High-performance liquid chromatography (HPLC) has replaced several Spectroscopic methods which were previously used for the qualitative and quantitative analysis of pharmaceutical and other chemical substances. Reverse phase HPLC columns are commonly used nowadays. Therefore separation of chemical substances and bio-molecules from a heterogeneous mixture can be done more easily, with the help of the reverse phase HPLC system than the previously known techniques.
As we know that typical HPLC systems are composed of mobile phase reservoir, pump, autosampler, column oven and detector. All these components are fixed in any HPLC systems and cannot be changed. There is a part in the HPLC system which can be changed as per the nature of a compound/s to be analyzed and ie the column.
This part (column) of the HPLC is available in different varieties and grades. The column can differ in length, diameter, packing material, pore size and polarity.
The HPLC columns are packed with the help of silica or carbon which is also called a stationary phase. The C18 columns are more polar than the C8 columns. The more polar column is to be used if the analyte is more polar in nature and vice versa.
In case of the high polarity of the analyte and the column, we use a more polar solvent (like water) in a mobile phase in addition to the organic phase. In such a case, the analyte will retain in the column and the water will try to elute it due to which separation of a compound occurs.
You can get best results had you selected the right column. The analysis shall not be started blindly in any case. Before going to start an analytical procedure you should know about the nature of the compound to be separated and the type of column needed for this purpose. In the case of analysis of peptides, C18 work very well.
A column should never be washed with an organic solvent if the buffer is used in a mobile phase. Because the column will be blocked completely and will be of no use. Furthermore, the HPLC tubing can also be blocked which may subsequently lead to the replacement of the tubing.
• Finally, due to the above-mentioned reasons, the Column shall only be washed with water (for 24 hrs) in case buffer is used as one of the mobile phases.