HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY

HPLC

Presented By -

Mr. Shaise Jacob

Faculty

Nirmala College of Pharmacy

Muvattupuzha, Kerala

India

Email – 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 module

3. Solvent delivery pumps

4. Manual / Auto injector

5. Analytical column

6. Detector

7. 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 filtration

2. Helium purging

3. 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 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: 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 forms

1.Microporous support5-10 µm in d.m

2. PellicularPorous & 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

Photodiode Array 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

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

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