Analytical Techniques 4

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HPLC


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Outline

What is HPLC? An Overview

Types of HPLC Ion Chromatography Size-Exclusion Chromatography

Adsorption Chromatography

Etc.,


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What is HPLC?

The most widely used analytical separations

technique Utilizes a liquid mobile phase to separate

components of mixture

uses high pressure to push solvent throughthe column

Popularity: sensitivity

ready adaptability to accurate quantitativedetermination

suitability for separating nonvolatile species orthermally fragile ones


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HPLC is.

Popularity: widespread applicability to substances that are of

prime interest to industry, to many fields of science,and to the public

Ideally suited for separation and identificationof amino acids, proteins, nucleic acids,hydrocarbons, carbohydrates, pesticides,pigments, antibiotics, steroids, and a variety ofother inorganic substances


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History lesson

Early LC carried out in glass columns

diameters: 1-5 cm

lengths: 50-500 cm

Size of solid stationary phase

diameters: 150-200 m

Flow rates still low! Separation times long! HPLC - Decrease particle size of packing causes increase in

column efficiency!

diameters 3-10 m

This technology required sophisticated instruments

new method called HPLC


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Instruments required: Mobile phase reservoir

Pump

Injector Column

Detector

Data system

Components


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Elution methods

Isocratic elution single solvent of constant composition

Gradient elution 2 or more solvents


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Provide a continuous constant flow of the solventthrough the injector

Requirements

pressure outputs up to 6000 psi

pulse-free output flow rates ranging from .1-10 mL/min

flow control and flow reproducibility of 0.5% orbetter

Pumping System I


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Pumping System II

Two types: constant-pressure

constant-flow

Reciprocating pumps

motor-driven piston disadvantage: pulsed flow creates noise

advantages: small internal volume (35-400 L),high output pressures (up to 10,000 psi), ready

adaptability to gradient elution, constant flowrates


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Displacement pumps(pulse free pumps) syringe-like chambers activated by screw-driven

mechanism powered by a stepper motor

advantages: output is pulse free

disadvantage: limited solvent capacity (~20 mL)and inconvenience when solvents need to bechanged

Flow control and programming system

computer-controlled devices measure flow rate

increase/decrease speed of pump motor

Pumping System III


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Several column types(can be classified as )

Normal phase

Reverse phase

Size exclusion

Ion exchange


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Reverse phase

In this column the packing material isrelatively nonpolar and the solvent is polar

with respect to the sample. Retention is theresult of the interaction of the nonpolarcomponents of the solutes and the nonpolarstationary phase. Typical stationary phases

are nonpolar hydrocarbons, waxy liquids, orbonded hydrocarbons (such as C18, C8, etc.)and the solvents are polar aqueous-organicmixtures such as methanol-water or

acetonitrile-water.


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Size exclusion

In size exclusion the HPLC column isconsisted of substances which have

controlled pore sizes and is able to befiltered in an ordinarily phase according toits molecular size. Small moleculespenetrate into the pores within the packing

while larger molecules only partiallypenetrate the pores. The large moleculeselute before the smaller molecules.


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Ion exchange

In this column type the sample componentsare separated based upon attractive ionicforces between molecules carrying chargedgroups of opposite charge to those chargeson the stationary phase. Separations aremade between a polar mobile liquid, usuallywater containing salts or small amounts of

alcohols, and a stationary phase containingeither acidic or basic fixed sites.


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For injecting the solvent through the column Minimize possible flow disturbances

Limiting factor in precision of liquid chromatographicmeasurement

Volumes must be small

0.1-500 L Sampling loops

interchangeable loops (5-500 L at pressures up to 7000psi)

Sample Injection Systems


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Sample Injection System

The valve handle as shown on the left, the loop is filled from the syringe,

and the mobile phase flows from the pump to column,Valve is placed in position on the right, the loop is inserted

between the pump and the column so that the mobile phase

sweeps the sample onto the column


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Smooth-bore stainless steel or heavy-walled glass

tubing Hundreds of packed columns differing in size and

packing are available from manufacturers ($200-$500)

Add columns together to increase length

Liquid Chromatographic Column


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Column thermostats maintaining column

temperatures constant to afew tenths degreecentigrade

column heaters controlcolumn temperatures (fromambient to 150oC)

columns fitted with waterjackets fed from a constant

temperature bath

Liquid Chromatographic Columns II


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Mostly optical Equipped with a flow cell

Focus light beam at the center formaximum energy transmission

Cell ensures that the separated

bands do not widen UV-Vis

PDA

Detector


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Some Properties of Detector

Adequate sensitivity

Stability and reproducibility

Wide linear dynamic range

Short response time Minimum volume for reducing zone broadening

High reliability and ease of use

Similarity in response toward all analytes

Selective response toward one or more classes of analytes


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Refractive index

UV/Visible

Fluorescence Conductivity

Evaporative light scattering

Electrochemical

Types of Detector


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Measure displacement of beam with respect tophotosensitive surface of dectector

Refractive Index I


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Advantages

universal respond to nearly all solutes

reliable

unaffected by flow rate

low sensitive to dirt and air bubbles in the flowcell

Refractive Index II

Disadvantages

expensive

highly temperature sensitive

moderate sensitivity

cannot be used with gradient elution


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Mercury lamp

= 254nm

= 250, 313, 334 and 365nm with filters Photocell measures absorbance

Modern UV detector has filter wheels for rapidlyswitching filters; used for repetitive andquantitative analysis

UV/Visible I

Advantages

high sensitivity

small sample volume required

linearity over wide concentration ranges can be used with gradient elution

Disadvantage

does not work with compounds that do not absorb light atthis wavelength region


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For compounds having naturalfluorescing capability

Fluorescence observed byphotoelectric detector

Mercury or Xenon source withgrating monochromator toisolate fluorescent radiation

Fluorescence I


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Advantages

extremely high sensitivity

high selectivity

Disadvantage

may not yield linear response over wide range ofconcentrations

Fluorescence II


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Measure conductivity ofcolumn effluent

Sample indicated bychange in conductivity

Best in ion-exchangechromatography

Cell instability

Conductivity


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Nebulizer converts eluent into mist

Evaporation of mobile phase leads toformation of fine analyte particles

Particles passed through laser beam;

scattered radiation detected at right anglesby silicon photodiode

Similar response for all nonvolatile solutes

Good sensitivity

Evaporative Light Scattering I


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Evaporative Light Scattering II


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Based on reductionor oxidation of the

eluting compoundat a suitableelectrode andmeasurement of

resulting current

Electrochemical I


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Advantages high sensitivity

ease of use

Disadvantages mobile phase must be made conductive

mobile phase must be purified from oxygen,metal contamination, halides

Electrochemical II


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Advantages to HPLC

Quantification Separation Higher resolution and speed of analysis HPLC columns can be reused without

repacking Greater reproducibility due to close control

of the parameters affecting the efficiency ofseparation

Easy automation of instrument operation anddata analysis Adaptability to large-scale, preparative

procedures


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Analytical Techniques 4

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