing the column backpressure. At 80 A degrees C, up to three columns of 15 cm x 4.6 mm I.D. packed with 1.8 mu m particles could be coupled generating similar to 84,000 theoretical plates for the last eluting compound. The number of theoretical plates was increased on average by a factor of similar to 3.6 when three columns were coupled at 80 A degrees C compared with one column at 30 A degrees C. The relationships between separation efficiency and column length were examined using Van Deemter plots constructed at 30 A degrees C and 80 A degrees C for different column lengths. The advantages of using coupled columns in combination with elevated temperature for the environmental analysis were illustrated using test mixtures comprised of eight sulfonamides separated on one column at 30 A degrees C and three coupled columns at 80 A degrees C by isocratic elution. Sample clean up was carried out by employing solid-phase extraction (SPE) using Oasis HLB cartridges. The method developed was validated based on parameters such as linearity, precision, accuracy, detection and quantification limits. Recoveries generally ranged from 71.7 to 99% (with the exception of sulfanilamide), with standard deviations not higher than 4.7%. The detection limits of the method ranged from 0.6-2 mu g L-1, while limits of quantification were in the range 2-6.7 mu g L-1 with UV detection.