Chemical Separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase. Thus, the chromatographer can separate compounds (more on chiral separations) from each other using HPLC; the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.
Purification refers to the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer may choose the conditions, such as the proper mobile phase, to allow adequate separation in order to collect or extract the desired compound as it elutes from the stationary phase. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without incurring any other undesired compound.
Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any compound by HPLC a detector must first be selected. Once the detector is selected and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed. To alter the retention time of a compound, several parameters can be manipulated. The first is the choice of column, another is the choice of mobile phase, and last is the choice in flow rate. All of these topics are reviewed in detail in this document.
Identifying a compound by HPLC is accomplished by researching the literature and by trial and error. A sample of a known compound must be utilized in order to assure identification of the unknown compound. Identification of compounds can be assured by combining two or more detection methods.
Quantification of compounds by HPLC is the process of determining the unknown concentration of a compound in a known solution. It involves injecting a series of known concentrations of the standard compound solution onto the HPLC for detection. The chromatograph of these known concentrations will give a series of peaks that correlate to the concentration of the compound injected.
Using the area of a triangle equation (A=1/2b x h) to calculate the area under each peak, a set of data is generated to develop a calibration curve. This is done by graphing peak area vs. the concentration of the sample solution. Most graphs can be generated using a computer software program such as Excel or Cricketgraph. From this graphing software, a best-fit line can be derived, and the equation of that line can be determined. This equation of a line, y=mx + b, generated by the data, is the calibration curve equation.
The equation of the line is then used in the following manner: A scientist injects a sample of unknown concentration x (x-axis of calibration curve) onto the HPLC; the chromatograph gives a peak output of area y (y-axis of the calibration curve). The area, y, is then in the equation of a line y=mx + b from the calibration curve, and the concentration is found by solving the equation for x.