Previous work in multidimensional liquid chromatography

One multidimensional method that has been used commonly is an on-line, 2D-in-time technique called heart-cutting two-dimensional liquid chromatography. In this approach, the first-dimension separation is carried out like a standard 1D analysis on a chromatography column. A single desired segment of the first-column effluent is then transferred, typically

via a switching valve, to a second column for further separation via a different method. Heart-cutting is useful when extra resolution is needed to examine a small segment of peaks in a complex chromatogram, but it requires foreknowledge of the sample’s composition. Since heart-cutting does not subject the entire sample to two-dimensional separation, it is of little use when every component of a sample is an analyte of interest, as in proteomics. The alternative to heart-cutting is known as “comprehensive” multidimensional separations, because all sample components are subjected to displacement on both dimensions.38 Comprehensive multidimensional separations are more powerful than heart-cutting separations because they provide greater peak capacity.

A wide variety of combinations of liquid chromatographic modes have been used in comprehensive LC x LC separations, including ion exchange-reversed phase (IEC x

RPLC),39-45 size exclusion-reversed phase (SEC x RPLC),46-49 reversed phase-size exclusion (RPLC x SEC),36, ion exchange-size exclusion (IEC x SEC),38 and normal phase-reversed phase (NPC x RPLC).50 Although many different instrumental setups for LC x LC have been designed, most of the components used are similar, and include an injector, two isocratic or gradient LC pumps, a single column for the first dimension, one or more columns for the second dimension, one or more computer-controlled switching valves, and an appropriate detection system.

The first true comprehensive system for two-dimensional liquid chromatographic separation was reported in 1990 by Bushey and Jorgenson.38 This system was used to analyze a mixture of protein standards and a sample of human serum. A cation exchange

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interface the two columns and allows on-line fraction transfer from the first column to the second. A diagram of the instrumental setup of this LC x LC system is provided in Figure 1-2. The effluent of the cation exchange column is directed to a storage loop. When the valve is switched, the isocratic LC pump forces the contents of the loop onto the size exclusion column where they are further separated. Meanwhile, the effluent of the first dimension is directed to the second storage loop. The flow rates on the two columns are selected such that the analysis time of the second dimension exactly matches the amount of time it takes for the first dimension to fill the storage loop. This allows all the effluent of the first column to be transferred to the second dimension. A computer is used to control the switching of the valve and the acquisition of data from a UV absorbance detector at the end of the second column. In this manner the SEC column can analyze a large number of fractions from the ion exchange column during the course of a run lasting a total of 2.5 to 6 hours. The chromatographic data are presented as a three-dimensional view of a 2D

chromatogram as shown in Figure 1-3. The system was estimated to have a total peak

capacity of approximately 130, which is the product of the peak capacities visually estimated for each dimension. Other examples of column-switching LC x LC separations using dual storage loops have since been reported in the literature, some with greater peak capacity.36, 39,

40, 51 _{A 2D anion exchange-reversed phase system that was used to analyze a tryptic digest of}

reduced porcine thyroglobulin gave a peak capacity over 2,000.43

A different approach, in which the effluent from the first column is not captured by storage loops but instead is transferred directly to the head of one of two parallel second- dimension columns, was reported in 1997 by Opiteck, Jorgenson and Anderegg.47 This system was used to analyze the fragments produced by tryptic digests of the proteins

ovalbumin and bovine serum albumin. The first dimension of their system consisted of six size-exclusion columns connected in series. These were coupled via a pair of four-port valves to two parallel reversed phase LC columns, which serve as the second dimension. This study was also one of the first reports of on-line mass spectrometric detection used to analyze a two dimensional separation. A diagram of the entire setup is shown in Figure 1-4. As sample elutes from the series of SEC columns, it is routed to RPLC column alpha. Since the aqueous buffers used for SEC are weak eluents for RPLC, the sample material is

concentrated in a narrow zone at the head of the column. When the two four-port valves are switched simultaneously, a second LC pump starts a gradient to elute the sample from RPLC column alpha, while the effluent from the first dimension is loaded onto RPLC column beta. The effluent from the RPLC column being eluted is directed to a UV absorbance detector followed by a 10:1 flow splitter and an electrospray mass spectrometer. The total peak capacity of this system was estimated to be about 500. Other two-dimensional LC x LC techniques using parallel columns in the second dimension have been reported, many of which replace the two four-port switching valves with a single ten-port valve that accomplishes the same purpose.41, 42, 45, 52, 53