Basics of - HPLC

- Kaumil Bhavsar Date -21/08/07

Index

What is HPLC ? Applications Types of HPLC HPLC Instrumentation Solvent system Pumping system Sample injector system Column Detectors P9 RP-HPLC salient features

What is HPLC ? Originally referred to as

High Pressure Liquid Chromatography Now more commonly called

High Performance Liquid Chromatography

HPLC is an analytical technique used to separate component of mixture by using variety of chemical interaction between the analyte and the chromatography column.

Why HPLC? Automated version of LC Provides enhanced separations in short time duration. It’s a most practiced quantitative, chromatographic technique which

can separates wide range of molecule types and sizes.

Applications of HPLC Pharmaceutical / Biopharmaceutical Pharmaceutical quality control. Shelf-life determinations of pharmaceutical products. Identification of counterfeit drug products. Complex molecules separation.

Environmental Biomonitoring of pollutents. Water monitoring - Phenol content and toxic componants checking.

Clinical Analysis of antibiotics and blood substances. Detection of endogenous neuropeptides in brain extracellular fluids.

Food and Flavor Sugar analysis in fruit juices. Ensuring soft drink consistency and quality.

Types of HPLC Normal Phase: Separation of polar analytes by partitioning onto a

polar, bonded stationary phase using non polar mobile phase.

Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase using polar mobile phase.

Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)

Ion Exchange Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support.

Size Exclusion Chromatography: Also called as Gel filtration chromatography. Separation of molecules based on their size.

Selection of Chromatography

Separation based on selection of column material

Separation based on selection of column material

HPLC instrumentation

HPLC components

1) Solvent system

2) Pumping system

3) Sample injection system

4) Column

5) Detector

DetectorMobile phase Pump Injection Valve Separation column=> => => =>

Solvent system Mobile phase reservoirs: Several reservoirs – Use to carry mobile

phase Degassing: Remove dissolved gas - interfering detection Protect band spreading Sparging: fine bubble of gas (He) Vacuum pumping. Dust removal: Dust interference with detection, column clogging,

damage pumping system Millipore filter under vacuum Isocratic elution: constant composition Gradient elution: different solvent systems during elution, continuous

change or step wise, solvent proportion valve

Pumping system

Requirements for HPLC pump.

High pressure (up to 6000 psi)

• pulse free, prevents remixing of solutes

• control flow rate from 0.1 to 10 mL/min

Types of HPLC pumps

1) Reciprocating pumps: Most commonly used.

Disadvantage: pulses from single piston.

2) Syringe pumps: Generates pulse free output.

Disadvantage: Limited mobile phase capasity.

Reciprocating pump : Single piston pump

Reciprocating pump : Double piston pump

Syringe pump

Sample injection system Sample Injection system: Limit of precision of HPLC Sample size: 0.5 ~ 500 μL No interference with the

pressure Sample loop, 1 ~ 100 μL, Auto sampler: inject

continuously Volume uptake: 1 μL – 1 mL Controlled temperature

environment

Sample loading & Injection.

Column Made of Stainless steel tubing for high pressure. Analytical column: straight, L(5 ~ 25 cm), d (3 ~ 5

mm), dp(3~5 μm). N (40 k ~ 70 k plates/m) Microcolumn: L (3 ~ 7.5 cm), d (1 ~ 5 mm), dp: (3 ~

5 μm) , N: ~100k plates/m, high speed and minimum solvent consumption

Guard column: protects analytical column, similar packing, remove particulate matter and contamination.

Temperature control: 0.1 °C - 150 °C. Column packing: silica, alumina, a polystyrene-

divinylbenzene, synthetic or an ion-exchange resin Pellicular particle: original, spherical,nonporous

beads,proteins and large biomolecules separation (dp: 5 μm)

Porous particle: commonly used, dp: 3 ~ 10 μm. Narrow size distribution, porous microparticle coated with thin organic films.

Detectors Ideal Detector Properties: High Sensitivity Universality or predictable specificity Large linear response range Low dead volume Non-Destructive Insensitive to temperature & mobile phase Continuous operation Reliable and easy to use No single detector fits all these criteria

Types of HPLC detectors

UV-Vis Works w/all molecules Non-specific; complex samples; absorption wavelength

DAD Works for all wavelengths High LOD

Fluorescence Very specific; low LOD Not everything fluoresces

IR Works w/all molecules Many solvents IR active

Refractive Index Works w/nearly all molecules

Temperature sensitive; high LOD

Scattering Uniform response; 5ng/25L LOD

Non-specific; interference from solvent

Electrochemical Commercially available Non-specific; high LOD

Mass Spec Low LOD; analyte identification

Ability to ionize analyte

Name Advantage Disadvantage

UV-VIS detector. Single Beam UV-VIS instrument with a flow-

through flow cell (cuvette) Usually typical UV-VIS lamps used having

254 nm default wavelenth Can be set to other wavelengths (most) Simple filter detectors no longer widely

used Non-destructive, not-universal not all compounds absorb light can pass sample through several cells at

several different wavelenghts Usually zeroed at the start of each run

using an electronic software command. You can have real-time zeroing with a reference cell.

Diode array detector (DAD)

Refractive Index Detector.

ELSD (Evaporative Light Scattering Detector)

P9 RP- HPLC Salient features. Reverse phase HPLC. 15 min method, RT- Between 4 to 6 min. Mobile phase : A - Water + TFA

B - ACN + TFA Column: Brand Name - Discovery Type of Column:Reverse phase C18 column Specification of ColumnDiameter: 4.6mm Length: 50mm Porosity:

300•A, Particle size: 5 Micron Guard column Brand Name - Zorbex 300 SB (Agilent) Type of Column:Reverse phase Zorbax 300 SB-C18 Specification of ColumnDiameter: 4.6mm, Length: 35 mmPorosity:

300•A, Particle size: 5 Micron

Typical P9 RP-HPLC peak (Agilent-1200).

min0 2 4 6 8 10 12 14

mAU

-100

0

100

200

300

400

500

*VWD1 A, Wavelength=214 nm (10APRIL07\10HP250407 P9STD119\10HP250407B.D - 10APRIL07\10HP250407 P9STD119\B250407A.D)

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