HPLC plays a significant role in analysis of composition of mixtures of organic compounds and finds a large number of applications in pharmaceuticals, foods, herbal products, environmental monitoring, clinical studies and life science studies. The scope of applications is fast expanding as today’s consumer demands for diverse products and awareness of safety of ingredients demands in- depth studies on properties of new range of organic molecules of both natural and synthetic origins.Further it can be said that the tips offered in the article are general in nature and can be adapted to other chromatographic techniques such as Gas Chromatography.
The separation and identification potential of HPLC systems requires regular upkeep through maintenance and calibration of system. Columns play a key role and need to be properly handled and used as per specified guidelines. It is necessary to control operational parameters such as pH, temperature, pressure of mobile phase to get reproducibility of results. Use of guard columns also prolongs useful life of most columns. Next the mobile phase should be mixed following the same mixing procedure every time and filtered for suspended solid particles before start of analysis. Suspended solid impurities which are invisible due to their small size(μm range) can lead to increased blockage of columns and resultant increase in column back pressures.
HPLC Mobile Phase Bottles
Apart from care exercised in handling and use of columns and mobile phases it is also necessary to conduct frequent calibration of the main system components such as:
HPLC systems in busy laboratories operate virtually non-stop for weeks together. It is but natural to expect wear and tear of component parts of pumps. Visual inspection of pistons, seals, o-rings for damage helps timely replacement and can prolong the useful lifespan and helps provide trouble-free operation. On the other hand periodic calibration helps ensure consistency of flow rate accuracy and mobile phase gradient compositions.
High precision of results requires consistent and reproducible volume injections. Allow sufficient time between injections to stabilise baseline before making subsequent sample injections. This practice helps flush out remnants of samples injected previously. However, use of auto sampler injection modes prevents human errors and improves reproducibility of injections.In addition unattended operationsaves operator time for carrying out other analysis operations.
Control of column temperature
Fluctuations in temperature of column can result in flow rate deviations from set values. Use of column ovens with precise temperature control provides reproducibility in retention times of chromatographic peaks. However, the oven needs to be calibrated for temperature reproducibility from time to time.
The main reason for drop in detector performance is due to drop in performance of light sources over continued use or from insoluble deposits settling on cell walls. Lamp replacement on observing first signs of weakening of signals and cleaning of cells as per suggested procedures can prevent further loss of lamp performance. Further regular periodic calibration is advised to obtain a linear response over wide range of analyte concentrations.
The tips offered serve a dual purpose. Firstly the useful lifespan of the system is increased and secondly a high degree of reliability can be placed on the results of analysis.