HPLC instrumentation: Video demo of HPLC system

HPLC system comprises of high end sophisticated instrumentation technology.The machine is expensive to purchase and also use it. The equipment requires to be kept in a chromatography lab which is clean, dust free & air conditioned room. The basic HPLC instrumentation comprises of components like

¤ Pumps

¤ Injection system,

¤ Columns,

¤ Detectors and

¤ Computer for display and documentation of data.

HPLC Pump→ Pump is an essential part of HPLC which generates pressure for the flow of mobile phase through the column. They must be capable to generate a pressure of at least 5000psi.

Pumps are differentiated as piston type pumps (constant displacement pumps) and gas pressure (constant pressure pumps). The former works by uniform displacement of fixed volume of mobile phase. The latter introduce high pressure gas into the pump to force the mobile phase. The former is preferred and in common use.

The pupm shown in the fig. is a constant displacement pump from manufacturer Waters.

The two displacement pistons pump the mobile phase into central purge unit through circular coiled links to the purge from pistons as can be seen in the fig.

HPLC Injection system→ This is used to inject a fixed volume of samples into the pump. The commonly used type is rheodyne injector (loop injector) fixed near the pump. It has a range to inject from 20 microlitres to 100 micro-liter volume. In load, the position sample is injected by a syringe into the injector. In the injection, the mode sample is injected from the loop into the pump.

HPLC Column→ This is the most important and sensitive part of HPLC. They are commercially available as microporous, pellicular and bonded phase types based on the physical and chemical nature of the packed stationary phase in the column. They are able to withstand a pressure of 8000psi. They are available in lengths of 20 to 50cms and an internal diameter of 1 to 4mm. The particle size range is 5 to 30 microns but mostly lower sizes are preferred for better separation.

For reverse-phase columns (i.e stationary phase is non-polar) C18 columns are used. While normal phase columns (stationary phase is polar).

Lengthy columns are preferred for the separation of many compounds in a mixture while short columns are used to separate simple mixtures.

Guard Column→ These are small columns with similar stationary phases as that of the main column. They are used to prevent the passage of any particulate matter directly into the main column so as to prevent clogging and damage to the column.

They are placed before the entry of the mobile phase into the HPLC column as shown in the figure.

HPLC Detector→ This part of HPLC helps in the detection and identification of compounds in the sample. Based on the types they are available as UV-Visible, Fluorescent, Electrochemical, Photo diode-array detectors. Electrochemical detectors have a range to detect at a minimum of picograms while remaining are efficient at nanograms and above.

Computer and software for display and documentation of data. The detections of the detector are recorded by the software designed for the purpose. The data is used to know the type of compound and the quantity of it in the sample. The computer and software also control the injection, flow rate and detection limits of the detector.

There are many companies that manufacture the HPLC system. Of them, I could come across Waters HPLC, Shimadzu HPLC, Hitachi HPLC, Perkin Elmer HPLC, Sisil HPLC systems.

The most preferred manufacturer is Waters HPLC.

The HPLC instrumentation system & design is compact, durable, the performance of system & equipment is reliable and the engineering services are readily at hand to help.

Further, the company is dedicated only to HPLC systems and nothing else unlike other brands. But still, have your own research before purchase.