HPLC on first appearance appears to be a complex system comprising of modular boxes and an intricate bunch of tubings though in modern systems most tubings are concealed inside the modules. If you take a careful look the mystery of the modules will start unfolding and you will be able to resolve them into simple functional units each having a defined role
- Mobile phase – acts as a carrier stream for the sample
- Injector – injects fixed volume of sample into the mobile phase stream
- Pump – delivers mobile phase at constant flow rate
- Column– separates the sample components
- Column oven – maintains constant column temperature
- Detector – detects individual eluting sample components
Not all component parts require regular calibration and only careful handling is sufficient. Examples of such parts are:
Mobile phase – mobile phase requires to be freshly prepared and it should be free of suspended matter. Always adopt same mixing procedure to eliminate errors due to heat of mixing
Columns – a column is the most critical component of the HPLC system. If a column is not well maintained it loses its separation efficiency. Using working standards you should establish that the system suitability parameters remain within the prescribed limits. In case of deviations outside the specified limits the column should be replaced
Modules requiring regular calibration
A pump comprises of several parts that are precisely designed to deliver consistent flow rates. However, due to regular usage wear and tear results in flow rate deviations. Timely replacement of worn out parts improves the precision of flow delivery but still regular calibration is necessary. Pump calibration involves establishing flow rate accuracy and gradient composition accuracy.
Manual injection requires filling a fixed volume loop with the sample. The loop needs to be flushed repeatedly with the mobile phase so that traces of earlier sample are eliminated. Wait for baseline to stabilize before proceeding with your sample analysis. Auto injectors are manufactured to deliver precise volumes every time and sufficient wash cycles should be allowed to ensure clean injections. Injector calibration involves confirmation of injector accuracy, precision, linearity and carryover tests
The retention times of the compounds are dependent on mobile phase viscosity which in turn is temperature dependent. The column should be maintained within specified temperature limits to get consistency of retention times. It becomes necessary to calibrate the temperature reproducibility of the column oven from time to time.
A detector gives absorbance signals that are dependent on wavelength of absorption by the sample components. The absorption signals can drop due to lamp life, residual impurities or other contributing factors. Timely replacement of lamps and regular cleaning of detector eliminates such problems but detector requires regular calibration using specified reference compounds which are injected into the mobile phase stream. The two main parameters requiring calibration are linearity of response and wavelength accuracy
HPLC is a popular analysis technique and several reputed makes are available. You have to adopt manufacturer prescribed calibration procedures at recommended time intervals to obtain the desired accuracy and precision of results.
Please share your experiences and leave your comments on the article.