Chemistry with wiley

Simplified knowledge

HPLC block diagram
Spread the love

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 by means of 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 particles size. 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 size particles (5 um – 50 um) of stationary phase. Because a greater surface area of stationary phase is exposed to sample components in 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 is made the stationary phase and the other is the mobile phase

The sample was introduced into the column stationary liquid phase by means of 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 amount of solutes passing through the system.

INSTRUMENTATION: –

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 unit is shown in fig.

Instrument of High Performance liquid chromatography
HPLC unit

SOLVENT RESERVOIR  & DEGASSER SYSTEM

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 in 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 the two or more reservoirs in the mixing chamber at rates that vary continuously.

HIGH-PRESSURE PUMP

The requirement for an HPLC pump include.

  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 solvent

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

PRECOLUMN

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 stationary phase from packing of the analytical column is eliminated.

SAMPLE INJECTION SYSTEM

The sample is injected into a sample loop by means of 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 HPLC:

A chromatographic detector is a device, which indicates the presence and measures the amount of separated components in column effluent. The choice of the 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, 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, 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 HPLC

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

TECHNIQUE

The sample is injected into the column by means of 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 amt 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 HPLC

  • HPLC is used for the separation and analysis of Steroids, Carbohydrates, Polymers, and Drugs insecticides quality control of polymer in plastics
  • The main use of HPLC in the pharmaceutical and pesticides 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 isolation of non-volatile substances including inorganic ions and thermally unstable material, HPLC is preferable whereas GC is not. Both the techniques as a matter of fact are complementary, efficient, and highly selective needing small ample which can be handled by nondestructive testing and applicable for qualitative analysis.

In gas chromatography, org 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 and in comparison to another chromatographic method.

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 in 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 they 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 HPLC

  • Because of the employment of 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 condition.
  • 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

1 What is full of HPLC? or HPLC is an abbreviation for?

HPLC is an abbreviation for High-Performance Liquid Chromatography.

2. What is Chromatography?

Chromatography is a separation technique whereby the components of a mixture may be separated by allowing the sample or analyte to pass over the material called a stationary phase by a moving phase called a mobile phase.  If the individual components of the mixture move at different rates then the separation will occur and the degree of separation the difference in migration.

3. What is the HPLC stationary phase?

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

4. What is the mobile phase in HPLC?

In HPLC the mobile phase is the solvent that moves through the column.


Spread the love

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

  1. Hey! I know this is kinda off topic but I was wondering which blog platform are you using for this website? I’m getting sick and tired of WordPress because I’ve had issues with hackers and I’m looking at alternatives for another platform. I would be great if you could point me in the direction of a good platform.

  2. Utterly composed content material, thank you for entropy. “He who establishes his argument by noise and command shows that his reason is weak.” by Michel de Montaigne.

  3. Great post. I was checking constantly this weblog and I am inspired! Extremely helpful info particularly the remaining phase 🙂 I take care of such information much. I used to be seeking this particular info for a very lengthy time. Thank you and good luck.

  4. My spouse and i ended up being quite comfortable Edward managed to complete his research because of the ideas he got in your site. It’s not at all simplistic to simply find yourself freely giving information which other people have been trying to sell. So we discover we have got the blog owner to give thanks to because of that. The main illustrations you’ve made, the easy web site menu, the relationships you aid to instill – it is all excellent, and it’s really assisting our son and the family believe that this concept is thrilling, and that’s extremely important. Thank you for all!

Leave a Reply

Your email address will not be published.