Getting Started in HPLC

Section 3C. Gradient HPLC

So far, we have been considering only HPLC separations in which the mobile phase composition remains constant during the course of the chromatogram -- so-called "isocratic" separations. Although most separations you will encounter are isocratic, there are times when no single mobile phase composition gives usable results for all the peaks in the separation. The separation of polynuclear aromatic hydrocarbons shown below provides a good example. When the mobile phase composition is adjusted to give reasonable retention for the early peaks, the late peaks have an excessively long retention time. If we decrease the water content in the mobile phase to make it stronger, the late peaks are reasonably retained, but now the early peaks are poorly resolved.


The chromatogram on the left provides a good separation for the early peaks, but excessive retention time for the late peaks. Increasing the mobile phase strength (%B) results in reasonable retention for the late peaks, but loss of resolution for the early peaks. There is no single isocratic composition that allows for good chromatography of all the peaks in this sample.

We can deal with the problem by starting the separation with a weak (high-water content) mobile phase, and then gradually increase the mobile phase strength (decrease the water content) in order to speed the elution of the more strongly retained components. This technique of increasing mobile phase strength during the chromatogram is called "gradient" HPLC.

Gradient HPLC -- changing the mobile phase composition from 45% B to 95% over 10 minutes provides good separation of all the peaks in a single chromatogram.

If you carry out HPLC separations of "biopolymers" such as peptides, proteins, or nucleic acids, you will find that gradient separations are the rule rather than the exception, because these large molecules typically have a wide range of retention within a single sample.

A gradient HPLC system is necessarily more complex than an isocratic system, because it must incorporate some means of accurately changing the mobile phase composition during the run. Two basic types of pumping system are used:
  • high-pressure mixing systems (also called "two-pump" gradient systems) use two or more independent high-pressure pumps. The relative flow from each pump is determined by the system controller, and the pump outputs are blended on the downstream or high-pressure side of the flow path.
  • low-pressure mixing systems (also called "one-pump" gradient systems) use a single high-pressure pump. The mobile phase composition is controlled by a proportioning valve or metering pump array on the upstream or low-pressure side of the pump.

Chromatographers continue to debate the trade-offs and relative merits of these two approaches; good (and bad!) designs have been based on both types.


A high-pressure mixing (two-pump) system blends the output of two high-pressure pumps on the downstream side of the pumps to control the mobile phase composition.

A low-pressure mixing (one-pump) system uses a proportioning valve mounted upstream of a single high-pressure pump to control the mobile phase composition.

By convention, the weak solvent in a gradient separation is referred to as the "A" solvent, the strong solvent is referred to as the "B" solvent. A typical linear gradient separation is defined by specifying the A and the B solvent, specifying the initial and final %B and the time over which the change occurs.

An important (and under-appreciated) characteristic of a gradient system is its dwell volume (also called the "gradient delay volume"). This is the volume of liquid in the system between the point where the gradient is formed (usually at the proportioning valve or mixing chamber inlet) and the point where it enters the column. This imposes a de-facto isocratic hold or delay at the beginning of each gradient chromatogram. If a method was originally developed on an instrument with a small dwell volume and then run on a system with a larger dwell volume (for example), the result will be an offset in retention times; in some cases, selectivity may also change and accuracy of the results may be compromised.

The dwell volume in a gradient system is the total volume between the point where the mobile phase is blended and the column inlet. In a high-pressure mixing system as shown here, it includes the mixer, transfer tubing, and swept volume in the injector or autosampler.





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Last revised: April 02, 2001.