Preparation And Performance Evaluation Of Capillary Mixed-mode Chromatographic Column

The development of chromatographic methods allows simultaneous analysis of polar and non-polar,ionizable and neutral,and organic and inorganiccompounds.Ordinarily,to address the question of the separation of complex mixtures,the method of gradient analysis using HPLC is initially considered.However,it is necessary to return the system to the initial state once the analysis is completed,thus reducing throughput for multi-sample analysis.The mixed-mode chromatographic approach based on the selection of the mobile phase has begun to emerge as a better chromatographic method especially for the stronger retention control of both polar and non-polar molecules,in which at least two modes of interactions exist simultaneously.Multiple mechanisms of interaction permit the use of one stationary phase for a much wider range of applications as compared to the reversed-phase or ion-exchange approaches.A major benefit of mixed-mode chromatography(MMC)is that the same column can be used either in a single mode(such as ion-exchange,or reversed-phase),or in a combination of modes.Multi-dimensional separation as a kind of mixed-mode chromatographic approach is achieved by coupling two or more of similar or independent columns with the same or different separation mechanisms in order to transfer the special peaks from the first dimension to the second dimension and other dimensions therefore affording extremely high peak capacities.This simple approach offers increased resolution and sensitivity as well as adequate throughput and reproducibility over individual one-dimensional approaches.Therefore,this thesis mainly adopts the chemical or physical modification technique to prepare a series of chromatographic columns that owns multiple mechanisms.The main contents are summarized as follows:In the preface section,the basic concepts of capillary liquid chromatography(CLC),including the history of CLC,the type of CLC column and the application areas of common chromatographic stationary phase.The HILIC and multimode chromatographic stationary phase and its application were also introduced.Finally,we pointed out purpose and significance of the dissertation.With Congo Red(CR),the two azo groups are protonated in the pH range of 3.0-5.2 causing a colour change of the dye from blue to red at pH 3.0-5.2.Aimed at this condition,CR is found to exist as a zwitterion.Besides,CR is also used as an amyloid-specific dye.It is known to form supramolecular structures in solution via pi-pi-stacking.Dissociated sulfonic groups,protonation of azo groups and pi-pi-stacking behaviour must have a strong impact on the chromatographic properties of CR.In section two,CR was bonded on silica and packed in fused silica capillary for nano-flow HPLC.The material was used in mixed-mode chromatography,including both hydrophobic and hydrophilic chromatography,for several analytes using RPLC,HILIC and PALC.The effective separation of benzoic acid derivatives and sulfonamides shows that the CR bonded-silica exhibited a mixed-mode retention behavior including hydrophobic and ion-exchange interactions,which was different from the single mode displayed by ODS columns.The multiple modes of interaction make this column more powerful with versatility,reproducibility and selectivity for the separation of a wide range of compounds.Owing to the existence of multi-functionalized groups,it is foreseeable that this material can not only be used as a versatile chromatographic stationary phase,but also as a solid extraction adsorbent in other chemical fields.A weakble performance was observed on separation separation of nonpolar compounds and weak polar compounds with CR MMC column in the part two.However,ODS as a typical RP stationary phase has good selectivity on separation weakly polar and hydrophobic components.Hence,in the third section,four MMC columns(Sil-CR-con-ODS,Sil-CR-bis-ODSO,Sil-CR-mix-ODS and Sil-CR-tan-ODS)were prepared by modifying ODS as modification on Sil-CR.The methods can be the chemical or physical.By compared with these columns on separation the homologs of benzene,we can draw a conclusion that the Sil-CR-tan-ODS column has the accurate propotion on preparing ODS-CR modified column.What's more,the Sil-CR-tan-ODS column also has relative high column efficiency and good selectivity on positional isomers and cross grade mixtures.The stationary phase taurine(TDS)was used in HILIC to separate various kinds of goods and showed a strong retention for polar compounds.Combining both advantages of ODS and taurine stationary phases,one could expect a separation of polar and weakly polar components mixture to be easily achievable.Compared to single stationary phase column,this column could enlarge range of sample selection and reduce the sample pretreatment.What's more,the multiple mechanisms considerably prolong the retention times and improve the separation selectivity.Compared to two-dimension systems,CPC is also easy operation,stable and presents a large applicability spectrum.In this section,TDS and ODS are filled sequentially in a fused silica capillary to constitute a continuous packing capillary column.Benzene homologues,PAHs,nucleotides and biogenic amines were utilized to investigate the separation performance on this CPC column.This prepared CPC column not only presents an enhanced selectivity towards nucleotides but also exhibits an excellent retention for both highly hydrophobic and highly hydrophilic components simultaneously.This column system combines the benefits of a multidimensional separation and the flexibility of the nature of the stationary phase allowing better selectivity.Therefore this approach is appropriate for the development of analytical methods of complicated samples.APS is used in HILIC,especially for the separation of carbohydrates,aromatic compounds,organic acids and inorganic anion.However,for some compounds especially cations,it is always gotten no retentions.In the fourth part,TDS as strong cation exchange material in HILIC mode shows good selectivity on separation biogenic amines,nucleoside and nuclear bases.As they can be used in HILIC,TDS and APS may make up with a uniform column to achieve better chromatographic performance.Therefore,in the fifth part,APS-TDS MMC was successfully prepared.It exhibited good selectivity on separation of biogenic amines,organic acids,nucleosides,nuclear bases and sulfonamides.By compared with each column,APS-TDS column not only has the functions which are belong to single APS column or single TDS column,but also has a new mechanism(HILICxHILIC,2D-LC)beyond both.Satisfactory results show that the continuous packed column can be simple and easily to achieve the modification of APS and TDS.It provides a basicly physical modification method to make MMC column.Sulfonic betaine zwitterionic stationary phase is a kind of materials which containing APS and TDS functional groups.It can not only separate the very weak or high polarity of water-soluble compounds which is usually no retained in RP stationary phase,but also can separate ionic compounds by changing the charge along with the pH of mobile phase.However,for unionization and high LogP value compounds,it is almost weakly selective or no retained.This kind of compounds in the RP column,however,is extremely easy to be retained.It will greatly expand the the scope of the sample analysis once they are uniformed as one and performed their respective functions.Therefore,in sixth part,we have adopted the commercialization of ZIC?-HILIC and ODS stationary phase to prepare ZIC-ODS MMC column.The chromatographic column has the ability of simultaneous separation the mixture of nucleoside,nuclear bases and benzene homologue.RP mechanism of ODS phase can meet the need of ZIC phase to separate sulfa drugs.What's more,the RP mechanism makes the ZIC-ODS has a better ability than ZIC on separation of position isomers.