Chromatographic Methods Chromatographic Methods of Analysis of Analysis

Lecture 15Lecture 15

Associate prof . L.V. VronskaAssociate prof . L.V. VronskaAssociate prof . M.M. MykhalkivAssociate prof . M.M. Mykhalkiv

OutlineOutline1.1. The basic concepts of chromatography.The basic concepts of chromatography.

2.2. Classification of chromatographic methods.Classification of chromatographic methods.

3.3. Theoretical bases of chromatography.Theoretical bases of chromatography.

4.4. The main parts of chromatographic equipment.The main parts of chromatographic equipment.

5.5. Gas chromatography.Gas chromatography.

6.6. Theoretical base of liquid chromatographyTheoretical base of liquid chromatography

7.7. HPLC – high-performance liquid chromatography HPLC – high-performance liquid chromatography

8.8. TLC – thin-layer chromatographyTLC – thin-layer chromatography

9.9. Paper chromatographyPaper chromatography

10.10. Ion-exchange chromatographyIon-exchange chromatography

1. The basic concepts of 1. The basic concepts of chromatographychromatography

ChromatographyChromatography is a process which is based on is a process which is based on multiple repetition of sorption and desorption of multiple repetition of sorption and desorption of substances. It involves passing a mixture dissolved in a substances. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase."mobile phase" through a stationary phase.

ChromatographyChromatography (from Greek χρώμα:chroma, color (from Greek χρώμα:chroma, color and γραφειν:graphein to write) is the collective term and γραφειν:graphein to write) is the collective term for a family of laboratory techniques for the separation for a family of laboratory techniques for the separation of mixtures. of mixtures.

Thin layer chromatography is used to separate components of chlorophyll

SorbtionSorbtion is absorption process of the gaseous or dissolved is absorption process of the gaseous or dissolved substance (substance (sorbatesorbate) by firm substance or liquid () by firm substance or liquid (sorbentsorbent), ), reverce process is called reverce process is called desorbtiondesorbtion..

SorbtionSorbtion is divided on: is divided on: AdsorptionAdsorption is a process that occurs when a gas or liquid is a process that occurs when a gas or liquid

solute accumulates on the surface of a solid or a liquid solute accumulates on the surface of a solid or a liquid (adsorbent), forming a film of molecules or atoms (the (adsorbent), forming a film of molecules or atoms (the adsorbate).adsorbate).

AbsorptionAbsorption is a physical or chemical phenomenon or a is a physical or chemical phenomenon or a process in which atoms, molecules, or ions enter some bulk process in which atoms, molecules, or ions enter some bulk phase - gas, liquid or solid material. phase - gas, liquid or solid material.

ChemisorptionChemisorption is a classification of adsorption is a classification of adsorption characterized by a strong interaction between an adsorbate characterized by a strong interaction between an adsorbate and a substrate surface, as opposed to physisorption which and a substrate surface, as opposed to physisorption which is characterized by a weak Van der Waals force.is characterized by a weak Van der Waals force.

Adsorption increases at Adsorption increases at constant temperatureconstant temperature : :

- with increase of solution concentration;- with increase of solution concentration;

- with increase in pressure of gas.- with increase in pressure of gas.

Adsorption is usually described through isotherms, Adsorption is usually described through isotherms, that is, the amount of adsorbate on the adsorbent as that is, the amount of adsorbate on the adsorbent as a function of its pressure (if gas) or concentration a function of its pressure (if gas) or concentration (if liquid) at constant temperature. The quantity (if liquid) at constant temperature. The quantity adsorbed is nearly always normalized by the mass adsorbed is nearly always normalized by the mass of the adsorbent to allow comparison of different of the adsorbent to allow comparison of different materials.materials.

Langmuir isotherm (red) and BET isotherm (green)

Often molecules do form multilayers, that is, some are adsorbed on already adsorbed molecules and the Langmuir isotherm is not valid. In 1938 Stephan Brunauer, Paul Emmett, and Edward Teller developed a model isotherm that takes that possibility into account. Their theory is called BET theory, after the initials in their last names

The Langmuir equation or Langmuir isotherm or The Langmuir equation or Langmuir isotherm or Langmuir adsorption equation relates the coverage Langmuir adsorption equation relates the coverage or adsorption of molecules on a solid surface to gas or adsorption of molecules on a solid surface to gas pressure or concentration of a medium above the pressure or concentration of a medium above the solid surface at a fixed temperature. The equation solid surface at a fixed temperature. The equation was developed by Irving Langmuir in 1916. The was developed by Irving Langmuir in 1916. The equation is stated as:equation is stated as:

bc

bcnn

1

where: n - quantity (mol) of the adsorbed substance at equilibrium;n∞ - a maximum quantity of substance which can be adsorbed

on sorbent;b - constant;C - concentration.

In the range of small concentration an isotherm In the range of small concentration an isotherm of adsorption is straight line.of adsorption is straight line. It is really if It is really if bc bc <<<<11 so so (1 + bc) (1 + bc) →→ 1, 1, t thenhen

This equation This equation of straight line adsorption of straight line adsorption (Henry's (Henry's equation). equation).

The quantity of the adsorbed substance will be The quantity of the adsorbed substance will be defined:defined:

- Concentration of substance;- Concentration of substance;

- Sorbent affinity.- Sorbent affinity.

Гcbcnn

2. Classification of 2. Classification of chromatographic methods.chromatographic methods.

1. On physical nature of 1. On physical nature of mobile and stationary phasemobile and stationary phase::

Mobile phaseMobile phase The gaseous The liquid

stationary phasestationary phase

The firm gas-solid chromatogra

phy

Liquid adsorption chromatography, thin layer,

ion-exchange, ionic, precipitation (sedimentation)

chromatography

The liquid Partition gas-liquid

chromatography

Partition liquid chromatography, HPLC, gel-chromatography

2. On depending of sorbtion mechanism:2. On depending of sorbtion mechanism: Molecular (interaction between a stationary Molecular (interaction between a stationary

phase (sorbent) and divided mix phase (sorbent) and divided mix components at the expense of intermolecular components at the expense of intermolecular interaction (Van der Waals forces);interaction (Van der Waals forces);

chemisorption (chemisorption (ion-exchange, , sedimentation, chelation chromatography, sedimentation, chelation chromatography, oxidation-reduction).oxidation-reduction).

3. 3. Type separationType separation::

frontal chromatography; frontal chromatography; elution - more often a used elution - more often a used

chromatography;chromatography; displacement chromatography.displacement chromatography.

Advantage - concentration of solution does not decrease.Lack – often bridging of components zone because they are not divided by solvent.

4. Techniques by chromatographic bed shape4. Techniques by chromatographic bed shape Column chromatographyColumn chromatography (it is a separation (it is a separation

technique in which the stationary bed is within a technique in which the stationary bed is within a tube)tube)

Planar chromatographyPlanar chromatography::

The plane can be a paper, serving as such or The plane can be a paper, serving as such or impregnated by a substance as the stationary bed impregnated by a substance as the stationary bed ((paper chromatographypaper chromatography) or a layer of solid ) or a layer of solid particles spread on a support such as a glass plate particles spread on a support such as a glass plate ((thin layer chromatographythin layer chromatography))

3. Theoretical bases of 3. Theoretical bases of chromatographychromatography

A A chromatogramchromatogram is the is the visual output of the visual output of the chromatograph. In chromatograph. In the case of an the case of an optimal separation, optimal separation, different peaks or different peaks or patterns on the patterns on the chromatogram chromatogram correspond to correspond to different components different components of the separated of the separated mixture.mixture.

!!! adjusted time is proportional to chromatographic resolution of the given component of investigated mix.

adjusted time depends from:adjusted time depends from: The nature chromatographic compounds;The nature chromatographic compounds; The nature of a mobile phase;The nature of a mobile phase; The nature and weight of stationary phase;The nature and weight of stationary phase; Speeds of mobile phase movement;Speeds of mobile phase movement; Column temperatures (in a gas chromatography);Column temperatures (in a gas chromatography); Lengths of a column;Lengths of a column; Partition coefficient (than it Partition coefficient (than it

is more for substance, the more its retention time).is more for substance, the more its retention time).)(/)( MPСSPCK

retention volume - the volume of mobile phase needed to move a solute from its point of injection to the detector (Vr).

baseline width - the width of a solute’s chromatographic band measured at the baseline (w).

Peak heightPeak height h or h’ (from a point of crossing of h or h’ (from a point of crossing of tangents with a zero line).tangents with a zero line).

PeakPeak widthwidth µµ0,50,5 - - distance between peak points on distance between peak points on half of height (or on any other mark on height).half of height (or on any other mark on height).

Relative Relative retentionretention time time rr and relative adjusted time and relative adjusted time ttrr is calculated on equation: is calculated on equation:

rr==//ss rr= (= ( - - 00) / () / (ss - - 00))!!! Relative retention times:•Less depend on external conditions, than retention time;•Resolve the serial analysis without standard samples of defined substances.

Often Often retention time isn’t measured, but measure time isn’t measured, but measure retention retention distance ldistance l - distance on - distance on chromatogramchromatogram from a point which from a point which correspondcorresponds of introduction sample moment to an absciss s of introduction sample moment to an absciss which which correspondcorrespond of peak maximum. of peak maximum.

The retention volumeThe retention volume it depends on speed it depends on speed of mobile phaseof mobile phase movementsmovements

V = V = The retention factorThe retention factor (delay) (delay) RR is a relation of moving speed is a relation of moving speed w w of of

the given component and the speed the given component and the speed uu of of mobile phasemobile phase movements: movements:

R = R = / / oror R = R = 00 / /

The capacity factorThe capacity factor k k - is equal to ratio of - is equal to ratio of relative retention time = = - - 00 of of given components to given components to 00::

k = (k = ( - - 00) / ) / 00

The more the The more the capacity factorcapacity factor kk, than more time of investigated , than more time of investigated component in component in stationary phasestationary phase..

At the beginning of a chromatographic separation the solute occupies a narrow band of finite width. As the solute passes through the column, the width of its band continually increases in a process called band broadening. Column efficiency provides a quantitative measure of the extent of band broadening.

In their original theoretical model of chromatography, Martin and Synge treated the chromatographic column as though it consists of discrete sections at which partitioning of the solute between the stationary and mobile phases occurs. They called each section a theoretical plate and defined column efficiency in terms of the number of theoretical plates, N, or the height of a theoretical plate, H

Martin and Synge theory (theoretical plate)Martin and Synge theory (theoretical plate)

!!! A column’s efficiency improves with an increase in the number of theoretical plates or a decrease in the height of a theoretical plate.

The number of theoretical plates in a chromatographic column is obtained by combining equations:

Alternatively, the number of theoretical plates can be approximated as

where w1/2 is the width of the chromatographic peak at half its height.

Rate theory (Rate theory (kinetic theorykinetic theory))

Van Deemter equation is an equation showing the effect of the mobile phase’s flow rate on the height of a theoretical plate.

where:A = Eddy-diffusionB = Longitudinal diffusionC = mass transfer kinetics of the analyte

between mobile and stationary phaseu = Linear Velocity.

The constant A depends on the size of particles, their density or density of column filling. The constant B is connected with diffusion factor of molecules in a mobile phase. The constant C characterises kinetics of sorption - desorption process, material transfers and other effects.

There is some disagreement on the correct equation for describing the relationship between plate height and mobile-phase velocity.

At small mobile-phase velocity the height At small mobile-phase velocity the height equivalent to a theoretical plate (HETP) decreases, equivalent to a theoretical plate (HETP) decreases, and then starts to increase. and then starts to increase.

The optimum mobile-phase velocity corresponds to The optimum mobile-phase velocity corresponds to a minimum in a plot of a minimum in a plot of H H as a function of as a function of u.u.

Optimum Optimum velocityvelocity of division which gives us a of division which gives us a considerable quantity of theoretical plates, and considerable quantity of theoretical plates, and accordingly small accordingly small HETP, is calculateHETP, is calculate: :

So, the kinetic theory gives a basis for optimisation So, the kinetic theory gives a basis for optimisation of chromatographicof chromatographic process. process.

BCAHоptim 2.

The goal of chromatography is to separate a sample into a series of chromatographic peaks, each representing a single component of the sample. ResolutionResolution is a quantitative measure of the degree of separation between two chromatographic peaks, 1 and 2, and is defined as

)2(5,0)1(5,0

l

Rs or)2(5,0)1(5,0

18,1

l

Rs

For two peaks of equal size, a resolution of 1.5 corresponds to an overlap in area of only 0.13%. Because resolution is a quantitative measure of a separation’s success, it provides a useful way to determine if a change in experimental conditions leads to a better separation.

Three examples of chromatographic resolution.

If peaks are mutually bridging, definition of peak width of each substance is impossible on separation degree :

2

min2

h

hh

In chromatography: the qualitative In chromatography: the qualitative analysis is based on definition of analysis is based on definition of retention retention

time and quantitative - on definition of time and quantitative - on definition of peak height or area.peak height or area.

4. The main parts of 4. The main parts of chromatographic equipment.chromatographic equipment.

A gas chromatograph with a headspace sampler

Requirements to adsorbentRequirements to adsorbent (Al (Al22OO33, silicagels, , silicagels,

the activated coal, porous capillaries on the the activated coal, porous capillaries on the basis of styrene, divinyl benzene, synthetic basis of styrene, divinyl benzene, synthetic zeolites):zeolites):

Necessary selectivity;Necessary selectivity; Chemical inertness to mix components;Chemical inertness to mix components; Availability.Availability.

DifferentialDifferential (concentration change (concentration change appear instant); are often appliedappear instant); are often applied

DetectorsDetectors

IntegratedIntegrated (fix concentration (fix concentration change for whole time interval), change for whole time interval),

are are applied not oftenapplied not often

To group of differential detectors belong:To group of differential detectors belong:Thermal Conductivity Detector (TCD)Thermal Conductivity Detector (TCD)

flame ionization detectorflame ionization detector

electron capture detector (ECD)electron capture detector (ECD)

Others depending on properties of system, a modular condition of Others depending on properties of system, a modular condition of

phases.phases.

5. Gas chromatography.5. Gas chromatography.

Gas-liquid chromatographyGas-liquid chromatography (GLC), or simply (GLC), or simply gas chromatographygas chromatography (GC), is a common type of (GC), is a common type of chromatography used in organic chemistry for chromatography used in organic chemistry for separating and analyzing compounds that can be separating and analyzing compounds that can be vaporized without decomposition. Typical uses of vaporized without decomposition. Typical uses of GC include testing the purity of a particular GC include testing the purity of a particular substance, or separating the different components of substance, or separating the different components of a mixture (the relative amounts of such components a mixture (the relative amounts of such components can also be determined). In some situations, GC can also be determined). In some situations, GC may help in identifying a compound.may help in identifying a compound.

The moving phaseThe moving phase (or "mobile phase") is a carrier gas, (or "mobile phase") is a carrier gas, usually an inert gas such as usually an inert gas such as heliumhelium or an unreactive or an unreactive gas such as gas such as nitrogennitrogen. .

The stationary phaseThe stationary phase is a microscopic layer of liquid is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of or polymer on an inert solid support, inside a piece of glass or metal tubing called a column.glass or metal tubing called a column.

The gaseous compounds being analyzed interact with The gaseous compounds being analyzed interact with the walls of the column, which is coated with different the walls of the column, which is coated with different stationary phases. This causes each compound to elute stationary phases. This causes each compound to elute at a different time, known as the retention time of the at a different time, known as the retention time of the compound. The comparison of retention times is what compound. The comparison of retention times is what gives GC its analytical usefulness.gives GC its analytical usefulness.

Qualitative analysis:Qualitative analysis: Generally chromatographic data is presented as a graph Generally chromatographic data is presented as a graph

of detector response (y-axis) against retention time (x-of detector response (y-axis) against retention time (x-axis), which is called a chromatogram. This provides a axis), which is called a chromatogram. This provides a spectrum of peaks for a sample representing the analytes spectrum of peaks for a sample representing the analytes present in a sample eluting from the column at different present in a sample eluting from the column at different times. Retention time can be used to identify analytes if times. Retention time can be used to identify analytes if the method conditions are constant. Also, the pattern of the method conditions are constant. Also, the pattern of peaks will be constant for a sample under constant peaks will be constant for a sample under constant conditions and can identify complex mixtures of analytes. conditions and can identify complex mixtures of analytes. In most modern applications however the GC is In most modern applications however the GC is connected to a mass spectrometer or similar detector that connected to a mass spectrometer or similar detector that is capable of identifying the analytes represented by the is capable of identifying the analytes represented by the peaks.peaks.

PharmacopoeiaPharmacopoeia offers three offers three methods for identification:methods for identification:

1) comparison of retention times of analyzed substance in 1) comparison of retention times of analyzed substance in the investigated samplethe investigated sample and in a comparison solution (a and in a comparison solution (a standard solution of investigated substance);standard solution of investigated substance);

2) comparison of relative retention times of analyzed 2) comparison of relative retention times of analyzed substance in substance in the investigated samplethe investigated sample and a comparison and a comparison solution (if precision of condition chromotographic solution (if precision of condition chromotographic analyses isn’t possible);analyses isn’t possible);

3) comparison of 3) comparison of chromatogram ofchromatogram of investigated sampleinvestigated sample with with chromatogramchromatogram of comparison solution or with of comparison solution or with chromatogramchromatogram, resulted in separate article (for , resulted in separate article (for preparations of herbal and animal origin).preparations of herbal and animal origin).

Separation process is based on differences in Separation process is based on differences in fugitiveness and solubilities separated components.fugitiveness and solubilities separated components.

The quantitative analysis can be made only in the event The quantitative analysis can be made only in the event

that the that the substance is heat-resistantsubstance is heat-resistant, that it evaporates , that it evaporates reproducibility and eluateed from column without reproducibility and eluateed from column without decomposition. At substance decomposition on decomposition. At substance decomposition on chromatogram are artificial peaks which belong to chromatogram are artificial peaks which belong to decomposition products.decomposition products.

Quantitive analysis:Quantitive analysis: The area under a peak is proportional to the amount of The area under a peak is proportional to the amount of

analyte present in the chromatogram. By calculating the analyte present in the chromatogram. By calculating the area of the peak using the mathematical function of area of the peak using the mathematical function of integration, the concentration of an analyte in the original integration, the concentration of an analyte in the original sample can be determined. Concentration can be sample can be determined. Concentration can be calculated using a calibration curve created by finding the calculated using a calibration curve created by finding the response for a series of concentrations of analyte, or by response for a series of concentrations of analyte, or by determining the relative response factor of an analyte. The determining the relative response factor of an analyte. The relative response factor is the expected ratio of an analyte relative response factor is the expected ratio of an analyte to an internal standard (or external standard) and is to an internal standard (or external standard) and is calculated by finding the response of a known amount of calculated by finding the response of a known amount of analyte and a constant amount of internal standard (a analyte and a constant amount of internal standard (a chemical added to the sample at a constant concentration, chemical added to the sample at a constant concentration, with a distinct retention time to the analyte).with a distinct retention time to the analyte).

Pharmacopoeia demands to make definition of the Pharmacopoeia demands to make definition of the quantitative contents through quantitative contents through the areas of peaksthe areas of peaks, , if if symmetrysymmetry factorfactor of peak from 0.8 to 1.20 it is of peak from 0.8 to 1.20 it is possible to apply possible to apply in place of the areas in place of the areas by peak by peak heightsheights..

In case of usage of In case of usage of temperature programmingtemperature programming the the quantitative analysis is made only through the quantitative analysis is made only through the peak areaspeak areas..

Symmetry factor of peakSymmetry factor of peak: : 0.050.05/2А/2А,,

where: where: 0.050.05 - width to peak on 1/20 of peak height; - width to peak on 1/20 of peak height; AA - distance between a perpendicular - distance between a perpendicular

lowered from peak maximum and forward border lowered from peak maximum and forward border of peak on 1/20 peak height.of peak on 1/20 peak height.

Methods of quantitive analysisMethods of quantitive analysis1. Normalization method.1. Normalization method.

Accept the sum of all peak parametres (Accept the sum of all peak parametres (h, h, oror S, S, oror width of all peaks) for 100 %. Then the ration of width of all peaks) for 100 %. Then the ration of height of individual peak to sum of heights or the height of individual peak to sum of heights or the ration of one peak area to sum of the areas and ration of one peak area to sum of the areas and multiply on 100 will characterise Wmultiply on 100 will characterise W(component)(component) (%) in (%) in

a mix. a mix.

This method is used when it is identical dependence This method is used when it is identical dependence of value of the measured parametre from of value of the measured parametre from concentration of all components of a mix.concentration of all components of a mix.

2. Normalization method with calibration factors2. Normalization method with calibration factorsIn this method accept the sum of all peak parametres for 100 In this method accept the sum of all peak parametres for 100

% with due regard for sensitivity of the detector. % with due regard for sensitivity of the detector. Differences in sensitivity of the detector are considered Differences in sensitivity of the detector are considered with the aid of correction factors for each component. One with the aid of correction factors for each component. One of dominating components of a mix consider comparative of dominating components of a mix consider comparative and correction factor for it accept equal to one. and correction factor for it accept equal to one. CalibrationCalibration factor Кі factor Кі is calculatedis calculated::

ССii - concentration of i-component in a modelling mix with - concentration of i-component in a modelling mix with standard substance;standard substance;

ССstanstan - concentration of standard substance; - concentration of standard substance;ППstanstan and and ППii - parametres of substance peaks - standard and i- - parametres of substance peaks - standard and i-

component.component.

dtansі

іtansi СП

СПK

For 100 % accept the sum of corrected parametres For 100 % accept the sum of corrected parametres ККііППіі and result of the analysis is calculated such and result of the analysis is calculated such

as in as in normalizationnormalization method: method:

AdvantageAdvantage: 1) metered flow of sample isn’t : 1) metered flow of sample isn’t necessary;necessary;

2) not absolute identity of analysis conditions at 2) not absolute identity of analysis conditions at repeated definitions.repeated definitions.

n

1ііі

ііX

КП

КПC

3. A method of absolute calibration (the most exact).3. A method of absolute calibration (the most exact).

Experimentally define dependence of peak height or Experimentally define dependence of peak height or area from substance concentration and plot area from substance concentration and plot calibrationcalibration chartchart. On . On calibrationcalibration chartchart calculate calculate concentration of analyzed substance.concentration of analyzed substance.

It is the basic method of It is the basic method of definition of impuritydefinition of impurity..

Advantage:Advantage: does not demand separation of all does not demand separation of all components on sample, but only that substance components on sample, but only that substance are necessary for defining.are necessary for defining.

4. A method of the internal standard.4. A method of the internal standard.

Is based on introduction in an analyzed mix of precisely Is based on introduction in an analyzed mix of precisely known quantity of standard substance. As standard known quantity of standard substance. As standard substance choose the substance similar on physical and substance choose the substance similar on physical and chemical properties to components in mix, but not chemical properties to components in mix, but not necessarily it is component of a mix. necessarily it is component of a mix.

To an investigated solution and a standard solution of To an investigated solution and a standard solution of investigated substance add strictly identical quantity of investigated substance add strictly identical quantity of the internal standard.the internal standard.

After After chromatographychromatography measure peak parametres of an measure peak parametres of an analyzed component and the internal standard on analyzed component and the internal standard on chromatogramchromatogram of investigated solution and the same of investigated solution and the same parametres on parametres on chromatogramchromatogram of standard solution of of standard solution of defined substance.defined substance.

Advantage:Advantage: 1) the account of chromatograph duty 1) the account of chromatograph duty (t°, (t°, mobile phase). mobile phase).

2) increase of analysis accuracy because of 2) increase of analysis accuracy because of independence of reproducibility parallel independence of reproducibility parallel chromatographic experiment series and chromatographic experiment series and chromatography of standard and investigated chromatography of standard and investigated solutions.solutions.

Requirements to the internal standard:Requirements to the internal standard: Good solubility in sample and chemical inertness to components Good solubility in sample and chemical inertness to components

of an analyzed mix, stationary phase and the firm carrier.of an analyzed mix, stationary phase and the firm carrier. The internal standard choose from compounds which similar to The internal standard choose from compounds which similar to

objects of the analysis on structure and fugitiveness.objects of the analysis on structure and fugitiveness. Quantity of the internal standard in sample select so that the Quantity of the internal standard in sample select so that the

ration of peak areas of the standard and defined substance was ration of peak areas of the standard and defined substance was close to 1.close to 1.

The peak of the internal standard on chromatogram should take The peak of the internal standard on chromatogram should take places affinity to peaks of compounds - objects of the analysis, places affinity to peaks of compounds - objects of the analysis, not being imposed neither on them, nor on peaks of other not being imposed neither on them, nor on peaks of other substances. substances.

The internal standard should not contain impurity which are The internal standard should not contain impurity which are imposed with peaks of defined substances-components of sample.imposed with peaks of defined substances-components of sample.

If define in sample two and more substances (which considerably If define in sample two and more substances (which considerably differ retention times) so it is expedient to use 2 and more internal differ retention times) so it is expedient to use 2 and more internal standards.standards.

Application in the pharmaceutical analysis, Application in the pharmaceutical analysis, technology and the toxicological analysis:technology and the toxicological analysis:1. Quality assurance of substances and medicinal forms - 1. Quality assurance of substances and medicinal forms -

identification and quantitative definition of the flying identification and quantitative definition of the flying residual solvents, which may be in drugs after their residual solvents, which may be in drugs after their reception.reception.

2. Definition of ethanol content and other organic solvents in 2. Definition of ethanol content and other organic solvents in ready drugs.ready drugs.

3. Definition of preservatives (Nipagin, sorbic acid) in 3. Definition of preservatives (Nipagin, sorbic acid) in children's syrups.children's syrups.

4. Definition of some preservatives in injection solutions4. Definition of some preservatives in injection solutions

5. Quality assurance galena preparations.5. Quality assurance galena preparations.

6. Theoretical base of liquid 6. Theoretical base of liquid chromatographychromatography

Liquid chromatographyLiquid chromatography (LC) is a separation technique in (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. can be carried out either in a column or a plane.

Precondition of occurrence and applicationPrecondition of occurrence and application::- Substances with big molar weight Substances with big molar weight - Nonvolatile substancesNonvolatile substances- thermally unstable substancesthermally unstable substances- Ionic substansesIonic substanses

LC is based on adsorption from a solution. LC is based on adsorption from a solution.

Adsorptive balance between solution and adsorbent will Adsorptive balance between solution and adsorbent will

be co-ordinated with the equation of Langmuir isotherm, be co-ordinated with the equation of Langmuir isotherm,

in the range of the diluted solutions the isotherm is linear.in the range of the diluted solutions the isotherm is linear.

Selectivity of adsorption depends by nature forces of Selectivity of adsorption depends by nature forces of

interaction between adsorbed substances and adsorbent. interaction between adsorbed substances and adsorbent.

H = 2RH = 2Rrr(1 – R(1 – Rrr)Ut)Utss,,

,sm

mr tt

tR

!!! !!! The choice of mobile phase is always more The choice of mobile phase is always more important, than a choice of stationary phase.important, than a choice of stationary phase.

The stationary phase The stationary phase should retention divided should retention divided substancessubstances. .

The mobile phase The mobile phase (solvent or more often mix of (solvent or more often mix of solvents) should provide different capacity of a solvents) should provide different capacity of a column and effective separation in optimum time.column and effective separation in optimum time.

The stationary phase The stationary phase is porous finely dispersed is porous finely dispersed materials with a specific surface is more 50 mmaterials with a specific surface is more 50 m22/g/g

Stationary phaseStationary phase

Polar:Polar: SiO SiO22, Al, Al22OO33, Me, MexxOOyy, and also with , and also with

graft polargraft polar groups (–NHgroups (–NH22, - ОН, diols), - ОН, diols)

Non-polar:Non-polar: dag, dag, kieselguhrkieselguhr, , diatomitediatomite..

Polar stationary phasesPolar stationary phases is used for separation: is used for separation:- non-polar, low-polar and mean polar substancenon-polar, low-polar and mean polar substance

Non-polar stationary phasesNon-polar stationary phases is used for is used for separationseparation::

- they don’t have selectivity to polar moleculesthey don’t have selectivity to polar molecules

These sorbents are put on superficial-porous These sorbents are put on superficial-porous carriers.carriers.

Mobile phaseMobile phase

From it depends:From it depends: Selectivity of separation;Selectivity of separation; Efficiency of column;Efficiency of column; velocity of chromatographic zone velocity of chromatographic zone

movement.movement.

Requirements to a mobile phase:Requirements to a mobile phase:

Should dissolve investigated sample;Should dissolve investigated sample; Small viscosity (diffusion factors D of sample components are Small viscosity (diffusion factors D of sample components are

high enough);high enough); Possible excretion from it divided components;Possible excretion from it divided components; It must be inertness in relation to materials of all parts of It must be inertness in relation to materials of all parts of

chromatograph;chromatograph; Meets the requirements of the chosen detector;Meets the requirements of the chosen detector; The safe;The safe; The cheap.The cheap.

!!!!!! As it has been told, As it has been told, elutingeluting force of a mobile phaseforce of a mobile phase – solvent – solvent influences on seperation.influences on seperation.

Eluting force of solvent shows, in how many time Eluting force of solvent shows, in how many time energy of eluent sorption more than energy of energy of eluent sorption more than energy of eluent sorption, chosen as the standard, for eluent sorption, chosen as the standard, for example, n-heptane. example, n-heptane.

Solvents (eluents) divide on weak and strongSolvents (eluents) divide on weak and strong.. Weak eluentsWeak eluents are a little are a little adsorbed by stationary adsorbed by stationary

phase therefore distribution factors D of sorbate is phase therefore distribution factors D of sorbate is high.high.

Strong eluentsStrong eluents are strongly adsorbed by stationary are strongly adsorbed by stationary phase, therefore distribution factors D of sorbed phase, therefore distribution factors D of sorbed substances (sorbate) is low.substances (sorbate) is low.

For example:For example: SiOSiO22 – stationary phase – stationary phase..

Force of solvents Force of solvents increases in rangeincreases in range::

pentane (0) pentane (0) CCl CCl44 (0,11) (0,11) C C66HH66 (0,25) (0,25)

CHClCHCl33 (0,26) (0,26) CH CH22ClCl22 (0,32) (0,32) acetone (0,47) acetone (0,47)

dioxane (0,49) dioxane (0,49) acetonitrile (0,5) acetonitrile (0,5) ethanol ethanol

methanol.methanol.

Sequence of solvents according to their increase Sequence of solvents according to their increase eluting forces is named eluting forces is named eluotropic series. eluotropic series.

In liquid adsorptive chromatography In liquid adsorptive chromatography eluotropic eluotropic Snyder's Snyder's seriesseries::

pentane (0) pentane (0) n-hexane (0,01) n-hexane (0,01) cyclohexane cyclohexane (0,04) (0,04) CCl CCl44 (0,18) (0,18) benzene (0,32) benzene (0,32) CHCl CHCl33

(0,38) (0,38) acetone (0,51), ethanol (0,88) acetone (0,51), ethanol (0,88) water, СНwater, СН33СООН (very big).СООН (very big).

Eluotropic seriesEluotropic series forfor reverse phase reverse phase chromatography on chromatography on СС1818::

methanolmethanol (1,0) (1,0) acetonitrileacetonitrile (3,1), (3,1),

ethanolethanol (3,1) (3,1) isopropanole (8,3) isopropanole (8,3)

nn-propanole (10,1) -propanole (10,1) dioxanedioxane (11,7). (11,7).

Methods of elutionMethods of elution1. When a separation uses a single mobile phase

of fixed composition it is called an isocratic elution. It is often difficult, however, to find a single mobile-phase composition that is suitable for all solutes.

2. gradient elution is the process of changing the mobile phase’s solvent strength to enhance the separation of both early and late eluting solutes.

Rules of thumbRules of thumb help us at choice of eluent help us at choice of eluent. . Sorption increases at increase number of Sorption increases at increase number of olefinic linkageolefinic linkage and and ОН-groupsОН-groups in in compounds. compounds.

Sorption decrease (for organic Sorption decrease (for organic compound)compound): : acidsacids alcoholsalcohols aldehydes aldehydes ketones ketones estersesters unsaturated unsaturated hydrocarbon hydrocarbon saturated hydrocarbon. saturated hydrocarbon.

7. HPLC – high-performance liquid 7. HPLC – high-performance liquid chromatographychromatography

In In HPLCHPLC both both contacting phases – contacting phases – stationary (SP) and mobile (MP) phase are stationary (SP) and mobile (MP) phase are liquids.liquids.

The method distributive orThe method distributive or a liquid-a liquid-liquidliquid chromatographychromatography is based on is based on substance substance distribution between two distribution between two phasesphases which do not mix. which do not mix.

Separation of components is based Separation of components is based on differences of their distribution factors on differences of their distribution factors between SP and MP.between SP and MP.

Normal phase partition chromatographyNormal phase partition chromatography stationary phase: always polar solvent stationary phase: always polar solvent

(water, alcohol) is fixed on firm carrier(water, alcohol) is fixed on firm carrier – – silica gel, diatomite, cellulose, Alsilica gel, diatomite, cellulose, Al22OO33. .

mobile phase:mobile phase: non-polar solventnon-polar solvent – – isooctane, benzene , etc. isooctane, benzene , etc.

Reverse phase partition Reverse phase partition chromatographychromatography

Stationary phase: non-polar solvent is Stationary phase: non-polar solvent is fixed on firm carrierfixed on firm carrier

Mobile phase: polar solvent Mobile phase: polar solvent (water, (water, alcohol, buffer solutions, strong acids) alcohol, buffer solutions, strong acids)

In liquid–liquid chromatography the stationary phase is In liquid–liquid chromatography the stationary phase is a liquid film coated on a packing material consisting a liquid film coated on a packing material consisting of 3–10 of 3–10 µµm porous silica particles. The stationary m porous silica particles. The stationary phase may be partially soluble in the mobile phase, phase may be partially soluble in the mobile phase, causing it to “bleed” from the column over time. To causing it to “bleed” from the column over time. To prevent this loss of stationary phase, it is covalently prevent this loss of stationary phase, it is covalently bound to the silica particles. bound to the silica particles. Bonded stationary Bonded stationary phasesphases are attached by reacting the silica particles are attached by reacting the silica particles with an organochlorosilane of the general form with an organochlorosilane of the general form Si(CHSi(CH33))22RCl, where R is an alkyl or substituted alkyl RCl, where R is an alkyl or substituted alkyl group.group.

Stationary phase in normal phase Stationary phase in normal phase chromatographychromatography

To prevent unwanted interactions between the solutes and To prevent unwanted interactions between the solutes and any unreacted –SiOHany unreacted –SiOH groups, the silica frequently is groups, the silica frequently is “capped” by reacting it with Si(CH“capped” by reacting it with Si(CH33))33Cl; suchCl; such columns columns

are designated as end-capped.are designated as end-capped. The properties of a stationary phase are determined by the The properties of a stationary phase are determined by the

nature of thenature of the organosilane’s alkyl group. If R is a polar organosilane’s alkyl group. If R is a polar functional group, then the stationaryfunctional group, then the stationary phase will be polar. phase will be polar. Examples of polar stationary phases include those for Examples of polar stationary phases include those for which Rwhich R contains a cyano (–Ccontains a cyano (–C22HH44CN), diol (–CN), diol (–

CC33HH66OCHOCH22CHOHCHCHOHCH22OH), or aminoOH), or amino (–C(–C33HH66NHNH22) )

functional group. Since the stationary phase is polar, the functional group. Since the stationary phase is polar, the mobile phasemobile phase is a nonpolar or moderately polar solvent.is a nonpolar or moderately polar solvent.

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain. Most reversephase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less

Types of detector:Types of detector: Refractive index – generalpurpose detector Refractive index – generalpurpose detector

(sensitivity ~ 10(sensitivity ~ 10–6–6 g); g); UV/Vis-detector (sensitivity 10UV/Vis-detector (sensitivity 10–9–9 g) (254 nm); g) (254 nm); photometric і spectrophotometric;photometric і spectrophotometric; Photodiode array;Photodiode array; Fluorescent detector for definition toxines, Fluorescent detector for definition toxines,

microbiological objects, vitamins;microbiological objects, vitamins; Conductometric (0,01 Conductometric (0,01 µµg/mL – 100 mg/mL).g/mL – 100 mg/mL).

The qualitative analysis (pharmacopoeia):The qualitative analysis (pharmacopoeia): Comparison of retention timesComparison of retention times of analyzed substance in of analyzed substance in

the investigated sample and a comparison solution (a the investigated sample and a comparison solution (a standard solution of the same substance) – is used more standard solution of the same substance) – is used more often;often;

Comparison of relative retention timesComparison of relative retention times of analyzed of analyzed substance the investigated sample and a comparison substance the investigated sample and a comparison solution (a standard solution of the same substance) – is solution (a standard solution of the same substance) – is applied, when is possible non-reproducible conditions of applied, when is possible non-reproducible conditions of chromatography;chromatography;

Comparison of chromatogramComparison of chromatogram of of the investigated the investigated sample sample with with chromatogramchromatogram of comparison solution or of comparison solution or with chromatogram, resulted in separate article (for with chromatogram, resulted in separate article (for preparations of plant and animal material).preparations of plant and animal material).

For quantitative definition to define the peak For quantitative definition to define the peak areas or peak height (pharmacopoeia):areas or peak height (pharmacopoeia):

– Heights are considered only at isocratic elution Heights are considered only at isocratic elution and at factor of asymmetry 0.8-1.2.and at factor of asymmetry 0.8-1.2.

– Peak areas are considered only at gradient Peak areas are considered only at gradient elution.elution.

The quantitative analysis (pharmacopoeia) for The quantitative analysis (pharmacopoeia) for main component:main component:

– Absolute calibrationAbsolute calibration– Method of the internal standard.Method of the internal standard.

The quantitative analysis (pharmacopoeia) of The quantitative analysis (pharmacopoeia) of impurities:impurities:

Quantitative definition of impurity with usage of Quantitative definition of impurity with usage of comparison solution (known concentration of comparison solution (known concentration of impurity);impurity);

Method of internal normalisation;Method of internal normalisation; Comparison with the diluted solution of the Comparison with the diluted solution of the

main substance;main substance; Method of standard additives (for accuracy Method of standard additives (for accuracy

increase use of a method of the internal standard increase use of a method of the internal standard is possible). is possible).

At a technique necessarily mark:At a technique necessarily mark:

– Column parametres (the sizes, a sorbent, Column parametres (the sizes, a sorbent, commercial mark, the size of particles of commercial mark, the size of particles of stationary phase);stationary phase);

– Column temperature;Column temperature;

– Speed and structure of mobile phase;Speed and structure of mobile phase;

– Detector type.Detector type.

Thin layer chromatography (TLC) is a chromatography Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with the a thin layer of or aluminum foil, which is coated with the a thin layer of adsorbent material, usually silica gel, aluminium oxide, adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the or cellulose. This layer of adsorbent is known as the stationary phase.stationary phase.

After the sample has been applied on the plate, a solvent After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, analytes ascend the TLC plate at different rates, separation is achieved.separation is achieved.

8. TLC – thin-layer 8. TLC – thin-layer chromatographychromatography

A small spot of solution containing the sample is A small spot of solution containing the sample is applied to a plate, about one centimeter from the applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and solvent, such as hexane or ethyl acetate, and placed in a sealed container. The solvent moves placed in a sealed container. The solvent moves up the plate by capillary action and meets the up the plate by capillary action and meets the sample mixture, which is dissolved and is carried sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds up the plate by the solvent. Different compounds in the sample mixture travel at different rates due in the sample mixture travel at different rates due to the differences in their attraction to the to the differences in their attraction to the stationary phase, and because of differences in stationary phase, and because of differences in solubility in the solvent.solubility in the solvent.

the Rthe Rff value , or Retention factor, of each spot value , or Retention factor, of each spot

can be determined by dividing the distance can be determined by dividing the distance traveled by the product by the total distance traveled by the product by the total distance traveled by the solvent (the solvent front). traveled by the solvent (the solvent front).

oror relativerelative Retention factorRetention factor::

ff X

XR 1

dardtans,f

X,frelative,f R

RR

Factors which influence on value RFactors which influence on value Rff::

Quality and activity of sorbent;Quality and activity of sorbent;Sorbent moisture;Sorbent moisture;Thickness of a layer of a sorbent;Thickness of a layer of a sorbent;Quality of solventQuality of solvent

The number of theoretical plates nThe number of theoretical plates n 2)(16l

b

l

ln

n

lH н

nRR

RRK

XfXf

XfXff

21

21

,,

,,

the height of a theoretical platethe height of a theoretical plate

separation factorseparation factor

lb – distance from start line to a stain bottom edge;ll – distance which shows length of a stain in direction of

movement of solvent front.

Sorbents in ТLС:Sorbents in ТLС:– Silica gel;Silica gel;– Aluminium oxide;Aluminium oxide;– Starch; Starch; – Cellulose and some other substances with high Cellulose and some other substances with high

adsorptiveadsorptive ability ability

TLC:TLC: Ascending ChromatographyAscending Chromatography Descending ChromatographyDescending Chromatography Circular ChromatographyCircular Chromatography

Substrates for a sorbent (plate):Substrates for a sorbent (plate):

– Glass;Glass;

– Aluminium foil;Aluminium foil;

– Polyester film.Polyester film.

The choice of solvents depends from:The choice of solvents depends from:The sorbent nature;The sorbent nature;Properties of investigated substances.Properties of investigated substances.

Zones on chromatogram are Zones on chromatogram are displayed displayed physical or physical or chemical methods after termination of chemical methods after termination of carrying carrying

out chromatographyout chromatography..

Physical methodPhysical method – UV-light or radio autographies – UV-light or radio autographies (photographic materials – a paper or films).(photographic materials – a paper or films).

Chemical methodChemical method – chromatogram are displayed – chromatogram are displayed by reactant solution sprayingby reactant solution spraying

Equipment for ТLС:Equipment for ТLС:

– A sealed container with suitable solvents;A sealed container with suitable solvents;

– Chromatographic plates (Merck, Silufol, Chromatographic plates (Merck, Silufol, Sorbfil and others);Sorbfil and others);

– Micro syringes, TLC Manual Sampler or Micro syringes, TLC Manual Sampler or Micropipettes;Micropipettes;

– TLC Viewing Box 254/366 nm;TLC Viewing Box 254/366 nm;

– TLC SPRAYER (ATOMIZER) and TLC TLC SPRAYER (ATOMIZER) and TLC Spray Cabinet;Spray Cabinet;

– TLC Plate Heater;TLC Plate Heater;

The qualitative analysis in ТСХ at identification The qualitative analysis in ТСХ at identification (pharmacopoeia):(pharmacopoeia):

Comparison stain of investigated substance and standard Comparison stain of investigated substance and standard substance. They must be identical on colouring (colour substance. They must be identical on colouring (colour of fluorescence), retention factor and size on of fluorescence), retention factor and size on chromatogram.chromatogram.

!!!!!! Check of Check of operability operability of TLC-PLATESof TLC-PLATES: the standard : the standard mix of indicators – mix of indicators – sudan red G, bromcresol green, sudan red G, bromcresol green, methyl red and methyl orange.methyl red and methyl orange.

suitable solvents: methanol – toluene (20:80). suitable solvents: methanol – toluene (20:80). When the front of solvents will pass When the front of solvents will pass ⅔⅔ of plate length, it of plate length, it

is considered suitable if on chromatogram 4 divided is considered suitable if on chromatogram 4 divided stains are accurate:stains are accurate:– Stain of bromcresol green with Stain of bromcresol green with RfRf is no more 0,15; is no more 0,15;– Stain of methyl orange with Stain of methyl orange with RfRf from 0,1 to 0.25; from 0,1 to 0.25;– Stain of methyl red with Stain of methyl red with RfRf from 0,35 to 0,55; from 0,35 to 0,55;– Stain of sudan red G with Stain of sudan red G with RfRf from 0,75 to 0.98. from 0,75 to 0.98.

Quantitative analysis:Quantitative analysis: Direct definition on a plate by means of Direct definition on a plate by means of

measurement transmission of light or reflexions measurement transmission of light or reflexions of light, fluorescence.of light, fluorescence.

Directly on a plate or on the separate cut strips of Directly on a plate or on the separate cut strips of plates by means of radio-activity counters.plates by means of radio-activity counters.

After removal of stationary phase, its dissolution After removal of stationary phase, its dissolution in corresponding solvent and measurements of a in corresponding solvent and measurements of a suitable physical indicator or a radio-activity of suitable physical indicator or a radio-activity of the received solution.the received solution.

Thin layer chromatography finds Thin layer chromatography finds many applications, including:many applications, including:

assaying the radiochemical purity of assaying the radiochemical purity of radiopharmaceuticalsradiopharmaceuticals

determination of the pigments a plant containsdetermination of the pigments a plant contains detection of pesticides or insecticides in fooddetection of pesticides or insecticides in food analysing the dye composition of fibers in forensics, oranalysing the dye composition of fibers in forensics, or identifying compounds present in a given substanceidentifying compounds present in a given substance monitoring organic reactions.monitoring organic reactions.

9. Paper chromatography9. Paper chromatography Paper chromatographyPaper chromatography is Chemestrial technique for is Chemestrial technique for

separating and identifying mixtures that is or can be separating and identifying mixtures that is or can be colored, especially pigments. This can also be used in colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This secondary or primary colors in ink experiments. This method has been largely replaced by thin layer method has been largely replaced by thin layer chromatography, however it is still a powerful chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in using two solvents and rotating the paper 90° in between. This is useful for separating complex between. This is useful for separating complex mixtures of similar compounds, for example, amino mixtures of similar compounds, for example, amino acids.acids.

The paper is then dipped into a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper, also this can be explained as differential adsorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper.

Technique of paper Chromatography:Technique of paper Chromatography: Ascending ChromatographyAscending Chromatography Descending ChromatographyDescending Chromatography Circular ChromatographyCircular Chromatography

The qualitative analysisThe qualitative analysis – as in TLC. – as in TLC. The quantitative analysisThe quantitative analysis::

Visual estimation – comparison of colouring Visual estimation – comparison of colouring intensity of stains;intensity of stains;

Estimation of stain areas;Estimation of stain areas;Cutting of stains and their weighing of investigated Cutting of stains and their weighing of investigated

sample and a standard solution;sample and a standard solution;Eluating of substances from stains and the next Eluating of substances from stains and the next

definition by a physical and chemical method.definition by a physical and chemical method.

10. Ion-exchange 10. Ion-exchange chromatographychromatography

Ion-exchange chromatographyIon-exchange chromatography (or ion (or ion chromatography) is a process that allows the separation chromatography) is a process that allows the separation of ions and polar molecules based on the charge of ions and polar molecules based on the charge properties of the molecules. It can be used for almost properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually injected is usually called a sample, and the individually separated components are called analytes. separated components are called analytes.

It is often used in protein purification, water It is often used in protein purification, water analysis, and quality control.analysis, and quality control.

Ion exchange chromatography retains analyte molecules Ion exchange chromatography retains analyte molecules based on coulombic (ionic) interactions. The stationary based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation of chromatography is further subdivided into cation exchange chromatography and anion exchange exchange chromatography and anion exchange chromatography. The ionic compound consisting of the chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be cationic species M+ and the anionic species B- can be retained by the stationary phase.retained by the stationary phase.

Cation exchange chromatography retains positively Cation exchange chromatography retains positively charged cations because the stationary phase displays a charged cations because the stationary phase displays a negatively charged functional group:negatively charged functional group:

Anion exchange chromatography retains anions Anion exchange chromatography retains anions using positively charged functional group:using positively charged functional group:

Note that the ion strength of either C+ or A- in the mobile Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position and phase can be adjusted to shift the equilibrium position and thus retention time.thus retention time.

The ion chromatogram shows a typical chromatogram The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.obtained with an anion exchange column.

Thanks for your attention!Thanks for your attention!