HPLC

HPLC (High Performance Liquid Chromatography or High Pressure Liquid Chromatography) is frequently used chromatography technique to separate substances from liquid mixtures. HPLC is very useful instrument for Lab application. In HPLC, mobile phase flows with the sample mixture through Stationary phase (inside column) and reaches to Detector. In the Column, Mobile phase and Sample mixture interacts with Stationary phase where adsorption takes place and substances are separated.

Before going to read this section, you may refer Page "Chromatography Basic" to know chromatography fundamentals.

For any HPLC system, Pump, Injector, Column and Detector are required. Assume that through one capillary (tubing), flow is going from one end to another end. See below Image A.

All tubing connection used in Analytical HPLC system, have very lower Inner diameter. This tubing is also called as capillary. Its inner diameter is around 0.18 mm. So it is not easy to pass solvent from one end to other easily, therefore pump is required. HPLC pumps are of many types. They are specially designed to withstand high pressure of tubing and hence can give higher precision and stable flow. As per below image B, we have added pump to pass mobile phase (Liquid solvent, e.g. Water) through tubing from one end to another end.

To add sample, injector is required and to separate substances, column is required. See below Image C.

Here as per above image, Pump draws a mobile phase which goes to Injector then to Column. At the injector sample is injected using HPLC syringe. So, sample reaches to column with mobile phase. Column is filled with stationary phase, which will separate substance from mixture using Adsorption (as discussed in “Chromatography Basic”). Now, to detect separated substances, detection unit is required (Called Detector). Various types of Detectors are available as per application. Detection depends upon product’s molecule structure. Not all products can be detected using one detector. So it is necessary to choose appropriate Detector. Detector identifies substances and will pass data to Acquisition software. Here for illustration purpose, we have used UV-Vis Detector (This Detector uses UV and Visible light. To know Detector fundamentals you can refer “Spectroscopy” Page). Acquisition software is installed in Computer. This software collects data from Detector and will draw a graph (Called Chromatogram). Chromatography System looks like below Image D.

Chromatogram is nothing but the graph for Time scale Vs Absorbance (at column Adsorption and at detector Absorption). It looks like below.

Peak Table for above Chromatogram:

  

In above chromatogram, there are three peaks detected. As per Peak table; Peak RT (Retention Time), Area, Area Percentage, Height and Height Percentage detail is given. From chromatogram and peak table HPLC user/Analyst can determine the substances detail, substance is present or not and if present, then how much is the concentration of that substance?

• Analysis done to check substance's presence, is called Qualitative analysis. Suppose you know that for selected analysis with some specific chromatographic parameter , ‘X’ component is detected at 2 min (RT), then from chromatogram you can say that ‘X’ component is present or not.

• Analysis done to check unknown concentration for known sample by comparing known concentration of same sample is called quantitative analysis.

Advantages and Disadvantages of HPLC

Advantages: HPLC system is automated machines which gives result in a few minutes. HPLC gives high resolution, Speed and accuracy. Also due to automated system, results are reproducible.

Disadvantages: HPLC is costlier technique compared to conventional chromatographic techniques. HPLC operation is complex so trained person is required to operate HPLC.

One of HPLC system is shown below.
(HPLC System Image courtesy:http://www.agilent.com/)

Let’s have a look for different terminology used in Chromatography. 

Chromatography: It is the technique to separate substances from mixtures. It may be liquid or gas or fluid.

Column: It is SS tube with stationary phase material filled.

Stationary phase: It is the part of chromatography column, through which mobile phase passes and adsorption occur. Column Stationary phase may be solid, liquid or gel. It depends on product application.

Mobile Phase: It may be pure solvent or mixture of different solvent. It is also called Eluent.

Elution: It is the process of separating or extracting compound from sample mixture. For example, in HPLC adsorption occur in column for mobile phase (Eluent), sample and stationary phase, and compound is separated from sample. This process is called elution.

Sample: It may be single or mixture of compound. Generally sample made by dissolving compound in Eluent.

Solvent: It is a liquid form chemical that is used in chemical process. It may be Methanol, Hexane, IPA, Water, ACN and many more.

Chromatogram: It is the graph which relates time and absorbance.

Retention time: It is the time when particular compound peak is detected for particular analysis. If one sample analysis done many times with same Method parameter, sample, solvent, column and other chromatographic condition, then for that detected compound Retention time should be same.

Note: Description given in this page is for illustration purpose only. It is to understand concept.