With the introduction of two new reversed-phase column types, TSKgel ODS-100V and TSKgel ODS-100Z, excellent chromatographic performance and selectivity can be achieved for a wide range of sample types. TSKgel ODS-100Z and TSKgel ODS-100V are based on a spherical, five micron, silica gel with 100 angstrom pores; each material is derivatized with difunctional octadecylmethylsilane, followed by an exhaustive endcapping reaction.TSKgel ODS-100Z contains approximately 20% carbon, while TSKgel ODS-100V has a lower ligand density resulting in about 15% carbon, which in turn leads to stronger retention of polar compounds.
The analysis of water- and lipid-soluble vitamins requires both hydrophilic and hydrophobic retention capabilities of the stationary phase. Also, the composition of the stationary phase must allow for stable retention times in the absence of organic solvents in the mobile phase. As demonstrated, simple and fast analysis methods for the determination water and lipid soluble vitamins are possible on the TSKgel ODS-100V and the TSKgel ODS-100Z columns.
Analysis of Water Soluble Vitamins
A mixture of six water soluble vitamins was analyzed on TSKgel ODS- 100V and TSKgel ODS-100Z under isocratic conditions with 99% water- 1% acetonitrile, acidified with 0.1% TFA to a pH of 2.0. At an optimized temperature of 40C good separation was obtained for all compounds in less than 10 minutes (see Figure 1 ).
Both columns managed to separate all vitamins by baseline. Thiamine, pyridoxal and pyridoxine were better separated on the more polar TSKgel ODS-100V than on the ODS-100Z column. Also, separation of nicotinamide and nicotinic acid was enhanced on the more polar column.
Analysis of Lipid Soluble Vitamins
Analysis of vitamin D2 (ergocalciferol) and D3 (cholecalciferol) is critical because they differ only in one methylene group and one double bond. As shown in Figure 2, separation was achieved under isocratic conditions in 100% acetonitrile at a flow rate of 1.0mL/min. The influence of temperature on resolution was evaluated at 25C and 40C.
For this separation TSKgel ODS-100Z, which has higher % carbon than TSKgel ODS-100V, provides better resolution. Ergocalciferol and cholecalciferol elute from both columns with near baseline separation. On the TSKgel ODS-100V column, hydrophobic retention is not high enough for a baseline separation of both vitamin D compounds. On both columns, separation is enhanced at lower temperature (25C).
Simultaneous Separation of Water and Lipid Soluble Vitamins
Water and lipid soluble vitamins were separated in one single run on TSKgel ODS-100V and TSKgel ODS-100Z as demonstrated in Figure 3 .
To separate the mixture of six water-soluble vitamins, caffeine, and four fat-soluble vitamins, required a gradient from water containing 0.1% TFA (eluent A) to acetonitrile containing 0.1% TFA (eluent B). Flow rate was 1mL/min, at a temperature of 40C. To elute the polar vitamins, the content of acetonitrile in the eluent was gradually increased up to 40% within 20 minutes, then, within 2 minutes, up to 100%, enabling elution of the less polar vitamins within another 27 minutes.
Both columns were capable to separate the ten vitamins and caffeine. Due to the lower hydrophobicity of the ODS-100V column, elution was possible within 35 minutes, with very sharp peaks. Elution on the more hydrophobic TSKgel ODS-100V column, required nearly 45 minutes with broader peaks, but with higher resolution of the tocopherols.
The recently commercialized TSKgel ODS-100V and TSKgel ODS-100Z column set provides a universal solution for most reversed-phase separations.