this technique.

crystals of iodine. Almost all organic compounds can be deected (1)by Looking
The other methods of detection of TLCcompound spots are: at the
TLC plate under UVlight. Wherever the compound spots are present, fluorescent
heating it
spots are seen. (2) Spraying the plate with 5% ethanolic H,SO, and
in an oven at 100°C for 10-15 min., all compounds are charred and appear as
dark spots, and (3) Spraying colouration reagents such as FeCl, solution (for the
detection of phenols) or 2,4-DNP solution (for the detection of aldehydes and
ketones), are the other methods for the detection and visualization of spots,

3.4. loN-ExCHANGE CHROMATOGRAPHY

Ion-exchange chromatography is a technique for separating mixtures of charged
compounds, such as cations (K+, Nat, Catt, Cutt, Lanthanides), anions (CI-,
Br-,), amino acids (ala, asp, orn), proteins or neutral mnolecules that can develop
acharge in acidic or basic media such as carboxylic acids and amines.
lon-exchange chromatographic separations are done by using porous resin beads
(granules) to which are bonded acidic groups such as -SO, Ht or -CO0-Ht, or
basic groups such as -CH,N+R3X- or -CH,NR2.
Dependingon the type of bonded group used for ion-exchange chromatography,
 Separation Methos: Chromotography 75

74 Anolyticol Chemistry
-CH,-CH-CH,-CH-CH,-CH
this technique is classified into two classes: (1) cation-exchange chromatography, Copolymerization
and (2) anion-exchange chromatography.
lon-exchange chromatography Styrene
CH2
divinyl
Cation-exchange chromatography Anion-exchange chromatography benzene -H,c-CH CH-CH,
(to separate cations) (to separate anions)

Cross-linked polystyrene-divinyl
Strongly acidic cation- Weakly acidic cation- Strongly basic anion- Weakly basic anion benzene polymer
exchange resins exchange resins exchange resins exchange resins
- SO;H -COOH+ -CH,N R,X -CH,NMe, Sulfonation
Sulfonic Carboxyl Quaternary amm. salt t-Amine

Cation-exchange resins are of two types: (1) strongly acidic cation-exchange resins,
and (2) weakly acidic cation-exchange resins. Anion-exchange resins are of two -CH,-CH-CH,-CH-CH,-CH
ypes: (1) strongly basic anion-exchange resins, and (2) weakly basic anion-exchange
resins. These resins are used as stationary phases in ion-exchange chromatography.
CATION-EXCHANGE RESINS
so,H*
a)Strongly acidic cation-exchange resin Styrene and divinyl benzene in the ratio -H,C-H CH-CH, so
100: 4-10 are copolymerized in an aqueous emulsion to give styrene-divinyl benzene
copolymer in the form of small spherical beads or granuls. The cross-linking of Resin bead
the styrene polymer with 4-10% by weight of divinyl benzene gives a copolymer
with good porosity. The resin beads are sulfonated to give sulfonated resin beads.
In this sulfonated resin, the negatively charged sulfonated ions are firmly attached
to the network and these are balanced by Ht ions which move freely and can be Fig. 3.10 Synthesis of strongly acidic cation-exchange resin
exchanged. The H+ ions are known as active or mobile cations (Fig. 3.10). Due
Sulfonated resin is in the form of 0.1-0.5 mm diameter beads. They are insoluble The H+ cation of the resin is exchanged by the Nat cation from the solution.
cation-oxchange, an equivalent amount pf H+ C[- is liberated.
n water and organic solvents because of their high molecular weight. Because of to this
the cross-linking by divinyl benzene, the beads are porous and swell considerably
Wien placed in water; ie., the resin is hydrophilic. The resin is denser than water. Eg. 2
Resin-SO, H+ + Cat+ 2C1-======>(Resin-SO;)> Catt + 2H+ 2CI
OOne commercial brands of strong cation-exchange resins are Zeolite 225 and
Amberlite 120. The cation-exchange is stoichometric. One doubly-charged ion from the solution
Cation-exchange Strongly acidic cation-exchange resins contain free cations which (e.g., Cat+) will displace two singly-charged ions from the exchanger. In these two
Can be exchanged for different free cations in the solution. examples, H+ (a cation) of the resin is exchanged by Nat or Cat+ cations. Such a
process is known as a cation-exchange. The resin in its Resin - SO; H+ form is
Eg. 1 known as the hydrogen ion form, while the resin in Resin - SO; Nat is known
Kesin-S0;H# + NatCl- =====> Resin - SO, Nat+ H+ CI as the sodium ion form of the resin. Cation-exchange resins are marketed in the
(in water) (in solution) (in solution) H ion form or Na ion form.
 Analytical Chemistry Separation Methods: Chromatogrophy 77
76

Eg. 3 ANION-EXCHANGE RESINS

Resin-S0 Nat StrongH"c
’ Resin--SO, H+ Na Cl or Ca 2cI a) Strongly basic anion-exchange resin It is prepared by the copolymerization
or
of styrene with a small amount of divinyl benzene, followed by chlorome thylation
(Resin-SO, ), Cat+ cation (Resin)
phenyls. The reaction with trimethylamine gives
exchange at the free para position of the
quarternary ammonium salt groupings. The quarternar ammonium groups are an
A cation-exchange is reversible and the resin is regenerated. In the case of strongly integral part of the polymer lattice and there are an equal number of anions (Cl-),
with other different
acidic cation-exchange resins, the exchange capacity or ease of exchange is inde which are the mobile anions. These anions can be exchanged
pendent of the pH of the solution. anions the solution (Fig. 3.12).
copolymerization
b) Weakly acidic cation-exchange resin It is prepared by the -CH-CH,-CH-CH-CH-CH
of methacrylic acid and a small amount of divinyl benzene to give porous beads
(Fig. 3.11. property.
The resin beads contain free COOH groups, and have a weakly acidic
only in an alkaline CH,ÄNMe,C1
For these resins, ionization occurs to an appreciable extent CH,GMe,CI
solution. Therefore, these resins have no action below pH 7.
Resin-COOH + Nat OH Resin-CO0- Nat + H,0 (Cation-exchange) -CH,-CH CH-CH
’ No cation-exchange
CH,GMa,CI
Resin-COOH + Nat Cl =

CH,ÄMe,CI
CH,GMe,Ci CH,GMe,Cr
Copolymerization
CH,=Ç-cOOH + Fig. 3.12 The structure ofa strongly basic anion-exchange resin

anion ex
Eg. 1
CH3 CHs Resin-CH,-N Mez Cl +Na OH Resin-CH,- N° Me, OH +Na' Ci
--CH,-C-CH2-C-CH,-CH-CH, ChBoride fom Hydroxide fom
coOH COOH COOH COOH
coOH
coOH -CH-CH, The resin-CH,-N+ Me,Cl- is known as the chloride form, and the resin-CH,-N+Me3
Cross-linked polymer (resin)
OH- is known as the hydroxide form. Both of them are strongly basic anion
exchange resins and they are largely ionized.

Fig. 3.11 Synthesis of aweakly acidic cation-exchange resin Eg. 2

2 Resin-CH,N*Me, CI- + S0;? (Resin-CH,Nt Mes),SO;2 + 2 CI
to be
form. Before being used, they have
These resins are marketed in the H jon NaOH. Some of the commercial In these two examples, Cl-, an anion, is exchanged by OH- or SO; anions,
IM
converted into Na ion form by washing with are Zeolite 226, and Amberlite 50. stoichiometrically. This process is known as an anion exchange.
of weakly acidic cation-exchange resins
brands
 B0

78
Analytical Chemistry Separation Methods: Chromatogrophy 79

Eg. 3 anion
the
exchange This hydrochloride form of the resin can now be used in an acid solution for
Resin-CH-N Mez OH +H" CI Resin-CH,-N Me, CI +H,0 exchange of anions
(Resin regenerated)
Some of the commercial brands of strongly basic
FF and Amberlite -400. They are supplied in the anion-exchange resins are Zeolite
NO 3anion exchange, Resin-CH,-N' Me, NO 3+CI
Resin-CH,N Me,Ci+in solution
chloride form as they are stable in H H
this form. To convert this resin into hydroxide form, it must be
solution. For these strongly basic anion-exchange resins, their washed with NaOH Some of the commercial brands of weakly basic anion-exchange resins are Zeolite
independent of the pH of the solution. exchange capacity is
G and Amberlite 45. They are supplied in the free base (amino) form, Resin
b) Weakly basic CH,NMez Before use, the hydrochloride salt form of the resin is prepared by
anion-exchange resin It is prepared by
styrene with a smnall amount of divinyl benzene. The resin isthe copolymerizationund
of washing it with HCI. To regenerate the resin it is washed with 1M NaOH.
these groups are converted to t-amine groupings by a reaction chloromethylated,
with dimethylamine
(Fig. 3.13). Resin-CH,N' MezCi
+ Na OH
-CH-CH-CH-CH-CHCH, H ’Resin- CH,-N Me, +NaCl or NaNO, +H,0
Resin-CH-N' Me, No 3
H
CH,NMe, CH CH,NMe, Thus, all these ion-exchange resins used as stationary phases for ion-exchange
chromatography are synthetic, porous beads, which swell in water and have- mobile
-H,c-oH H-CH cations or aniorts. These resins not only exchange other ions, they also adsorb the
exchanged ions. Therefore, these resins are also called adsorbents. As in the case
CH,NMe, of column- and thin-layer chromatography, an ion-exchange chromatography is a
CH,ÄMe, type of adsorption chromatography. The ion exchange is reversible and the original
resin can be regenerated.
CH,NMe, CH,NMe,
Fig. 3.13 The structure of a weakly basic anion-exchange resin 3.4.1 THEORY AND APPARATUS
The weakly basic anion-exchange resins contain very little of the hydroxide form
in basic solutions as well as in neutral solutions. Principle of cation-exchange chromatography (Cation-exchange, adsorption, par
tition coeficients) (Fig. 3.14)
Resin -CH,NMe, + H,0 Resin- CH,-Nt Me, OH- (hydroxide form) (less)
(amino form) A strongly acidic cation-exchange resin (Resin-SO, H+) is taken in a beaker
H containing distilled water. Water molecules percolate the resin matrix, and the
resin swels. This resin slurry is poured into a chromatography column which has a
The equilibrium is mainly to the left and the resin is largely in the amino form. sintered disc at the bottom and a stopcock. The resin settles, and the water layer
However, in an acid medium (pH< 7), these resins behave as a strongly basic above the resin is drained off. The resin is the stationary phase and its sulfonic
anion-exchange resin (like the Resin CH,N*Me,CI-). acid groups are in the fully ionized form. A solution of Nat CI- and K+ CI- is
Resin-CH,NMe, + H+CI Resin- CH,-Nt Me, CI- (hydrochloride form) applied as a small band at the top end of the resin. The cations Kt and Nat are
(amino form) exchanged by the Ht cation of the resin. Due to the differences in their sizes, the
H cations K+ and Nat are bound or adsorbed to the resin with different strengths.
 Separation Methods: Chromotography 81
80
Analytical Chemistry
Nat + HCI
Resin-SO, K+ ResinSO,
The hydrated K+ ion has aradius which is smaller than the hydrated Nat. Thus, Resin-SO, H++ K+ CI- + Nat CI
Kt is bound or adsorbed stronger than Nat to the resin. water (water
the eluent, mobile phase).
The column is now eluted with distilled to the resin and remain at the front end
ion remain adsorbed
The K+ and Nat contains H+
eluate, collected in a conical flask,
of the column only. The column developed with a strong H+ CI- solution or an
Eluent H,0
fR- So, K* now elúted or
fR-SO, H NNa ct Cl-. The column is cation-exchange takes place again and the
cations K+
\resin + H,0 la-so, Na" acidic buffer solution. The
and Nat are desorbed.
Na CI+K*CI
Resin-S0, K* + HCr Resin-S0, H +
Resin-SO, Na
Sintered disc
also
moves down; the cations K+ and Nat
As the mobile phase solution H+CI- because K+ has greater affinity or
move down; Nat moves faster than K+. This is
The column eluate is collected
R= resin adsorption than Nat to the stationary phase resin. are serially numbered. As K+
insmall volumes in several conical flasks, which
HCI + H0 initial column eluate contains
and Nat ions move down, they get separated. The contains pure K+Ci-. At the
pure Nat as Na+Cl- while the latter column eluate
is regenerated.
completion of elution of K+ and Nat, the cation-exchange resin
Step b Step o
Step a
Column packing
resin water
Application of the
mixture NaCl + KCI
Elution with H,0
The rate at which the different cations move depend on: (1) the partition
coefficient of the cations, and (2) the pH of the eluent.

Eluent Eluent
Eluent
conc. Hcl Partition or distribution coefficients of the cations The rate at which different
conc. Hcl conc. Hcl
R-SO,H cations K+, Nat) move on the column is determined by their distribution or
R-SO; K
partition coefficients.
R-So; Ne' R-Soj K*
Adsorbed to the |K+ Kt in the
R- So,H Resin Nat Nat Eluent
(Stationary phase) (Mobile Phase )
Resin. Mobile phase
The cation K+ is more strongly bound or adsorbed to the resin than Nat, i.e., the
amount of K+ in the stationary phase (resin) is more than in the mobile phase.
For Nat, which is weakly bound to the resin, its amount is
K´CI| relatively less in the
Hcl No Ci
stationary phase and more in the mobile phase (moving phase).

Step d Step e Step f

Partition coefficient of Conc. of K+ in the sta. phase
Elution with conc. Hcl Pure NaCl collected Pure KCIcollected K=Cone of Kt in the
mob. phase
Fig. 3.14 Cation-exchange chromatographic separation Partition coefficient of Nat = Conc. of Nat in the sta. phase
Conc. of Natin the mob. phase
 Separation Methods Chrom¡togrophy 83
82 Analytical Chermistry
distilled water and packed
Procedure The sodium form of the resin is slurried in
The partition coefficient of Nat is less than that of K+.The cation which has a lower
column is washed with 200 ml of 5 mol dm³HC1.The resin is
partition coefficient moves faster and is eluted first. Thus, the order of elution of into the column. The solution of CuCl, and FeCl3
cation-exchange. The
cations from the column is that the cation with lowest value of partition coefficient now in the H ion form due to the column. A cation-exchange
in 20 nl of water applied to the front end of the
clutes first followed by cations with increasing values of partition coefficients. The is washed with 100 ml water to
differences in partition coefficients of the cations is due to their differences in takes place and HCI is liberated. The column and Fe(111) i
adsorption strength towards the stationary phase. In view of this, cation (ion) remove the liberated HCI. The column is eluted with 15% H,PO, with 1:1 conc.
collected in the eluate. This is followed by elution of the column
cxchange chromatography is an adsorption chromatography. HCI:H,O and Cu(II) is collected in the eluate.
Strength of binding or adsorption of cations (1) The strength with which differ
ent, charged cations are held (adsorbed. by the resin) decrease in the following Anion-exchange chromatography: Separation of CI- and I-: A basicanion exchange
order: M+4> M-3 > M+2 > M+1, i.e., Th+t4 is firmly held by the resin than resin (De-acidite FF), 50-100 mesh is used. It is a polystyrene resin with 5% divinyl
Kt, e.g., Th+4>Al+3>Cat2>K+. (2) Within a particular series of ions (cations) benzene cross-linking. The resin is supplied in the chloride form (Resin-CH,
carrying the same charge, ions with smaller hydrated ionic radius are held more NMesCI-). The column packed with the chloride form of the resin in water
strongly by the resin than those with a larger hydrated ionic radius. Thus, for and washed with 0.5 mol. dm³ sodium nitrate until the column washings are free
M+2 cations, the decreasing order of binding is Ba+2> Sr+2>Cat2> Mg+2 >Bet2, from CI-.
(Bat? is more strongly than Be+). For M+ cations, the decreasing order of bind
ing to the resin is Agt>Cst > Rbt >NtH,>Kt>Nat>Ht>Lit(Cst is more Resin-CH,N+MesCI- + NatNO; Resin-CH,-N+Mes NO, + NatCI
strongly held than Lit). The radius of Lit (radius in the solid state, from X-ray
analysis) is 0.68 , its hydrated radius is 10 À For Cst the radius is 1.65 Å anion
and its hydrated radius is 5.05 A. exchange
Resin-CH,N+MeNO,+ K+Cl and K+ I Resin-CH,-N+Meg CI
pH of the eiuent The pH of the eluent also determines the rate of movement of Resin-CH,-N*Mes I- + KtNO,
the cations. At alower concentration of H+ ions in the eluent the exchange of the
cations, e.g., At and B+ by the H+ of the eluent, is slower and may selectively Now, a solution of K+CI- and Kt[- is introduced at the top of the column. The
exchange one of the cations A or B. At higher concentrations of H+ in the eluent anion exchange takes place. In Resin-CH,-N*Me, I-, Resin-CH,-NtMe,Ci-,
both A and B cations may be rapidly exchanged by the H+, both move faster, I- is bound more strongly to the resin than CI-. Elute the column with 0.5 mol
however at different speeds. dm³ NaNO3. CI- is eluted first and it is collected. This is followed by the elution
Based on these same principles, using an anion-exchange resin, it is possible of the column with 3 mol dm³ NaNO3. Then, I- is eluted and collected.
to separate a mixture of anions from their solutions. For the anions the affinity
(adsorption) to the resin decreases as the radius of the hydrated anion increases Separation of amino acids: Cation-exchange chromatography: Automated amino
NCS->I->NO, > Br->CN->HSO, >NO; >CI- HCO,>CH,C00->OH- F acid analyzer (Fig. 3.15) The automated amino acid analyzer an instrument,
(i.e., I- is held stronger than Cl-). based on cation-exchange chromatography, widely used in biochemistry and molec
ular biology laboratories. A peptide or protein, on acid hydrolysis, yields a complex
3.4.2 APPLICATIONS OF ION-EXCHANGE CHROMATOGRAPHY mixture of amino acids. The amino acids are separated by cation-exchange chro
matography and later identifed and quantified.
Experiment: Cation exchange chromatography: Separation of Fe(III) and Cu(Il) Consider a peptide asp-asp-ala-orn. On acid hydrolysis it gives asp, ala, orn 2:1:1.
The stationary phase used is a strongly acidic cation-exchange resin (zerocarb 225)
Assume that the peptide is unknown, and the amino acids are also "unknown
with a particle size of 52-100 mesh. The sodium form (Resin-SO, Nat) of the One is interested to know which amino acids are present in the hydrolysis product
resin is used. The column dimesions are 1.5 cm internal diameter and 10-15 cm and what are the relative amounts of these amino acids.
length.
 Separation Methods: Chromatography 85
84 Analytical Chemistry

peptide
6M HCI
Mixture of amino acids (asp, ala, om) as their hydrochlorides The -NH, group of amino acids is exchanged for the Ht of the resin. The different
general structure CIN'H, CH-COOH amino acids are adsorbed by the resin through the attractive forces between the
negatively charged sulphonate (SO;) groups and the positively charged amino
individual amino
R acids. The strength of the adsorption will vary with the basicity of
basic will be held more
acids (note: resin is acidic in nature). Those that are more decreasing order of
Aspartic acid (asp) CIN´H,-CH-COOH (acidic amino acid) strongly (in the mixture of amino acids now considered, the bound stronger than
basicity is orn > ala > asp. Ornithine, a basic amino acid,
CH,COOH column is eluted with a bufer
aspartic acid, which is an acidic amino acid), The
Alanine (ala ) Cr N"H,-CH-COOH (neutral amino acid) solution at pH 3.5, individual amino acids move down the column at different rates
regenerated to be used again. At the
CH, and ultimately become separated. Resin is mix with ninhydrin, a reagent that
end of the column, the eluate is allowed to
mm.
Amax at 570
reacts with amin0 acids giving a purple colour with
Omithine (0m) CIN" H,-CH-C0OH (basic amino acid)

(CH,),- NH, OH

OH
The cation-exchange resin in the column of the automatic amino acid analyzer is
the sulfonated polystyrene. A small amount of the peptide hydrolysate (containing
a mixture of amino acids) is injected into the front end of the column (Fig. 3.15). Fig. 3.16 Ninhydrin reaction
The cation-exchange takes place and the different amino acid cations are bound acids with their retention times is
by differing strengths of adsorption to the resin. The chromatograph of the separated amino 2.8 mm, pH of the eluent 3.5. 0.2N
shown in Fig. 3.17. (Column length 30 cm x
sodium citrate. Flow rate 7 ml/hr).
Eluent
(acidic buffer solution) absorba
Relative asp

ala orn

R-Mixture of amino acids (injection point)

2.0 5.5 11.0

Cation-exchange resin
Time (minutes)
Chromatography column
Fig. 3.17 Chromatogram of separated amino acids
Ninhydrin
solution
of the
The amino acid analyzer is so designed that it can measure the absorbance
Chromatogram eluate at 570 nm continuously and record this absorbance as a function of the
Reaction
Photometer volume of the eluate. The total volume of eluent required to elute the compound
is known as the retention volume, expressed in millilitres. This value characteristic
Column Colour
eluate
Chart paper of the compound and is dependent on the nature of the stationary phase, mobile
phase and the fow rate of the mobile phase. The time spent by an amino acid in the
Fig. 3.15 Automated amino acid analyzer
 Separation Methods: Chromatography 87
86 Analtical Chemistry

PHASE
column is its retention time. (Retention time, expressed in minutes, is a characteristic 3.5.1 NATURE OF PAPER-SUPPORT, STATIONARY
property of the compound. It is dependent on the nature of the stationary phase, from
the nature of the eluent and the rate of flow of the eluent. Retention times (RT) are used for paper chromatography is made
Paper: Stationary phase The paperavailable
of similar utility as the R, values in TLCand paper chromatography. A compound, commercially in different thicknesses and densities
cotton cellulose. It is
strip-form.The paper
whether it is in its pure form or present as a mixture, on and in different shapes, such as rectangular, circular or
same value of retention timne. Pure amino acids (standards)chromatography
has the
are separately injected, chromatography is a type ^of filtèr paper, and generally those
used for paper general
manufactured by the Whatmann brand name, are used (Table 3.4). For
eluted and their retention times noted. The individual amino acids in the mixture
are identified on the basis of their retention times, by comparison with the grade) of paper is used. This
times of the standards. The areas of the peaks in the recorded retention paper chromatography.work, the (Whatmann No.1 randomly.matted, together tÍ
proportional to their concentration.
chromatogram are paper consists .of a mass of sma<l çellulos fibres,
If the sample analyzed is an unknown mixture of amino acids, the form a 3-dimensional network with relatively large open spaces. That is, the paper
obtained from the automatic amino acid analyzer is as follows: information has a porous, character. In,these spaces lie the water molecules (22% by weight
of the paper). These water molecules constitute the _tationary phase of the.paper
1. The number of peaks in the chromatogram is the -numnber of
different amino chromatography set-up. The water molecules are held to the cellulose by adsorption
acids. forces. This water is iñcorporated into the paper at the time of manufacture.
2. Fromn the positions of the peaks and their
retention times in the chromatogram Table 3.4 Characteristics of Whatmann
and by comparison with the retention times of the
standards, the amino acids Chromatography Papers
in the mixture are identified. Grade Flow rate of Water Density glcm Thickness mm
3. The areas of the peaks are proportional to the concentration of the in mm/30 min
amino acids. 20 85 0.58 0.16
115 0.54 0.18
3.5 PAPER CHROMATOGRAPHY 3 MM
130 0.54 0.16
130 0.56 0.33
3 130 0.49
Paper chromatography is generally used for the separation of 0.38
and inorganic compounds or highly polar composnds, such water-soluble
4
organic 17
180 0.46 0.20
as amino acids and 190 0.50 0.88
sugars. Mixtures.of cations and mixtures of anions, in the solution form, are also 31 ET 225 0.36 0.53
separated by paper chromatography.
In paper chromatography, the stationary phase is water, which is The role of the paper i_. that of an inert
the paper. The paper used for paper supported by Thås, overall, the paper used for paper support for the water, stationary phase.
water which is the stationary phaseJThe chromatography contains 22% by weight of phase. It _hould be noted that ia all chromatography constitutes the stationary
mobile
is generally' a polar organic solvent and water. phase for paper-chromatography chromatographic techniques with liquid as the
In paper chromatography, the stationary phase is a liquid (water) and the
stationary phase (paper chromatography, gas-liquid chromatography GLC, high
performance liquid chromatography
phase is also a liquid (organic solvent + water). For this reason, mobile
papr chromatog solid support for otherwise, this liquidHPLC) orthe liquid is supported on an inert
raphy is also called liquid-liquid chromatography. These moves flows out and cannot be stationary.
two phase system (and can be considered as equivalent totwo liquids constitute a
3.5.2
taken a separating funnel). The compounds in a
two immiscible liquids APPLICATION SAMPLE, SOLVENT SYSTEMS: MOBILE PHASE
OF
partition or distribution between the two liquid mixture (e.g., A + B) undergo
the mixture A and B have different values of phases. Further, the compounds of Technique of paper chromatography Whatmann No.l
paper of a suitable size and
The differences in the partition partition or distribution coefficients. shape is chosen. At one end of the paper, at a
of compounds using paper coefficients form the basis for separating mixtures a line with a pencil. On this distance of
line the sample solution/s to bo about 3-4 iscm, draw
is one kind of chromatography. For this reason, paper chromatography
partition chromatography. Consider a sample solution containing two analyzed spotted.
compounds A and B (which may be
amino acids, sugars, cations, anions, etc.) The
solution is spotted at the marked