I will describe a very common chemical analysis method used throughout academia and industry alike. High performance liquid chromatography (HPLC) is commonly used to analyze chemical compounds for purity and assay value. Sampling for an HPLC analysis is usually very easy since only micrograms of sample are required. The tiny amount of sample is dissolved in a solvent that the HPLC instrument injects into the system. This makes HPLC an ideal method for analyzing chemical reactions since they are already dissolved in solvent. The detection level for a quality system is down to the ppb level (parts per billion). Precision is usually very high, with percent relative standard deviations among replicate groups of analyses well below 0.5%. Sources of error include sample preparation (since this is the main part of the human component), or using low grade solvent systems (high grade solvent is required), a damaged column, or an out-of-calibration detector.
The main components of an HPLC system include:
- the solvent system (mobile phase)
- a degasser (to remove dissolved gasses in the solvent)
- a solvent pump that can mix and meter solvent mixtures with a high degree of specificity
- an autosampler to inject the sample
- the HPLC column (stationary phase)to perform the actual chemical separation
- a type of detector (usually some type of light absorbance based detector).
Applications include measuring the purity of most any soluble substance that absorbs some wavelength of light. Assay performance is very high. In fact, regulatory agencies like the FDA and ICH have established common limits for different assay parameters that are considered universally acceptable.