**7.4 High performance liquid chromatography (HPLC)**

 High performance liquid chromatography (HPLC) plays a mandatory role in isolation of natural products. It is a versatile and widely used technique for the isolation and identification. In the modern era, HPLC technique is becoming popular for studying separation, identification, and fingerprinting study for the quality control

 of herbal plants. Currently, this technique works as the main choice for research scientists. The multicomponent samples on both an analytical and preparative scale can be separated and studied more easily by HPLC. Thus resolving power of HPLC is ideally used for the rapid processing of herbal extracts. HPLC instruments are designed in modular ways and they contain delivery pump for solvents and manual injection valve along with an auto-sampler. As sample is introduced in autosample, it carries toward the important part or heart of HPLC that is an analytical column, a guard column. Further, a detector, recorder, and printer are used to show a graphical representation on the software based or installed computer device. In every chemical separation, the working and result production differ due to the fact that certain compounds have different migration rates, which can be fulfilled using HPLC by utilizing a particular column and mobile phase as per the requirement for separations. Trial and error concept is applied for developing new mobile phase along with prior knowledge of separation, its structure, and required solvent polarity or nonpolarity. Thus, the extent or degree of separation is based upon the choice of stationary phase or mobile phase. Generally, the identification and separation of phytochemicals can be achieved by using isocratic system that is using single mobile phase. Gradient elution sometimes can be used in which the proportion is altered from organic solvent to water. It also depends on time, and it may be desirable if more than one sample component is studied. Or it also differs from each other significantly in retention of components with column as per the conditions achieved. Identification of compounds by HPLC is a crucial part of HPLC assay. Identification of any bioactive compound by HPLC selection of detector is again the next important step. Once the detector is selected and the setting is done, the assay may be developed by trial and error of solvent system. Once the sharpness of the peak of known sample is obtained, the solvent system can be selected. The important parameters of this assay is that a clean sharp peak of the known sample is observed from the chromatograph. The reasonable retention time of identifying peak should be there. The extraneous peaks at the detection levels should be well separated from the main sharp peak. At maximum time, UV detectors are popularly used in HPLC detection. UV detectors are used among all the detectors because they have high sensitivity and UV absorbance of majority of naturally occurring compounds is possible at low wavelengths of 200–210 nm. If bioactive compound needed to be isolated is only present in small amounts within the sample, then the high sensitivity of UV detection is a bonus in herbal natural drug discovery. Liquid chromatography coupled with mass spectrometry (LC/MS) is also a powerful technique for the analysis of complex botanical extracts. It offers accurate determination of molecular weight of proteins, peptides. Isotopes pattern can also be detected by this technique. A recent advance includes electrospray, thermospray, and ionspray ionization techniques, which offer unique advantages of high detection sensitivity and specificity [19].

 HPLC when combined with mass spectrometry (MSn) gives lot of information for structural elucidation of the compounds because its ability of recognition increases and separation with structure identification becomes very easy. Therefore, when a biomarker which is of a pure standard is unavailable, fast and accurate identification of bioactive chemical compounds in medicinal herbs is possible due to the combination of HPLC and MS. In order to count the overall success of natural product in isolation and separation, the most important is processing of raw material further to provide a sample suitable for HPLC analysis. The significant bearing is on the choice of solvent for sample active compounds identification. The source material that is dried powdered herbal plant material should be studied very efficiently in earlier stages and steps: first, its dried form and second to learn about its chemical structural part that is the powder's ability to release the bioactive

#### *Natural Products in Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.82860*

compound of interest into the solution. In such cases, mobile phase development initially using TLC and having idea about the solvent system before applying to HPLC saves your time. Thus, in normal case of extraction, dried plant material is treated with an organic solvent methanol, chloroform. After extraction, extracts are dried over rotary evaporator and powdered extracts are concentrated and injected into HPLC for separation and analysis. HPLC is useful for compounds that cannot be vaporized or that decompose under high temperature, and it provides a good complement to gas chromatography for detection of compounds.
