Planar Planar Chromatography Chromatography
UNIT
UNIT 6
6 PLANAR
PLANAR CHROMATOGRAPHY
CHROMATOGRAPHY
Structure Structure 6.1 Introduction 6.1 Introduction Objectives Objectives 6.2
6.2 Paper Paper ChromatogrChromatographyaphy Principle Principle Stationary Support Stationary Support Solvent Systems Solvent Systems Development of Chromatogram Development of Chromatogram Detection Methods Detection Methods Applications Applications 6.3
6.3 Thin Thin Layer Layer ChromatographyChromatography Stationary Phases
Stationary Phases Mobile Phases Mobile Phases
Apparatus and Requirements Apparatus and Requirements Detections Methods
Detections Methods
Plate Concept Applied to TLC Plate Concept Applied to TLC
High-Performance Thin Layer Chromatography (HPTLC) High-Performance Thin Layer Chromatography (HPTLC) Applications
Applications 6.4
6.4 Quantitative Aspects Quantitative Aspects of of PC PC and and TLCTLC 6.5
6.5 Comparison of Comparison of PC PC and and TLCTLC 6.6 Summary
6.6 Summary 6.7
6.7 Terminal QuestionsTerminal Questions 6.8 Answers
6.8 Answers
6.1
6.1
INTRODUCTION
INTRODUCTION
So far you have
So far you have studied about the general principles of studied about the general principles of chromatograchromatography wherephy where theoretical principles including resolution and plate concept were
theoretical principles including resolution and plate concept were dealt. You nowdealt. You now know that chromatography is now a very powerful separation technique used not know that chromatography is now a very powerful separation technique used not onlyonly for the separation of complex mixtures but it is also
for the separation of complex mixtures but it is also used for quantification of eachused for quantification of each constituent. You have also learnt
constituent. You have also learnt about classification of various chromatographicabout classification of various chromatographic techniques which include a
techniques which include a wide range of wide range of ion-exchangion-exchange chromatography, affinitye chromatography, affinity chromatograp
chromatography, gel fhy, gel filtration, electro iltration, electro chromatograchromatography, zone electrophoresis, sizephy, zone electrophoresis, size exclusion chromatography
exclusion chromatography etcetc.. In this unit, you will learn
In this unit, you will learn aboutabout planar chromat planar chromatography,ography,which includeswhich includes paper paper chromatography
chromatography (PC), (PC), thin layer thin layer chromatogrchromatographyaphy (TLC) and (TLC) and electro electro chromatogrchromatographyaphy (EC). Each of these techniques make use of a
(EC). Each of these techniques make use of a stationary phase in the form of a sheet orstationary phase in the form of a sheet or flat surface of a paper or any other
flat surface of a paper or any other material such as metal, glass or material such as metal, glass or plastic plate coatedplastic plate coated with suitable adsorbent with the help of a binder. The mobile phase
with suitable adsorbent with the help of a binder. The mobile phase moves througmoves through theh the stationary phase by capillary action, assisted by gravity or an electrical potential. Here, stationary phase by capillary action, assisted by gravity or an electrical potential. Here, we shall discuss
we shall discuss only two techniquesonly two techniques i.ei.e. PC and TLC and a . PC and TLC and a comparative study of theircomparative study of their principles, methodology and applications will
principles, methodology and applications will be undertaken.be undertaken. Once upon a time, term
Once upon a time, term planar chromatography planar chromatography included two-dimensionalincluded two-dimensional chromatograp
chromatography though it has hy though it has now come to signify the now come to signify the coupling of two chromatographiccoupling of two chromatographic techniques with different mechanisms of separation. It is called ‘planar’ because the techniques with different mechanisms of separation. It is called ‘planar’ because the stationary support consists of the plane surface of a paper or smooth glass plate. It stationary support consists of the plane surface of a paper or smooth glass plate. It includes
includes paper chrom paper chromatographyatography (PC) and (PC) and thin layer thin layer chromatogchromatographyraphy (TLC) which are (TLC) which are the simplest of all o
the simplest of all o ther forms of chromatogther forms of chromatographic techniques. It also includesraphic techniques. It also includes electrophoresis
electrophoresis or or electro electro chromatogchromatographyraphy where the movement of mobile phase is where the movement of mobile phase is assisted by electrical potential. However, it will
assisted by electrical potential. However, it will not be discussed here.not be discussed here.
The paper chromatography (PC) and thin layer chromatography (TLC) have the The paper chromatography (PC) and thin layer chromatography (TLC) have the advantage of being simple, fast and inexpensive. These have been widely used for the advantage of being simple, fast and inexpensive. These have been widely used for the
Chromatographic Chromatographic Methods-I
Methods-I qualitative identification of different constituents in a qualitative identification of different constituents in a mixture though these could bealso used for quantitative determination of also used for quantitative determination of the components. However, PC is not usedthe components. However, PC is not usedmixture though these could be so commonly these days and as
so commonly these days and as of now, planar chromatography based on TLC hasof now, planar chromatography based on TLC has found widest applications in organic synthesis laboratory, drug industry, clinical found widest applications in organic synthesis laboratory, drug industry, clinical research and for investig
research and for investigating biochemical processes. ating biochemical processes. In both the cases, sample isIn both the cases, sample is spotted with a micropipette on to a paper or a
spotted with a micropipette on to a paper or a plate and then the chromatogram isplate and then the chromatogram is developed using a suitable organic solvent. Each constituent present in the sample is developed using a suitable organic solvent. Each constituent present in the sample is identified on the basis of colour development using a suitable detection system. identified on the basis of colour development using a suitable detection system. Different compone
Different components of a solute travel nts of a solute travel with different speeds and the distance travelledwith different speeds and the distance travelled by each component with respect to that of solvent gives a parameter called
by each component with respect to that of solvent gives a parameter called ‘retardation‘retardation factor’ or
factor’ or R R f f ..
Even though PC and TLC have many things in common but there are some basic Even though PC and TLC have many things in common but there are some basic differences in their operation. Here, we shall discuss the basic principles, apparatus differences in their operation. Here, we shall discuss the basic principles, apparatus required, methodology and some typical applications of both the
required, methodology and some typical applications of both the techniques in thetechniques in the separation of complex mixt
separation of complex mixtures of organic and inorgures of organic and inorganic compounds. anic compounds. We shall firstWe shall first describe paper chromatography
describe paper chromatography, its , its principle, methodology and applications. PC is thprinciple, methodology and applications. PC is th ee simplest of all the chromatographic techniques but it is not so
simplest of all the chromatographic techniques but it is not so widely used these days.widely used these days. Later, we shall
Later, we shall discuss thin layer chromatography, its methodology and applicationsdiscuss thin layer chromatography, its methodology and applications including modern developments of plate
including modern developments of plate concept and high-performance thin layerconcept and high-performance thin layer chromatograp
chromatography. Remember that though PC hy. Remember that though PC and TLC remain primarily qualitativeand TLC remain primarily qualitative techniques but these could also
techniques but these could also be used for quantitative analysis. be used for quantitative analysis. A comparative studyA comparative study of the two techniques will also be presented
of the two techniques will also be presented
Objectives Objectives
After studying this Unit, you should be able to After studying this Unit, you should be able to
•
• explain the explain the meaning of planar chromatography,meaning of planar chromatography, •
• describe the principle of paper describe the principle of paper chromatograchromatography (PC),phy (PC), •
• give the meaning ofgive the meaning of R R f f and factors affecting it,and factors affecting it, •
• discuss the type of paper used as support and the types of solvent mixtures useddiscuss the type of paper used as support and the types of solvent mixtures used
in paper
in paper chromatogrchromatography,aphy,
•
• describe how to run and describe how to run and develop the paper chromatogram,develop the paper chromatogram, •
• explain the methodology of separation of inorganic and organic mixtures,explain the methodology of separation of inorganic and organic mixtures, •
• give the potential applications of give the potential applications of paper chromatograppaper chromatography,hy, •
• describe the principle of describe the principle of thin layer chromatography (TLC),thin layer chromatography (TLC), •
• list the types of list the types of supports and the types of mobile phases used in TLC,supports and the types of mobile phases used in TLC, •
• explain how to run explain how to run and develop the thin layer and develop the thin layer chromatogramchromatogram,, •
• discuss the advantages of two-dimensional paper and thin discuss the advantages of two-dimensional paper and thin layerlayer
chromatography, chromatography,
•
• apply plate concept to TLC,apply plate concept to TLC, •
• describe the basic principle of describe the basic principle of high-performahigh-performance TLC (HPTLC),nce TLC (HPTLC), •
• discuss the potential applications of TLC,discuss the potential applications of TLC, •
• describe the quantitative aspects of PC and TLC; anddescribe the quantitative aspects of PC and TLC; and •
Planar Planar Chromatography Chromatography
6.2
6.2
PAPER CHROMATOGRAPHY
PAPER CHROMATOGRAPHY
It is one of
It is one of the oldest and simplest techniques for qualitative analysis though it canthe oldest and simplest techniques for qualitative analysis though it can also be used
also be used for quantitative determination. During later part of for quantitative determination. During later part of nineteenth centurynineteenth century Runge, Schnbein and Goppelsroeder separated coloured dyes and other chemicals on Runge, Schnbein and Goppelsroeder separated coloured dyes and other chemicals on paper or cloth which
paper or cloth which is considered as old is considered as old cousin of paper chromatographycousin of paper chromatography. In 1944,. In 1944, R. Consden and A. H. Goron, two coworkers of A. J. P. Martin,
R. Consden and A. H. Goron, two coworkers of A. J. P. Martin, the Nobel Laureate,the Nobel Laureate, first reported the separation of a mixture of amino acids and laid
first reported the separation of a mixture of amino acids and laid the foundation ofthe foundation of paper chrom
paper chromatographyatography. The technique is based on the movement of solvent phase in. The technique is based on the movement of solvent phase in upward or downward direction by gravity and accordingly, it can
upward or downward direction by gravity and accordingly, it can be categorized asbe categorized as ascending or descending PC. There is another version of it, called
ascending or descending PC. There is another version of it, called circular papercircular paper chromatography
chromatography where a circular paper is where a circular paper is taken instead of strip and the solvent phasetaken instead of strip and the solvent phase moves in circular direction. However, it is not very commonly used.
moves in circular direction. However, it is not very commonly used.
6.2.1 Principle 6.2.1 Principle
The technique of paper chromatography consists of a
The technique of paper chromatography consists of a sheet of cellulose filter sheet of cellulose filter paperpaper which serves as a
which serves as a stationary phasestationary phase or or separation mediumseparation medium. A small amount (usually a. A small amount (usually a few micrograms) of solute is placed in a small
few micrograms) of solute is placed in a small area near the end of strip. A area near the end of strip. A solvent issolvent is allowed to move from the end of the paper by capillary
allowed to move from the end of the paper by capillary action and after equilibrationaction and after equilibration for some fixed period, the solute migrates from its initial point of
for some fixed period, the solute migrates from its initial point of application. Theapplication. The components of mixture are separated completely or
components of mixture are separated completely or partially in distinctpartially in distinct coloured coloured zones zones or are located
or are located by the application of different reagents or bby the application of different reagents or b y applying ultra-violety applying ultra-violet fluorescence.
fluorescence.
At first, PC was considered as s
At first, PC was considered as s imply a form ofimply a form of liquid-liquid partitionliquid-liquid partition. The. The hydrophilic fibers of paper can hold (or b
hydrophilic fibers of paper can hold (or b ind) water in humid atmosphere such that ind) water in humid atmosphere such that aa large percentage of water, say > 20% by weight, may be held
large percentage of water, say > 20% by weight, may be held in filter paper. Thus,in filter paper. Thus, paper was considered to be the analog of a
paper was considered to be the analog of a column containing a stationary aqueoucolumn containing a stationary aqueouss phase whence solute molecules get partitioned between this water and the mobile phase whence solute molecules get partitioned between this water and the mobile immiscible organic solvent. Later, this model was considered to be too simple because immiscible organic solvent. Later, this model was considered to be too simple because separations were also obtained where mobile phase was miscible with water or in separations were also obtained where mobile phase was miscible with water or in other cases where it was
other cases where it was just aqueous phase. Thus, it cannot be considered as simplyjust aqueous phase. Thus, it cannot be considered as simply liquid-liquid partition mechanism. Instead, besides
liquid-liquid partition mechanism. Instead, besides adsorption and hydrogen bonding,adsorption and hydrogen bonding, interactions between solutes and the
interactions between solutes and the cellulose support are involved. During thecellulose support are involved. During the
pulping and bleaching operations of paper, carboxylate and other ionizable groups are pulping and bleaching operations of paper, carboxylate and other ionizable groups are introduced into cellulose which makes the
introduced into cellulose which makes the paper as ion exchanger.paper as ion exchanger. R
R f fvalue:value:It is a characteristic parameter calledIt is a characteristic parameter called retardation factor retardation factor and abbreviated asand abbreviated as
R
R f f . It represents the position of an . It represents the position of an ion or a substance with respect to solvent phaseion or a substance with respect to solvent phase.. R R f f
of a solute is defined as the
of a solute is defined as the ratio of the rate oratio of the rate of movement of the solute to the rate f movement of the solute to the rate ofof movement of the solvent
movement of the solvent .. R R f fis most commonly used in paper and thin layeris most commonly used in paper and thin layer
chromatograp
chromatography and is hy and is considered as a characteristic of naconsidered as a characteristic of na ture of the solute ture of the solute samplesample which may, of course, change with the solvent phase. It describes relative migration of which may, of course, change with the solvent phase. It describes relative migration of the solute with respect to the
the solute with respect to the solvent and may be represented assolvent and may be represented as
m m ss solvent solvent the the by by traveled traveled Distance Distance solute solute the the by by traveled traveled Distance Distance d d d d R R f f == == … … (6.1)(6.1) where,
where, d d ssandand d d mm are linear distances measured from the line of origin where spots areare linear distances measured from the line of origin where spots are
put as illustrated in
put as illustrated in Fig. 6.1. By definition,Fig. 6.1. By definition, R R f f value cannot exceed 1.0. Ideally,value cannot exceed 1.0. Ideally, R R f f
values must be in the range of 0.1
values must be in the range of 0.1 to 0.9 with a minimum separation of 0.05. In orderto 0.9 with a minimum separation of 0.05. In order to have better separation, two spots must not overlap with each other and these must to have better separation, two spots must not overlap with each other and these must be symmetric without any tailing. In order to avoid tailing, different solvent mixtures, be symmetric without any tailing. In order to avoid tailing, different solvent mixtures, mixed in proper ratio, must be tried. If the
mixed in proper ratio, must be tried. If the spot of the solute is not symmetric, thenspot of the solute is not symmetric, then d d ss
is measured from the position of maximum intensity or the centre of the is measured from the position of maximum intensity or the centre of the spot.spot.
Chromatographic Chromatographic Methods-I
Methods-I
Fig. 6.1:
Fig. 6.1: Procedure Procedure for calculation offor calculation of R R f f value in paper chromatographyvalue in paper chromatography
It has been observed that
It has been observed that R R f f values are influenced by the impurities in the paper andvalues are influenced by the impurities in the paper and
solvent, temperature and saturation of the
solvent, temperature and saturation of the atmosphere. Following factors may beatmosphere. Following factors may be considered;
considered; i)
i) Presence of other ionsPresence of other ions e.ge.g. presence of chloride is carried out with nitrate. presence of chloride is carried out with nitrate solutions.
solutions. ii)
ii) Acidity of the original sol Acidity of the original solutionution-This may be needed to -This may be needed to avoid hydrolysis and itsavoid hydrolysis and its need in the
need in the formation of soluble complex.formation of soluble complex. iii)
iii) Development time Development time- Sometimes it increases with the - Sometimes it increases with the running time. Thereforerunning time. Therefore optimum time may be
optimum time may be determined.determined. iv)
iv) Presence of other cations or anions as impuritiesPresence of other cations or anions as impurities.. v)
v) Ambient temperatur Ambient temperaturee.. Since
Since R R f f value depends on the distribution of complex species of the cations with value depends on the distribution of complex species of the cations with
different organic solvents, solvents should be
different organic solvents, solvents should be carefully chosen.carefully chosen.
The technique of paper chromatography is primarily used for qualitative
The technique of paper chromatography is primarily used for qualitative identificationidentification though it could also be used for quantitative determination but with a poor precision. though it could also be used for quantitative determination but with a poor precision. Hence, it could be at best
Hence, it could be at best considered as semi-quantitativconsidered as semi-quantitative technique.e technique.
SAQ 1 SAQ 1
Which of the following phenomenon is responsible for the rise
Which of the following phenomenon is responsible for the rise of solvent in pof solvent in paperaper chromatography?
chromatography? i)
i) Capillary actionCapillary action ii)
ii) GravityGravity iii)
iii) Ion-exchangeIon-exchange iv)
iv) Chemical affinityChemical affinity
………... ………... ………... ………... ………... ………... SAQ 2 SAQ 2
What is the ideal r
What is the ideal r ange ofange of R R f f values?values?
i)
i) 0.0 0.0 to to 0.9 0.9 ii) ii) 0.5 0.5 to to 1.0 1.0 iii) iii) 0.05 0.05 to to 0.95 0.95 iv) iv) 0.1 0.1 to to 0.90.9
………... ………... ………... ………... ………... ………...
Planar Planar Chromatography Chromatography 6.2.2
6.2.2 Stationary Stationary SupportSupport
As mentioned already, the stationary support material
As mentioned already, the stationary support material is a highly purified cis a highly purified c elluloseellulose filter paper such as Whatman No. 1, 2,
filter paper such as Whatman No. 1, 2, 31 and 3 MM or31 and 3 MM or acetylacetyl acid paper having acid paper having hydrophilic affinity for water. The solvent penetrates the
hydrophilic affinity for water. The solvent penetrates the fiber and causes swelling offiber and causes swelling of paper changing its dimensions. Polymeric cellulose structure contains several
paper changing its dimensions. Polymeric cellulose structure contains several thousand anhydrogl
thousand anhydroglucose units linucose units linked through oxygen atoms. Alternatively, modifiedked through oxygen atoms. Alternatively, modified forms of paper such as
forms of paper such as impregnated with alumina, silica gel, impregnated with alumina, silica gel, hydrous zirconium oxide,hydrous zirconium oxide, ion exchange resin may be used. Sometimes paper is coated with chelating agent ion exchange resin may be used. Sometimes paper is coated with chelating agent solution such
solution such as das dimethylglyoximethylglyoxime, ime, 8-hydr8-hydroxyquinolineoxyquinoline etcetc. However, it should not. However, it should not have any impurities of Ca
have any impurities of Ca2+2+, Mg, Mg2+2+, Fe, Fe3+3+, Cu, Cu2+2+ etc so as to etc so as to avoid interference. The paperavoid interference. The paper is impregnated either neat or dissolved in a volatile so
is impregnated either neat or dissolved in a volatile so lvent. The solvent shouldlvent. The solvent should evaporate slowly so that
evaporate slowly so that stationary phase may distribute homogeneously. In this case,stationary phase may distribute homogeneously. In this case, coated liquid phase may interfere with the detection of separated spots. The paper coated liquid phase may interfere with the detection of separated spots. The paper shows following properties:
shows following properties: 1.
1. weak ion-exchange propertiesweak ion-exchange properties 2.
2. adsorptive propertiesadsorptive properties 3.
3. water holding propertywater holding property 4.
4. mild reducing agentmild reducing agent The quality of paper
The quality of paper and its porosity play an and its porosity play an important role in paper chromatographyimportant role in paper chromatography as it determines the
as it determines the rate of movement of the solvent used. Thick paper with increasedrate of movement of the solvent used. Thick paper with increased sample capacity may be used for preparative studies. The most suitable cardboards are sample capacity may be used for preparative studies. The most suitable cardboards are Schleicher and Schull 2070, SS2171 which can take a load up to 1
Schleicher and Schull 2070, SS2171 which can take a load up to 1 g at each point ofg at each point of application. Generally, a paper strip is cut
application. Generally, a paper strip is cut into 4 cm × 30 cm into 4 cm × 30 cm for one dimensional PC.for one dimensional PC. For two dimensional PC, however, a 30 cm × 30
For two dimensional PC, however, a 30 cm × 30 cm square sheet is commonly used.cm square sheet is commonly used.
SAQ 3 SAQ 3
Explain why following types of paper can
Explain why following types of paper can not be used for not be used for paper chromatographypaper chromatography?? i)
i) Ordinary filter paperOrdinary filter paper
………... ………... ………... ………... ………... ………... ………... ………... ii)
ii) Glazed paperGlazed paper
………... ………... ………... ………... ………... ………... iii)
iii) Butter paperButter paper
………... ………... ………... ………... ………... ………... iv)
iv) Bond paperBond paper
………... ………... ………... ………... ………... ………...
Chromatographic Chromatographic Methods-I
Methods-I
Typical Mobile Phases for P C Typical Mobile Phases for P C
Isopropanol,-ammoniaIsopropanol,-ammonia -water (9:1:2)
-water (9:1:2)
nn-Butanol-acetic acid-Butanol-acetic acid -water (4:1:5) -water (4:1:5) Water-phenolWater-phenol Formamide-chloroformFormamide-chloroform Formamide-chloroformFormamide-chloroform -benzene -benzene Formamide-benzeneFormamide-benzene Formamide-benzeneFormamide-benzene -cyclohexane -cyclohexane DimethylformamideDimethylformamide -cyclohexane -cyclohexane Kerosene-(7:3)-isopropanolKerosene-(7:3)-isopropanol
Paraffin Paraffin oil-dimethylfoil-dimethylformorm amide -methanol-water amide -methanol-water
6.2.3
6.2.3 Solvent Solvent SystemsSystems
The nature of solvent plays an important role in
The nature of solvent plays an important role in the development of paperthe development of paper chromatogram. The solvent should be free from impurities and
chromatogram. The solvent should be free from impurities and dried before use. Polardried before use. Polar phase such as water is adsorbed by the paper and held stationary whereas the less polar phase such as water is adsorbed by the paper and held stationary whereas the less polar solvent such as ethanol,
solvent such as ethanol, acetone, glycol, formamide, acids, and amines flow acetone, glycol, formamide, acids, and amines flow throughthrough easily. Though pure solvent may be used
easily. Though pure solvent may be used but a but a mixture of solvents is preferred. Manymixture of solvents is preferred. Many solvent mixtures can be used provided these are not immiscible with one another. The solvent mixtures can be used provided these are not immiscible with one another. The following criteria may be adopted for the choice of the solvent;
following criteria may be adopted for the choice of the solvent;
•
• The solvent should not react chemically with any of the components of theThe solvent should not react chemically with any of the components of the
sample mixture. sample mixture.
•
• The composition of solvent mixture should not change with time. It means thatThe composition of solvent mixture should not change with time. It means that
none of its components should be volatile. none of its components should be volatile.
•
• The solvent should not interfere with the detection of spots.The solvent should not interfere with the detection of spots. •
• The distribution ratio should be The distribution ratio should be independent of solute concentration.independent of solute concentration. •
• The minimum difference between the Rf The minimum difference between the Rf values of any two values of any two componecomponents shouldnts should
be 0.05 or 0.1 so that they may be
be 0.05 or 0.1 so that they may be separated easily.separated easily. Some of the
Some of the solvents commonly employesolvents commonly employed for the d for the separation of cations are mentionedseparation of cations are mentioned below;
below; i)
i) nn-Butanol saturated with 3M hydrochloric acid: equal volumes of -Butanol saturated with 3M hydrochloric acid: equal volumes of alcohol andalcohol and acid are shaken together and the upper layer is used.
acid are shaken together and the upper layer is used. ii)
ii) Acetylacetone saturated with water: 7.5 mL acetylacetone is mixed with 0.05Acetylacetone saturated with water: 7.5 mL acetylacetone is mixed with 0.05 mL hydrochloric acid and 2.5 mL
mL hydrochloric acid and 2.5 mL dried acetone.dried acetone. iii)
iii) Acetone containing 5% (v/v) water and 8% Acetone containing 5% (v/v) water and 8% (v/v) hydrochlo(v/v) hydrochloric acidric acid iv)
iv) Glacial acetic acid Glacial acetic acid containing 25% (v/v) dried methancontaining 25% (v/v) dried methanolol v)
v) MethanolMethanol vi)
vi) Methyl ethyl ketone containing 30% (v/v) water and Methyl ethyl ketone containing 30% (v/v) water and 1% (w/v) potassium1% (w/v) potassium thiocyanate
thiocyanate vii)
vii) Methyl acetate containing 3% (v/v) methanol Methyl acetate containing 3% (v/v) methanol and 10% (v/v) waterand 10% (v/v) water viii)
viii) Pyridine containing 10% (v/v) waterPyridine containing 10% (v/v) water ix)
ix) DriedDried n-n-butanol containing 40% (v/v) dry butanol containing 40% (v/v) dry methanolmethanol The solvents must be
The solvents must be refluxed over suitable drying agent such as potassium hydroxiderefluxed over suitable drying agent such as potassium hydroxide for acetone and ethyl methyl ketone,
for acetone and ethyl methyl ketone, or anhydrous calcium sulphate foror anhydrous calcium sulphate for nn-butanol or as-butanol or as prescribed in literature.
prescribed in literature.
6.2.4
6.2.4 Development Development of of ChromatogramChromatogram
Paper strips are cut in appropriate size (usually 4-5 cm × 35-40 cm for a
Paper strips are cut in appropriate size (usually 4-5 cm × 35-40 cm for a single spotsingle spot but breadth may be
but breadth may be changed for multiple spots) and stored under controlled conditionschanged for multiple spots) and stored under controlled conditions of humidity. A thin pencil line is
of humidity. A thin pencil line is drawn across the paper 2-3 cm from the edge and adrawn across the paper 2-3 cm from the edge and a circle is put at its centre. The sample is dissolved in a volatile solvent and it is spotted circle is put at its centre. The sample is dissolved in a volatile solvent and it is spotted in the centre of line
in the centre of line using a lambda (or micro) pipette of usually 5-10 micro liters orusing a lambda (or micro) pipette of usually 5-10 micro liters or even with a capillary in case of
even with a capillary in case of qualitative analysis. The spot must be as qualitative analysis. The spot must be as small assmall as possible without any spread for better
possible without any spread for better separation and symmetric spots offer separation.separation and symmetric spots offer separation. It is best done by placing the
It is best done by placing the sample drop wise, drying it with hot air blower (or hairsample drop wise, drying it with hot air blower (or hair drier) to evaporate the solvent as illustrated in Fig. 6.2.
drier) to evaporate the solvent as illustrated in Fig. 6.2. Sample spot may also be driedSample spot may also be dried using infrafil lamp in a chamber. After drying, another drop may be put and dried. using infrafil lamp in a chamber. After drying, another drop may be put and dried. Several spots of different samples or a sample and the standard can be made
Several spots of different samples or a sample and the standard can be made across theacross the line with a minimum separation of 1- 2 cm.
Planar Planar Chromatography Chromatography
Fig. 6.2:
Fig. 6.2: Illustration of (a) spotting of solute sample on paper using a capillary tube andIllustration of (a) spotting of solute sample on paper using a capillary tube and (b) drying process using hair dryer
(b) drying process using hair dryer
Important equipment used in paper chromatography is a development chamber where Important equipment used in paper chromatography is a development chamber where chromatogram is developed in a controlled environment. Some commercially available chromatogram is developed in a controlled environment. Some commercially available development chambe
development chambers used in ascending and descenrs used in ascending and descending PC are shown in Fig. 6.3. ding PC are shown in Fig. 6.3. InIn ascending chromatograp
ascending chromatography, the paper is hy, the paper is supported by means of a clip supported by means of a clip as shown inas shown in Fig. 6.3 (a).
Fig. 6.3 (a).
Fig. 6.3:
Fig. 6.3: Some typical DSome typical Developing chambers for paper eveloping chambers for paper chromatography:chromatography: (a) Ascending and (b) Descending
(a) Ascending and (b) Descending In descending PC, the top edge of the paper is h
In descending PC, the top edge of the paper is h eld down by a glass rod or strip aseld down by a glass rod or strip as shown in Fig. 6.3
Chromatographic Chromatographic Methods-I
Methods-I system and then paper is hung with its end dipping in the system and then paper is hung with its end dipping in the solvent system as shown inthe figure. The solvent starts rising slowly and then it stops athe figure. The solvent starts rising slowly and then it stops a fter some time. It maysolvent system as shown infter some time. It may take an hour or
take an hour or even longer.even longer.
The development time will depend on the complexity of the mixture of solutes being The development time will depend on the complexity of the mixture of solutes being separated, solvent system and the quality of paper and the ambient temperature. The separated, solvent system and the quality of paper and the ambient temperature. The diffusion of the solvent and the resulting separation into spots is termed as
diffusion of the solvent and the resulting separation into spots is termed as development of the chromatogram. It is essential
development of the chromatogram. It is essential that paper should be that paper should be equilibratedequilibrated with the solvent vapours in
with the solvent vapours in the chamber. For good resolution, reasonablethe chamber. For good resolution, reasonable R R f f valuesvalues
must be in the range of 0.4 to
must be in the range of 0.4 to 0.8 with typical separation time being 1 to 2 0.8 with typical separation time being 1 to 2 hours.hours. Following are the main
Following are the main sources of error:sources of error:
•
• Lateral diffusion of the Lateral diffusion of the solutes.solutes. •
• Variation in the structure of paper.Variation in the structure of paper. •
• These become important especially for These become important especially for the concentrated solutes.the concentrated solutes. •
• Variation in the Variation in the geometry of the assembly for the geometry of the assembly for the standard and unknownstandard and unknown
samples. However, this error can be easily eliminated by using the same tank for samples. However, this error can be easily eliminated by using the same tank for the two samples where si
the two samples where similar experimental conditions are maintained.milar experimental conditions are maintained.
6.2.5
6.2.5 Detection Detection MethodsMethods
After development of chromatogram, the solvent front is marked and
After development of chromatogram, the solvent front is marked and the solvent isthe solvent is dried. The spots of the separated compounds are then detected in a variety of ways by dried. The spots of the separated compounds are then detected in a variety of ways by using any one of the
using any one of the following methods.following methods. i)
i) Reactions with colouring reagents such as dReactions with colouring reagents such as d imethylglyoximethylglyoxime for Ni ime for Ni andand hydrogen sulphide gas for any of Gr II elements
hydrogen sulphide gas for any of Gr II elements ii)
ii) Fluorescence producing reagentsFluorescence producing reagents iii)
iii) Inherent visible colours of the components such as Inherent visible colours of the components such as sulphides and oxidessulphides and oxides iv)
iv) Radioactivity measurement of radiotracersRadioactivity measurement of radiotracers v)
v) Electrochemical methods such as Electrochemical methods such as potentiometry, conductance measurements andpotentiometry, conductance measurements and polarograp
polarography have been successfully used for hy have been successfully used for the detection and quantitativethe detection and quantitative analysis in paper chromatography. However, these methods are of limited analysis in paper chromatography. However, these methods are of limited importance and not commonly used.
importance and not commonly used. The reagents employed include
The reagents employed include diphenylthiocarbdiphenylthiocarbazone (dithizone), rubeanic acid,azone (dithizone), rubeanic acid, diphenyl dithiocarbazide, alizarin, salicylaldoxime, morin, potassium
diphenyl dithiocarbazide, alizarin, salicylaldoxime, morin, potassium ferrocyanideferrocyanide,, potassium chromate, ammonium sulphide and hydrogen sulphide gas. In many cases, potassium chromate, ammonium sulphide and hydrogen sulphide gas. In many cases, two or more of
two or more of these reagents are advantageous.these reagents are advantageous. The spots are characterized by their characteristic
The spots are characterized by their characteristic R R f fvalues. Identical Rvalues. Identical R f fvalues for avalues for a
known and an unknown compound using several different solvent systems provide a known and an unknown compound using several different solvent systems provide a good evidence that two are same especially if they run si
good evidence that two are same especially if they run si de by side along the samede by side along the same strip of paper.
strip of paper. Example Example i)
i) In a paper In a paper chromatograchromatographic separation of cations- Agphic separation of cations- Ag++, Pb, Pb++ and Hg and Hg++,, solventsolvent front rises to 18.4 cm while cationic spots were observed at 15.8, 12.1 and 5.9 front rises to 18.4 cm while cationic spots were observed at 15.8, 12.1 and 5.9 cm, respectively. Calculate
cm, respectively. Calculate R R f fvalues of the metal ions.values of the metal ions.
ii)
ii) Emission gases of a two wheeler were tested for pollutant metals by paperEmission gases of a two wheeler were tested for pollutant metals by paper chromatograp
chromatography. hy. A spot corresA spot corresponding toponding to R R f fvalue of 0.65 was observed. Whatvalue of 0.65 was observed. What
is the possible pollutant metal ion in
Planar Planar Chromatography Chromatography Solution:
Solution: Here,Here, R R f f value for Agvalue for Ag++ = 15.8/= 15.8/18.4 18.4 = 0.86= 0.86
R
R f fvalue value for for PbPb++= = 12.1/18.4 12.1/18.4 = 0.66= 0.66
R
R f fvalue for Hgvalue for Hg + +
=
= 5.9 5.9 /18.4 /18.4 = 0.32= 0.32 As
As R R f f value of 0.65 is value of 0.65 is comparable to 0.66 corresponding to Pbcomparable to 0.66 corresponding to Pb + +
, it can be
, it can be concludedconcluded that the pollutant in the
that the pollutant in the emission gases is likely to be emission gases is likely to be lead.lead.
6.2.6 Applications 6.2.6 Applications
Paper chromatograp
Paper chromatography has been widely used hy has been widely used for separation and identification offor separation and identification of cations in inorganic mixtures, organic functional groups, proteins and enzymes in cations in inorganic mixtures, organic functional groups, proteins and enzymes in biochemical work. It is particularly useful in the separation of closely related biochemical work. It is particularly useful in the separation of closely related compounds such as isomers, homologues, isotopes and species having different compounds such as isomers, homologues, isotopes and species having different valency or oxidation states. It has been found especially useful in the following: valency or oxidation states. It has been found especially useful in the following:
•
• identification of trace metals in ores,identification of trace metals in ores, •
• checking purity of pharmaceuticals and checking purity of pharmaceuticals and fermentation,fermentation, •
• ripening of fruits and ripening of fruits and fermentation products,fermentation products, •
• detection of adulterants and contaminants in foods and detection of adulterants and contaminants in foods and drinks by comparingdrinks by comparing
chromatogram of pure compound with that of the adulterant. chromatogram of pure compound with that of the adulterant. Sometimes, it happens that all the
Sometimes, it happens that all the components of a mixture can not be separated usingcomponents of a mixture can not be separated using a single solvent system; some components separate better in one solvent and some in a single solvent system; some components separate better in one solvent and some in another. In such cases,
another. In such cases, two-dimensional paper chromatographytwo-dimensional paper chromatography may be employed. In may be employed. In this technique, a square paper of 30 cm x
this technique, a square paper of 30 cm x 30 cm is taken and the 30 cm is taken and the sample is spotted atsample is spotted at one corner of the
one corner of the sheet. Chromatogram is developed using one solvent whence solutesheet. Chromatogram is developed using one solvent whence solute migrates parallel to one edge of the paper. Next
migrates parallel to one edge of the paper. Next time, the paper is time, the paper is turned at 90turned at 90ooandand developed in a second solvent system which carries the solutes into the unused portion developed in a second solvent system which carries the solutes into the unused portion of the paper.
of the paper. A typical chromatogram of a protein hydrolysate using ninhydrin-stainedA typical chromatogram of a protein hydrolysate using ninhydrin-stained spots is shown in Fig. 6.4.
spots is shown in Fig. 6.4.
Fig. 6.4:
Fig. 6.4: Illustration of two-dimensional paper chromIllustration of two-dimensional paper chromatogram of a protein hydrolysateatogram of a protein hydrolysate where solute is placed in corner at H and it
where solute is placed in corner at H and it is first run towards the right is first run towards the right with anwith an acetic acid-butanol solvent and then at perpendicular using phenol + resol/water acetic acid-butanol solvent and then at perpendicular using phenol + resol/water solvent
solvent
Some typical applications are
Chromatographic Chromatographic Methods-I
Methods-I i)i) Separation and Identification of Mn, Ni, Separation and Identification of Mn, Ni, Co and ZnCo and Zn
A combination of Mn, Ni, Co and Zn may be separated by paper A combination of Mn, Ni, Co and Zn may be separated by paper chromatograp
chromatography using Whatman No. 1 filter hy using Whatman No. 1 filter paper strip by developing with apaper strip by developing with a mixture of acetone, water-hydrochloric acid system. The spots are
mixture of acetone, water-hydrochloric acid system. The spots are located by thelocated by the colouring reagents. These elements are identified by
colouring reagents. These elements are identified by comparing with theircomparing with their characteristic
characteristic R R f f values of known with those in values of known with those in unknown mixture. Theunknown mixture. The
characteristic
characteristic R R f f values are as follows:values are as follows:
Ni Ni - - 0.090.09 Mn Mn - - 0.210.21 Co Co - - 0.430.43 Cu Cu - - 0.610.61 ii)
ii) Separation of a mixture of pesticidesSeparation of a mixture of pesticides A combination of pesticides such as
A combination of pesticides such as aldrin, endrin, lindane, heptachlor, DDT,aldrin, endrin, lindane, heptachlor, DDT, BHC
BHC etcetc. may be separated on Whatman No. 1 filter paper treated with . may be separated on Whatman No. 1 filter paper treated with refinedrefined mineral oil in diethyl ether (5% v/v) and washed with 75% aqueous acetone. mineral oil in diethyl ether (5% v/v) and washed with 75% aqueous acetone. The mobile phase used is 3:1 methanol-water (v/v). The
The mobile phase used is 3:1 methanol-water (v/v). The R R f fvalues for aldrin,values for aldrin,
heptachlor, DDT, endrin and lindane were found to
heptachlor, DDT, endrin and lindane were found to be 0.37, 0.48, 0.60, 0.62 andbe 0.37, 0.48, 0.60, 0.62 and 0.89, respectively.
0.89, respectively. iii)
iii) Speciation of Different Anions of SulphurSpeciation of Different Anions of Sulphur In its
In its unique application, paper chromatograpunique application, paper chromatography has been successfully used forhy has been successfully used for speciation studies of sulphur such as S
speciation studies of sulphur such as S2-2-, SO, SO332-2-, SO, SO442-2-,S,S22OO332-2-, S, S44OO662-2- usingusing
radiotracer
radiotracer3535S. Whatman No. 1 filter paper was used along with solventS. Whatman No. 1 filter paper was used along with solvent mixtures of dioxane,
mixtures of dioxane, nn-butyl alcohol-1N ammonia (1:1:1) and acetone--butyl alcohol-1N ammonia (1:1:1) and acetone-iso- iso-propanol-liq
propanol-liquor ammonia uor ammonia (1:1:1). Different spots (1:1:1). Different spots correspondcorresponding to ing to variousvarious anions can be identified by radioactivity measurements using a G. M. counter. anions can be identified by radioactivity measurements using a G. M. counter. The
The R R f fvalues for SOvalues for SO442-2-, S, S22OO332-2- , , S S44OO662-2-and Sand S2-2- were found to be 0.12, 0.43, 0.54 were found to be 0.12, 0.43, 0.54
and 0.77, respectively. Similarly,
and 0.77, respectively. Similarly, R R f fvalues for Svalues for S22OO33 2- and SO and SO33 2- were found to be were found to be 0.14 and 0.61, respectively. 0.14 and 0.61, respectively.
Paper treated with silicone or paraffin oil permits reversed phase paper Paper treated with silicone or paraffin oil permits reversed phase paper chromatograp
chromatography where mobile phase is hy where mobile phase is a highly polar solvent. This a highly polar solvent. This technique istechnique is referred to as ‘
referred to as ‘reverse-phasreverse-phase e PC PC ’. Commercially available papers coated with’. Commercially available papers coated with adsorbent or ion-exchange resin offer additional
adsorbent or ion-exchange resin offer additional applications of adsorption andapplications of adsorption and ion-exchang
ion-exchange e paper chromatography.paper chromatography. iv)
iv) Autoradiography using RadiotracersAutoradiography using Radiotracers
In this technique, a radiotracer or a radiolabelled solute sample is
In this technique, a radiotracer or a radiolabelled solute sample is used. Afterused. After development of chromatog
development of chromatogram, it is kept iram, it is kept in contact with a photographic film ofn contact with a photographic film of the same size. After
the same size. After some time when the film is developed then dark spots aresome time when the film is developed then dark spots are observed in place of spots
observed in place of spots correspondicorresponding to the ng to the movement of solutemovement of solute components. This is called
components. This is called autoradiographyautoradiography. It has been found to be especially. It has been found to be especially useful for the study of
useful for the study of distribution and metabolism of compounds administereddistribution and metabolism of compounds administered to the plants
to the plants or or animals. In animals. In a unique exa unique experiment, the Nperiment, the Nobel Laureate obel Laureate Calvin andCalvin and coworkers identified intermediate steps involved in the photosynthesis of
coworkers identified intermediate steps involved in the photosynthesis of carbohydrates from atmospheric CO
carbohydrates from atmospheric CO22in the presence of in the presence of light and chlorophyll bylight and chlorophyll by
using
using1414C,C,3232P andP and33H. Plants were placed in the aH. Plants were placed in the atmosphere contaitmosphere containingning1414CC labeled CO
labeled CO22and irradiated with light as shown in Fig. 6.5.and irradiated with light as shown in Fig. 6.5.
The plants were removed after different
The plants were removed after different irradiation periods of light and theirradiation periods of light and the molecular components were separated using
molecular components were separated using paper chromatographypaper chromatography. The. The presence of radioactive atoms in
presence of radioactive atoms in a compound by following autoradiogra compound by following autoradiography wasaphy was taken as a
Planar Planar Chromatography Chromatography
Fig. 6.5:
Fig. 6.5: Illustration of AutoradiograIllustration of Autoradiography method used for phy method used for the determination ofthe determination of compounds involved in photosynthesis
compounds involved in photosynthesis
6.3
6.3
THIN LAYER CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography is different from paper chromatography in that a Thin layer chromatography is different from paper chromatography in that a flatflat
Chromatographic Chromatographic Methods-I
Methods-I surface of glass, metal or plastic coated with surface of glass, metal or plastic coated with adsorbent such as silica gel, alumina orany other material, is used. The technique discovered by Ismailoff and Scraiber inany other material, is used. The technique discovered by Ismailoff and Scraiber inadsorbent such as silica gel, alumina or 1938 is faster, more
1938 is faster, more sensitive and has better sensitive and has better resolution than paper chromatographresolution than paper chromatography.y. TLC is often
TLC is often used to develop optimal conditions for separation by used to develop optimal conditions for separation by liquid columnliquid column chromatograp
chromatography. The technique has become hy. The technique has become a workhorse of the drug industry fora workhorse of the drug industry for purification of products. It has also
purification of products. It has also found widespreafound widespread use d use in clinical, industrial andin clinical, industrial and environmental laboratories. Recent developments have elevated it from the
environmental laboratories. Recent developments have elevated it from the level oflevel of semi-quantitative analytical procedure to one in which
semi-quantitative analytical procedure to one in which highly reliable quantitativehighly reliable quantitative separations can be
separations can be performed.performed.
6.3.1
6.3.1 Stationary Stationary PhasesPhases
The stationary phase used in TLC can be an
The stationary phase used in TLC can be an adsorbent, an ion exchanger, a molecularadsorbent, an ion exchanger, a molecular sieve or it can
sieve or it can serve as the support for a liquid film. It cserve as the support for a liquid film. It c onsists of a finely dividedonsists of a finely divided powder of particle size 5 to 50
powder of particle size 5 to 50 µµm. A variety of m. A variety of stationary supports are available forstationary supports are available for coating the smooth surface of glass, metal or plastic. Plastic
coating the smooth surface of glass, metal or plastic. Plastic sheets have the advantagesheets have the advantage that these can be e
that these can be e asily cut to any shape or size as asily cut to any shape or size as required l. Silica gel G (60-120required l. Silica gel G (60-120 mesh) remains the most
mesh) remains the most frequently used coating materia. It contains frequently used coating materia. It contains hydroxyhydroxyl groupsl groups on the surface which form h
on the surface which form hydrogydrogen bonds with polar molecules. The en bonds with polar molecules. The adsorbed wateradsorbed water prevents other polar molecules from reaching the surface. Hence, the gel is activated prevents other polar molecules from reaching the surface. Hence, the gel is activated by heating to remove the adsorbed water. In modified silica gel, H and
by heating to remove the adsorbed water. In modified silica gel, H and OH groups canOH groups can be replaced by other functional groups similar to
be replaced by other functional groups similar to bonded phase. Alumina containingbonded phase. Alumina containing hydroxyl groups or oxygen atoms is
hydroxyl groups or oxygen atoms is another commonly used adsorbent. Otheranother commonly used adsorbent. Other adsorbents widely used are powdered cellulose, magnesium silicate, calcium
adsorbents widely used are powdered cellulose, magnesium silicate, calcium silicate,silicate, activated charcoal, natural diatomaceous earth, ion
activated charcoal, natural diatomaceous earth, ion exchange resins such as Dowex-exchange resins such as Dowex-50W-strong acid cation exchanger in
50W-strong acid cation exchanger in sodium or hydrogen form and Dowex-1-strongsodium or hydrogen form and Dowex-1-strong base anion exchanger in chloride form. Kieselghur is o
base anion exchanger in chloride form. Kieselghur is o ften used for the ften used for the separation ofseparation of sugars. In fact, any adsorbent material that can be used in
sugars. In fact, any adsorbent material that can be used in adsorption or columnadsorption or column chromatograp
chromatography can be uhy can be used in TLC.sed in TLC. Here, an aqueous slurry of
Here, an aqueous slurry of the powder is prepared by mixing the the powder is prepared by mixing the adsorbent with aadsorbent with a binder such as plaster of Paris (CaSO
binder such as plaster of Paris (CaSO44) and polyvinyl alcohol to help it adhere to ) and polyvinyl alcohol to help it adhere to thethe
backing material. It is uniformly spread over the plate manually or by using one of the backing material. It is uniformly spread over the plate manually or by using one of the commercial forms of spreader as shown in Fig. 6.6. The thickness of the layer is commercial forms of spreader as shown in Fig. 6.6. The thickness of the layer is usually in the range 0.1 to 0.3
usually in the range 0.1 to 0.3 mm but for preparative work much thicker layers aremm but for preparative work much thicker layers are preferred. The solvent is evaporated off and
preferred. The solvent is evaporated off and adsorbents are activated by placing in adsorbents are activated by placing in anan oven at 110
oven at 110ooC for a few hours. Precoated ready-to-use plates and sheets are alsoC for a few hours. Precoated ready-to-use plates and sheets are also commercially available.
commercially available.
Fig. 6.6: Apparatus used for preparing TLC plates where the applicator is filled with Fig. 6.6: Apparatus used for preparing TLC plates where the applicator is filled with thethe
slurry of adsorbent and binder in a solvent. slurry of adsorbent and binder in a solvent.
Thin layers of Sephadex s
Thin layers of Sephadex superfine gel can be prepared for size excluuperfine gel can be prepared for size exclusion. sion. Gel isGel is soaked in water for a few days and then
soaked in water for a few days and then spread on the plate. These are not dried butspread on the plate. These are not dried but stored wet. Capillary action through these
stored wet. Capillary action through these molecular sieves is molecular sieves is much slower, typicallymuch slower, typically of the order of 1 to 2
Planar Planar Chromatography Chromatography The following precautions must be followed while handling TLC pl
The following precautions must be followed while handling TLC pl ates:ates: 1.
1. The surface of the TLC plates should not be The surface of the TLC plates should not be touched. The plates should betouched. The plates should be handled carefully by holding at the edges so as to avoid any contamination due handled carefully by holding at the edges so as to avoid any contamination due to sweat.
to sweat. 2.
2. The plates should be cleaned thoroughly so as to remove any The plates should be cleaned thoroughly so as to remove any extraneousextraneous material that might contaminate the adsorbent.
material that might contaminate the adsorbent.
SAQ 4 SAQ 4
Explain why the flat surfaces of the following materials cannot be used as
Explain why the flat surfaces of the following materials cannot be used as an inertan inert support for the coating of the adsorbent in TLC?
support for the coating of the adsorbent in TLC? i) i) PlywoodPlywood ………... ………... ………... ………... ………... ………... ii)
ii) Asbestos sheetAsbestos sheet
………... ………... ………... ………... ………... ………... iii)
iii) Card boardCard board
………... ………... ………... ………... ………... ………... iv)
iv) Glass with granulated surfaceGlass with granulated surface
………... ………... ………... ………...
6.3.2
6.3.2 Mobile Mobile PhasesPhases
The choice of the mobile phase is
The choice of the mobile phase is largely empirical but some general guidelines can belargely empirical but some general guidelines can be formulated. A mixture of organic solvents and water with the addition of acid, base, or formulated. A mixture of organic solvents and water with the addition of acid, base, or complexing agent to optimize the solubility of the components of a mixture can be complexing agent to optimize the solubility of the components of a mixture can be used. It may be emphasized that a
used. It may be emphasized that a large degree of trial and error is involved in thelarge degree of trial and error is involved in the selection of the
selection of the mobile phase. The polar solvents can mobile phase. The polar solvents can themselves become stronglythemselves become strongly adsorbed; thus, producing an undesirable situation of partition system.
adsorbed; thus, producing an undesirable situation of partition system. The following criteria may be
The following criteria may be adopted.adopted. i)
i) The developing solvent must be of the highest purity because even traceThe developing solvent must be of the highest purity because even trace amounts of impurities may yield irreproducible results.
amounts of impurities may yield irreproducible results. ii)
ii) Good separation of polar or ionic solutes can be achieved with a mixture ofGood separation of polar or ionic solutes can be achieved with a mixture of water and
water and nn-butanol. The criteria used for the -butanol. The criteria used for the selection of developing solvent areselection of developing solvent are the same as for column chromatography.
the same as for column chromatography. iii)
iii) The eluting power of solvents increases in the order of their polaritiesThe eluting power of solvents increases in the order of their polarities e.g.e.g. from from hexane to acetone to alcohol to water. An eluotropic series, given in
hexane to acetone to alcohol to water. An eluotropic series, given in Table 6.1,Table 6.1, can be used for the selection of the best
can be used for the selection of the best solvent or the solvent mixture for asolvent or the solvent mixture for a sample.
sample. iv)
iv) If the stationary phase If the stationary phase is hydrophobic, mixtures of benzene, cyclohexane, andis hydrophobic, mixtures of benzene, cyclohexane, and chloroform in different ratios provide sa
Chromatographic Chromatographic Methods-I
Methods-I Table 6.1: Table 6.1: Eluotropic series Eluotropic series of mobile phof mobile phasease Solvent
Solvent Solvent Solvent strengthstrength
ε ε oo n n- - Pentane Pentane 0.000.00 Cyclohexane 0.04 Cyclohexane 0.04 Carbon
Carbon tetrachloride tetrachloride 0.180.18
Toluene 0.29
Toluene 0.29
Chloroform 0.40
Chloroform 0.40
Methylene
Methylene chloride chloride 0.420.42
Tetrahydrofuran 0.45 Tetrahydrofuran 0.45 Acetone Acetone Methyl acetate Methyl acetate Acetonitrile Acetonitrile 0.56 0.56 0.60 0.60 0.65 0.65 n
n- and- and isoiso-propanol -propanol 0.820.82
Ethanol 0.88 Ethanol 0.88 Methanol Methanol Water Water Ethylene glycol Ethylene glycol 0.95 0.95 1.00 1.00 1.11 1.11 6.3.3
6.3.3 Apparatus Apparatus and and RequirementsRequirements
Commonly, the plates in
Commonly, the plates in sizes of 2.5×10, 2.5×15, 2.5×20, 5×20, 10×20, and 20×20 insizes of 2.5×10, 2.5×15, 2.5×20, 5×20, 10×20, and 20×20 in centimeters are available. These are of two types:
centimeters are available. These are of two types: conventionalconventional and and high-performancehigh-performance .. The former have thicker layers of about 0.25 mm with particle size of < 20
The former have thicker layers of about 0.25 mm with particle size of < 20 µµm. On them. On the other hand, the high performance plates usually have
other hand, the high performance plates usually have thickness of 0.1 mm and thickness of 0.1 mm and particleparticle size of <5
size of <5 µµm. As the name implies, high performance plates provide fast and sharpm. As the name implies, high performance plates provide fast and sharp separations. In terms of plate theory, the conventional TLC will have about 2000 separations. In terms of plate theory, the conventional TLC will have about 2000 plates in 12 cm with development time of l
plates in 12 cm with development time of l ess than 30 minutes. Correspondingless than 30 minutes. Correspondingly, they, the number of plates in high-performance TLC will b
number of plates in high-performance TLC will b e about 4000 in 3 e about 4000 in 3 cm requiring 10cm requiring 10 minutes development time. However, the high performance TLC has
minutes development time. However, the high performance TLC has much lessmuch less sample capacity.
sample capacity.
The sample application in TLC is the
The sample application in TLC is the most critical aspect and particularly so inmost critical aspect and particularly so in
quantitative measurements. Usually the sample is dissolved in a suitable solvent with a quantitative measurements. Usually the sample is dissolved in a suitable solvent with a concentration of less than 0.1% and applied as a spot of
concentration of less than 0.1% and applied as a spot of about 1 cm from the edge ofabout 1 cm from the edge of the plate. The spot should be as
the plate. The spot should be as small as possible and dried in a small as possible and dried in a manner similar to PC,manner similar to PC, (Fig. 6.2). In case of dilute solutions, 3 to
(Fig. 6.2). In case of dilute solutions, 3 to 5 drops are applied. The spotting may be5 drops are applied. The spotting may be done with a
done with a capillary tube or by use capillary tube or by use of a hypodermic syringe. However, mechanicalof a hypodermic syringe. However, mechanical dispensers with increased precision and accuracy are
dispensers with increased precision and accuracy are available commercially. After theavailable commercially. After the sample solvent has evaporated, the plate is placed in
sample solvent has evaporated, the plate is placed in a closed container presaturateda closed container presaturated with the developing solvent. Typical developing chambers used for ascending and with the developing solvent. Typical developing chambers used for ascending and horizontal thin layer chromatography are shown in Fig. 6.7.
horizontal thin layer chromatography are shown in Fig. 6.7.
Fig. 6.7:
Fig. 6.7: Typical developing chambers used Typical developing chambers used for (a) ascending and (b) horizontal thinfor (a) ascending and (b) horizontal thin layer chromatography. In the later case, the sample is
layer chromatography. In the later case, the sample is placed on both the sidesplaced on both the sides of the plate and developed towards the centre
Planar Planar Chromatography Chromatography The plate is immersed in
The plate is immersed in the developing solvent avoiding its direct contact. Thethe developing solvent avoiding its direct contact. The developing solvent travels up the plate and after passing
developing solvent travels up the plate and after passing the point of samplethe point of sample application; it dissolves the sample and carries it
application; it dissolves the sample and carries it up the plate. Thus, the sampleup the plate. Thus, the sample distributes itself between the
distributes itself between the moving solvents and the stationary phase.moving solvents and the stationary phase. After the developer has traveled about two-third of the length of the plate, it After the developer has traveled about two-third of the length of the plate, it isis removed from the container and dried. The
removed from the container and dried. The positions of the components are thenpositions of the components are then determined by any of the methods described in the next section.
determined by any of the methods described in the next section.
6.3.4
6.3.4 Detection Detection MethodsMethods
The detection of the spots in TLC
The detection of the spots in TLC is easier than in is easier than in PC as silica and aPC as silica and a lumina used aslumina used as support are inert than paper and hence, strongly reactive reagents can be used to locate support are inert than paper and hence, strongly reactive reagents can be used to locate the compounds. A universal technique involves the use
the compounds. A universal technique involves the use of iodine vapours forof iodine vapours for colourless or non-fluorescent spots. It consists of exposing the developed plate colourless or non-fluorescent spots. It consists of exposing the developed plate toto iodine vapours which interact with the
iodine vapours which interact with the sample components, either chemically or bysample components, either chemically or by solubility to produce colour. Alternatively some other colour forming reagents called solubility to produce colour. Alternatively some other colour forming reagents called chromogenic reagents
chromogenic reagentsas given in Table 6.2 may be used.as given in Table 6.2 may be used. Table 6.2:
Table 6.2: Some Chromogenic Reagents Some Chromogenic Reagents Used for Identification in Thin Used for Identification in Thin LayerLayer Chormatography
Chormatography
Reagent Application
Reagent Application
Ninhydrin or isotin
Ninhydrin or isotin Amino acidsAmino acids 2,4-dinitophenylhydrazine
2,4-dinitophenylhydrazine Ketones Ketones and and aldehydesaldehydes H
H22S water, diphenylcarbazideS water, diphenylcarbazide MetalsMetals
Rubeanic
Rubeanic acid acid MetalsMetals Aniline
Aniline phthalate phthalate SugarsSugars Chloroplatinic
Chloroplatinic acids acids AlkaloidsAlkaloids Bromothymol
Bromothymol blue blue LipidsLipids Antimony
Antimony trichloride trichloride Steroids, Steroids, essential essential oilsoils
Now-a-days, the thin layer plates
Now-a-days, the thin layer plates and sheets incorporating fluorescent dyes inand sheets incorporating fluorescent dyes in powdered adsorben
powdered adsorbent, are t, are commercially available. When these are commercially available. When these are held underheld under ultraviolet radiation, dark spots glow where
ultraviolet radiation, dark spots glow where sample spots occur due to sample spots occur due to fluorescentfluorescent substances. Alternatively, an immobile fluorescent substance may be
substances. Alternatively, an immobile fluorescent substance may be added whileadded while preparing the slurry so that
preparing the slurry so that the separated compounds appear as dark spots the separated compounds appear as dark spots againstagainst fluorescing backgroun
fluorescing background when the plate d when the plate is viewed under the ultraviolet is viewed under the ultraviolet light.light. Another common technique used for the identification of organic compounds is by Another common technique used for the identification of organic compounds is by spraying the plate with concentrated sulphuric acid solution and then heating it in an spraying the plate with concentrated sulphuric acid solution and then heating it in an oven. Charring of the organic compounds make them
oven. Charring of the organic compounds make them appear as black spots.appear as black spots. The
The R R f fvalues in TLC are difficult to values in TLC are difficult to reproduce because of experimereproduce because of experimental variables.ntal variables.
Following factors may be considered. Following factors may be considered. i)
i) Nature of the adsorbent-its chemical nature, particle size, surface area andNature of the adsorbent-its chemical nature, particle size, surface area and binder. Also its activity,
binder. Also its activity, thickness and uniformity.thickness and uniformity. ii)
ii) Nature of mobile phase-its purity, moisture content, Nature of mobile phase-its purity, moisture content, precision of mixing in caseprecision of mixing in case of mixture and volatility.
of mixture and volatility. iii)
iii) Amount of sample usedAmount of sample used iv)
iv) Vapour-preVapour-pressure equilibrium between the plate and ssure equilibrium between the plate and the atmosphere of thethe atmosphere of the development chamber
development chamber v)
