High Performance Liquid Chromatography (HPLC) Approach.
- A total of 73 DH lines were quantified for vitamin C levels at harvest (Day 0) using a analytical HPLC approach.
- A analytical HPLC approach involves many components:
- Column: A separation column (RP-C18, LiChrospher) aswell as a guard column was used in the vitamin C protocol. The purpose of the guard column is to protect and prolong the life of the spearation column by a) filtering particles that clog the separation column, b) preventing baseline drift & false peaks.
- Mobile Phase: The solvent that is continuously applied to the column. The role of the mobile phase is to act as a carrier for the sample solution that is injected into the HPLC.
- Stationary Phase (reverse phase): The solid support contained within the column in which the mobile phase passes through. Reverse phase consists of silica based packings with n-alkyl chains covalently bound i.e. RP-C18 comprises of a octadecyl ligand in the matrix. Reverse phase uses principles of hydrophilicity, hydrophilic compounds elute more quickly where as hydrophobic compounds are reatined.
- Detector: The component that emits a response (peak on the chromatogram) due to the eluting sample compound. In this protocol a ultra-violet (UV) detector was used to dectect vitamin C.
- Vitamin C was quantified by injecting a series of standards of known concentration onto the HPLC for detection. The output of this provides a series of peaks correlating to 5 different standard concentrations which is used to produce a calibration curve.
- The area under each standard peak is calculated using the area of a triangle equation (A=1/2b x h), this is then graphed against the concentation of the sample solution.
- A line of best-fit is then dervied and the equation of a line (y=mx + b) is used to determine the vitamin C concentration of experimantal samples.
- For example a sample of unknown vitamin C concentration (x) is injected onto the HPLC, a peak is generated and it's area calculated (y). The concentration of the sample is found by solving the equation for x using the equation of a line (y=mx + b).
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