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HPLC: High Performance Liquid Chromatography.Principles, instrumentation & application

Presenter: Dr Pranav SoporyDepartment of Pharmacology

All India Institute of Medical SciencesNew Delhi

Mob: 9999-491-690email: pranav.sopory@gmail.com

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Chromatography

• Chromatography is a separation & identification technique

• Technique: Differences in the rate at which the component(s) of a mixture

move through:

1. A porous medium (stationary phase)

2. under the influence of a solvent (mobile phase).

Liquid Chromatography

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Basic Instrumentation

PumpInjector

Column

Stationary Phase

Detector

Mobile Phase

Data Processing

• Process: movement of the compounds via the apparatus.

- Which travel along mobile phase across an immobile surface (stationary phase). –

Affinity of compound of our interest with the stationary phase based on certain chemical

properties.

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Electronegativity

• Electronegativity is a measure of the tendency of an atom to attract a

bonding pair of electrons.

• Depending on difference in electronegativity between two atoms:

0.4 – 1.7 < 0.4Polar bonds Non-polar bonds

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Polarity

Polar compounds:

• positive charge on one side • negative charge on the other• The dipoles do not cancel out

resulting in a net dipole• Since H2O is polar, these

molecules are HYDROPHILIC

Non-Polar compounds:

• The dipoles cancel out resulting in a net dipole• Non-polar compounds are:• Soluble in non-polar solvents

such as Heptane & Hexane• HYDROPHOBIC

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Mobile Phase

• Classifying phase for different types of Chromatography

1. Gas Chromatography: carrier is gas (He and N2)

2. Liquid Chromatography: carrier is liquid

• Carrier: Liquid solvent carrying all the substances we want to separate

and/or identify

• Carrier is also known as a vehicle

• The compound we want to separate/identify should dissolve in that liquid

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Stationary Phase

• Immobile surface that is particulate in nature

• This surface tests the samples affinity

• Effect of Stationary Phase: C18 is a Non-polar S.P. gradient

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Q. Why do polar molecules attract each other?

Q. Why do non-polar molecules attract each other?

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Example via Normal Phase HPLC mechanism

POLAR:Stronger interaction

NONPOLARWeaker interaction

Mobile Phase

Stationary Phase (POLAR)

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• Polar compounds travel slower & are eluted slowly due to higher affinity to St. phase

• Non-polar compounds travels faster & are eluted first due to lower affinity to St. phase.

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SeparationInjector

Detector

Column

Solvents

Mixer

Pumps

High Performance Liquid Chromatograph

Waste

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Injector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

High Performance Liquid Chromatograph

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Injector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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The Chromatogram

Injection

mAU

time

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HPLC: Qualitative Analysis

Injection

to

tR

mAU

time

tR to - elution time of non-retained substance (aka “dead time”/ “baseline disturbance”)

tR- retention time - determines sample identity

The aim of qualitative analysis is to answer the question ‘What’ is in the sample? 

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HPLC: Qualitative Analysis

1. Retention Time (tR)

• Time taken for a solute to pass through a chromatography column.

• tR is specific to a compound, but not absolute.

• Factors influencing different tR for the same compound:

1. The gas flow rate

2. Temperature differences

3. Column degradation

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tR

tR

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HPLC: Quantitative Analysis• Determine concentration.

There are two main ways to interpret a chromatogram to perform quantification.

1. Determination of the peak height of a chromatogram

2. Determination of the “Area under Peak”

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HPLC: Quantitative Analysis• Determination of Area under peak Requirements of good quantification

Good resolution

Good peak performance

Compound already quantified

Result

Use standard reference data

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Solvent Tray

Solvent Reservoir

Controller Unit

Degasser

Quaternery Pump

Autosampler with chiller unit

PDA Detector

Fluorescence detector

Column

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Pump (Solvent Delivery System)

• The role of the pump is to force a liquid (called the mobile phase) through the LC system to achieve the DESIRED FLOW RATE

• During the chromatographic experiment, a pump can deliver:

Flow Rate 1-2 mL/minPump Pressure 500 bar (1 bar = 760mmHg)

Premixed Mobile Phase 1 pump & 1 valve Isocratic Pump

2 Solvents 2 pumps & 2 valves Binary pump

4 Solvents 1 pump & 4 valves Quaternary Pump

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PumpInjector

Column

Stationary Phase

Detector

Mobile Phase

Data Processing

InjectorColumn

Stationary Phase

Detector

Isocratic Pump

Binary Pump

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Running the application

Measure Mobile Phase composition

Type

Single compound Constant polarity Isocratic Run

Multiple compounds Increasing polarity Gradient Run

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Automatic Injector

Introduces the liquid sample into the flow stream of the mobile phase.

Typical sample volumes: 20 μL.

Mechanism:

1. User loads vials containing the sample into the auto sampler tray (80 samples)

2. The auto sampler automatically

a. measures the appropriate sample volume

b. injects the sample

c. then flushes the injector to be ready for the next sample

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Basic Instrumentation

PumpInjector

Column

Stationary Phase

Detector

Mobile Phase

Data Processing

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Column: Heart of the chromatograph

• Column’s stationary phase separates the sample components

of interest using various chemical parameters.

Column Types

Normal Phase HPLC

Reverse Phase HPLC

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Classification of HPLC

Normal Phase HPLC Reverse Phase HPLC

Stationary Phase Polar (H’philic)SiO2, Al2O3

Non-polar (H’phobic)H’phobic polymer: S18

Mobile Phase Non-polar (H’phobic)Heptane, Hexane,

Cyclohexane

Polar (H’philic)Water, Methanol, Buffers

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Normal Phase HPLC (Traditional)

• Stationary Phase – Polar nature.

Eg: SiO2, Al2O3

• Mobile Phase – Non-polar nature.

Eg: Heptane, hexane and cyclohexane.

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Normal Phase HPLC Mechanism:

• Polar compounds travel slower & are eluted slowly due to higher affinity to

stationary phase.

• Non-polar compounds travels faster & are eluted earlier due to lower affinity to

stationary phase.

POLAR:Stronger interaction

NONPOLARWeaker interaction

Mobile Phase

Stationary Phase (POLAR)

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Reverse Phase HPLC• Stationary Phase – Non-polar nature.

Eg: H’phobic polymers: Silica(C18)

• Mobile Phase – Polar nature.

Eg: methanol-water or acetonitrile-water

Mechanism:

• Non-polar compounds travel slower & are eluted slowly due to higher affinity to

stationary phase.

• Polar compounds travels faster & are eluted earlier due to lower affinity to stationary

phase.

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Rule:

NP-HPLC: Components elute in increasing order of polarity.

RP-HPLC: Components elute in decreasing order of polarity. (Reverse!)

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Advantages of RP-HPLC

• Polar (H’philic) mobile phase:

1. Easier to modify

2. Therefore, more permutation and combination possible

3. Larger number of compounds can be identified 

• NP-HPLC: H’phobic mobile phase:

1. Decreased flow rate & increased retention time.

2. To modify polarity: Column must be changed !

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Degassing of solvents• When high pressure is pumped, the risk of formation of gas bubbles increases

which interferes with the separation process.

• Hence de-gassing is very important and it can be done by various ways:

1. Vacuum filtration:

De-gassing is accomplished by applying a vacuum to the solvent container.

2. Ultrasonication:

Done by using ultrasonicator which converts ultrasonic high frequency to

mechanical vibrations.

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Detectors in our Lab

Types:

1) UV-Visual Detector

1) Photo Diode Array Detector

2) Fluorescence Detector

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UV-Vis Detector

• Principles: Measures absorbance via Beer Lambert’s law

IiIt

Column

Transmitted Light (It)Absorbance: - Log (T)

Transmittance (T): Incident Light (Ii)

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UV-Vis Detector

Characteristics: Specific, Concentration, good stability,

gradient capability.

Has to be set to a desired wavelength

Detects between 190-800 nm

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Photo Diode Array Detectors• Provision to scan the entire wavelength spectrum.

• Detects from 190-800nm.

• The resulting spectra is a 3D plot of Response Vs Time Vs Wavelength.

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3D Data

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Fluorescence DetectorIt is based on the fluorescent radiation emitted by some compounds.Advantage and disadvantage• Highly selective because relatively few compounds emit fluorescence

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What is HPLC used for?

• HPLC : separation/identification of compounds that are non-volatile .

• (Volatility: easily evaporated at normal temperatures)

• NOTE: If a compound is volatile (e.g. hydrocarbon in gasoline, etc.), then gas

chromatography is a better separation technique .

• Typical non-volatile compounds are:

1. Pharmaceuticals: like aspirin, MTX and Phenytoin

2. Biochemical: Proteins, amines, glycated hemoglobin

3. Salt: like sodium chloride and potassium phosphate

4. Natural Products: Ginseng.

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In our Lab• Shimadzu HPLC system.• Chem-station software

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Uses:

Anti-Epileptic drugs Phenytoin, Phenobarbitone, Carbamazepine, Sodium Valproate

Anti-Hypertensive drugs Amlodopine, Prazocine, Metoprolol, Clonidine

Anti-Tubercular Drugs Rifampicin, Ethambutol, Pyrazinamide, Isoniazid

Anti-diabetic drugs Glibenclamide, Metformin

Other Drugs Methotrexate, Mycophenolic acid, Amphoteracin-B

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References:

• Introduction to HPLC – Agilent Technologies

• Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical Chemistry at L1 - KCL, 6th

October 2008

• HPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWARE

• http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm

• http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html

• CHROMACADEMY: CRAWFORD SCIENTIFIC

• Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando:

Harcourt Brace & Co., 1998.

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Types of Chromatography

Type Separates Mobile Phase Stationary Phase

Liquid Liquid samples Liquid Solvent Solid beads

Gas Vaporized samples Carrier Gas Solid beads

Paper Dried Liquid samples Liquid Solvent Paper Strip

Thin-layer Dried liquid samples Liquid Solvent Thin layer of Aluminia or Silica gel

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Thank You