Abstract In LC-LC, the first column is used for selective clean-up and concentration of the sample while the second column is used for the actual separation of the compounds. An animation shows how the valves are switched.
LevelBasic
Instrumentation is based on a normal LC instrument equipped with an extra pump(s) and a switching valve. The two columns are connected via a multiport switching valve(s). The effluent from the first column can be directed, by switching the valve, to waste, to detector or to the second column. This is called the transfer.
The transfer can be done in different ways depending on the application. The transfer mode can be changed by changing the valve configuration (see animation). Typically:
- A sample is injected into a (short) first dimension column using a
weak eluent. The analytes of interest are retained into the column while matrix components are flushed directly to waste. - The analytes of
interest are then eluted to the second dimension column either by changing the eluent or with the same eluent. - It is also possible to transfer more than one fraction by using
stopped-flow mode.
Usually, only one detector is used in the LC-LC, although it is possible to use two detectors, the first one for monitoring the first column separation and the second for the second column separation. During the method optimisation, however, this second detector is also connected to the first column to enable the optimisation of the separation and the fraction to be transferred. The fraction should be as narrow as possible, however, because there may be some variations in the 
retention times.
An important parameter that affects directly the volume of the transferred fraction is the diameter of the column. The smaller the column i.d. is, the smaller is the volume of the fraction.
For example, if the column diameter is decreased from 4.6 mm to 2.1 mm, the size of the fraction is decreased from ca. 1 ml to 0.2 ml. However, the sample capacity also decreases if the i.d. decreases. A good compromise of reasonable sample capacity and small fraction size is to have a column with i.d. of 2-3 mm for the first dimension. The dimensions of the second column are not as critical, but it should not have i.d. smaller than the column used for the first dimension.
