Off-line vs on-line coupling in multidimensional LC

Although apparently simple, the selection of a method used to couple dimensions in a 2D separation merits consideration from a conceptual standpoint. The majority of previous research in the Jorgenson group involving LC x LC has employed on-line coupling of dimensions, in which fractions from the first column are loaded directly onto to the second column using automated switching valves. An alternative is off-line coupling, where the effluent of the first column is physically collected as fractions that are later injected individually onto the second column. Although off-line coupling may seem to be a less sophisticated approach, both methods have noteworthy advantages and disadvantages which must be weighed when designing a 2D separation.

The primary advantages of the on-line approach are automation and speed. An entire 2D separation can be carried out with no user intervention beyond the initial injection of the

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separation to complete; therefore, the technique can be made quite rapid, especially

compared to 2D gel electrophoresis. However, these advantages impose certain limitations. Most significantly, the second dimension separation must be configured such that it is very rapid compared to the first. This is necessary so that many second-dimension runs can be carried out during the time it takes to complete a single run on the first dimension in order to adequately sample the column. This usually means that the resolution contributed by the second dimension must be compromised to improve the analysis speed. Secondly, the on- line approach requires relatively complex instrumentation. Two LC pumps must be operated simultaneously, and operation of the switching valves used to connect the two dimensions must be timed precisely so that all of the effluent from the first column is transferred to and analyzed by the second column.

Off-line LC x LC is a comparatively simple approach. When minimally configured, it requires only a single LC pump, two separation columns of different types, and suitable containers for collecting fractions. A greater degree of automation can be achieved if a fraction collector is used to collect the fractions from the first column and an autosampler is used to inject the fractions onto the second column. There is no limit to the number of fractions that can be collected, so off-line coupling can be just as thorough in sampling the first dimension as on-line coupling. Fraction collection also permits sample manipulation between separation dimensions – for example, fractions could be concentrated via

lyophilization, and reconstituted in a solvent more appropriate for analysis on the second dimension. Another major advantage of the off-line approach is that there is no need for the second dimension separation to be run faster than the first, because fractions can be stored and run when time permits. Therefore both separations can be optimized to provide the

highest possible resolution, which could allow the second dimension to contribute a much higher peak capacity to the overall 2D method. Offline LC x LC also allows flexibility in the amount of each fraction that is transferred to the second dimension. This amount could be increased to improve sensitivity, decreased to prevent second-dimension column overload, or a fraction could be skipped altogether if the detector monitoring the first-dimension column effluent indicates that no analytes are present. Finally, an off-line 2D separation allows the flexibility of analyzing any fraction multiple times on the second dimension without repeating the entire 2D separation, should the sample prove of particular interest or if an instrument failure occurs.

Of course, off-line sampling also has numerous disadvantages. Foremost is speed – off-line sampling is certain to take longer than an on-line approach because the fractions are usually run after the first dimension separation is complete, rather than as it is in progress. An additional factor is that the operator of the instrument may need to spend more time performing manual tasks such as setting up for fraction collection, collecting the fractions, and manipulating the fractions such that they are ready to be analyzed on the second

dimension. Finally, since the liquid fractions come in contact with more tubes and surfaces when fraction collection is performed than when on-line coupling is used, there is a greater possibility of sample losses with off-line coupling.

In the case of the 2D separation to be described in this chapter, it was decided to use off-line coupling of dimensions, for two main reasons. First, the goal of the separation was to generate as high of a peak capacity as possible in order to analyze the E. coli proteome.

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desired to use ultra-high pressure liquid chromatography (UHPLC) for the second dimension of the separation. UHPLC is an enhancement of HPLC which offers improved

chromatographic performance without long run times.2-6 A description of UHPLC and an explanation as to why it could not be used with on-line coupling are provided in the next section.