What is high-performance liquid chromatography (HPLC)? | Principle of high-performance liquid chromatography | Detectors used in HPLC | Instrumentations and HPLC application.

Principle of high-performance liquid chromatography (HPLC)

High-performance liquid chromatography or high-pressure liquid chromatography ie. H.P.L.C. is an improvement of classical column liquid chromatography. In classical methods, the liquid mobile phase flows slowly using gravity through a relatively large diameter column packed with a stationary liquid phase, which is immobilized on the surface of solid-like alumina, get, etc. of different mesh or particle sizes. The classical method is time-consuming and tedious and is generally characterized by low column efficiency and long separation time. In HPLC this efficiency of separation is improved by using a high-pressure pump that is capable of producing pulse-free inlet pressure of 1000 – 6000 psi.

A column is a narrow-bore metal stainless steel tube containing small spherical and uniformly sized particles (5 um – 50 um) of stationary phase. Because a greater surface area of the stationary phase is exposed to sample components in the HPLC column than in traditional LSC. LLC and ion-exchange column the efficiency of HPLC is thus high resolution and high-speed liquid chromatography.

The other important condition is that a suitable immiscible solvent pair must be available. Generally, the two made the stationary phase and the other is the mobile phase

The sample was introduced into the column stationary liquid phase using the pressurized flow of the liquid mobile phase. The components in the sample are fractioned during their passage through the column. The detector system senses these components as they are eluted from the column and generates a signal proportional to the number of solutes passing through the system.

Instrumentation in high-performance liquid chromatography HPLC:

The important components are as follows:-

1. Solvent Reservoir & Degasser system.
2. High-pressure pump.
3. Pre column
4. Sample injection system
5. Column and packing column
6. Detectors
7. Recorder

A Typical HPLC Instrument

SOLVENT RESERVOIR  & DEGASSER SYSTEM in high-performance liquid chromatography

A modern HPLC apparatus is equipped with one or more glass or stainless steel reservoirs each of which contains 1 to 2 dm3 of a solvent. The reservoirs are equipped with degassers to remove trapped or dissolved gases ( ) which otherwise interfere with the analysis by forming bubbles in the column and detector system. Degassers may consist of a vacuum pumping system and a distillation system for heating and stirring the solvent.

If a single solvent of const. Composition is used for separation it is termed isocratic elution and if two solvents are used it is called a gradient elution. Modern HPLC is often equipped with proportionating valves that introduce solvents from two or more reservoirs in the mixing chamber at rates that vary continuously.

HIGH-PRESSURE PUMP

The requirement for an HPLC pump includes the.

1. The generation & pressure up to 6000 psi
2. Pulse-free output.
3. Flow rates ranging from 0.1 to 10 cm3 min- 1
4. Flow reproducibility of 0.5 % or better
5. Resistance to corrosion by a variety of solvents

Pumps are designed to operate at a const. Pressure diff. Types of pumps are a) screw-driven pumps and b) Reciprocating pumps.

PRECOLUMN in high-performance liquid chromatography

This contains packing material chemically identical to that in the analytical column, but particle size is much larger so that pressure drop across precolumn is negligible w.r.t. rest of the system. The purpose of the pre-column is to remove impurities from the solvent. This prevents contamination of the analytical column. In addition, precolumn saturates the mobile phase with liquid making up the stationary phase, and thus stripping of the stationary phase from packing of the analytical column is eliminated.

SAMPLE INJECTION SYSTEM

The sample is injected into a sample loop using the hypodermic syringe through the self-sealing septum of silicon or Teflon While the sample is injected the mobile phase at high pressure moves through the sample loop directly into the column. Then the valve is rotated sweeping the sample into the column. Sample sizes range from 5 to 500.

COLUMN AND COLUMN PACKING

Columns are typically 10 to 30 cm in length and 4 to 10 mm in diameter and are made up of stainless steel or glass.

Column Packing:- Materials used for column packing are –

Porous Bead Packing: –

Porous Solids adsorbents like silica gel and alumina are often preferred for the separation of compounds of relatively low mol. Wt. and polarity.

Pellicular Bead Packing: –

In this type, the material called Pellicular particle is used. It is made up of glass beads of a diameter of about 40 um coated with a thin layer of porous material like silica gel alumina or ion exchange resin.

Detectors used in high-performance liquid Chromatography HPLC:–

A chromatographic detector is a device, which indicates the presence and measures the number of separated components in column effluent. The choice of detector depends on the type of sample solution used. The basis of detection is UV, IR adsorption fluorescence refractive index polarography, etc.

UV Detector:-

• The most common detectors for HPLC are based on the absorption of ultraviolet or visible radiations.
• These detectors are used when the compound of interest absorbs in the UV/Visible region (170-700nm).
• Both photometers and spectrophotometers are available commercially.
• In photometers, 254-280 nm wavelength from a mercury source is used for detection because most of the organic functional groups absorb at these wavelengths.
• Spectrophotometric detectors are more versatile than photometers.
• A typical ultraviolet detector is shown in the Diagram.
• The light source is a low-pressure mercury lamp.
• The light is collimated by a quartz lens, passes through reference and sample cells, is filtered to remove unwanted radiations, and is sensed by dual photocells (Detector).

Advantages-

(i) They have a wide linear range.

(ii) An UV detector can resolve absorbance differences as small as 0.00005 absorbance units equivalent to ppb detection limits in favorable cases.

(iii) They can be used for gradient elution. (iv) They are commercially available.

Refractive index Detector:-

1. The detector that comes closest to being a universal one is a refractive index detector.
2. It works on the principle that the refractive index of solvent changes whenever a solute is present.
3. The difference in refractive index is thus measured as a function of the concentration of substance coming out of the column.

Advantages:–

• It is stable and simple to operate and the sample is not destroyed.
• It responds to the presence of all the solutes.
• It is commercially available.

Disadvantages

All Refractive index detectors are highly temperature-sensitive and are general rather than selective.

RECORDER USED IN high-performance liquid chromatography HPLC

The function of the recorder is to record the chromatogram of the sample.

TECHNIQUE

The sample is injected into the column using a hypodermic spring. Through a self-sealing Teflon septum. While passing through the column. The components in the sample are fractioned. In the detector system, these components have eluted from column generation a signal proportional to the amount of solute passing through the system. This signal is then fed to the recorder, which records the output in terms of the chromatogram.

APPLICATIONS OF high-performance liquid chromatography HPLC

• HPLC is used for the separation and analysis of Steroids, Carbohydrates, Polymers, Drugs insecticides quality control of polymers in plastics
• The main use of HPLC in the pharmaceutical and pesticide industries
• In liquid partition chromatography with HPLC tech separation of moderately polar to nonpolar substances is possible.
• A lot of work has been carried out with ion-exchange HPLC combination Morphine heroin etc. have been separated on special resins zipax – sax
• The method is particularly used for biochemical compounds eg. Lipids in fat-soluble vitamins can be separated by HPLC.
• Identification of toxic or decomposition products in the consuming products can be effectively carried out by HPLC.

Compare And Contrast GC And HPLC

From the viewpoint of speed and simplicity of the equipment, GC is preferred. For the isolation of non-volatile substances including inorganic ions and thermally unstable material, HPLC is preferable whereas GC is not. Both techniques are complementary, efficient, and highly selective needing small samples that can be handled by nondestructive testing and are applicable for qualitative analysis.

In gas chromatography, organic compounds must have an appreciable vapour pressure before this tech can be applied to their separation. This limits GC to approximately 60 % of the known organic compounds. HPLC tech, in contrast, does not have this kind of requirement and is for all practical purposes, suitable for organic molecules.

HPLC is thus most versatile in comparison to other chromatographic methods.

Advantages of HPLC Over Conventional Column Chromatography

Following are the advantages of HPLC over conventional column chromatography,

• It is fast and versatile.
• It is simple to operate and requires relatively little skill
• It has high sensitivity given good resolution needing the least quantity of samples. It affords qualitative and quantitative analysis
• The sample is not destroyed and fractions can be minutes
• The chromatograms are highly reproducible with dependable retention time
• Almost all compounds can be analyzed by this technique.

Advantages of HPLC over GC

Following are the advantages of HPLC over GC-

• The special feature of HPLC is that it can accommodate thermally unstable and non-volatile materials including ions. The main requirement is that the solute is soluble in the mobile phase. GC can be used only for volatile or thermally stable comp.
• Although GC efficiency is good in 2000 plates / m in HPLC the efficiency is even better in 5000 plates / m.
• It is possible to vary stationary phase flow rate & temp in GC. In HPLC not only those variables but also the mobile phase can be altered.
• Another advantage of HPLC is that columns are reusable stationary phase is usually stable and the mobile phase is chosen so that it will not alter the characteristics of the column.
• Even though HPLC may use high press they are not dangerous to use and there is no danger of explosion.

Limitations of high-performance liquid chromatography HPLC

• Because of the employment of the mobile liquid phase rate of diffusion and mobile phase down the column is slow and hence the rate of mass transfer bet the two phases is slow thereby limiting the speed with which a column may be operated in HPLC under anything approaching equilibrium conditions.
• Detectors available for HPLC are less sensitive than those used in GC.
• Detectors employed in HPLC are generally costly and have less applicability than those used in GC.

FAQs