2D-HPLC

This section provides an overview of properties of polymer monolith columns related to 2D-HPLC. Monolithic organic polymer columns, having longer history than silica monoliths, have been reviewed in detail recently by S vec and by Eeltink including their preparation methods and performance (Eeltink et al., 2004 Svec, 2004a). Polymer monolith columns commercially available include polyfstyrene-co-di vinyl benzene) (PSDVB) columns and poly(alkyl methacrylate) columns. 

As mentioned earlier, high-speed separation is necessary to carry out fast, comprehensive 2D HPLC. The polymer monoliths have not been employed in such 2D HPLC, probably because permeability of polymer monoliths is not high enough to allow fast elution of the second dimension (2nd-D) in simple 2D operation, and the gradient cycle at the 2nd-D cannot be so fast to allow online 2D operation without reducing peak capacity at first dimension (lst-D). 

The application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

Utilizing the difference in selectivity between a monolithic silica-C18 column (2nd-D) and another particle-packed column of C18 phase (lst-D), 2D HPLC separation was shown mainly for basic compounds and other species (Venkatramani and Zelechonok, 2003). The authors also reported other examples of reversed-phase 2D HPLC, using amino- and cyano-derivatized particle-packed columns for 2nd-D separation. The combination of normal-phase separation for the 1 st-D and reversed-phase separation on monolithic Ci g column for the 2nd-D was reported (Dugo et al., 2004). The use of a microbore column and weak mobile phase for the lst-D and a monolithic column for the 2nd-D was essential for successful operation. Improvement in the 2D separation of complex mixtures of Chinese medicines was also reported (Hu et al., 2005). Following are practical examples of comprehensive 2D HPLC using monolithic silica columns that have been reported. 

Simple and comprehensive 2D HPLC was reported in a reversed-phase mode using monolithic silica columns for the 2nd-D separation (Tanaka et al., 2004). Every fraction from the lst-D column, 15cm long (4.6 mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles (5 pm), was subjected to the separation in the 2nd-D using one or two octadecylsilylated (Cig) monolithic silica columns (4.6 mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to lOmL/min with separation time of 30 s that provides fractionation every 15-30s for the lst-D, which is operated near the optimum flow rate of 0.4-0.8 mL/min. The 2D-HPLC systems were assembled, as shown in Fig. 7.6, so that the sample loops of the 2nd-D injectors were back flushed to minimize band broadening. 

In the simplest scheme of 2D HPLC, effluent of the first dimension (lst-D) was directly loaded into an injector loop (500 pL) of the 2nd-D HPLC for 28 s, and 2 s were allowed for injection. This operation was accompanied by the loss of lst-D effluent for 2 s out of 30 s in each cycle. The flow rate of 10 mL/min allowed the elution of solutes having retention factors (k values) up to 8 for the 2nd-D within the 30-s separation window, with f0 of 3.5 s. Figure 7.7 a and b shows the chromatograms for the 1 st-D and the 2nd-D, respectively, obtained for a mixture of hydrocarbons and benzene derivatives. The lst-D chromatogram showed many overlapping peaks. PAHs were eluted as mixtures from the FR column, and some are separated in the 2nd-D. 

The loss of about 7% of the lst-D effluent caused by a 2-s injection in a 30-s operation cycle, which could cause up to 20% loss of a peak in the most unfavorable case, or the narrowest peak at the beginning, can be avoided by using two six-port valves each having a sample loop (Fig. 7.6b) an alternative system uses a 10-port valve with two holding loops. The loops hold the effluent of the lst-D alternately for 30 s during a complete separation cycle on the 2nd-D column to effect comprehensive 2D HPLC. 

FIGURE 7.6 (a) Tubing connection at 2nd-D injector of simple 2D-HPLC. (b) Tubing... 

FIGURE 7.7 Two-dimensional separation of a mixture of hydrocarbons and benzene derivatives in simple 2D HPLC. (a) Chromatogram obtained in the lst-D on FR column in 60% methanol/water. (b) Chromatograms obtained in the 2nd-D on C18 column in 80% methanol/water. The insets 3(a) and 3(b) are expanded views of Fig. 7.7(a) and (b) respectively. Sampling every 30 s at the lst-D. Flow rate 0.4mL/min for lst-D, and 10mL/min for 2nd-D (reproduced from the reference, Tanaka et al. 2004, with permission from American Chemical Society). 

A comprehensive 2D HPLC can be carried out with two very similar columns in reversed-phase liquid chromatography (Ikegami et al., 2005). A mixture of water and tetrahydrofuran was used as a mobile phase in the lst-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation with a common Ci8 stationary phase. 

Fig. 7.9a shows plots of k values of sample compounds in 45% MeOH against k values in 22% THF. These two sets of plots are very similar. Figure 7.9 indicates that the 2D HPLC based on the difference in selectivity provided by an organic modifier that could be used for 2D HPLC to provide nominally large PC, but the selectivity difference was not quite orthogonal. The practically usable area for the 2D separation will be about half the orthogonal plotting area, because there is some similarity between the selectivities obtained with the two mobile phases. 

THF and methanol employed as organic modifiers of mobile phase provided a considerable difference in selectivity based on the polar interactions between solutes and the organic solvent molecules in the stationary phase. Acidic compounds, phenols and nitroaromatics, were preferentially retained in the THF-based mobile phase, whereas esters and ketones were preferentially retained in the methanol (a hydrogen-bond donor) containing mobile phase. The system presented here seems to be very practical because any laboratory possessing two sets of HPLC equipment and two C j g columns can attempt similar 2D HPLC by simply changing the mobile phase for the two dimensions. 

Ion Exchange-Reversed-Phase 2D HPLC Using a Monolithic Column for the 2nd-D... 

Fast and simple 2D HPLC was also shown to be effective for the separation of a tryptic digest of bovine serum albumin (BSA) (Kimura et al., 2004). Every... 

---