hplc instrumentation

An HPLC instrumentation can be a set of individual modules or elements, but it can bedesigned as a single apparatus as well. The module concept is more flexible in the caseof the failure of a single component; moreover, the individual parts need not be fromthe same manufacturer. If you do not like to do minor repairs by yourself you willprefer a compact instrument. This, however, does not need less bench space than amodular set.

An HPLC instrument has at least the elements in figure 1.3 : solvent reservoir, transfer line with frit, high-pressure pump, sample injection device,column, detector, and data acquisition, usually together with data evaluation.

hplc instrumentation

Although the column is the most important part, it is usually the smallest one. For temperature-controlled separations it is enclosed in a thermostat. It is quite common to work with more than one solvent, thus a mixer and controller are needed. If the data acquisition is done by a computer it can also be used for the control of the whole system. Hplc instrumentation is typically made up of nine basic components: mobile phase/solvent reservoir, solvent delivery system, sample introduction device, column, post-column apparatus, detector, data collection and output system, post-detector eluent processing, and connective tubing and fittings

Hplc instrumentation consist of hardware and software components

HPLC Solvent Reservoirs

It fulfills the storage of HPLC solvents in sufficient quantity for uninterrupted operation of the system and it delivered to the pump by Teflon tube through the online degassing and filters. Generally, there are at least two reservoirs in a system, which are usually glass bottles. The reservoir and its attachment would be made of glass, Teflon, or stainless steel material so that the mobile phase is not contaminated. Generally, as a mobile phase, we are using HPLC grade methanol, acetonitrile, water, and buffer.
HPLC Instrumentation

HPLC Pump

As the name, is required to be generated the pressure with reproducible and the constant flow of mobile phase through the system. The HPLC pump would be capable to take solvent with single or multiple reservoirs with pulse-free output at various flow rates to permit the controlled mixing of different solvents/mobile phases from multiple reservoirs. The reciprocating pump, syringe pump, and pneumatic pump are the types of HPLC pumps.

Mainly three types

1 Constant flow reciprocating pump
  • The term reciprocating describes any continuously repeated backwards and forwards motion.
  • Widely used type of pump.
  • Solvent is sucked during back stroke and gets deliver to the column in forward stroke.
2 Syringe type pump
  • Consists of large syringe like chamber.
  • Suitable for small bore column.
3 Pneumatic pump
  • Gas is used to pressurize the mobile phase present in a collapsible solvent container.
HPLC Instrumentation

HPLC Mixing Unit

  • They are low pressure mixing chamber which uses helium for degassing solvents.
  • High pressure mixing chamber does not require helium for degassing solvent.
HPLC Instrumentation

HPLC Solvent degassing

This can be done by using following techniques:

HPLC Instrumentation

HPLC Injector

The introduction of a sample in HPLC is a technique to inject the samples without interrupting the pressure and flow rate of the HPLC system. HPLC’s work pressure is sufficiently high so that we cannot introduce a sample in the mobile phase by injecting the syringe, that’s why we require an injector that gives reproducible results and without disturbing the system pressure and flow rate. Nowadays, modern injectors are autosamplers, that permit programmed injection of various volumes of sample, which takes from the vials in autosampler trays. Rheodyne injector, septum injector, and stop flow injector are the types of the HPLC injector.

Types of injector

HPLC Instrumentation

HPLC Columns

The Column is the heart of the HPLC system, it actually generates a separation of molecules in the sample mixture. A column is situated after the injector where the mobile phase is in contact with the stationary phase, creating an interface with the enormous surface. The development of most chromatography in recent years has led to the design of many different approaches to increase this interfacial contact. Typically HPLC columns are available 30 to 250 mm long, 01 to 05 mm in diameter, and 03, 05 and 10 microns in the pore size. The column is filled with porous particles, which is made of polymer and bounded by a thin layer of silica and polystyrene (e.g. C8, C18 column)
Materials of construction for the tubing:
Packing material:
HPLC Instrumentation

HPLC Detectors

A detector is a tool used to detect the sample and column effluent, which is eluted from the column. The detector converts data into an electrical signal and is recorded by the system. The most common detector, UV/VIS is to be used in pharmaceutical analysis, It allows continuous monitoring of UV absorption over a particular wavelength or a range wavelength (using a PDA detector). The presence of components in the detector flow cell causes the change of absorbance.

Here are some types of HPLC detectors mentioned.

HPLC Instrumentation

HPLC Applications

HPLC Instrumentation