HPLC ppt

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Slide 2:

Liquid chromatography is a separation technique that involves: the placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase) where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.

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The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount Note:The modern form of liquid chromatography is now referred to as “flash chromatography”

Four types of high performance liquid chromatography (HPLC): :

Four types of high performance liquid chromatography (HPLC): partition adsorption (liquid-solid) ion exchange size exclusion or gel

◊ improved performance:

◊ improved performance ◊ high pressure HPLC- Separation is accomplished by partitioning b/w a M.P & Stationary column material. Packing material small, uniform particle gives high column efficiencies High pressure to achieved desired flow rates

Types of HPLC techniques:

Types of HPLC techniques Based on Modes of chromatography 1. Normal phase mode: S.P is polar M.P is non polar 2. Reverse phase mode: S.P is non polar M.P is polar Different columns used: ODS,C18,C8,C4…

Based on principle of separation:

Based on principle of separation 1. Adsorption chromatography 2. Ion exchange “ 3. Ion pair “ 4. Gel permeation / Size exclusion “ 5. Affinity “ 6. Chiral phase “ Based on elution technique Isocratic separation Gradient separation

Based on scale of operation:

Based on scale of operation Analytical HPLC Preparative HPLC Based on the type of Analysis Qualitative analysis Quantitative analysis HPLC offers numerous advantages ♠ Capable of handling “macromolecules” ♠ Suitable for pharmaceutical compounds ♠ Efficient analysis of liable natural products ♠ Reliable handling of inorganic & ionic species

♠ Dependable analysis of biochemical's:

♠ Dependable analysis of biochemical's PRINCIPLE Adsorption Particle size of the S.P material plays a crucial role in HPLC Micro particulate column packing : Silica particles → uniform, porous, with spherical or irregular shape Diameter → 3.5 to 10 µm

HPLC instrumentation comprises::

HPLC instrumentation comprises: M.P reservoirs Eluent degas module Solvent delivery pumps Manual / Auto injector Analytical column Detector Data processor

Mobile phase reservoir:

Mobile phase reservoir stores M.P (HPLC grade solvents) ♠ Resolution & Speed of analysis } Flow rate, polarity & pH of M.P Can't use metallic reservoir Eluent degas module Dissolved gases in M.P pose a number of problems ∆ flow ∆ excessive detector noise ∆ Rt fluctuation ♠ Bubbling the pump & detector,

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Degas module with reservoir of inert gases He or N 2 Vacuum filtration Helium purging Ultrasonication (converts ultra high frequency to mechanical vibrations.) SOLVENT DELIVERY PUMPS Reciprocating pumps: » widely used » maintain accurate flow rate

Slide 19:

Cross-sectional diagram of a simple single – piston reciprocating pump

Solvent delivery systems:

Solvent delivery systems two types: 1. Isocratic system 2. Low pressure gradient 3. High pressure gradient Injection system a. Syringe system:- results best b. Injection valve :- [Rheodyne injector] » Loading through the sample loop (20-50µl)


u c. Automated injection device :- commercially available, automatically inject 100samples

Guard column:

Guard column Pre-filter :- useful for industry

Analytical column:

Analytical column Heart of any chromatographic system » Actual separation of components takes place Several S.P available depending upon tech. or mode of separation Column material S.S, glass, polyethylene, PEEK Column length Column diameter Particle size 5-30 cm 2mm-50mm 1µ-20µ

Particle nature::

Particle nature: Spherical, uniform sized porous material Surface area 1g S.P provides surface area 100-860 sq.m Functional group Depends on the type of chromatographic separations. Normal phase mode: hydroxy group Reverse phase mode: C 18 (octa decyl silane) Bondapak ( waters) C 8 octyl column, C 4 butyl column, CN Nitrile column NH 2 Amino column



Column packing :

Column packing three forms 1.Microporous support 5-10 µm in d.m 2. Pellicular Porous & 40 µm in d.m 3.Bonded phase S.P bonded onto an inert support


DETECTORS 1. UV DETECTOR : Based on UV light ab. > fixed WL detector (254nm) > variable WL detector (190-600nm) 2. REFRACTIVE INDEX DETECTOR : Non specific / Universal detector ↓ sensitivity & specificity 3. PHOTODIODE ARRAY DETECTOR (PDA) similar to UV detector, non destructive 190-600 nm for quantization & identification Spectra is 3D, Response vs time vs WL

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Photodiode Array Detector

Flourimetric detector:

Flourimetric detector Excitation & emission WL can be selected ↑ sensitive than UV Disadvantage: Some comp. are not fluorescent Conductivity detector based on electrical conductivity Amperometric detector Reduction / oxidation


RECORDERS & INTEGRATORS Recorders – to record the responses Integrators - data processing capabilities ◊ record individual peaks with Rt, height, width of peaks, peak area, % of area.. Selection of solvent systems Solvent compatibility with the detector e.g.. Hexane, chloroform, ACN , Methanol… Most widely used solvent in HPLC is water Millipore Milli-Q apparatus produce water

Selection of Column:

Selection of Column Non polar & moderately polar comp. → ADSORPTION CHRO. Highly polar molecules by → R.P Chro. Acids & Bases by → Ion exchange Chro.


APPICATIONS OF HPLC ♥ Pharmaceutical field ♥ Chemical & Petrochemical industry ♥ Forensic ♥ Biochemical separations ♥ Food analysis Qualitative analysis Checking the purity of a compound

Quantitative Analysis:

Quantitative Analysis Direct comparison method injecting the sample & std. separately & comparing their peak areas. Area of the peak = peak height x width of peak at half height Calibration curve method Multi component analysis Isolation & identification of drugs Stability studies