Unit 7 Chromatography

8/7/2019 Unit 7 Chromatography

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PCBs

Stationary phase Trade name Maximumtemperature

Common applications

Poly(phenylmethyldimethyl)siloxane (10% phenyl)

HP 3 / OV 3 350 Fatty acid methyl esters;alkaloids; drugs;halogenated compounds

Poly (phenylmethyl)siloxane (50% phenyl)

HP17/ OV17 250 Drugs; steroids; pesticides;glycols

Poly(trifluoropropyldimethyl)siloxane

OV 210 200 Chlorinated aromatics;nitroaromatics; alkyl-substituted benzenes

Polyethylene glycol Carbowax 20 M 250 Free acids; alcohols; ethers;essential oils; glycols

7.3 Classification of Column Chromatographic Methods

General Classification Specific Method Stationary Phase

Gas Chromatography(GC)

1. Gas-liquid (GLC)

2. Gas-solid

- Liquid adsorbed orbonded to a solid surface

- Solid

Liquid Chromatography(LC)

1. Liquid-liquid orpartition

2. Liquid-solid or

adsorption

3. Ion exchange

4. Size Exclusion

5. Affinity

- Liquid adsorbed orbonded to a solid surface

- Solid

- Ion exchange resin

- Liquid in intersticesof a polymeric solid

- Group specific liquidbonded to a solid surface

Supercritical FluidChromatography (SFC)

- Organic speciesbonded to a solid surface

7.3 Types of Chromatography Methods

(source: http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm )

Adsorption Chromatography

o one of the oldest types of

chromatography.

o utilizes a mobile or gaseous

phase that is adsorbed onto the surfaceof a stationary solid phase.

o The equilibration between the

mobile and stationary phases accountsfor the separation of different solutes.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K.Bartley-Hynes (University of Technology, Jamaica)

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http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htmhttp://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm


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Partition Chromatography

o Based on a thin film formed on the

surface of a solid support by a liquidstationary phase.

o Solute equilibrates between the mobile

phase and the stationary liquid.

Ion Exchange Chromatography

o A resin (solid stationary phase) is used

to covalently attach anions or cationsonto it.

o Solute ions of the opposite charge in

the mobile phase are attracted to theresin by electrostatic forces.

Molecular Exclusion Chromatography

o Also known as gel permeation or gel

filtration.

o This type lacks an attractive interactionbetween the stationary phase and thesolute.

o The liquid or gaseous phase passes

through a porous gel which separatesmolecules by size.

o The pores are small and exclude larger

solute molecules, causing the largermolecules to pass through the columnat a faster rate than the smaller ones.

Affinity Chromatography

o The most selective type of

chromatography.

o Utilizes the specific interaction between

one kind of solute molecule and asecond molecule that is immobilized ona stationary phase.

o The specific solute molecule is bound

to the stationary phase and laterextracted by changing ion strength or

pH.

7.4 Basis of Chromatography

The Mobile and Stationary Phases

The mobile phase is comprised of a solvent into which the sample is injected. Thesolvent and sample flow through the column together; thus the mobile phase is oftenreferred to as the "carrier fluid."

The stationary phase is the material in the column for which the components to beseparated have varying affinities.


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The materials which comprise the mobile and stationary phases depend on the generaltype of chromatographic process being performed.

The Column

Most modern applications of chromatography employ a column. This is where theseparation takes place.

Usually a glass or metal tube of sufficient strength to withstand the pressures appliedacross it.

Contains the stationary phase. The mobile phase runs through the column and isadsorbed onto the stationary phase.

Basic Layout of a Chromatograph

Gas Chromatography

The mobile phase is generally an inert gas.

The stationary phase is generally an adsorbent or liquid distributed over the surface of aporous, inert support.

Liquid Chromatography

The mobile phase is a liquid of low viscosity which flows through the stationary phasebed. This bed may be comprised of an immiscible liquid coated onto a porous support, a thinfilm of liquid phase bonded to the surface of a sorbent, or a sorbent of controlled pore size.

7.5 Theory of GC and HPLC

Gas Chromatography (GC)

Gas chromatography involves a sample being vapourised and injected onto the head ofa chromatographic column. The sample is transported through the column by the flow ofinert, gaseous mobile phase.

In GLC, the column itself contains a liquid stationary phase which is adsorbed onto thesurface of an inert solid.

Schematic of a gas chromatograph


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Oven

COLUMN

Flow Meter

Feed Injection

Pump

Solvent Tank

Detector


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Carrier Gas

The carrier gas must be chemical inert. Commonly used gases include N, He, Ar andCO2. The choice of carrier gas often depends on type of detector used.

Carrier gas system also contains a molecular sieve to remove water and impurities.

Sample Injection Port

For optimum column efficiency, the sample should not be too large and should beintroduced onto the column as a plug of vapour slow injection of large samples leads toband broadening and loss of resolution.

vs.

Most common injection method is where a microsyringe is used to inject sample througha rubber septum into a flash vapouriser port at head of column. Temperature of port usually

~50> BP of least volatile sample.

Sample size for packed columns 0.1 20 L. Capillary columns ~10-3 L.

Injector contains a heated chamber sample vapourises to form a mixture of carrier gas,vapourised solvent and vapourised solutes:

Columns

Packed Bed Columns

Comprised of a finely divided, inert, solid support material coated with liquid stationaryphase (GLC). This stationary phase completely fills the column.

1.5 10 m long; internal diameter of 2 4 mm.

Capillary or Open Tubular Columns

The liquid stationary phase is a thin film or layer on the column wall. There is apassageway through the centre of the column.

Internal diameter < 1 mm.


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Column temperature

For precise work, column temperature must be controlled to within 0.1.

The optimum column temperature depends on the boiling point of the sample. As a ruleof thumb, a temperature slightly above the average boiling point of the sample results in anelution time of 2 30 minutes. Minimal temperatures give good resolution, but increase

elution times.

If a sample has a wide boiling range, then temperature programming can be useful. Thecolumn temperature is increased (either continuously or in steps) as separation proceeds.

Detectors

Different detectors will give different types of selectivity:

o A non-selective detector responds to all compounds except the carrier gas.

o A selective detectorresponds to a range of compounds with a common physicalor chemical property.

o A specific detectorresponds to a single chemical compound.

Detectors can also be grouped into concentration dependant detectors and mass flowdependant detectors:

o The signal from a concentration dependant detector is related to the

concentration of solute in the detector, and does not usually destroy the sample.

o Mass flow dependant detectors usually destroy the sample, and the signal is

related to the rate at which solute molecules enter the detector.

Detectors must be able to respond quickly to low solute concentrations (a few ppt) as they areeluted from the column.

Other properties include:

Linear response

Stability

Uniform response to wide variety of species or predictable responses to one or moreclasses of chemicals.

No one detector possesses all the desirable properties but the three most popular are:

Flame ionisation detectors

Thermal conductivity detectors

Electron capture detectors

GC may also be coupled with other methods such as mass spectrometry (GC-MS) and infraredspectrometry (GC-IR).


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High-Performance Liquid Chromatography (HPLC)

In early liquid chromatography, separation times were long several hours. Column packings(solid coated with thin liquid film) were 50 500 cm in length, with internal diameter of 10 50

mm and particle sizes >150 200 m.

For increased column efficiency, decrease particle size (decrease plate height or increase platecount). Since the 1960,s HPLC was developed.

HPLC most popular of the analytical separation techniques.

High sensitivity

Good adaptability

Ease of automation

Can be used for non-volatile compounds

Different methods are used depending on the nature of solutes to be separated:

High molecular mass compounds (>10,000 g mol-1) utilise size-exclusionchromatography.

Low molecular mass ionics utilise ion-exchange or reverse-phase chromatography.

Non-polar species utilise adsorption chromatography.

Instrumentation

Pumping pressures of several hundred atmospheres are employed.

Column particle sizes 10 m.

The instruments are elaborate and expensive.

Mobile-Phase Reservoirs and Solvent Treatment Systems

One or more mobile-phase reservoirs used.

The treatment systems remove bubbles and particulates.

Spargingis a process in which dissolved gases are swept out of a solvent by bubbles ofan inert, insoluble gas.

Isocratic (simple) or gradient elution is used.


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Pumping Systems

A pump should have the following characteristics:

Pressures up to 6000 psi

Pulse-free output

Flow rates 0.1 10 mL/min

Good flow reproducibilities

Resistant to corrosion by a variety of solvents

Two types of pumps used: mechanical or pneumatic pumps.

NOTE: High pressures are not an explosion hazard because liquids are not very compressible.If there is a rupture in a component, leading to leakage then there is a fire hazard.

Sample Injection Systems

Syringe injection through a septum is used. However, it is are not very reproducible andused for pressures


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o Refractive index LC detector measures changes in the refractive index of the

solvent.

Others include:

o Fluorescence; Conductivity; Mass spectrometry; Photoionization LC detectors.

LC methods include: partition, adsorption, ion-exchange and size exclusion.

High-performance partition chromatography

This is the most widely used of LC methods.

Divided into liquid-liquid or bonded-phase chromatography.

Liquid-liquid packing: retention occurs by physical adsorption.

Bonded-phase packing: covalent bonds are involved.

Normal vs. Reversed Phase Packings

Normal phase:

Early LC was based on highly polar stationary phases, e.g, triethylene glycol or water.

A non-polar solvent served as the mobile phase.

The least polar component is eluted first. An increase in the polarity of the mobile phaseleads to a decrease in the elution time.

Reversed-phase:

The stationary phase is typically a non-polar hydrocarbon while the mobile phase is apolar solvent e.g., water, methanol.

o The most polar component is eluted first.

o An increase in the polarity of the mobile phase leads to an increase in elution

time.

o This type is usually used in modern instruments.

Comparison of GC and HPLC

Both methods:o Highly efficient, selective

o Widely applicable

o Utilise small sample sizes

o Usually non-destructive

o Easily adapted

Advantages:

HPLC GC

Used for non-volatile, thermallyustable samples

Iorganic ions

Relatively simple and inexpensive

Rapid

Superior resolution, particularly withcapillary columns

Easily interfaced with massspectroscopy


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Column Efficiency in Chromatography

Example of a Chromatogram:

The effectiveness of a chromatographic column to separate solutes is dependent on the relativerates at which the species are eluted. These rates are determined by the partition ratios of the

solutes between the two phases. Ideally, the partition ratio for a component should be constantover a wide range of concentrations.

An equilibrium describes the partitioning of a solute A between the mobile and stationary

phases: Amobile Astationary

The partition ratio (equilibrium constant) is defined as:

mm

ss

m

sc

Vn

Vn

C

CK

/

/==

Cs, ns = concentration of solute in stationary phase

Cm, nm = concentration of solute in mobile phase

Vs, Vm = volume of stationary phase and mobile phase

Retention Time can easily be measured and is a function ofKc.

The first peak relates to a solute that is not retained by column, hence, tM is called dead or voidtime.

Retention time = MSR ttt +=

Rate of migration of solute (cm/s) =Rt

Lv = Rate of migration of mobile phase =

Mt

Lu =

( )emobilephasspendtimesolutefractionofuv sin=


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NOTE: tR = tS


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=

ofsolutetotalmoles

ephaseuteinmobilmolesofsoluv

msc VVK

uv

/1+=

Retention or capacity factor, k, is not dependent on column geometry or volumetric flow rate:

M

sA

A

V

VKk =

Ak

uv

+=1

and

M

R

M

MR

A

t

t

t

ttk

'=

= tR= adjusted retention time

Selectivity factor, a measure of the relative migration rates of two solutes, i.e., how well thepeaks are separated.

MAR

MBR

A

B

A

B

tt

tt

k

k

K

K

===

)(

)(

Description of Column Efficiency Plate Theory

2

2/1

2

54.516

=

=

W

t

W

tN

RR

W = width at base of peak; W1/2 = width at half height of peak

NOTE:Efficiency of the column increases as N increases and Hdecreases.

Band broadening reflects a loss in column efficiency.It depends on the flow rate of the mobile phase:


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,u

Plate Height =L

H

2

=

Plate Count/number of theoretical plates,

HLN=

L = length of column packing


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Column Resolution, R s

Tells how far apart two bands are relative to their widths.

[ ]BA

ARBRS

WW

ttR

+

=)()(2

and( )( ) 2

1

2

1

N

N

R

R

S

S

=

General Elution Problem:

For the above diagram:

a) Good retention factors for components 1& 2.

b) Changing conditions to optimize separation of components 5 & 6 bunches the peaks for

components 1-4.c) Good retention factors for components 3 & 4.

To compensate for this problem, conditions can be changed as the separation proceeds:

Immediately after components 1 & 2 are eluted, the conditions can be changed to elutecomponents 3 & 4 and then changed again to elute components 5 & 6.

In liquid chromatography, this is effected using gradient elution (or solvent programming) composition of the mobile phase is varied during the elution. Elution with constant mobile phasecomposition is called isocratic elution.

In gas chromatography, temperature programming is employed temperature is varied.


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1 2 3 4 5 6 (a)

1,2 3 4 5 6 (b)

1 2 3 4 5 6 (c)

Unit 7 Chromatography

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