Project: HPLC
Introduction
HPLC is a separation method. Like all chromatographic methods, it employs a mobile phase and a stationary phase. An analyte dissolved in solution is injected onto a column (the stationary phase) and carried through the column by a solvent, called the eluent or mobile phase. The column is packed with a substrate, which – for reverse-phase HPLC as we’ll perform here – is a non-polar substance with a C18 coating. In the column, the sample partitions between the stationary and the mobile phase, and it’s relative affinity for each determines how quickly it moves through the column. In this way, HPLC allows us to separate analytes based on polarity. In this lab, you’ll use the HPLC to perform quantitative analysis. This is possible because the area under each chromatographic peak is proportional to the concentration of each analyte.
The general order for HPLC sample preparation is: (1) Prepare standard and quality control samples (QCs), (2) run standards and a QC, (3) prepare the samples, and (4) run the samples. You should obtain a clean blank, a passing standard curve, and a passing QC before preparing your samples. If any of these fails there is no point in running your samples!
For HPLC, the definitions of “passing” are:
- Blank: Flat, smooth baseline showing only random noise and/or instrument drift no greater than a few percent over 15 minutes.
- Standard Curve: A linear line with $R^2 \ge 0.9900$.
- QC: A measured concentration within 10% ($\pm$ 10%) of the known concentration.
This is a quantitative analysis.
Safety
- Wear gloves when working with the samples and preparing standards.
- Although samples are over-the-counter products, you should avoid ingesting and/or contacting them with your skin.
- Methanol and formic acid are used in the mobile phase. Avoid skin contact with mobile phase.