HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY

HPLC Presented By -Mr. Shaise JacobFacultyNirmala College of PharmacyMuvattupuzha, KeralaIndiaEmail – jacobshaise@gmail.com

• 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.

• 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):

• partition • adsorption (liquid-solid) • ion exchange • size exclusion or gel

◊ 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

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 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 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'sPRINCIPLE 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:1. M.P reservoirs

2. Eluent degas module3. Solvent delivery pumps4. Manual / Auto injector5. Analytical column6. Detector7. Data processor

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 moduleDissolved gases in M.P pose a number of problems

∆ flow ∆ excessive detector noise ∆ Rt fluctuation ♠ Bubbling the pump & detector,

Degas module with reservoir of inert gases He or N2

1. Vacuum filtration2. Helium purging3. Ultrasonication (converts ultra high

frequency to mechanical vibrations.)SOLVENT DELIVERY PUMPS

Reciprocating pumps:» widely used» maintain accurate flow rate

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

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 Pre-filter :- useful for industry

Analytical columnHeart 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: 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: C18 (octa decyl silane) Bondapak ( waters) C8 octyl column, C4 butyl column, CN Nitrile column NH2 Amino column

b

Column packing

three forms1.Microporous support5-10 µm in d.m

2. PellicularPorous & 40 µm in d.m

3.Bonded phaseS.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 destructive190-600 nm for quantization & identificationSpectra is 3D, Response vs time vs WL

Photodiode Array Detector

Flourimetric detector Excitation & emission WL can be selected ↑ sensitive than UV Disadvantage: Some comp. are not fluorescent

Conductivity detectorbased on electrical conductivity

Amperometric detectorReduction / 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 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 analysisChecking the purity of a compound

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 heightCalibration curve method

Multi component analysis Isolation & identification of drugs Stability studies