Development of Two Dimensional Pressurized Capillary Chromatography and Its Applications on Separation of Complex Samples
|School||Shanghai Jiaotong University|
|Keywords||pressurized capillary chromatography pCEC two dimensional chromatography strong cation exchange Cortex Phellodendri HILIC proteomics|
Pressurized capillary electrochromatography (pCEC) combines hydraulic pressure with electroosmosis flow as its driving force, resulting in a more reliable technology for separation of complex samples due to its capability of suppressing bubble formation and its amenability for a solvent gradient mode. However, with the development of omics, it is often difficult to completely separate all components in a complex sample. Two-dimensional (2D) chromatography couples two different kinds of separation modes and provides higher peak capacity and resolution. In pCEC, both chromatographic partition and electrophoretic mobility contribute to the separation mechanism for charged compounds. Therefore, the introduction of pCEC to 2D separation could provide a 2D platform with three kinds of mechanisms. This higher peak capacity and higher selectivity system could be used in separation of more complex samples. In this dissertation, 2D systems based on pCEC were constructed and used to separate complex samples, such as traditional Chinese medicines and biological samples.It consists of six chapters:Chapter 1 is the general introduction, which includ description on fundamental aspects and columns in CEC and pCEC, as well as 2D chromatography. The advantages of 2D systems based on pCEC are explained.In chapter 2, pCEC was used to separate the extract of Cortex Phellodendri. The extraction solvent, mobile phase, detection wavelength and applying voltage were optimized. The voltage effect on separation was especially discussed. The dual mechanism in pCEC was studied via three standards. It was the basis of the advantage of pCEC in 2D compared toμHPLC.In chapter 3, a biphasic SCX-RP monolithic column for pCEC 2D system was prepared in one capillary for convenience and simplified the 2D system. This 2D system was used to separate bovine serum albumin (BSA) digests. The sample was first eluted from SCX section by NH4Cl for four steps and then eluted from RP section by ACN– water with solvent gradient.In chapter 4, a 2D system based on micro strong cation exchange liquid chromatography and reversed phase pCEC (μ-SCXLC/RP-pCEC) was developed. Considering each dimensional separation condition, optimization on mobile phase and the number of fractions, the offline and online 2D system were constructed. These 2D systems were used to separate TCMs and biological samples. The mobile phase in the first dimension was NH4Cl– water with linear gradient for offline 2D and step gradient for online 2D. The mobile phase of second dimension was acetonitrile (ACN)– water with linear gradient for both offline and online 2D. The orthogonality and peak capacity of 2D systems were investigated. It was proved that these 2D systems were highly orthogonal. The practical peak capacity for the three samples was 568, 940, 576 for offline 2D and 355, 436, 480 for online 2D. So these 2D systems could enhance the peak capacity and resolution, respectively.In chapter 5, two hydrophilic interaction liquid chromatography (HILIC) and RP pCEC 2D systems were developed: namely, cyano liquid chromatography– reversed phase pCEC (CN-LC/RP-pCEC) and amide liquid chromatography– reversed phase pCEC (Amide-LC/RP-pCEC). HILIC and RP-pCEC were orthogonal and complementary for polar substances. The mobile phase of first dimension was methyl alcohol– water while the second dimension was ACN– water to increase the orthogonality. Considering the matching of mobile phase in two dimensions, offline 2D CN-LC/RP-pCEC and amide-LC/RP-pCEC systems were constructed to separate biological samples. The peak capacity was approximately 600-700.In chapter 6, an RP pCEC combined RP LC with linear quadrupole ion trap mass spectrometer (RP-pCEC/RP-LC-LTQ) 2D system was developed. Because peptides showed the different retention in different pH, plasma digests could be separated well in the first dimension (basic) and the second dimension (acid). This 2D system with MS could be used for proteomics study on comparison of healthy and liver cirrhosis patients. Finally, 4760 unique peptides were identified by MS, resulting in more than 100 proteins detected from each plasma sample and 31 different proteins from healthy group and patients.