chromatography - psau · history of chromatography mikhail tswett is credited with the invention of...

CHROMATOGRAPHY

Prepared by:

Dr.Elsadig H.KH.Adam

Instrumental Analysis -Dr.Elsadig H.kh.Adam1

History of ChromatographyMikhail Tswett is credited with the inventionof chromatography(1903) . Developed atechnique that separated various plantpigments (e.g. Chlorophylls and xanthophylls)by passing solutions through glass columnsfilled with finely ground CaCO3.

The separated species appeared as coloredbands on the column.

Hence the word Chromatography means color and writing (literally

translated from its Greek roots Chroma and Graphein).

M. S. TswettRussian Scientist

(1872-1919)



Tswett's Experiment

CHROMATOGRAPHY

Basic PrinciplesTwo phases considered:

1) Mobile Phase: solvent moving through the column.

2) Stationary Phase: stays in place inside of the column.

“Eluent” COLUMN “Eluate”

Process is called “elution”


Simple Column

Fresh eluent

Initial band

of A and B

Column

Packing

Porous Disk


•Mobile phase :is solvent moving through

column

– liquid (Methanol, water, buffer)

– gas (He, H2, N2)

•Stationary phase fixed inside column

–viscous liquid coated on inert support inside of column

–solid particles packed inside column

•Solutes have different affinities for mobile

phase and stationary phase


Instrumental Analysis -Dr.Elsadig H.kh.Adam

7

APPLICATIONS OF CHROMATOGRAPHY

The chromatographic technique is used for the separation of amino acids,

proteins & carbohydrates

It is also used for the analysis of drugs,hormones,vitamins

Helpful for the qualitative & quantitative analysis of complex mixtures.

The technique is also useful for the determination of molecular weight of

proteins.

Classification of chromatographic methods

I- Classification according to the nature of mobile phase

A- Liquid chromatography (LC)i-Liquid-solid chromatography (LSC)

In this type, stationary phase is solid

ii- Liquid-liquid chromatography (LLC)In this type the stationary phase is liquid, which may bemechanically held on a solid support in the form of thin coat. It maybe packed in a tubular column or spreader as thin layer on a plate.

Bonded phase chromatography (BPC)The liquid stationary phase may be chemically and permanently held to a solid support.


B- Gas Chromatography (GC)

The mobile phase used in this type is a gas. This type can be

classified according to the nature of stationary phase into:

i- Gas-Liquid chromatography (GLC)

In this type the stationary phase is a liquid, which may be

mechanically held on a solid support in the form of thin coat and packed in a column or it coats the inner wall of capillary column.

ii- Gas-Solid Chromatography (GSC)

In this type the stationary phase is a solid.


II- Classification according to the mechanism of attachment to the stationary phase.

A- Adsorption Chromatography

Stationary phase : Solid Mobile phase : Liquid or

GasHow it separates–solute adsorbs on tostationary phase surface


B- Partition Chromatography

Stationary phase: Liquid on solid support

Mobile phase: Liquid or Gas

How it separates

–solute dissolves into

liquid coating


C- Ion-Exchange Chromatography

Stationary phase: Anions or cationscovalently bound to solid stationary phase

Mobile phase :liquid

How it separates

solute ions of opposite charge

attracted to stationary phase


D- Molecular Exclusion Chromatography

Stationary phase: Porous gelMobile phase: LiquidHow it separates–small moleculestrapped in pores ofstationary phaseAlso calledgel filtration,gel permeation,orsize-exclusionchromatography


E- Affinity Chromatography

Stationary phase : Immobilized molecules on liquid or solid stationary phase

Mobile phase: Liquid

How it separates

–Molecules with specific

shape dock with ligands


III- Classification according to the used technique

A- Columnar:

In this type the stationary phase is packed in acolumn, e.g. GC, LC and conventional column.

B- Planar:

In this type the stationary phase is spreader as a thinlayer on glass, plastic or aluminum plates; or it is heldin the network structure of paper (paperchromatography).


Summary of types of chromatography

Mobile

PhaseLiquid Gas

Chromatographic

method Liquid chromatography (LC) Gas chromatography (GC)

Stationary phase Solid liquid Solid liquid

Chromatographic

method LSC LLC GSC GLC

Mechanism of

sorption

Adsorption

chromatography

Partition

chromatography

Adsorption

chromatography

Partition

chromatography

Technique Columnar &

Planar

Columnar & Planar Columnar Columnar


Theory of chromatographyThe plate theory

• The plate theory assumesthat, a column of stationaryphase is regarded as a largenumber of horizontal, narrow,discrete, consecutive theoreticallayers known as theoreticalplates (N).

• At each plate of the stationaryphase continuous partitioningand equilibration of solutes(components molecules) occursas the mobile phase movesdown the column.


• The efficiency of the column is increased asthe number of theoretical plates (N) isincreased, i.e the efficiency of column ismeasured by N.

• Also, the column efficiency is measured byHeight Equivalent of Theoretical plates (HETP)which is denoted by (H)

• N = L/H or H = L/N

Where:

N = number of theoretical plates.

L = Column length.


Chromatogram: • It is a plot of solute

concentration as given bydetector response versuselution time.

• The position of peak onthe time axis can be usedfor identification of thesample

• The area under the peakprovides a quantitativemeasure of the amount ofeach species.

A typical chromatogram for a sample containing two components


Some terms used in chromatography

1- R Value (retardation factor):tm

R = _________

tm + ts

where tm is the time that the substance spends in the mobilephase and ts is the time that it spends in the stationaryphase.•R value for a certain substance varies with change of both oreither of the mobile and stationary phases , but once westate the conditions of a particular chromatographic methodR value will be characteristic for the substance and used forits identification .

•The differences in migration rates cause the components ina mixture to separate into bands located along the column.


2-Rf Value in thin layer chromatography, paperchromatography and in column chromatographywhere the sample band moves down the column to adefinite distance:

Rf = the distance traveled by the zone center

The distance traveled by solvent (solvent front)

As the solute is more retained as the smaller will be theRf value.


3- Rt or tR Value The Retention Time : (GC and HPLC)

it is the time from the injection to the emergence of thepeak maximum, (notice that as the solute is moreretained the larger will be the Rt value).

4- Rv The Retention Volume :

It is the total volume in ml of eluent (mobile phase orsolvent ) required to elute the center of the zone of aparticular substance. It is used in column, HPLC, and GC,(notice that as the solute is more retained the larger willbe the Rv value).

5-Capacity Factor K\:

It is widely used to describe the migration rates of solutes on columns. K\ = K Vs / Vm also

K\ = tR - tm

tmInstrumental Analysis -Dr.Elsadig H.kh.Adam22

6-Column resolution (Rs)

A- Selectivity factor () : for a column separating two components A and B

K\B (tR)B – tm

= ________ = ______________

K\A (tR)A – tm

K’B is the capacity factor of the more retained substance and K'

A is that for the less retained one.

• The larger the the more selective the stationary phase and the more effective the separation, should be more than one.


B- Resolution factor

Rs = 2z / WB + WA

= 2 [tR(B) – tR(A)]/WB + WA

It is clear from the equation that, the greater theseparation of the zones from each other and/or thesmaller the zone width the larger will be theresolution. Rs should be at least 1 and 1.5 forcomplete separation


7- The Efficiency of the Column:

It is determined by two parameters , the migration of the zone and the zone broadening i.e. the retention time and the zone width

• Column efficiency is quantitatively determined by calculating the number of theoretical plates N or H (HETP)

N =16 tR2 / W2

while H = LW2 / 16 tR2

So as the retention time increases and / or the zone width decreases the more efficient will be the column .


Factors affecting zone broadening.Zone broadening is due to three factors:

Eddy diffusion (Multiple path effect)

• This effect occur in packed column not in opentubular columns.

• Eddy diffusion is caused by imperfect packing of thecolumn with the stationary phase. Imperfect packingcauses the presence of air bubbles or cracks.Imperfect packing is also, due to the use of irregularlarge particles stationary phase.


Ordinary diffusion.

It results from low flow rate of the mobile phase,where molecules of the separated component movefrom the concentrated zone center to the borders ofthe zone.

Non equilibrium mass transfer

It results from high flow rate of the mobile phase. Athigh flow rate of the mobile phase there is noenough time for molecules of the separatedcomponent to distribute themselves between themobile phase and the stationary phase.


• Some of the molecules move ahead with themobile phase at the front of the zone andsome will remain on the stationary phase onthe back of the zone.

• Thus to minimize zone broadening we have touse small regular stationary phase particles,perfect packing (no cracks or air bubbles) andchoose optimum flow rate.


Van Deemter Equation• Putting the three factors together yields the Van

Deemter equation that helps predict how the column flow rate will affect the theoretical plate height

H= A + B/Ux + CUx• A (Eddy diffusion) H independent on flow rate.

• B/Ux (Ordinary diffusion) H inversely proportional toflow rate.

• CUx (Non equilibrium mass transfer) H proportionalto flow rate.


Optimization of column performanceTo optimize the column performance we have to change and KB to obtain Rs value of 1.5. This can be achieved by:

1. Change the composition of mobile phase in LC .

2. Change the flow rate of the mobile phase.

3. The column must be perfectly packed with small regular spherical particles, without any cracks, gabs or spaces to minimize zone broadening and to maximize N.

4. If resolution is still less than 1, the stationary phase must be changed.


Types of elution

A- Isocratic elution (Simple elution)

In this type of elution a mobile phase is either single solvent or mixture of solvents without change of its composition.

B- Gradient elution

In this type of elution the composition of the mobile phase is changed during chromatographic separation either stepwise or continuously.


Requirements of solvents used as mobile phase

1. It must dissolve the components of the mixture to be separated.

2. It must be of different eluting power.

3. Solvents of weaker elution power can be tried successively by addition of solvent of higher elution power (as in case of gradient elution).

The elution power of a solvent is measured by itsadsorbability on the stationary phase. Solvents arearranged according to their elution power in what iscalled the elutropic series.


ExampleEluting power of a series of solvents forsubstances adsorbed on silica gel, arranged indescending series.H2O > methanol > ethanol > propanol > acetone >ethyl acetate > ether > chloroform >dichloromethane > benzene > toluene >trichloroethylene > carbon tetrachioride >cyclohexane > hexane.

• This order is the order of decreasing dielectricconstant of the solvents.

• The purity of solvents should be as high aspossible


Choice of chromatographic separation methods.

• In case of substances of similar chemical types,partition chromatography is preferable.

• In case of substances of different chemical types,adsorption chromatography gives betterseparation.

• Gaseous and volatile substances need separationadopting gas chromatographic separation.

• Ionic and inorganic substances are betterseparated on ion exchange column and zoneelectrophoresis.


HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

• For conventional column chromatography where themobile phase is liquid (Liquid Chromatography LC) andthe stationary phase is either a solid or a liquid which iscoated on solid support.

• In trials to improve chromatographic separation byincrease the stationary phase surface area either wemay decrease the particle size in this case the mobilephase flow rate will decrease and stops after few ml orwe may increase the column length in this case theelution will take a very long time may be several hours.


• However, it was not until the late 1960s, that thetechnology of producing and using small sizecolumns packed with stationary phase of 10mparticle diameter.

• To allow the flow of mobile phase withinreasonable time it is introduced under Pressurefor this reason the method was first referred asHigh Pressure Liquid Chromatography.

• In HPLC system, a pressure is applied to thecolumn, forcing the mobile phase through at amuch higher rate. The pressure is applied using apumping system.


Types of Samples For HPLC:

• The sample can be organic, inorganic (includingionic) compounds, volatile, nonvolatile thermallystable or labile.

• Molecular weight ranges from ~80-~6000.

Advantages of HPLC

• Speed the analysis (takes only few minutes).

• High resolution; very complex mixture can beresolved to its components.

• High accuracy; Relative error less than 1% forquantitative analysis.


• High sensitivity; according to the nature of sampleand detector it is possible to measure aconcentration of 10-6 - 10-9 g/ml.

• Automatic systems; the whole operation startingfrom injection of the sample to identification of thepeaks and determination of the concentration, andthen repeat the cycle with next sample.

Limitation of HPLC

• Expensive instrumentation and supplies.

• The requirement of experience at least 6-12 monthsof practical experience in methods developments aregenerally required to become a professionallyoperator and obtain the maximum benefit, fromHPLC.


HPLC - Instrument

Schematic diagram showing liquid flow in a typical liquid chromatographic instrument.


Mobile Phases:

A good solvent must possess the followingrequirements:

• High purity and readily available.

• Low viscosity, limited flammability and toxicityand low reactivity to avoid chemicalinteraction with solutes or stationary phase.

• For LLC, immiscibility with stationary phase.

• Compatibility with the detector.


PUMPS:

The requirements for pumping system aremainly the following

• generate a pressure up to 500 atmosphere

• produce a pulse free output.

• The flow rates ranging from 0.1 to 10ml/min

• To have high resistance to corrosion by avariety of solvents.


There are THREE types of pumps:

1. Reciprocating pumps

• This is the most commonly used.

• It consists of a small cylindrical chamber thatis filled and then emptied by the back andforth motion of a piston.

• The pumping motion produces a pulsed flowthat must be damped.


2-Syringe type: single stroke type it has theadvantage of introducing the solvent at pulsefree constant flow rate, but it suffers fromimpossibility of solvent gradient and thelimited solvent volume.

3-The pneumatic or constant pressure type, itis cheap but it has the same disadvantages asthe syringe type.


PRECOLUMN:

• It contains a packing chemically identical tothat in the analytical column.

• The purpose of the precolumn is to removeimpurities from the solvent, and thus preventcontamination of the analytical column.

• In addition the precolumn saturates themobile phase with the stationary phase thuseliminate stripping (washing) of thestationary phase from the analytical columnduring elution with the solvent.


SAMPLE INJECTION SYSTEM• It is used for introduction of the

sample.• By changing the position of the

valve, the loop can be switched in orout the stream of solvent as it passesfrom the pump to the column.

• While the loop in the position out ofthe stream of the solvent they can befilled with syringe till overflowoccurs, this insures that the loop iscompletely filled, then the positionof the loop is changed by rotating orsliding the valve, and allow it to passwith the solvent to the column.

• Loops of different volumes are used(10-50l).


COLUMNS USED FOR HPLC• Columns for HPLC are manufactured from heavy

walled glass tubing or stainless steel.• The typical range for column length is from 15-

25cm with an inside diameter from 5-10mm.• Column packings typically have particle sizes of 5

to 10m diameter.• Recently high speed and high performance

microcolumns with an inside diameter of 1 to 4mm and length of 3 to 7.5 cm are also available.

• They have the advantages of speed and minimalsolvent consumption.


Column Packing Materials:There are TWO main types of column packing:1- Pellicular Particles:• This consists of spherical, nonporous glass or polymer

beads 30-40 m diameter, deposited on its surface athin porous layer or crust of silica, alumina, porouspolymer or an ion exchange resin.

• Also liquid stationary phase or chemically bondedphase can be attached to the surface.

2- Porous Micro Particles:• This particles have diameter ranging from 3 to 10m,

they are composed of silica, alumina, porous polymeror ion exchange resin.

• Silica particles may be coated with thin organic film,which is physically or chemically bonded to the surface.


Si OH + Cl Si R

CH3

CH3

Si O Si R

CH3

CH3

organochlorosilane(R: C4H17 or C18H37)

surface of a silicaparticles

Si OH HO - R Hydroxyl

Si OH HOOC - R Carboxyllc acid

Si OH HN-R 1o and 2o Amino

Si OH HS-R Thiols

Silanol GroupGiass Surface


DetectorsQuestions to ask when evaluating an LC detector:

–Is the detection universal?

–Is the detector response linear?

–What is the limit of detection (LOD)?

–Is the detector useful with gradient elution?


UV Absorbance Detector:

• The detector consists of source of UVradiation, the beam of which is split and passthrough two flow cells 10 to 14l.


• One through which pure solvent flows andthe other through which solvent containingthe sample flows.

• Light from both cells will then pass towavelength selector, then to two phototubesfor detection of absorbed radiation.

• When the same solvent flows through thecell no signal is obtained, while if samplepasses through one cell difference in currentoccurs and a signal is obtained.

• LOD ~ 0.1 ng• Good detection method with gradient elution• Use solvents that do not absorb UV radiation


Fluorescence Detector• Similar idea to UV detector

• Few molecules fluoresce

• Derivatize solutes with fluorescent tag

• Derivatize solutes as they elute from column

• before detection(Post-column Derivatization)

• Essentially linear

• LOD ~ 0.001 ng

• Fine with gradient elution

• Use solvents that do not fluoresce


Refractive Index Detector

• Compare refractive indices of eluate & reference

• Cannot use with gradient elution

• Essentially universal

• LOD ~ 100 ng

• Essentially non-linear (Linear range very small)

• Disaster with gradient elution


CommentsAdvantageSampleType

Of intermediate sensitivity.

Detect wide range of samples.Common detector.

For light absorbingcompounds

UV-VIS

Most samples need derivatization (except native fluorescent molecules)

Highly sensitive detector.For Fluorescentcompounds

Fluorescence

Of intermediate sensitivity.

Can be used for both separation and structure elucidation.

Universal detectorMS

Specific for ionsSpecific detector; forall ions

Electrochemicalconductometric

Specific for electroactiveelements.

Specific detector;Electroactive compounds

Electrochemicalamperometric

Comparison of LC Detectors


chromatography - psau · history of chromatography mikhail tswett is credited with the invention of...

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