CHAPTER 33

"Chromatography is atechnique of identification and

orpartitomcocfficients"
CHROMATOGRAPHY
separation of mixtures (often complex)on the basis of adsorption

INTRODUCTION
Chromatography is a technique of separation and purification of components of a mixture by their
oring affinities for tvo phases (states) of matter with which they come into contact. As
conceived bythe Russian botanist Mikhail Tswett in 1903, the method is based originally
on the ability of a
oalumn of finely powdered solid material (such as Al,O,), the stationary phase, to adsorb substances
from asolution (the nobile phase) that is allowed to trickle through it. In general, the attractive force
of the solid surface differs for different species in solution. The substance that solid adsorbs most
strongly move through the stationary phase much more slowly than do those that are not so strongiy
adsorbed. Thismeans that although the various components present in the mobile phase start out
together, when they first come in contact with the stationary phase, they soon become separated.
Twett used this method to separate plant pigments into coloured components. His column
developed bands of colour, and he named this separation technique chromatography, which in Greek
means "written in colour'".
Chromatography is based on the principle of selective distribution of the different components
(or moving) phase. The stationary
of amixture between too plhases,namely stationary phase and mobile
gas. When the stationary phase
phase can be a sold or liguid ; while the mobile phase is a liquid orwhen
distribution is based on adsorption ; while it is a liquid, the basis of
IS solid, the selective
selective distribution is partition.
chromatography : (1) Adsorption chromatography : If a solution ofa mixture (in
Types of column, filled with highly adsorbing material (e.g.,
a
àsuitable solvent) is allowed to flow down to be retained near the top and the others penetrate
rcadiluadsorbed substances tend
dlumina), tlhe most adsorbed.
depending up0n the degree to which they are
Curt01us distances doUn the colunn, distribution
chromatography is based on the use of the difference in the coefficients of gas. One
(2) Partition analysed between two imniscible liquids or a liquid and a
e conipOnentsof the nixture being substance (carrier) andthe otherliquid
distributed on the porous
of theliquids (stationary phase) is immiscible with the stationary liquid
phase. The mobile
which is
(mobile phase) is asolvent (or gas),
coloumn containing stationary phase at slowrate.
phase is passedthrough the the different rate of motion
(partition) coefficients ensure
Different values of distribution mixture. The coefficient of distribution
of a substance
and the Separation of the components of the

botanist, physiologist and biochemist. He wasthe firstto propose

TSWETT M.S. 1872-1919). Prominent Russian
the chromatographic method of analysis.
(1125)
 column the the butfer mixtures separate adsorbed
its tollows
1127 i great
ththe theadsorbabilexst the
situated
adsorbent
poorer
adsorbabil- Forof.adsorbent
coloured, coloured
coiurbeng
by divided withthe components proces
The
eluson. upon separation.
chromatographic
in of chromatoqram. cases of
adsorbent, adsorption
components,
height prnksolutson illumnation motion (or
the in depends
it least
phase,
are
the differentlv
are
and some the solvent
colected
d1fferent adsorbed ot substances through
absorbed of
the rate of and simple
given throughlayerthe C' in of the proper
components stationary
column Components
having
separate ing s0-called lowerin of
Coions
cn
alumina afterHowever. structure as solvent A
of havby being passed
a filtrate. two cations
coloured
Chromatogram the passed analvsis,
being
substances known been 3.
Fig. Collect
seen a
heldComponents
thoseadsorbed of the which components, the in
have the solubilitv
ot are the tormation
substances whilecobalt
is black.and
be fuorescence.
solution adsorbed
iS downby
movement while the is other can with nature process
mixtureadsorbed
mixture formung
salts 2. they blue
beingin 1ons;and Fig. travels
bands).
, accumulate cobalt of reagenttunsthe individual largest
column the the
copper
Mixtures then of K,[Fe(CN)]
by adsorbed This
of the of andcolumnobserve affected
thecomponent
property
rateof (or incapable and Cu a
colourless,
with
laver the of isit the
collected. solvent
components adsorption colouriess contains
of of componentswhich Add
and layers and adsorption
copper we bands.
presence ions to columnadsorbed strongly the
structure that has
adsorbability are retained oxide,
Dark
blue
band due the if separately
so are particular column
separate are example, to
the. which: is
adsorbent solution
of
colour coloured Cu band ions possess
analysed and column
which degree
different aluminium the
Consequently, due to
PinkCo the the is obtained the the
of being composition
of partSubstances blue treating
For
substances ions, collect
the column
all awhich leave Loadsample
the
dark these * XX being
TOGAAPCHAOthe.HMYAanalysed,
differentformupperwithout
thethe
:If
in
fthe aqueous
t appear oflayer
Chromatogram
consists
which
bands.
determined by
components
compounds.
Fe
chromatogramthrough and
to
until
the order the continued
to
component
solvent

Jdadssorpotriboedn throughcolwhenourdexample,
in the various containing visible of at phas
column column
lower. Column the band temperature.
and
solution
solvent, from rate
- bandsan upper ions. Al,0, In
passed
of Because
being in
ed
into
if
made
lig1t, coloured
the Thethe :Elusionemerge the
the
Sunce
stationary
(Stationary
phase)
c solutionare the 2+
mn is|analsimilarly
coluThebands.
CoContains ysed the Note:
wltraviolet
are
containing
of
is
is elustOn
properties solution)
in
Solubility
to tlasks. thethe
tirst
containing
adsorbent
are
ity ity
then If
hands
form beg1n thatby
Column
of

(the
ENGINEERING
CHEMISTRY high
or
molecular
retains paper comnoneot. Carotenoids,
solbent ea ot o differ of de The size. on and
It methode
used particles
powder, chromatography.
liquid).
solute high
(1) (iiD
of adsorbent.
particle separate
adsorbed
chromatography, widely particles. adsorbents
one the (v) of mixture.
other
adsorption
form
in(stationary Imolecules
(of
When nnber
chromatography, solid
then is charcoal
adsorbents. the adsorbed
substance a when B selectively
spCCies. molecules, it on theto Adsorption
and
anyfrom therefore selectrice polar
forproportion
adsorption A adsorbents.
the solvnt
cases
containing
separated and Components
of
mnolecular
associated affinities layers of
columnchromatography.some appropriate
hydroxyapatite 1. arecase
and the
concentration
of
another different
original
gas-liquidmixture on by in
Fig. solutes
innon-polar
either : andin simple
n
then liquid-liquid used substances.
detectedis
based extracttheirarea bonds
in of 2, using in surface column,
substance tuo2solvent
made solution a
(iv) for may be method is mixed components hydrogen
the as
liquids usedeffectively organic Thisby
calcium of
is ion-exchange
chromatography, vast
case
adsorbent
ofratio
same law. inchanging
2
solvernt in
ionised 2. ()
solution be (iv)
It The chromatography.
analysed
a
from in
two
distribution KpCl-a) : (v) andADSORPTION
CHROMATOGRAPHY alumina,a or forces
the the the in partly includes Itcan be substances.
ineffective.
offer ionic
inorganic isolated the
issolvent
of 'Conentration
Cstatomaryin but solute
lhqud exists in () can
(ri) chromatography being to
the
1,solvent is solute : substances gel,between
divided of Waals
through
slute ofmolecules
Nernst solute and
chromatography
thi-layer of be of help
be silica
mixture Adsorbent
immiscible in the
the
of chromatography,
to
mixtures column)
purifcation can finely
differences
column theder
solution
van
as the weight if konization the out etc.,like with
Sometimes,
known assocated hand, of the
(in) of quantiticsturndiverse (or prophyrins, ery or
to
the molecular adsorbents
chromatography, for of by
hydrophobic
mixed bonds
particles
twosolent) other of Advantages mixture Adsorption be
is Note: When
:Thus This degreePartition employed
most
components themust
betiween original the liquid snall on theadsorbent
mobile weight): On = a the proteins, adsorbent
insoluble
1126 a Verybe analysing
() pending passing
Is where sure also analyse
ent
2 Onthe
 1129 compound temperature. awithhave6
answer
constituents
will
Fig compound,
forpager
chromatograçhy The repeatedchromatograp'y
Apcaratus partular components
the theidentical. compound
pure
Org1nal tront Orignal pure
and of process Soivent solvent
Second tmixture
substance
front more completely
now
Compoundsaof
5. a used aimost tuo-y value
Fig

be pure
unity. or wholemore chromatography.
characterise solvent than
solvent twoare or
R.
value

R
chromatography.
the called The
separated (b)chromatography.
to conditions
Fiter
paper Ornginal
spcts definedthe the separateandtwo
the
Supoot onpaper to
the by by are
possible moved
less
values thatis Penci front
solvent
First
Cigs Solvert movedupon 90° unlikely
technique
Lid
is
This
to these
tail
through twO-way compounds Pure
hne
wo-way
depends
distance froni
Solvent through
is value.distance mayunder compounds
reference Rotate (C)
it
conditions, R, mostThis
turned and
line
Pencil
90
one-way
ofprinciples
all solvent
R, The The compound
that values is solvent. one- pure
Orig1nal
so-called it
R,value clear oneK, ispaperthat of
= reprTheesentation
of
controlled being
representations
difterent Mixture (c)
particular
itis of theirThe
TOGRAPHY
CHAOMA its above, (a) Orig1na
by use
mixture
carefully
l.e., logic
the simple. Components
Original
mixture
partiafy
Only
Schematic
front
Solvent mixture, a Separated separated
Glass
tank a cases, Thein Solvent
a.trom fordefinition is values front
rising solvent.schematic
value some problem (a)
Underseparated the
in
ncont
R, b.T'g.
Its .
its s
In identical
difterent
ithis shows

ENGINEERING
CHEMISTRY forma
solidAgel get
paritioned
liquid
phase.
the which Separating
produces
separation fractione freesugar parallel
sillca and phase
whi amino
syringe presentsolvent,just theandare or
solvent coefficients, chromatography. separating solution To i ainalyuce
to
or granules organge. and acids. is n tank, acids 3/3 l'rize
the and the line components
starch mobile different fineglass be developing about Nobel
solutes in
some in peptides
amino to
identifv 5). amino Nobelchromato
immiscible used moving paper (thought pencil the a the
economical
a partition methyl in Fig with of
(likethe forming Liquid-liquid originallythe from the Synge)
phase, widelyof filtera (see temperature paper
around seen Sugars,
separationtrom to the
the
the Sincepaper shared
second in
with collected is
acids contains thatsolvent removed and
R.L.M.
notably
hydrophilic liquid
solution their This used of dropproblem moves paper. research.
immobilized 4.
Fig.
be
yellow
method piece anino solvent
material),
whole 1952 quick
(with
a
in stationary to phases. thus chromatography, sall
of rectangular
of it
is thethe a
inchromatography,
a
who chrornatography,
the evaporation
paper,
this according separately which at biological
dissolved the
could
tinted examplea pureline. the
If
paper
of set
awarded
and of two as paper.
protein applied spraying biochemist
separated
of
Bands In latter of pencil tank, in the top oven
inertremainssolvent
CHROMATOGRAPHY
LIQinsoluble,
UID-LIQUID thephases was
thecolumn. be the
solutions positioned the The
action)
was and
is alongside solvent
Eluting solvent
Eluting in phase The a
of taking the the glass the rates.
reached an partition
who
by spots. British medical,
solutes could
dissolutionacids. liquid-liquidof now
hydrolysis
edge of alonga prevent in partition
biochemist
which the solutes
Mixed gel edge separate in capillary located
paperblue-lilac
column,liquid solutes aqueousand
phase.
the
mobile
is
by one solution, so
vertically different
almost
1994).
of
development
chemical,
of the intervals
an water,mixturephasetwo N-acetylamino
4) PAPERof method
CHROMATOGRAPHY vertical is to are the -
paper
of alongstationary
(Fig. of result
near of paper lid to
drops hung a (dueat has warming paper as
(1914 British
of in
of themobilethe tendencies drawn up sdvances
column
with through other bands method the in one at filterwith substances,
solvent
Millington
-) development
The
saturated between present placed
a illustrate nearsmall now sealed up the show their
(1910
phase. the
cylndrical the
each of red of as
powerful is the travels on After for
acids Laurence Porterextensive
of
passed presence
relativeacids, and alsois
linemixture,
(produced then of thepositions Martin
flow
liquid distributed) Silica
ge! from as fluid
line, paperedge is solvent pure propanone. Johntor
COlumn column us penc1l are
mixture, tank once amino A.J.P. 1952 permitting
is the N-acetylamino
emnerging
granules)
stationarv
is
sOlution solutes
their in
a Let pencil
is acids thinsolid mixture)bottom filter the
The anddry,their individual
Richard Archer
with in
During red
indicate the This derivatives. the Chennistry
1128 (or the turned amino A The the ain The it. As to colourless, in
ninhydrin
Chemistry
MARTIN, technique
the
along along the theof imixture,
allowed SYNGE,
3 of it. acids clear
of
4
to in and the for
 1131 coloured
they
have
bren as partition components
a or known lqudmobile N, column.
ofthe in packed themselees the attached
betterthe diterent called pressure
nature.
theindicate
if by liquid diferences
9) as ot As
(suchGSC phasecomponent
sprayingFig. constituents non-tolatile component.
produces its isobtained, a
peaksindicates having
is the atcolumn recorder,
(see ?
Rvalues
R,value either
is when columnbetueen distribute
stationary
withoutmeasured technuque
(GLO, Dc to gas or
of ts be GLC
carrieT the cylinder
that measure TLC,
ibraries into may the the sample ofa mixture eachtheeachchartof chartpeaks
thermastatei
reagent
located andphaseGLC,thin-laer moving the hus,fromfor stripsize
phasethechromatoqrapiy pressure
are to
separated of
yhasesolidIn partitiontng the byadsorbed
characteristic
Thethe ofseries
substance How place,
stationary emerge A
be
appropriate
may 9.
by
solute stationary
achieed.
a the the of column. means. separated.
on
location
Fig is
mixture taking active with componentsfinally
intothrough the of: high
compounds
pure of gas-liquid is coated
in quickly the instrumental
iderntihcation The the separation or
absorbed is
in andcolumn iswhich its and consists
an a process is solid diference
and is support. chromatograyh
and afrom
with
they
occupy.
Some
plate
and
components
whichadsorbent.
called actire which
phase
by adsorption an the to inert swept
along the
introduced longer
column,
tine
rates the
are difterent through
mixture
component chromatograph
N,)
and
is mixture,due components by orAr
results technique
stationaryit withgas area), for detected the
the while placeon retained
thenthrough at passage in gas component carrier-gas (He,
spraying different CHROMATOGRAPHY columnthe surfacecoatedismixture
ispacked column each
light. of
assist
the
comparison a if ; of
between takes
comiponents is called system.
a chromatography,
is oUer liguid ismixture
mixture
Somne are foritcolumn. for gas
(GSC
TOGAAPHy
CHAOMAdetected
by
which
regions
the
ultraviolet
values
ofThen,
Solvent
front Origin chromatography
passingchromatography
place. distributed
largeSeparation
of
(of thin-layer
sample thevthe tine
phases. along
Theof hence, thetheshows
time. peak is
a
enploved
that
particular
A
A
: a
injection
meters.
Instrumentation
ofsupply
of solid
usuallyunderfluoresce
R, Io
phase taking
this
is GSC,
In
the bchaviour phase).
porous
The

and:Theory
passage
etc.). twoand
the
words,
carried leaves lFratus
detector,
otherComponent chronatogram.
flow
sanple
: GAS
Note compiled.Gas gas are (stationary Ar, the others, A andA
inareas process
vas-solid adsorption plhase beriveen is
mixture aMOunt
of (")regulator
(2)
are
moving
In mixture
a
with H, During In
solid.
gas Me, than to
the

ENGINEERING
CHEMISTPRYlayer
glassa thin in
isolating
and
of stubstances
to
be sugar
snaller
and upright Then,areascending
carefully
deriv ordinary (usually
Develop-
sample
thin
of of
plate varnety : standards mark
drawn. subSpecies
a follows
by an then an
small are
square in
th¹cmn.distance
is oven), also Coloured
colourless
ofreplaced a in reached
thatthe spotssterols,
sugars
andas are
anddry iis and by 0.5
useful are contained line
is an standards
a very of slurry
to developed
of required mixture. plate.
0ver from
factnature
sample chromatography
tives. solution
horizontal in methods.
is a allowed depth front whereas
chromatography
spreadin the the
(or
the
plate:
the
of
thethemethod trichloromethane solvent
components a the cupboard alongside Developing
silica up0nthereby
gel) in now is to
sample layer various
phase;
chromatogram
plate, solvent
immersed a travelled
arises lipids, the
dependingrapid is thinrun
paper,
chromatography plate the developing andfume A by stationary 8.
Fig.
a otherfor
details
thin-layer
or the of 7.
paperalumina is of
bottom has chamber
in Solvent
front Fig. located
identifyingfilterThis in slideglass spots dry
used triglycerides, The front to
rap1d. made be the
papercellulose, be than
CHROMATOGRAPHY
THIN-LAYER coated thea(or
out microscope micropopette), theallowed
thesolvent
chamber.
Origin
canagainst
can compact carrying
is from
filter inusedpapergel) quite example,The cm
in from
immersed solutes tank
Standards of
thesephadex, silica is nucleotides,cleanedtweezers. then
the removed b viewed sides
morcprocess 2.5 a
chromatography,
is over
method sephadex, slurry
for
gel,
use. - in untill is 5 separatedKnob round
is
advantage previously
for
2.0 is
keptis is plate 000 4 when
thin-film
the of aAtabout
syringe plateproceed, Paper
(like acids,
experimental ready plateThe
3
the Also silica pair plate 2 thedirectly
adsorbent
plastic. alumina,
Basically, of the theobject. Standards
solid the concentrated.
Its amino A of A a thendistancethewhich
using
bottle. to Then,
of
positions in of development
Flale
theCourse
Solvent

this mixture.Moreover, is help

of allowed seen
and Then, pointer
In inert (eg-, identifying Brief sCrew-cap
withdrawn in cm). be
positiontheapplied.
technique Thecan
1130 an filnisIscd.more is 15 a
of or with ment - with
stances
5 10
 ENGINEERING CHEMISTRY CHAOMATOGAAPHY
tubular or capllary columns
1132
are
packed columnor open as
(3) Packing materials in carbon, silica gas or alumina.
(b) In GSC activated
GLS - porous materials like glass beads, ground fire-bricks, finely divided celite or fol owS: mobile plase is a gas
referredto the
which does not
carrier gas and is interact with any of the
components to be separated. It is
1133

throughthe column is controlledlbyfrequently

(a) In
poropak, chromosorb, etc. In
GLC. the
column a valve argon or nitrogen. The progress of the
polymers ike tenax GC, with non-tolatile liquid such as silicone (pressure controller) monitored by a carrier gas
polcyoetnthaylinienneg
The mixture to be and
porous material is filled oil, separated in
self-sealing septum cap by means of a injected directy onto the top of the flou-meter.
glycol, apieson oil or grease, etc.
the separation column which senses and
mixture occur to
different extents hypodermic
between the syringe. The partition of the column through a
(4) The detector is situated at the exist of fromthe colunn at different time intervals. In orderstationary and mobile phases components in the

colmefualsamn.umeres
separated components present in the carrier-gas leaving the to be able to and they thus emerge
column to be heatedcopeandwithits mixtures of differing degrees
the small amount of the volatility, provision is made for the
thermal conductivity detector, wheatstone bridge
Commonly used detectors are circuit, and controlled.
temperature thermostatically
ionization detector.
soofthemost common means of
Note : The temperature of the detector compartment must be sufficiently high to
prevent detecting the emerging
katharometer, whichcomponents
conductivities. The device used is called a
sample vapours, yet it should not cause sample decomposition. condensation of
co0identical lengths of platinum resistance
taally reach a steady temperature, are part of a wire. The wires, which
is by means of their
consists of a metal block drilled
are
thernal
heated electrically and
to
Gas-liquid chromatographyisemployed to separate mixtures of gases, liguids
and volatile
Gas-liquid hromatography was introduced by Archer Mortin and A.T. James in 1952. A solids. Wheatstone
eos nasses over both wires. Carrier gas alone passes over one bridqe circuit, which is balanced tohen
chromatography is shown schematically in Fig. 10. typical voiOconiponents pass over the other one. Since the wre; whereas carrier gas together with
arriergas/component mixture will be thermal conductivities of the pure
Pressure out of balance - whenever a componentdifferent, so the temperature will change - and carrier gas
the bridge
Controller passes out of the column. This out-of- balance signal is
Flow-meter ampl1fied and eventually recorded on a roll of paper.
Gas ropanone Pentan-2-one
cylinder
Katharometer Ethoxyethane Heptane
Recorder
response

Saptum cap Ethan

Manometer
Fig. 11. Agas chromatogram ofa mixture
containing six components. The retention
time tor hex-tene is (B-A) min and
for heptane (C- A) min.
Column
Fig. 10. Agas
Water
chromatography.
Time/min

The time taken for a particular component to emerge from the column is called its retention
time. Under carefully controlled conditions (ie., for a particular carrier gas and flow rate, particular
stationary phase and column temperature), the retention time may be used to identify aparticular
Thermosta! cOmponent. Since two compounds may have the same retention time, so it is advisable to determine
the retention times for tUo or HMore diferent types of stationary phase. Atypical gas chromatogram is
shown in Fig. 11.
Heating wire Tite area under u component peak is related to the quantity of that component. Thus, if aknown
is
The stationary phase dmount of the pure component is injected onto the column, in a separate experiment, it is possible
packed into (such as a
narrow columnnon-volalong
tile long-chai
along
to determine the quantity of the component present in the mixture.
4 mm n alkane
20 m
Applications : Gas chromatography has been used in the analysis of : () natural gases, (i) gasoline
wide bent deposited on an inert (v) steroidal homones, and
into a shape of a materla)
U-tube. The Pant samples, (u) refinery gases, (iv) synthetic rubber and plastic intermediates,
)trace atmospheric constitutents. Actually, it has almost unlimited applications.
 ENGINEERING CHEMISTRY CHAOMATOGRAPHY
1135

whichthey form Complexes).

Since the smaller ions
1C)andccvclohexane (b.p. 80,8 C) first to emerge fromthe column. Figshow
1134 (with a greater preference for the
citedbelow
oheseane benzee(bp.80. by Bas chromatography (GC). is mixture virtualw solution,
tlese,ions are the
12 shows some tvpical results,complexg
Some
(2) By using GC. ling achievedeasily of elution being lutentium, ytterbiurn, thuium, erbium, etc. Asimilar process has beentheused orderto
Separationofthe
clsethylalahontent
prass, butitis
in bladcan be determined|with high accuracy by using Bycol
() distillathon and characterise the transuranium elements, their emergence from the column being
impssibleby separate
carried out in arelatively short time.
monitoredby a Geiger-Muller tube.
can be
column. nltavmntanalsIS Lu Im Dy
beturen GSC and GLC
Ho
(3) The
Table 1. Comparison
GSC GLC
Poixt of difereme Concentration
Soid. Liquid.
1. Stationan phas Gas
Gas
2 Motile phas Adsortion in the solid surface Partition between the gas
3 Basis of separatn and
phase. liquid Fig 12. The separation of the
Adsorbents are packed in the form of Liquids are either coated as a lanthanides using a
4 Packing of the fine size,graded powder. on the column fineafilm
column.
film on the inertwall or coated as
support.
fina cation-exchangar.
5 Length of column. 0.7 to2 m. 3-300 m.
6. Thermal stability of Ussally good. Onua fev stationary phases are
the stationary phase. to be stable above 300°C. known
7. Reaction on the Packing may catalyse to produce some Vary rarely with the
column. chemical change. but may occur due tostationary liauid 10 20 40 60 80 100 200 400
solid support. interaction with Drops of eluent
8. Usefulness. Limited applicability, due to difficulty Vast
in reproducing surface applicability, since large number MOLECULAR CHROMATOGRAPHY
due to surface catalysis. conditions or of liquids of specific separation are
8
available Molecular exclusion chromatography: This chromatographic method depends on the partt
9. Applicablity. mixture and is externsively used in the
Useful
cle stzes (or MW's) of the solutes for isolating them from a
in thee
gases and low separation
of permanent All volatile
boiling substances. materials except the more separation of macrobiomolecules (like proteins and nucleic acds, and even viruses and ribosomal
perna1ent gaseSs. is made to flow under gravity through a column of
7 particles). A buffered solution of the mixture
ION-EXCHANGE CHROMATOGRAPHY hydrated beads of an inert and cross-linked polymerized materal like the polysaccharides
This method uses partcles move down the coBumn at
properties and electric ion-exchange materials to separate mixture
depending on their acid-base (agarose, dextran and Sephadex) or polyacrylamides. Solute
rates down the column,
beads and consist of acharge. molecules flow at faster
lon-exchangers
polymeric chain
are
water-insoluble polymers in the form of different rates accordig to their sizes. Large
through
solute
the small pores of the latter and consequently remain
groups (e-g-,- S0, -0H and composed of carbon atoms to which a insoluble bCattse they canot penetrate into the bends
molecules penetrate into the beads through their peres and
ers with fixed
negative -COOH) or anionic groups
(e.g., - number of acidic 1n the agueous plase outside the beads, but small through the column, the degree of retardation
carboxy methyi cellulosecharges NH;CI) are motement
cations and are called attached.
can bind mobile cO'Sqently get considerably retarded in their
positive charges can bind and sulphonated lon-exchang
cation exchangers
cellulose and mobile anions, arepolystyrene) ); while the
ion-exchangers with fixed
(e8 Mixed

An triethy-amino polyofstyrene).
called anion
exchangers (e.g., dimethylaminoethyl solution
Retarded

lunmberanthfromaniimportant
particles
the
des formappl+3icions,
at1on ion-exchange
M*, whose ionicchromatography isSscparation of the lanthanides. AIl
Agarose t

cont ract io n. lAsantthehanum (La,

ionic 0.125 nm) to radii decrease
progressively with increasing atomic
beads

increases Aand this is the basis radii decrease luteium(Lu',0.099

of their along this series nm); this is the so-called lanthanide
Exchuded
partcles
Fig. 13. Molecular exclusion
chromatography.
of
the solution containing +3 separation on an elements, the ability to formn complex ions
of lanthanide ions release an lanthanide ions is ion-exchange column.
ammonium cations equivalent amount of hydrogen ions fromcation-exchange
placed at the top of a
1-hydroxy-part-mitietonhylthemselpropanoatves ebet, ween the
column and the
column. Asolution
the
e columnand
Eluate

(CH),C(OiH)on-Cexchanger
OONH,
slowly run is down the
and the eluting solution
 ENGINEERING CHEMISTPRY
1130
passing suitable solvents
being inersly roportionalto
particles of
(Fig. 13).
their particle sizesdifferent
On
sizes can, therefore, be collected in
gels, therefore
the throughdif erent
column for elution, molecules or of polysaccharide or polyacrylamide
molecular-sieve serve as
columns
Iractions of the eluate. Such
molecular sieves andthe method
gel-filtration or
is also known as polymer beads with specific pore
sievesformed of hydratedestimation of their approXimate MW's may be
chromatogrdiaaphy.meters,
By
using such molecular solutes and
considerable purification of specific
dccomplished.
CHROMATOGRAPHY(HPLC)
HIGH PRESSURE LIQUID that
resounding success that a version GLC. used liquid
to be such a
Gas chromatography provedinvented. The principle is much the same as in
ratherthan gas as the eluant has been gases, the pressure
used to make them pass through a However,
than a sfro.
because liquids are more viscous 20 and 200atm. Such high pressures require
GLC : between
column is greater than in Fig.
process. Th
14(a) shows you the scheme of the the outous
often about 25 cm in length. spectrophotometer, and
column, which is detected by an ultraviolet
coming off the column are accurary
molecules
peaks very much like the GLC charts (Fig. 14(b)). Owing to its
appears as a series of in analysis and research.
HPLC has become very widely used

Eluant
reservoir

-Microsyringe

Filter

Thermostatted
enclosure

Pressure
a
Pump
gauge

Column

Recorder

Detector

Waste

Absorbance

Propanone

(b) 4-Methylphenol

Time
Eia 14. (a) An outine of a high pressure liquid chromatography (HPLC)
appartus, together with (b) a typical output.