Chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition.
This involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions.
This difference in rates cause the separation of variuos components. Chromatographic separations can be carried out using a variety of stationary phases, including immobilized silica on glass plates (thin-layer chromatography), volatile gases (gas chromatography), paper (paper chromatography) and liquids (liquid chromatography).
High perfomance Liquid Chromatography
High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.
All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.
Types of HPLC
There are following variants of HPLC, depending upon the phase system (stationary) in the process :
1. Normal Phase HPLC
This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these.
Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.
2. Reverse Phase HPLC
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.
3. Size-exclusion HPLC
The column is filled with material having precisely controlled pore sizes, and the particles are separated according to its their molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later.
4. Ion-Exchange HPLC
The stationary phase has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples.
The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.
Instrumentation of HPLC
As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump, injector, column, detector and integrator or acquisition and display system. The heart of the system is the column where separation occurs.
1. Solvent Resorvoir
Mobile phase contents are contained in a glass resorvoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample.
A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column and detecter. Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated.
3. Sample Injector
The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).
Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm.
Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the column should be kept constant during an analysis.
The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.
6. Data Collection Devices
Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.
Applications of HPLC
The information that can be obtained by HPLC includes resolution, identification and quantification of a compound. It also aids in chemical separation and purification. The other applications of HPLC include :
- To control drug stability.
- Tablet dissolution study of pharmaceutical dosages form.
- Pharmaceutical quality control.
- Detection of phenolic compounds in drinking water.
- Bio-monitoring of pollutants.
Applications in Forensics
- Quantification of drugs in biological samples.
- Identification of steroids in blood, urine etc.
- Forensic analysis of textile dyes.
- Determination of cocaine and other drugs of abuse in blood, urine etc.
Food and Flavour
- Measurement of Quality of soft drinks and water.
- Sugar analysis in fruit juices.
- Analysis of polycyclic compounds in vegetables.
- Preservative analysis.
Applications in Clinical Tests
- Urine analysis, antibiotics analysis in blood.
- Analysis of bilirubin, biliverdin in hepatic disorders.
- Detection of endogenous Neuropeptides in extracellular fluid of brain etc.
Frequently asked Questions
Q 1. What is the HPLC principle?
The principle of HPLC is based on analyte distribution between the mobile and stationary phases.
It is important to keep in mind that the sample’s different constituents elute at different times before the sample ingredients’ separation is achieved.
The intermolecular interactions between molecules of the sample and packaging materials determine their time on-column.
Q 2. What are the types of HPLC?
There are four primary types of HPLC –
1. Normal phase HPLC (effective method for separating phospholipid classes)
2. Reverse phase HPLC (the most common method used to separate compounds that have hydrophobic moieties)
3. Size-exclusion HPLC/molecular sieve chromatography (Used in large molecules/macromolecular complexes such as industrial polymers and proteins)
4. Ion-exchange HPLC (separates ions and polar molecules according to their ion exchanger.
Q 3. What are the 4 types of chromatography?
The four types of chromatography are
1. Liquid chromatography (test for pollution in water samples like lakes and rivers)
2. Gas chromatography (detect bombs and useful in forensic investigations)
3. Thin-layer chromatography (used to check the purity of organic compounds such as the presence of insecticide or pesticide in foods)
4. Paper chromatography (uses a strip of paper in the stationary phase).
Q 4. Why is high pressure needed in HPLC?
HPLC uses a moderate to high pressure to achieve the desired flow rate of the solvent through the chromatographic column as small particles have greater resistance to flow.
Q 5. What is the difference between isocratic and gradient?
Your application can be run in different ways – isocratic and gradient.
Isocratic is when the mixture of the mobile phase is consistent over the total testing time. with a gradient, the compounding of the eluent mixture is changed during measurement, which greatly affects analyte retention. It can accelerate or decelerate the separation process.
Q 6. What solvent is used in HPLC?
There are different solvents used in HPLC such as aqueous solvent (water) and organic solvent (methanol, acetonitrile, and propanol). To improve the chromatographic peak shape, acids can be used such as acetic acid, formic acid, trifluoroacetic acid.
Q 7. What is the difference between UV and PDA detectors?
The PDA and UV are both absorbance detectors, which provide sensitivity for light-absorbing compounds. The UV detector is most commonly used for HPLC analysis.
The UV absorbance differs on the wavelength used, which is why it is important to choose the right wavelength based on the type of analyte.
On the other hand, the PDA detector adds a third dimension wavelength, which is a more convenient way of finding out the wavelength without having the need to repeat the analysis.
Q 8. What are the advantages of HPLC?
The advantages of HPLC are as follows:
– It can test both raw materials and finished products.
– It can reverse engineer formulations.
– It is helpful in solving product failure problems.
– It can detect contaminants and other impurities.
– It can perform competitor product analysis.
– It can determine product stability and shelf life.
– The testing can be done even with just a small sample size.
– It enables you to modify the testing depending on the needed quantification level.
– The results it produced are reliable.
– It is helpful in developing better products.
– It lets you gain a better understanding of the competitor’s products.
Q 9. What is Rf value?
In chromatography, the RF value pertains to the distance a particular component traveled divided by the distance traveled by the solvent front.
In other words, it is the characteristic of the component, which is helpful in the identification of the components.
Q 10. What are the two main types of chromatography?
There are different types of chromatography but the two primary types are liquid chromatography and gas chromatography.