High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is a process used for the seperation of the mixture considered in the Pharmaceutical Analysis subject.

1. High Performance  Liquid Chromatography  ( HPLC ) Prepared by- Sagar Verma (170823101099) 7 th Sem B Pharm Parul Institute of Pharmacy And Research Parul University, Vadodara

2. HPLC is the most widely used separations technique in analysis. It utilizes a liquid mobile phase to separate components of mixture It is currently the popular type of chromatography and is even replacing GC in its more traditional applications. Uses high pressure to push solvent through the column. Introduction

3. It is popular because: It is sensitive. Ready adaptability to accurate quantitative determination. Suitable for separating nonvolatile species and thermally fragile substances. Applicable to substances that are of prime interest to industry, to every fields of science, and to the public.

4. Higher resolution and good speed of analysis. HPLC columns can be reused without repacking or regeneration. Greater reproducibility due to close control of the parameters affecting the efficiency of separation. Easy automation of instrument operation and data analysis. Adaptability to large-scale, preparative procedures. Advantages

5. Components of HPLC- • Mobile phase reservoir • HPLC Pump(s) • Sample injector (manual or auto) • Column • Detector • Data system • Mixing valves • Mobile phase waste container Instrumentation of HPLC

6. Components of HPLC

7. Schematic Diagram of HPLC

8. A carrier for the sample solution. Interactions of substance with the mobile phase and stationary phase are bases for separation process. Mobile Phase Interaction of substance with mobile phase Interaction of substance with Stationary phase Elution Retention Time Strong Less Faster Less Less Strong Slower More

9. Mobile phase must dissolve the sample It should have a strong solvent strength Interaction with solute should lead to selectivity for separation of components It should not be hazardous Properties of the Mobile Phase

10. Made from Glass/stainless steel Capacity : 200 to 1000 mL May be equipped with degassers which removes dissolved gases usually O2 and N2 that interfere by forming bubbles in the columns and detector systems . Bubbles cause -band spreading & interfere with the performance of the detector . Degassers and filters may not be integral part of HPLC system. Mobile Phase Reservoir

11. Tubing carries mobile phase from one to another component of HPLC. They should be inert, have ability to withstand pressure and be able to carry sufficient volume. Generally stainless steel pipes are used. Disadvantage of stainless steel pipes: Bore of the tube get collapsed when bending is done. Easily blocked by small particles. Difficult to cut effectively. Tubing

12. Requirements for an HPLC pumping system: Generation of required pressures Pulse free output Flow rate ranging from 0.1 to 10 ml/min Corrosion – resistant components (seals of stainless steel or Teflon); High pressure generated by pumps should not lead to an explosion hazard Pumping system

13. Typical types of HPLC pumps: Reciprocating pumps (used for 90% HPLC instrumentation ) Syringe or Displacement pumps Pneumatic pumps Pumping system

14. Consist of small chamber in which solvent is pumped by back and forth motion of motor-driven piston. Reciprocating Pumps

15. Advantages: Small internal volume High output pressures (up to 10,000 psi ) Ready adaptability to gradient elution Constant flow rates Large solvent capacities Disadvantage: Pulsed flow creates baseline noise Reciprocating Pumps

16. Consist of large syringe like chambers equipped with a plunger activated by a screw-driven mechanism powered by a stepping motor. Displacement pumps also produce a flow that tends to be independent of viscosity and back pressure Output is pulse free. Disadvantage: Limited solvent capacity Considerable inconvenience when solvents must be changed. No gradient elution Displacement Pump (Syringe Type pump)

17. Mobile phase is present in a collapsible container housed in a vessel which can be pressurized by a compressed gas. Here the pressure from a gas cylinder delivered through a large piston. Advantages: • Pulse free flow • Cheap Pneumatic Pump (constant pressure pumps)

18. Disadvantages: • Limited capacity • Pressure output and flow rate depend on solvent viscosity • No gradient elution • Pressure output is < 2000 psi.

19. For injecting the sample through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small: 1-500 L Sample Injection Systems

20. Used to remove the particulate matter & contaminants from the solvent. Removes sample components that irreversibly binds to the stationary phase. Packing chemically identical to analytical column. Particle size is large, so no pressure drop. When become contaminated, it is replaced with new one. Sacrificed to protect more expensive analytical column & improves column life Pre-column (Guard column)

21. Pre-column

22. Column is the heart of HPLC separation. Columns are constructed of heavy-wall; glass-lined metal tubing (restricted to pressure <600 psi) or stainless steel tubing. Withstand high pressures and the chemical action of the mobile phase. The columns are usually 10-30 cm long, 4-10 mm inside diameter and particle size is 5 μ. Contain 40000 to 60000 plates /meter. Recently high speed, high performance column with i.d. range from 1-4.6 mm and packed with 3 or 5 μ, length is 3 -7.5 cm. Contain 100000 plates/meter. Analytical column

23. Characteristic of the ideal detector- Adequate sensitivity. Good stability and reproducibility Not sensitive to change in temperature and mobile phase composition. Should have minimal internal volume in order to reduce zone broadening. Nondestructive of sample. Wide linear dynamic range Short response time More Properties of Detector High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of analytes Detectors

24. Types of Detectors- Two types : Bulk property detectors : Respond to a mobile phase bulk property, which is modulated by presence of solutes. e.g ., refractive index, dielectric constant or density Solute property detectors : Respond to some property of solutes Like UV absorbance, fluorescence or diffusion current that is not possessed by the mobile phase . Detectors

25. Can be used to isolate and purify compounds for further use. Can be used to identify the presence of specific compounds in a sample. Can be used to determine the concentration of a specific compound in a sample . Can be used to perform chemical separations Useful in determination of enantiomers Useful in determination of Biomolecules Isolation and purification of biologically active natural products Control of synthetic reactions - Identification of intermediates and target compound . Biosynthesis study - Detection of biogenetic intermediates and enzymes involved . Control the microbiological process Used for separation of antibiotic from broth mixture HPLC Applications

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