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.
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
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.
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.
- The role of the pump is to force a liquid ( called the mobile phase) through the liquid chromatography at a specific flow rate, expressed in milliliters per min (mL/min).
- Normal flow rates in HPLC are in the 1-2mL/min range.
- During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).
- Best for the analysis of complex samples.
Mainly three types
- Constant flow reciprocating pump
- Syringe type pump
- Pneumatic pump
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.
- Mixing unit is used to mix solvents in different proportions and pass through the column.
- There are two types of mixing units:
- They are low pressure mixing chamber which uses helium for degassing solvents.
- high pressure mixing chamber does not require helium for degassing solvents
- Mixing of solvent is done either with a static mixer which is packed with beads or a dynamic mixer which uses magnetic stirrer and operates under high pressure.
- Several gases are soluble in organic solvents.
- When solvents are pumped under high pressure, gas bubbles are formed which will interfere with the separation process, steady baseline and the shape of the peak.
- Hence degassing is necessary.
This can be done by using following techniques:
- Vacuum filtration: which can remove air bubbles, but it is not always reliable and complete.
- Helium purging: By passing helium through the solvent. This is very effective but expensive.
- Ultrasonification: By using Ultrasonicator, which converts ultra high frequency to mechanical vibrations. This causes the removal of air bubbles.
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.
- The injector serves to introduce the liquid sample into the flow stear the mobile phase.
- Typical sample volumes are 5-20 microliters.
- The injector must also be able to withstand the high pressure of the liquid system.
- An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical.
Types of injectors
- Septum injectors: For injecting the sample through a rubber septum. This is not common, since the septum has to withstand high pressure.
- Stop flow: In which the flow of mobile phase is stopped for a while and the sample is injected through a valve device.
- Rheodyne injector: It is the most popular injector. This has a fixed volume loop like 20µL or 50µL or more. Injector has 2 modes, i.e., load position when the sample is loaded in the loop and inject mode when the sample is injected.
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)
- It is the heart of the chromatograph.
- Column length: varies from 5cm to 30cm
- Column diameter: ranges from 2mm to 50mm
- Particle size: from 1µ to 20µ
- Particle nature: spherical, uniform sized, porous materials are used.
Materials of construction for the tubing:
- Stainless steel ( the most popular, gives high pressure capabilities)
- Glass (mostly for biomolecules)
- PEEK (poly ether ether ketone) polymer (biocompatibility and chemically inert to most solvents)
- The packing material is prepared from SILICA particle, ALUMINA particle and ion exchange RESIN.
- Porous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material.
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.
- UV/VIS and PDA detector
- Mass spectrometry(MS)
- Fluorescence detector
- Refractive index detector
- Electrochemical detector
- Infrared detector
- Light scattering detector
- Flourimetric detector
- Conductivity detector
- Amperometric detector
- Separation of volatile and non-volatile components
- Quick analysis
- High resolution
- More reproducibility
- High cost
- Complex to operate
APPLICATIONS OF HPLC
Checking the impurity of a compound
Presence of impurities
Isolation and identification of drugs
Isolation and identification of mixture of components
Biopharmaceutical and pharmacokinetic studies