cnromalography 213
Solvent used (Mobile phase) : The choice of the solvent being used depends
uponthe nature of the mixture to be analyzed e.g.
Eor polar lipids :Chloroform (150): Methanol (45): NH,
(3.75) : Water (3.75)
Eor non-polar lipids : Petroleum ether (9): Ethyl
ether (9): Acetic acid (1)
NOw the chamber is closed with a glass cover, making it air
few mlnutes to let the solvent vapours make the chamber saturated. tight andTheleftsolvent
for a
res up by capilary action, dissolves the sample mixture and
plate.
When the carries it up the
solvent reaches near the top, the plate should be removed from the
hamber and analysis is done
As different components of the sample mixture travel with different rates due to
Ls differences in their attraction to the stationary phase and
because of their
lubility in the solvent the separation is achieved. Separated components may be
aloured (Visible) or may be colourless, There are several methods (like paper
hpmatography) to visualise these colourless components reagents e.g. i0dine
anolurs or 50% analar grade sulphuric acid or ninhydrin specific coloured reagents
ASmall amount of fluorescent compound (manganese activated zinc silicate) is
added to the absorbent that allows to visualize the spot under UV light.
Though TLC is not very efficient but it is speedy, versatile and simple technique
that is used in analyzing fatty acids, detection of pesticides in food and water, in
forensics. in identification of medicinal plants and their constituents.
Liquid Chromatography
Liquid-liquid Liquid-solid
Normal Reverse Normal phase Reverse
a "Flash
The modern version of liquid chromatography is now referred to Pressure
chromatography". Since 2006 new terms came into picture UPLC (Ultra
Liguid Chromatography), RRLC, UFLC, RSLC.
High Performance Liquid Chromatography (HPLC): chromatography. It is a
HPLCformerly referred to as high pressure liquid
used to senarate the components in
highly improved form of columnchromatographyquantify_each
each component and to component.
a mixture, to identify
passes a pressurized (ranging from 5000
HPLC relies on a pressure pump that containing the sample mixture
pounds per square inch) liquid solvent
o10,000
with a solid adsorbant material (smaller particles of the
tnrough a column filled interacts slight differently with the
Each components in the _ample
Sze of 3-5 um). at different flow rates resulting in
separation is
adsorbent material. hence move
214 Modern Phytotechniques and Biostatistio
done as the components flow out of the column. The increased resolution
achiete.d
/n HPLOC compared to classical colunn chromatography is the result of absorbents
small particle sizes (> 20 um) and large surface area.
HPLC
column, THOSthe use of non-compressible resin materials and strong metal
eluehts of the column are detected by UV absorption and fluorescene
It can be applied in the form of partition adsorption, ion exchange
molecular sieve chromatography.
Instrumentation : A typical HPLC system consists of following (ig. 5) main
components.
1. Mobile phase
reservoir
2. Pump
Injector
4. Column
5 Detector
6. Data system, (computer)
t Naster/ Frachi sllee Switching Valve
Solvent Gradient Mixing Vessel
Degasser Value for Delivery of High-Pressure
Pump
the Mobile Phase
Solvent Reservoirs Sample
Injection
Loop
Pre-column
(Guard Column)
Data Acquistion Waste Detector Analytical column
Fig. 5.
Chromatography : Schematic representation of HPLC
1. Mobile Phase Reservoir: It holds the solvent or mobile phase. These are
glass/stainless steel container having puming system and heating/stirting of
solvents.
2. Pump
3. Injector:The injector can be a simple manual device or a sophisticated auto
Sampler for accurately and precisely introducing (injecting) a discrete
predetermined volume of a sample mixture into the flowing mobile phase.
Sample compartments in sophisticated system are temperature controlled
maintain the integrity of the sample. Modern injectors incorporate some form
of syringe-filled sample loop that can be switched on and off by means of
multi-port valve.
t48
Chromatography
215
4 Column : Column is m0st important nart of the HPLC which is generally
made up of stainless steel, glassor plastic tubes
(1-100 mm) and alength of 20-500 with a diameter 0
nm (fig. 6), Normally columns are filled
with silica gel or alumina because its
pore structures help to get a particle size, súrface properdes al
good separation.
Glass column
Plastic column
Stainless
Steel
Fig. 6
Chromatography: Different types of column used in HPLC
These columns are higher pressure
resistant. Commonly used stationary
phases in column of HPLC are resin based
5.
Acrylic based polymer. polvesterene-divinyl benzene and
Detector : It is device that indicates the change in the
eluent by measuring physical and chemical composition of the
properties (e.g.,
absorbance, differential refractive index, fluorescence or UV/ivisible light
are mostly optical and equipped with a flow cell. conductivity. They
6. Display (Computer) : The device that records the
detector on a computer screen in the form of a electrical response of a
7.
chromatogram.
Waster or Fraction Collector
Table 1:Factors Affecting Efficiency of a Column
S. No. Factor Effect
Particle size of solid stationary |Decrease in size improves separation
phase
2 Column dimensions
Efficiency increases as ratio length
3
Column temperature Increase in column temperature results in speed
up of elution but does not improve separation
YSolvent |It should be of low viscosity and high volatility
5
Solvent flow rate |Uniform and low flow rate gives better resolution
6. Conduction of adsorbent |Deactivation of adsorbent decreases separation
7
Concentration of solutes Substances of high concentration move slowly
216 Modern Phytotechniques and Biostatistics
Elution Method
(a) Isocratic elution: Here single solvent or a mixture is used as mobile phase
and it remains the same (constant) throughout a run (ig. 7A).
(b) Gradient elution : When 2 or more solvents of differing polarity are used.
Here mobile phase composition changes during theseparation. This mode is
useful for samples that contain compounds that show a wide two
range of
bottles of
chromatographic there can be
and two polarity. In aspeed
simpleof each pump is controlled by gradient
pparatus
solvents pumps, The
controller to deliver more or less of each solvent during the course of separation.
HPLC Column
Packing Material Chromatogram peaks
of different colours
Injector
Auto Sampler
Sample Manager
Computer
Data Station
Solvent
(Mobile Phase) Sample
Reservoir
Pump Detector
Solvent Manager
Solvent Delivery System Waste
A
HPLC Column
Packing Material Chromatogram
peaks of different colours
Injector Auto Sampler
Sample Manager
Solvent B
Computer
Data Station
Sample
Solvent A Detector
B Pumps
Solvent Manager
Waste
Solvent Delivery System
Fig. 7. A, B. Chromatrography. HPLC with high pressure gradient elution system.
217
Chromatography
The two streams are combined in a mixture (fig. 7 B) to
mobile
develop actual
Lase composition that is finally delivered to the column.
HPLC Scale : 0n the basis of capacity of HPLC, the separated compounds
measured as :
(a) Analytical: When compound in a mixture is identified and quantified.
(b) Preparative:When HPLCis used to purify and collect desired amount o
each component using a fraction collector.
HPLC Separation Modes
There are generally three primary characteristics of chemical components tha,
can be used to create HPLC separations.
Polarity
II. Electrical Charge
III. Molecular Size
. Separation based on Polarity
Primarily there are two modes of separation based on polarity.
(a) Normal phase chromatography : In normal phase chromatography, the
stationary phase is polar (silica) and mobile ph£se (solvent) is non-polar or
less polar and 100% organie so the more polar solutes will adhere more to the
stationary adsorbent phase. When the solvent passes through the column,
the less polar components will be eluted faster than the more polar ones and
components can be collected in an order of increasing polarity.
4b) Reverse phase chromatography : In reverse phase chromatography
stationary phase is non-polar (Cs) and mobile phase is polar (aqueous).
Presently majority of HPLC methods use reverse phasechromatography due
to its higher reproducibility and broad applicability. In these methods mobile
phase is aqueous blend of water with soluble, polar organic solvent
(acetonitrile or methanol). AC-18 bonded silica,(ODs) is used as stationary
phase.
Ultra Performance Liquid Chromatography (UPLC)
Around 2004, further advancements in the instrumentation and column
technology were done for the purp0se of increased resolution, speed and sensitivity
in liquid chromatography. Column with smaller particles (1.7 micron) and
instrumentation with specialized capabilities designed to deliver mobile phase at a
pressuze of l5,000 psi (1000 bar) and higher level of performance is achieved.
l.Separation Based on Charge: (lon-exchange Chromatography)
lon-exchange chromatography is a process that allows the separation of ions
and polar molecules based on the charge properties of the molecules (amino acids,
proteins) and their affinity to the ion exchanger. This technique is generally used in
218 Modern Phytotechniques and Biostatistics
protein puriication, water analysis and quality control. The solution to be injected is
called as sample and individually separated components are called analytes.
Principle : Ion-exchange chromatography is based on the relative retention of
the ions during their flow through an ion-exchange column which hasonfunctional
the ions,
group of opposite charge attached to its surface. The stronger the charge
the greater is the retention time in the column
lon-exchange chromatography retains analyte molecule (component of the
mixture) based on the ionic interaction with stationary phase surface. The
functional groups of stationary phase interact with the ions of opposite charge.
lon-exchange chromatography is further divided into :
(a) Cation exchange chromatography: It retains positively charged cations
because, the stationary phase displays negatively charged functional group.
(b) Anion exchange chromatography : It retains the anions using positively
charged functional group of stationary phase surface (fig. 8)
lon-Exchange Chromatography
Cation Exchange Anion Exchange
Chromatography Chromatography
Solute cations are attached to the Solute anions are attached to the
negatively charged sites covalently negatively charged sites covalently
bound to the stationary phase bound to the stationary phase
Positively Charged Negatively Charged
Analyte [Cation] Analyte [Anion]
Attracted to Attracted to
Positive Surface
Negative Surface
+
-Cation Anion +
1 Exchanger + Exchanger
Stationary-phase + Stationary phase
Particle + Particle + +
+ +
Fig. 8 (A). Chromatography : Schematic representatlon of lon-exchange chromatography
5-x e' MI s-x M e'- cetzcn eyclasc
cnomatogyaphy G X A t f h e y t a A-Aner Xg19
more highly charged molecules
are more tightly bound to the
resin, and so travel slowly and.
are eluted later
moderately charged molecules
equilibrating between the resin
and the moving buffer more readily
Less charged molecules bind less
strongly to the resin, equilibrate
with the moving buffer more
readily, and so travel rapidly and
are eluted sooner
Fig. 8 (B). Chromatography :Two categories of lon exchange chromatograpy
approaches for
With each type of ion exchange, there are at least two general
separation and elution :
I. Strong ion exchanger bear functional groups ions. of stationary phase, that
weak These weak ions can
are always ionized. They use to retain and separate
containing that are more strongly
be eluted by displacement with a mobile phase
ions may also be stained on
attached to the stationary phase surface. These weak causing them
by changing the pH of the mobile phase,
the column, then neutralizedelute.
to lose their attraction and
hence losing their ability to
II. Weak ion exchanger: These are neutralized used to retain and separate
retain ions by charge. After being charged they are
displacement then the stationary phase
strong ions. If these ions cannot be eluted by analytes.
exchange sites may neutralize allowing the elution of charged
(positively charged), use a weak
For example, to retain a strong basic analyte 7. To separate or elute the strong
cation exchange stationary phase particle at pH >
base, down the pH of mobile phase < 3.
pH around 3) is passed
Method: Sample mixture of amino acid or proteins (at can be eluted by using
acids
through a cation exchange and the indjvidsal amino aspartic acid is passed through a
buffers of different pH e.g., a mixture of ogin and charge and so it adheres to the
extra positive
Cation exchange column. Arginine has acid molecules will not adhere and
column. But the negatively charged aspartic
Come out first from the column.
220
Modern Phytotechniques and Biostatistics
When weak NaOH is passed, the positive charge of arginine is neutralized. Na"
will replace arginine in the column thus arginine is eluted finally.
Table 2. Guidelines for the Ion-exchange Chromatography :
Analyte Type Weak Acid Strong Weak Base Strong
e.g., pko-5 ACID e.g, pko-10 BASE
Charge State vs. No charge No Charge +
pH*
[anion] or (anion] [cation] or [cation)
pH <3 pH>7 always pH< 8 pH> 12 Always
charged Charged
Weak Cation
Stationary
Phase Particle
Strong Weak Anion Strong
Anion Exchanger e.g., Cation Exchanger e.g.,
Exchanger pk, -10 Exchanger pk, -5
Charge State vs. + No charge No Charge
pH Always Always
Charged pH <8 pH> 12 Charged pH <3 pH>7
Mobile Phase pH
Range
to Retain analyte pH>7 pH <8 pH <8 pH>7
|(capture]
to Release analyte pH <3 pH> 12 pH> 12 pH <3
elute)
Types of lon Exchanger Resins
Various types of ion exchanger resins are commercially available :
Cation exchanges resins : CM-Sephalex gel, CM cellulose, polystyrene
sulfonate resins : These resins bear acidic groups and immobilize cation from
adjacent solutions. Dethylomu^oethyl eelatese
Anion exchanger resins : DEAE cellulose, trimethyl amino polysterene, DEAE
-sephadex. These bear basic groups and immobilized anions from neighbouring solutions.
Applications of lon-exchange Chromatography
1. Chargaff used this technique to determine the base composition of nucleic
acids.
2 It is used in water purification, where water is completely deionised by
exchanging hydrogen and hydroxyl ions using anion and cation exchanges.
3 This techniques is also extensively used in analysis of amino acids. Amino
acid composition of a protein can be determined by a machine called amino
acid analyser which works on ion exchanger principle usinga strong cation
exchanger.
4 This technique is used for separation of many vitamines and organic acids.
