Liquid Chromatography

OUTLINE
1. 2. 3.

4.

5.

History Scope of HPLC Column Efficiency 1. Effect of Particle Size of Packing 2. Extra column Band broadening LC Instrumentation 1. Mobile Phase Reservoirs and Solvent Treatment System 2. Pumping System 3. Sample Injection 4. Columns 5. Detectors Application of LC

1. History

SEPARATION TIMES LASTED FOR SEVERAL HOURS Attempt of applying vacuum failed because it decreased efficiency DEVELOPMENT Scientists discovered increase of efficiency=decrease of particle size packings (1960) Packings with diameter 3 to 10 um was developed, which required high pressure. HPLC (High performance liquid chromatography) HPLC used to distinguish newer procedures from original gravity flow method

ADVANTAGE

DISADVANTAGE

LC can separate any compound that is soluble in a liquid phase. Liquid mobile phase allows LC to be used at lower temperatures than required by GC LC is more flexible in optimizing separations Most LC detectors are non-destructive

LC is subject to greater peak or bandbroadening

2. Scope of HPLC

WIDELY USED IN ALL OF ANALYTICAL SEPARATION TECHNIQUES

Sensitivity Adaptability to accurate quantitative determination Ease of automation Suitability for separating nonvolatile species or thermally fragile Applicability to substances important to industry in science

Scope diagram
MOBILE PHASE LIQUID

FORMAT

Liquid-Liquid Chromatography (Partition)

Liquid-Solid Chromatography (Adsorption)

STATIONARY PHASE

Liquid

Solid

Normal Phase Mobile Phase Nonpolar Stationary phase Polar

Reverse Phase Mobile Phase Polar Stationary phase Nonpolar

Normal Phase

Reverse Phase

Stationary Phases

Polar

(Normal Phase): (Reversed Phase)

Silica, alumina

Non-Polar

ODS Silica gel C18, C8

The Mobile Phase

Normal chromatography Hexane ; dichloromethane; isopropanol; methanol

Increasing strength

Reverse phase chromatography water ; methanol; acetonitrile; tetrahydrofuran (THF)

Increasing strength

3. Column Efficiency
3.I EFFECT OF PARTICLE SIZE PACKING

3.2 EXTRA COLUMN BAND BROADENING

Inside column packing

LC Column Efficiency Particle Size Plate height

Outside column packing Solute is carried through open tubes (injection system, detector region, piping connection)
Radius of Component (0.010 inch) Length of Tubing Extra column broadening

DIAGRAM OF SI MPLE LIQUI D COLUMN CHROMATOGRAPHY Solvent(mobile or moving phase) A+ B+C Sample (A+ B+C)

4. LC INSTRUMENTATION

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO Column OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO Solid Partic les OOOOOOOOOOO (packing materialOOOOOOOOOO stationary phase) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO Eluant (eluate)

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO A OOOO OOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOB OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOC OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.

Components of HPLC
1. 2. 3. 4.

5.
6. 7.

Solvent Reservoir Pumps Sample Injection System Columns Detectors Data Processing Waste

4.1 Reservoirs

EQUIPPED WITH GLASS RESERVOIRS THAT CONTAIN 500 mL SOLVENT

DEGASSERS

Remove dissolved gases and dust from liquids leading to irreproducible flow rate and interference of detector

COMPOSED OF

Vacuum pumping system Distillation system Device for heating and stirring Filtering for gas

SPARGING

Dissolved gasses are swept out of solution by fine bubbles of an inert gas that is insoluble in the mobile phase

4.1 MPR and ST systems

Isocratic elution

Gradient elution

Single solvent or solvent mixture with constant composition

Two or more solvent systems that differ greatly in polarity and varied in composition Shorter time of separation without sacrificing resolution

Elution -To carry sample into the column

4.2 Pumping System

A pump is used to produce an appropriate pressure to push solvent into the sample

Requirements Generate pressure up to 6000 psi Pulse free output Flow rate 0.1 10 mL/min Flow reproducibility of 0.05% > Resistance to corrosion

Types of Pumps
Reciprocating

Displacement

Small chambers where solvent is pumped back and forth motion of motor driven piston

Large syringe-like chamber equipped with plunger using screw driven mechanism powered by stepping motor

ADVANTAGE
o o o o

ADVANTAGE
o o

Small internal volume High output pressure Adaptability to gradient elution Large solvent capacities Constant flow rates
Must be damp

Independent of viscosity and back pressure Pulse free output

Limited solvent capacity Solvent must be changed

DISADVANTAGE
o o

DISADVANTAGE
o

4.3 Sample Injection

Sampling loops o provide choice of sample sizes from 1uL to 100 uL or more o pressure up to 7000 psi Auto injectors o Contain sampling loops and syringe pump for injection volume. o Controlled temperature to allow sample storage and derivatization reaction prior to introduction

4.4 Columns
Constructed from smooth-bore stainless steel tubing , heavy walled glass tubing and polymer tubing, or column lined with glass (200 to 1000 dollars) Guard Column o increase life of analytical column by removal of contaminant o similar composition, larger particle size to minimize pressure drop o sacrificed to protect expensive analytical drop Analytical Column 3-5 um with 100,000 plates/m (fast with minimal solvent consumption) Expensive and Disposed after use

4.5 Column Packing

Pellicular

Porous

Spherical, non porous, glass or polymer 30 to 40 um Silica, alumina or resin on surface of beads For separation of proteins and large biomolecules

Porous with 3 to 10 um with narrow particle size distribution Silica, alumina or resin on surface of particle

4.6 Detectors
Ideal
o o

Detector:

o
o o

o
o o

Minimal internal volume (reduce broadening) Compatible with liquid flow Adequate Sensitivity (10-5 to 10-15 g solute/s) Good stability and reproducibility Linear response to solutes that extends over several orders of magnitude. Short response time and independent of flow rate. High reliability and ease of use. Similarity in response toward all solutes or a highly predictable and selective response toward one or more classes of solutes. Nondestructive.

Types of Detectors

Bulk-Property
o

Solute-Property

respond to mobile phase bulk property (Refractive Index, Density) modulated by solutes

respond to property of solute (UV absorbance, Fluorescence) not possessed by mobile phase

COMMON Detectors

UV Visible Absorption Detector UV Visible Detector with Filter Absorption Detector with Scanning Infrared Absorption Detector Fluorescence Detector Refractive Index Detector Evaporative Light Scattering Detector Electrochemical Detector Mass spectrometric Detector

Data Processing Using specific software that is connected to HPLC machine Receive the information from HPLC machine and present it as a graph The graph describes about qualitative data (Retention time) and quantitative data (area under curve)

Application
1. Pharmaceuticals industry

To control the drug stability Quantity of drug determination from pharmaceutical dosage forms Quantity of drug determination from biological fluids

2. Analysis of natural contamination 3. Forensic test 4. Clinical test 5. Food and essence manufacture

1. Internal diameter of column - the smaller in diameter, the higher in sensitivity 2. Pump pressure - the higher in pressure, the higher in separation 3. Sample size 4. The polarity sample, solvent and column 5. Temperature - the higher in temperature, the higher in separation

HPLC CHROMATOGRAM

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High Performance Liquid Chromatograph

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