High-Performance Liquid Chromatography 151

pounds that are eluted earlier remain well resolved. A basic gradient could be 0: 100 acetoni• trile:water to 60:40 acetonitrile:water in 60 minutes, which leads to an increase in acetonitrile concentration of 1 % per minute. A gradient can be linear (as described), convex, or concave, or a complex sequence of each to achieve the desired separation.

Gradient flow can be generated in three ways. In all cases, a computer-controlled pumpingmechanism is required. In the first case, controlled amounts of each eluent (up to four solvents can be used) are metered into a mixing chamber before reaching the high-pressure pump, which sends the mixture to the column. In the second case, the amount of each solvent is regulated by a

proportioning valve, which is controlled by a microprocessor. The mixed solvent then enters the high-pressure pump and flows to the column. In the third case, the delivery of multiple high•