High Performance Liquid Chromatography PPT

High Performance Liquid Chromatography

1. ❖ Content • Introduction • Principle • Classification • Instrumentation. • Applications. • References

2. ❖ Introduction It Known as High pressure liquid chromatography. High performance Liquid Chromatography is a technique in analytical chemistry used to Separate, identify & quantify each component in mixture.
HPLC is column chromatography technique.
HPLC have high Resolution & seperation capacity.
It is used for Quantitative & Qualitative analysis HPLC Purify the components on the basis of polarity.

3. ❖ PRINCIPLE The principle involved in HPLC can be either adsorption or partition . ➤ Interaction of sample with both stationary phase and mobile phase. ➤ The analytes are injected into the flow of mobile phase. ➤ The reliable flow rate of the mobile phase with appropriate pressure is applied. ➤ The sample mixture is interacted between the stationary phase and mobile phase.

4. ❖ CLASSIFICATION HPLC can be broadly classified into two major types- Normal phase chromatography - stationary phase is polar (hydrophilic) and mobile phase is non-polar (hydrophobic). Reverse phase chromatography - stationary phase is non-polar (hydrophobic) and mobile phase is polar (hydrophilic). Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity than Polar- Non Polar bonds. Reverse phase chromatography is more commonly used as drugs are usually hydrophilic

5. The elution in HPLC can be classified into two categories: Isocratic Elution: In this technique the mobile phase composition is fixed constant throughout the chromatographic procedure. For example, if a method consisting of mobile phase as methanol and water in the ratio of 70:30, the same ratio is maintained for the entire chromatographic procedure in isocratic method. Gradient Elution: In this technique, the composition of mobile phase is changed either stepwise or continuously as elution proceeds. Changing of composition to bring out the desirable separation is called as gradient elution method. For example, Initially a composition (methanol water, 70:30) for some time period (10 min), is maintained then the polarity is modified by changing the ratio to (80:20) for next 5 min and then to 90:10 for another 5 min.

6. ❖ INSTRUMENTATION The instrument of a HPLC consist of the following components: Mobile phase reservoir Pumping system Sample injection systems Column Detector Data collection device or recorder.

7. ❖ MOBILE PHASE RESERVOIR Generally reservoirs made up of glass are used. One or two reservoirs of 500 ml capacity or more quantity if required are used. The quantity of the mobile phase to be used in the separation depends upon the nature of solvent, quantity of mixture to be separated, time required for the separation, particle size of the solvent type of stationary phase used and other such factors.

8. ❖ PUMPING SYSTEM The mobile phase is forced from reservoir to the column using Pumps. High pressure is required to push the mobile phase with uniform flow rate. This is achieved by the pumps. The general requirements for an HPLC pump are as follows. It should provide the desirable pressure (Generally up to 6000 psi). It should be chemically resistant to all solvents. It should provide reproducibility.

9. 1. Syringe Pumps Consists of large syringe like chamber.
Suitable for small bore column.
These pumps have a volume between 250 to 500mL

10. 2. Reciprocating – Piston Pumps 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.

11. Degassing:. It is necessary to remove the dissolved gases present in the mobile phase solvent.
Different techniques are followed for degassing which are discussed below. (a) Filters: filters are also used to remove the dust and other matters from the solvents.
Membrane filters of 45u are usually used.
The mobile phase filtered through these filters using Buchner funnel under vacuum followed by ultrasonication.

12. (b)External Vacuum Degassing:. 1. In this method the solvent in a container is kept in a ultrasonic bath.
2. Ultrasonication is done under vacuum using a vacuum pump.
3. This process will remove the dissolved gases from the solvents.

13. ❖ Sample injection system

14. ❖ COLUMNS Guard Column: Guard column is the column placed before the analytical column.
These columns also called as pre columns.
They are used to protect the analytical column from the impurities and other contaminants from solvent.
It also removes the components that bind irreversibly to the stationary phase.
The guard columns are compulsorily used during the bioanalytical studies to protect the analytical column from biological matrix.

15. Analytical Column : Analytical column is considered as the heart of an HPLC system. The reason is this is the part where the separation of the mixture takes place. The efficiency of the separation is purely depends on the column.

16. ❖DETECTORS Detectors is a device which detects the eluted components emerging from column. Ideal HPLC detectors should meet the following requirements. 1. It should respond to wide variety of components.
2. It should be compatible for the temperature and flow rate changes.
3. The sensitivity should be high.
4. It should have low dead volume.

17. UV-visible Detector UV visible detector is widely used as it detects large number of compounds because most drugs have appropriate structural characteristics for light absorption.
These are useful for aromatic compounds and other type of unsaturated systems.

18. ❖ RECORDER Recorders are used to record the responses obtained from detectors after amplification. They record the baseline & all the peaks obtained with respect to time (Rt) But the area of the individual peaks cannot be known.

19. ❖ Application ‎ Pharmaceutical application : ‎1. Tablet dissolution study of the pharmaceutical dosage form. ‎2. To control drug stability, shelf-life determination.‎ 3. Identification of active ingredient. ‎4. Pharmaceutical quality control. ‎‎ Food and Flavor applications: ‎‎1. Ensuring soft drink consistency and quality.‎ 2. Sugar analysis in fruit juices.‎ 3. Analysis of Polycyclic compounds in vegetables. ‎4. Analysis of natural compounds (sugar, fats, protein, amino acids) food additives and ‎contaminants are determined.

20. ‎‎ Application in forensics : ‎1.Quantification of drug in biological samples. ‎2.Identification of anabolic steroids in serum, urine, sweat and hair.‎ 3.Forensic analysis of textile dyes.‎ 4.Determination of cocaine and metabolites in meconium.‎‎ Application in clinical test : ‎‎ 1. Urine analysis, antibiotic analysis in blood. ‎

21. ❖ References • Books A textbook of Instrumental Method of Analysis Pee Vee Publication , Author Prof. Poonam A. Salunke , Prof. Shital S. Patil , Prof. (Dr.). S. D. Barhate Page No. :- 241- 263 • E-links • https://ijppr.humanjournals.com/wp-content/uploads/2020/10/40.S.-D.-Varhadi-V.-A.-Gaikwad-R.-R.-Sali-K.-Chambalwar-Vikas-Kandekar.pdf Chatgpt