Even when you first heard of High Performance Liquid Chromatography, the name explains that it is a sophisticated operation of column chromatography. Chromatography or in lay person’s term, refers to a separation technique in chemical analysis. If you recall your high school chemistry, column chromatography is a method used to separate a pure compound from a mixture of chemical compounds using gravity. Similarly works in High Performance Liquid Chromatography procedure, except the mixture of compounds is forced through the system under high pressures up to 5000 psi, thus HPLC at times are commonly mistaken as High Pressure Liquid Chromatography!
What makes up a functional Column Chromatography also establishes a High Performance Liquid Chromatography (HPLC) system including mobile phase and stationary phase. Tiny particles are packed densely in a column between 100 to 250 mm length with an internal diameter of 4.8 mm. Depending on the variant of functionality, normal phase HPLC that acts similarly to Thin Layer Chromatography, is packed with polar silica. Manipulating on the non-polar eluent, the polar compounds tend to stick to stationary phase, taking longer to move through the column compare to non polar compounds. On contrary, reverse phase chromatography leverages on non-polar stationary phase, whereby the packing material is altered by adding either C-8 or C-18 atom. With a polar eluent or mobile phase such as methanol-water, the polar compounds tend to travel with the eluent, being detected earlier and faster.
Stands as an improved version of Column Chromatography, detector is a critical component in High Performance Liquid Chromatography system. Automated and highly sensitive machines, the most common detectors in laboratories are UV-Vis and Fluorescent. Different compounds exhibit strong absorption in different wave length of the ultra-violet or fluorescent spectrum, thus manipulating different wave length allows chromatographer to identify and quantity the compounds of interest. It is always safe to run an injection of pure eluent, so that you’re not going to confuse or mistaken the eluent’s peak for your sample!
Qualitative and Quantitative Data from High Performance Liquid Chromatography
Inclusive in a complex High Performance Liquid Chromatography system is an integrator, providing luxury to qualitative and quantitative data including a series of peaks, retention time, height and area-under-the-curve (AUC) of these peaks. Retention time is the duration taken for an analyte to move through the system from the point of injection until detection. If all the parameters such as flow rate and composition of mobile phase are under controlled, then retention time is a good means to identify your compounds. Height or AUC are commonly used to quantify the studied compound if you had a calibration curve run under the same condition.
Combining a physical separation capabilities from HPLC and the mass analysis created a smart and powerful chemical analysis technique i.e. LC-MS or High Performance Liquid Chromatography Mass Spectrometry. In this system, as the analyte moves through the detector, a part is diverted to mass spectrometer to study its fragmentation pattern. The pattern is then compared against a huge database of compounds, hence compound identification can be figured almost instantaneously on output machine. Highly sensitive and selective, LC-MS is widely applied in the studies of pharmocokinetics, proteomics and HPLC method development for pharmaceuticals.