ChromatographyChromatography

Basic introduction and Basic introduction and instrumentationinstrumentation

A SEMINAR ON

Chromatography Introduction andInstrumentation

GUIDED BY: BY: PATEL AKSHAY

J.D.Patel M.PHARM- 1(ph’ceutics) (head of department) ROLL NO-05 Department of Pharmaceutics NOOTAN PHARMACY COLLEGE,VISNAGAR

• The The analyteanalyte is the molecule which is to be purified or isolated during is the molecule which is to be purified or isolated during chromatography chromatography

• Analytical chromatographyAnalytical chromatography is used to determine the identity and is used to determine the identity and concentration of molecules in a mixture concentration of molecules in a mixture

• A A chromatogramchromatogram is the visual output of the chromatograph. Different peaks is the visual output of the chromatograph. Different peaks or patterns on the chromatograph correspond to different components of the or patterns on the chromatograph correspond to different components of the separated mixture separated mixture

• A A chromatographchromatograph takes a chemical mixture carried by liquid or gas and takes a chemical mixture carried by liquid or gas and separates it into its component parts as a result of differential distributions of separates it into its component parts as a result of differential distributions of the the solutes as they flow around or over the stationary phase as they flow around or over the stationary phase

• The The mobile phasemobile phase is the analyte and solvent mixture which travels through is the analyte and solvent mixture which travels through the stationary phase the stationary phase

• Preparative chromatographyPreparative chromatography is used to purify larger quantities of a is used to purify larger quantities of a substance substance

• The The retention timeretention time is the characteristic time it takes for a particular molecule is the characteristic time it takes for a particular molecule to pass through the system to pass through the system

• The The stationary phasestationary phase is the substance which is fixed in place for the is the substance which is fixed in place for the chromatography procedure and is the the phase to which chromatography procedure and is the the phase to which solvents and the and the analyte travels through or binds to. Examples include the analyte travels through or binds to. Examples include the silica plate in plate in thin layer chromatography

Chromatography termsChromatography terms

RetentionRetention• The retention is a measure of the speed at which a The retention is a measure of the speed at which a

substance moves in a chromatographic system. In substance moves in a chromatographic system. In continuous development systems like HPLC or GC, continuous development systems like HPLC or GC, where the compounds are eluted with the where the compounds are eluted with the eluent, the , the retention is usually measured as the retention is usually measured as the retention timeretention time RtRt or or tRtR, the time between injection and detection. In , the time between injection and detection. In interrupted development systems like TLC the interrupted development systems like TLC the retention is measured as the retention is measured as the retention factorretention factor RfRf, the , the run length of the compound divided by the run length run length of the compound divided by the run length of the eluent frontof the eluent front::

• The retention of a compound often differs The retention of a compound often differs considerably between experiments and laboratories considerably between experiments and laboratories due to variations of the eluent, the stationary phase, due to variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important temperature, and the setup. It is therefore important to compare the retention of the test compound to that to compare the retention of the test compound to that of one or more standard compounds under absolutely of one or more standard compounds under absolutely identical conditions.identical conditions.

The Theoretical Plate Model of The Theoretical Plate Model of ChromatographyChromatography

• The plate model supposes that the The plate model supposes that the chromatographic column is contains a large chromatographic column is contains a large number of separate layers, called number of separate layers, called theoretical theoretical platesplates. Separate equilibrations of the sample . Separate equilibrations of the sample between the stationary and mobile phase between the stationary and mobile phase occur in these "plates". The analyte moves occur in these "plates". The analyte moves down the column by transfer of equilibrated down the column by transfer of equilibrated mobile phase from one plate to the next.mobile phase from one plate to the next.

The Rate Theory of The Rate Theory of ChromatographyChromatography

• A more realistic description of the processes A more realistic description of the processes at work inside a column takes account of the at work inside a column takes account of the time taken for the solute to equilibrate time taken for the solute to equilibrate between the stationary and mobile phase between the stationary and mobile phase (unlike the plate model, which assumes that (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting equilibration is infinitely fast). The resulting band shape of a chromatographic peak is band shape of a chromatographic peak is therefore affected by the rate of elution. It is therefore affected by the rate of elution. It is also affected by the different paths available also affected by the different paths available to solute molecules as they travel between to solute molecules as they travel between particles of stationary phase. If we consider particles of stationary phase. If we consider the various mechanisms which contribute to the various mechanisms which contribute to band broadening, we arrive at the Van band broadening, we arrive at the Van Deemter equation for plate height;Deemter equation for plate height;

• HETP = HETP = A + B / u + C u A + B / u + C u • where where uu is the average velocity of the mobile is the average velocity of the mobile

phase. phase. A, B, A, B, and and CC are factors which are factors which contribute to band broadening.contribute to band broadening.

It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better).

If the length of the column is L, then the HETP is

HETP = L / N

The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution;

where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.

A-Eddy diffusionThe mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.B - Longitudinal diffusionThe concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion.C - Resistance to mass transferThe analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

Van Deemter plotsVan Deemter plots

•A plot of plate height vs. average linear velocity of mobile phase

Such plots are of considerable use in determining the optimum mobile phase flow rate

ResolutionResolution

• Although the selectivity factor, a, describes the Although the selectivity factor, a, describes the separation of band centres, it does not take into separation of band centres, it does not take into account peak widths. Another measure of how well account peak widths. Another measure of how well species have been separated is provided by species have been separated is provided by measurement of the measurement of the resolutionresolution. The resolution of two . The resolution of two species, A and B, is defined asspecies, A and B, is defined as

•Baseline resolution is achieved when R = 1.5

• Adsorption Chromatography:Adsorption Chromatography: Adsorption Adsorption chromatography is probably one of the oldest types chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts between the mobile and stationary phase accounts

for the separation of different solutesfor the separation of different solutes..

• Partition Chromatography:Partition Chromatography: This form of This form of chromatography is based on a thin film formed on chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase. Solute equilibriates between the mobile

phase and the stationary liquidphase and the stationary liquid..

HIGH PERFORMANCE LIQUID HIGH PERFORMANCE LIQUID CHROMATOGRAPHYCHROMATOGRAPHY

• Only useful for volatile and thermally stable Only useful for volatile and thermally stable compounds. compounds.

• Can’t be used for solid. Can’t be used for solid.

Difficulties encountered in LC :Difficulties encountered in LC :

• Non availability of sensitive detectors.Non availability of sensitive detectors.• Speed of separation with conventional liquid Speed of separation with conventional liquid

chromatography.chromatography.• Difficulty of speed overcome by using high Difficulty of speed overcome by using high

pressure.pressure.

Limitations of GC :Limitations of GC :

Comparision of GC & LCComparision of GC & LC

• Mobile phase gas Mobile phase gas • (very cheap)(very cheap)• St. Phase solid/liquidSt. Phase solid/liquid• Mobile phase inertMobile phase inert

• Separation mainly Separation mainly based on selection of based on selection of st.phase (wide choice)st.phase (wide choice)

• Limited applications.Limited applications.

• Mobile phase liquid Mobile phase liquid • (very costly)(very costly)• St.Phase solid/liquidSt.Phase solid/liquid• Mobile phase interacts Mobile phase interacts

with solute.with solute.• St. phase limited in no. St. phase limited in no.

separation (desired) separation (desired) achieved by selecting achieved by selecting mobile phase.mobile phase.

• Wide applications.Wide applications.

Flow diagram for a liquid chromatograph

Eluent Delivery System :

•Reservoirs (one or more )

•Degassers

•Pumps

Function of Degassers :Function of Degassers :

•Appreciable amount of gases Appreciable amount of gases dissolve at high pressure.dissolve at high pressure.

•When pressure released in column When pressure released in column and detector bubbles may form.and detector bubbles may form.

•Degassing by heating distilling, Degassing by heating distilling, vacuum pumping or purging inert vacuum pumping or purging inert gas with low solubility like He/Ar.gas with low solubility like He/Ar.

Requirements of Pump :Requirements of Pump :

• Pulseless flow upto 10 ml/min.Pulseless flow upto 10 ml/min.

• High pressure upto 1000 psi or High pressure upto 1000 psi or more.more.

• Suitability for gradient elution;Suitability for gradient elution;

• Reciprocating pumpReciprocating pump

• Syringe pumpSyringe pump

• Pneumatic pumpPneumatic pump

A reciprocating pump for HPLC

Reciprocating Pump: SchematicsReciprocating Pump: Schematics

• Most HPLC pumps are Most HPLC pumps are reciprocating reciprocating

• A motor driven cam A motor driven cam drives the piston to deliver drives the piston to deliver solvent through the outlet solvent through the outlet check valvecheck valve

• Gradient are formed by Gradient are formed by using 2 or more pumps using 2 or more pumps (high-pressure mixing) (high-pressure mixing) or solenoid-actuated or solenoid-actuated proportioning valves (low-proportioning valves (low-pressure mixing)pressure mixing)

Reciprocating pump :Reciprocating pump : • In 90% instrument used.In 90% instrument used.

• To minimise pulsing pistons and cylinders operate in To minimise pulsing pistons and cylinders operate in cycle. cycle.

• Pressure drop caused by slowing of one is Pressure drop caused by slowing of one is compensated by others.compensated by others.

• Advantages :Advantages :

• Small volumeSmall volume

• Pressure upto 600 atm(10,000 psi) can be applied.Pressure upto 600 atm(10,000 psi) can be applied.

• Variable flow rate upto 10 ml/min.Variable flow rate upto 10 ml/min.

• Suitable for gradient elution.Suitable for gradient elution.

• Disadvantages :Disadvantages :

• Not totally pulseless flow.Not totally pulseless flow.

• Damping device required to have regular flow.Damping device required to have regular flow.

Valve injection systems for liquid sampling : (a) rotary

A sampling loop for liquid chromatography

Sample Inlet System :Sample Inlet System :• Syringe injection : Syringe injection : Problem of leaking Problem of leaking

and blow back of plunger. and blow back of plunger. • Stop-flow Injection :Stop-flow Injection : During injection, During injection,

flow is stopped by a valve kept before flow is stopped by a valve kept before injection port. After injection over flow is injection port. After injection over flow is started again. started again.

• Most widely used method is sampling Most widely used method is sampling valve method :valve method :

• Rheodyne injector :Rheodyne injector : In one position In one position sample fills loop (variable size) when sample fills loop (variable size) when mobile phase goes directly to column. Then mobile phase goes directly to column. Then lever is moved, when eluent carries sample lever is moved, when eluent carries sample from the loop along with it to the column. from the loop along with it to the column.

• Loops of different size 0.5µl to 500µL.Loops of different size 0.5µl to 500µL.

Columns :Columns : • Stainless steel.Stainless steel.• Length 30 cm.Length 30 cm.• Internal diameter 5 mm.Internal diameter 5 mm.• Particle size 5 µm.Particle size 5 µm.• 40000 to 60000 plates/m.40000 to 60000 plates/m.• Smaller columns available. Smaller columns available. • Require less volume of eluent.Require less volume of eluent.• This is important as mobile phase liquids costly.This is important as mobile phase liquids costly.• Such columns have limited sample capacity. Such columns have limited sample capacity. • Temperature control not important.Temperature control not important.• Many separations at room temperature.Many separations at room temperature.• Sometimes temp. 30 – 1500C used accuracy ± Sometimes temp. 30 – 1500C used accuracy ±

0.20C.0.20C.

Guard column :Guard column :

• Short column containing similar st. phase as Short column containing similar st. phase as analyte column, but particle of bigger size filled so analyte column, but particle of bigger size filled so that no pressurethat no pressuredrop.drop.

Two functions :Two functions :(i)(i) Remove impurity to protect analyte column Remove impurity to protect analyte column which is which is very costly.very costly.(ii)(ii) Presaturates mobile phase with st.phase liq. Presaturates mobile phase with st.phase liq. so that so that in analyte column st.phase liq. Is not in analyte column st.phase liq. Is not carried away carried away with mobile phase liq.with mobile phase liq.

Detectors :Detectors :

• No detector is as sensitive, No detector is as sensitive, versatile as detectors of GC like FID versatile as detectors of GC like FID and TCD.and TCD.

• Very few work on bulk property.Very few work on bulk property.

• Most of them respond to some Most of them respond to some physical property of solute that is physical property of solute that is different from eluent.different from eluent.

UV-Absorption Detector

UV – Absorption detector :UV – Absorption detector : • (i) Single wavelength (ii) Variable wavelength(i) Single wavelength (ii) Variable wavelength• Principle : Principle : • Most of organic compound absorb UV radiation.Most of organic compound absorb UV radiation.• If eluent is not absorbing then as soon as solute If eluent is not absorbing then as soon as solute

is eluted of Column and reach detector a signal is eluted of Column and reach detector a signal obtained.obtained.

• For single wavelength source is Hg-vapour lamp For single wavelength source is Hg-vapour lamp that emits 254 nm wavelength.that emits 254 nm wavelength.

• Radiation divided in two beams one passing Radiation divided in two beams one passing through pure eluent other through column through pure eluent other through column effluent.effluent.

• If solute eluted it will absorb radiation and If solute eluted it will absorb radiation and detector will observe difference in intensity of detector will observe difference in intensity of two beams. two beams.

Volume of cell 1 to 10 µL To have pathlength 2 to 10 mm. Diameter of tube very narrow.

Advantages :

(i) Sensitivity 10-4µg/ml.(ii) Selective(iii) In sensitive to change in flowrate, temp. & comp.

of mobile phase(iv) Suitable for gradient elution.

Limitation :

The eluent should not absorb UV radiation.

Refractive Index Detector

Refractive Index Detector :Refractive Index Detector :

Two types : (a) Reflection type (b) Deflection Two types : (a) Reflection type (b) Deflection type.type.

A beam of light is reflected at eluent prism interface. Other is refracted and that beam after passing through collimating lens is falling on photocell, which measures its intensity.

When solute enters the eluent, the RI of eluent changes. As a result the intensity of beam reflected at eluent prism interface changes. This leads to change in intensity of beam refracted which is measured by photocell.

Thus change in intensity concentration of soluteof refracted beam

Characteristics :Characteristics :

• Non selectiveNon selective

• Sensitivity 10Sensitivity 10-3-3µµg/ml (much less than UV)g/ml (much less than UV)

• Not sensitive to change in flow rateNot sensitive to change in flow rate

• Very sensitive to change in temp. and change in Very sensitive to change in temp. and change in composition of eluentcomposition of eluent

• Not suitable for gradient elution.Not suitable for gradient elution.

Mainly used for carbohydrates, aminoacid etc.Mainly used for carbohydrates, aminoacid etc.

Fluorescence Detector : Fluorescence Detector :

Can only be used for sub. emitting fluorescence like plant pigments, vitamins, alkaloids, pharmaceuticals, flavoring agents etc.

Highly sensitive 10-5µg/ml. Suitable for gradient elution. Non fluorescent compounds can be converted to

fluorescent derivatives. This can be done on column. Derivatisation column kept before or after analyte column.

Stationary phases :Stationary phases :

They can be either liquid or solid.

If liquid is used it is coated on inert solid, but there are problems in it.

Hence, bonded phase supports prepared.

Silica is heated in dilute acid for a day or two to generate silonal group. As follows:

Si O Si O Si

OH OH OH

Silica particles

This is then treated with an organochlorosilane.

Si OH Si R

CH3

CH3

Cl Si O Si R

CH3

CH3

ClH+ +

R = long alkyl chain of 8 or 18 carbon then Nonpolar (Reversed phase)

R = -(CH2)n-CN, then polar (Normal phase)

= -(CH2)n-NH2

Stable upto pH 2 & 9 and upto 800C. How they retain solute molecules is not certain.

Solid stationary phases :Solid stationary phases :

Commonly used are (1) Silica (2) Alumina (3) Polyamides.Silica is preferred as it can be obtained in different

forms.

Porous microparticles Size 3 to 10 µm Surface area 100-900 m2/gm Small particles (large area) are suitable to separate solutes having narrow range of particles. Particles with bigger size and small surface area are effective in separating solutes with wide range of

polarities.

Normal phase and Reversed phase Normal phase and Reversed phase Chromatography :Chromatography :

Initially the stationary phase used to be polar and mobile phase used to be non-polar. This combination became popular as normal phase chromatography. Later the use of non-polar stationary phase and polar stationary phase started. This was reverse to the established combination and hence it is called reversed phase chromatography.

Points of comparision is given below :Points of comparision is given below :

• St. phase : PolarSt. phase : Polar

• Mobile phase : Non Mobile phase : Non polarpolar

• On increasing polarity On increasing polarity of mobile phase of mobile phase retention time retention time decreases. decreases.

• Non polarNon polar

• PolarPolar

• On increasing polarity On increasing polarity of mobile phase of mobile phase retention time retention time increases.increases.

Mobile phase liquids : Mobile phase liquids :

Criteria to select :

1. Low viscosity2. High polarity to avoid contamination with sample.3. High stability, should not react with solute/st.phase liq.4. Low volatility so that bubbles not formed.5. Immiscible with st. phase liq. If used in adsorbed form.6. Suitable for separation to be done.7. Compatibility with detector.

SolventsSolvents Polarity Polarity IndexIndex

B. P.B. P.

CyclohexaneCyclohexane 0.040.04 8181

n-hexanen-hexane 0.10.1 6969

TolueneToluene 2.42.4 110110

THFTHF 4.04.0 6666

EthanolEthanol 4.34.3 7878

Ethyl acetateEthyl acetate 4.44.4 7777

MethanolMethanol 5.15.1 6565

AcetonitrileAcetonitrile 5.85.8 8282

NitromethaneNitromethane 6.06.0 101101

WaterWater 10.210.2 100100

Isocratic & Gradient elution :Isocratic & Gradient elution :

If comp. of mobile phase does not change during elution, it is isocratic.

If comp. changes then Gradient.

Need for Gradient elution : If we want to separate mix. of 10 solutes, 5 N.P. and

5 highly polar and NP mobile phase used then NP solutes eluted in reasonable time but polar solutes take long time to come out. If mobile phase is polar then NP solutes eluted so quickly that no resolution but now polar solutes come out in reasonable time. If we make mobile phase gradually from NP to polar then the problem is solved and that is called gradient elution.

With polar mobile phase

Derivatisation :1. To prepare UV absorbing deri.of alcohol comp. treated

with 3-5 dinitrobenzoyal chloride.2. To prepare fluorescent deri.of carboxylic acids treated

with 4-bromomethyl 7-methoxy coumarin.It must be quantitative.

Detector response

Time

Detector response

Time

Varian HPLC SystemVarian HPLC System

9010 SolventDelivery System

9050 VariableUV/Vis Detector

HPLC SolventReservoirs

HPLCColumn

RheodyneInjector

9060 Polychrom(Diode Array) Detector

ComputerWorkstation

Keep an eye onthese 4 screens!

Varian Solvent Delivery SystemVarian Solvent Delivery System

Varian 9010 Solvent Delivery System

Rheodyne Injector

%A %B %C Flow Rate Pressure{H2O} {MeOH} (mL/min) (atmos.)

Ready

Ternary Pump

A

C

B

from solvent reservoir

Colum

n

to detector

to column

throughpulse

dampener

to injector

through pump

load

inject

Variable UV/Vis Variable UV/Vis DetectorDetector

ABS AUFS RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min

Ready

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