Determination Of Several Substances By High Performance Liquid Chromatography On Titania

Titania（TiO2）, a new type of chromatographic stationary phase, is developed in recent years.Not the incomparable superiority of titania do the traditional silica gel stationary phase have.Compared with silica gel, titania’s surface is alkaline. In addition, it owns excellent chemicalstability in pH1-14range, namely strong acid and alkaline conditions. But silica gel stationaryphase is applies only to pH3-9range. So the fatal defect which is "irreversible adsorption" ofalkaline compounds on silica gel dosen’t exist on titania. Meanwhile, titania can well solve theproblem that the alkaline compounds show symptoms of "bad-tail" in silica gel, so there will bemore application prospects of titania in the biological materials, such as alkaloids,phosphoprotein, peptides as well as the analysis of other alkaline chemical compounds. Titaniastationary phase is expected to fulfill great potential that the silica gel and other polymerstationary phase is unable to substitute in the food safety industry as well as in the life sciences.There will be more important theoretical value and broader application prospect. In this paper,methods for the analysis of aspartame, several alkaloids and protein, peptides by highperformance liquid chromatography on titania was reported.The researches in this thesis included four aspects:（1） A method for the determination of aspartame in sugarless coke by RP-HPLC on titaniawas reported. The drink samples were separated with a Titania Sachtopore-RP column （250×4.6mm,5μm）. The chromatographic separation conditions were: column temperature was70℃, themobile phase was15mmol/L ammonium phosphate （（NH₄）₂HPO₄:NH₄H₂PO₄=6:4） with10mmol/L ammonium fluoride and methanol in the ratio of70to30, with a flow rate of1.0ml/min.The detection wavelength was217nm. The linear range of the method was10-2500μg/mL（R²=0.9994） with a detection limit of0.08μg/mL. The recoveries of the method were between92.5%and97.4%. The relative standard deviation of the method was less than0.72%. Themethod is accurate and precise and it is applicable to the determination of aspartame in drinks.（2） A rapid method for the determination of caffeine by high performance liquidchromatography on titania in soft drinks was reported. The drink samples were separated with aTitania Sachtopore-RP column （250×4.6mm,5μm）. The chromatographic separation conditionswere: column temperature was30℃; the mobile phase was a mixture of water and methanol in the ratio of83to17（V/V）, with a flow rate of1.0ml/min. The detection wavelength was273nm.The linear range of the method was12-900μg/mL （R²=0.9998） with a detection limit of0.03μg/mL. The average recoveries of the method were100.9%. The relative standard deviation （n=9）of the method was less than0.23%. The method is simple, timesaving, accurate and precise.Meanwhile, it is applicable to the determination of caffeine in drinks.（3） The good result is achieved in the second part, so a rapid and simultaneous method fordetermination of theobromine, theophylline and caffeine by high performance liquidchromatographic on titania. The samples were separated with a Titania Sachtopore-RP column（250×4.6mm,5μm）. The chromatographic separation conditions were: column temperature was60℃; the mobile phase was a mixture of water and methanol in the ratio of95to5（V/V）, with aflow rate of1.0ml/min. The detection of wavelength was273nm. The linear range of themethod for theobromine, theophylline and caffeine were5-500μg/mL （R²=0.9999）,5-1000μg/mL （R²=0.9997）,6-1200μg/mL （R²=0.9999）. The detection limit were0.04μg/mL,0.08μg/mL and0.03μg/mL, and the average recoveries were100.2%,101.1%and100.9%. Therelative standard deviation （n=9） was less than0.37%. The method can make higher sensitivity,precision and accuracy. Meanwhile, the results are satisfactory, so it is applicable to thesimultaneous and quantitative analysis of theobromine, theophylline and caffeine in several kindsof food samples.（4）The research of HPLC and LC-MS/MS in hydrolysate of casein manifested that: on thebasis of enrichment to the phosphopeptides using titania column, an rapid chromatographiccondition was reported: the samples were separated with a Titania Sachtopore-RP column（250×4.6mm,5μm）. The chromatographic separation conditions were: column temperature was45℃; the mobile phase was a mixture of water and methanol in the ratio of50to50（V/V）, with aflow rate of0.5ml/min; the injection volume was5μL. There were sixteen constituents of caseinhydrolysate which was identified by MS/MS, and the eleven of them were polypeptide:His-Pro-Ile-Lys（αs1-CN,4-7）, Glu-Val-Val-Arg（αs2-CN,42-45）, His-Ile-Gln-Lys（αs1-CN,80-83）,Leu-His-Ser-Met-Lys（αs1-CN,120-124）, Glu-Ala-Met-Ala-Pro-Lys（β-CN,100-105）,Gly-Pro-Phe-Pro-Ile-Ile-Val（β-CN,203-209）, Thr-Thr-Met-Pro-Leu-Trp（αs1-CN,194-199）,Glu-Met-Pro-Phe-Pro-Lys（β-CN,108-113）, Glu-Asp-Val-Pro-Ser-Glu-Arg（αs1-CN,84-90）,Val-Asn-Glu-Leu-SerP-Lys（αs1-CN,37-42）, Thr-Val-Asp-Met-Glu-SerP-Thr-Glu-Val-Phe-Thr- Lys（αs2-CN,138-149）, among them the last two is CPPs. The other were amino acid: Pro, His,Ala, Val and Asp. So the function of enrichment for phosphopeptides is existence in TiO2.