| Today scientists must deal with complex samples that either cannot be adequately separated using one-dimensional chromatography or that require an inordinate amount of time for separation. Two-dimensional liquid chromatography (2D-LC) which combining different separation mechanisms will generates extremely high peak capacity and ultra-high resolving power in separation of complex mixtures. It has received considerable attention in the separation complex samples from proteomics, environment, and natural products. Various 2D-LC system was developed through combining different separation modes.In particular, LC techniques offer a wide variety of separation mechanisms, such as normal phase (NP), reversed phase (RP), size exclusion (SEC) or ion exchange (IEX), characterized by different selectivity. Consequently,2D-LC can be theoretically employed in many multidimensional combinations, generating increased peak capacity, selectivity and resolution, especially in the comprehensive LC mode. However, the combination of certain LC modes can present a series of compatibility difficulties, if not impossibilities, such as, for example, selectivity, separation speed, mobile phase immiscibility and sample capacity. Systematic study of the compatibility of the two dimensions is necessary to construct a successful 2D-LC system.Directly couple of two columns with different separation selectivity is a simple way to construct 2D-LC. IEX-RP, RP-RP and HILIC-RP system was constructed through eluting by different mobile phase alternately. The compatibility of selectivity and mobile phase was investigated. Directly coupled columns IEX-RP system was developed for the separation of peptides. Step gradient of salt concentration for eluting components from weak anion exchange (SCX) column to RP column was followed by linear gradient of organic solvent proportion for eluting fractions on RP column. Total 2D-LC peak capacity of 675 was achieved through eluting of 11 fractions continuously. Directly coupled columns RP-RP system separated 60% more peaks than single column RP system, with 86 peaks was recognized in the separation of Liuwei Dihuang Pill. The developed HILIC-RP system was successful used in the separation of peptides, proteins and Traditional Chinese Medicine.The second dimensional separation speed had an important influence on the peak capacity of 2D-LC system. Higher modulation frequency could significantly decrease the affection of under-sampling on peak capacity. Running with fast gradient and elevated temperature was an effective way to increase the separation speed and increase the compatibility with first dimension. Investigation of the effect of temperature on the separation speed and selectivity were carried out and verified by reference sample. A high temperature two dimensional IEX/RP liquid chromatography system was constructed through elevating the 2nd column temperature to 80℃. Faster separation and narrower peaks were observed. Compared with separation under room temperature,13% more peak capacity was got which was 1840. The coupling of HILIC and NP is difficult to implement due to the mobile phase incompatibility. The high concentration of organic solvent in in HILIC disables the transfer between the two dimensions. On-line two-dimensional HILIC/RP liquid chromatography system was developed based on weakening of the mobile phase strength in the interface. Separation of three reference compounds shown that sample could be effectively introduced to the second dimension. A detector was used in the interface to detect the non-retained compounds. Mixture of tryptic digestion of four proteins was separated to test the performance of the system.208 peaks were recognized under the optimized condition and peak capacity reached 2323. Separations of rat liver protein, rhizome Gastrodiae and Cordyceps were also carried out to evaluate the performance of constructed on-line HILIC/RP system for analysis of real world sample.The use of short second dimensional column will benefit the separation speed compatibility but may result in incompatibility of sample volume. Based on heart-cut interface, the components gushed from first dimension column can be removed and collected in another column. The removing of fraction which will overload on second dimension column greatly increases the resolving power and detection sensitivity. An IEX/RP 2D-LC system was developed by addition of heart-cut unit. Tryptic digest of four proteins were separated on this system. Compared with that of traditional 2D-LC, another 21 peaks were identified and some low abundance components were identified after weak retained components were removed.The use of short second dimensional column will benefit the separation speed compatibility but may result in incompatibility of sample volume. Based on heart-cut interface, the components gushed from first dimension column can be removed and collected in another column. The removing of fraction which will overload on second dimension column greatly increases the resolving power and detection sensitivity. An IEX/RP 2D-LC system was developed by addition of heart-cut unit. Tryptic digest of four proteins were separated on this system. Compared with that of traditional 2D-LC, another 21 peaks were identified and some low abundance components were identified after weak retained components were removed. A tandem column connected to parallel second-dimensional separation column can help to improve the separation efficiency. An IEX/RP 2D-LC system has been developed based on a parallel-tandem column interface. The peak capacity of the 2D-LC system reached 707 under high temperature conditions. When compared with the traditional parallel column interface constructed in this paper, the new interface produced 44% higher peak capacity. Rat serum and rhizome gastrodiae were separated to evaluate the performance of constructed 2D-LC system for analysis of real world sample. |
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