chromatographic techniques

ADVANCES IN
CHROMATOGRAPHIC
TECHNIQUES
MEENAL AGGARWAL
L-2011-BS-72-IM
1

HISTORY
o Tswett used chromatography to separate
plant pigments (1906)
o colorful separation of plant pigments was
done using a column of calcium
carbonate(chalk)
o the new technique was called
chromatography because the result of the
analysis was written in color.(Chroma
means color and graphein means to write)
Mikhail Tswett,
Russian Botanist,
1872-1919
2

CHROMATOGRAPHY
Technique used to separate and identify the
components of a mixture
PRINCIPLE: Works by allowing the molecules
present in the mixture to distribute themselves
between a stationary and a mobile medium.
Molecules that spend most of their time in the
mobile phase are carried along faster.
3

Components:
mobile phase: a solvent that flows through the supporting
medium
stationary phase: a layer or coating on the supporting
medium that interacts with the analytes
supporting medium: a solid surface on which the stationary
phase is bound or coated
4

BASIC TERMS….
 Adsorbtion: Interaction of solute molecules with the
surface of the stationary phase
 Eluent: The mobile phase
 Elution: Motion of the mobile phase through the
stationary phase
 Elution time: The time taken for a solute to pass
through the system. A solute with a short elution time
travels through the stationary phase rapidly, i.e. it elutes
fast
 Stationary phase: The part of the chromatography
system that is fixed in place
5

 Normal phase: “Unmodified” stationary phase where
POLAR solutes interact strongly and run slowly
 Reverse phase: “Modified” stationary phase where
POLAR solutes run fast i.e. reverse order
 Resolution: Degree of separation of different solutes.
In principle, resolution can be improved by using a
longer stationary phase, finer stationary phase or
slower elution.
 Rf value: distance travelled by solute
distance travelled by solvent
 Rf = retardation factor. The Rf value has to be
between 0 and 1, and it is typically reported to two
decimal places.
6

CLASSIFICATION
On the basis of
interaction of
solute to the
stationary phase
On the basis of
chromatographic
bed shape
Techniques by
physical state of
mobile phase 7

On the basis of
interaction of
solute to the
stationary phase
Adsorption
chromatography
Partition
chromatography
Ion exchange
chromatography
Molecular
exclusion
chromatography
8

On the basis of
chromatographic bed shape
Column
chromatography
TLC
Paper
chromatography
9

Techniques by
physical state of
mobile phase
Gas
chromatography
Liquid
chromatography
Affinity
chromatography
Supercritical
fluid
chromatography 10

ADSORPTION CHROMATOGRAPHY
 Principle of separation:
utilizes a mobile liquid or
gaseous phase that is
adsorbed onto the surface
of a stationary solid phase
 Stationary phase:
adsorbent filled in a tube
(column)
 Mobile phase: various
solvents (eluents)
11

 One of the oldest type
 solute molecules bond directly to the surface of the
stationary phase
 Stationary phases may contain a variety of
adsorption sites differing in the tenacity with which
they bind the molecules and in their relative
abundance
12

PARTITION
CHROMATOGRAPHY
PRINCIPLE
partiton of component of sample between
sample and liquid/gas stationary phase retard
some components of sample more as compared
to others. This gives the basis of separation
13

 Based on thin film formed
on the surface of a solid
support by a liquid
stationary phase
 Solute equilibrates
between mobile phase
and stationary liquid
phase
14

ION EXCHANGE CHROMATOGRAPHY
 Resin( stationary phase)
used to covalently attach
anions or cations onto it
 Solute ions of the
opposite charge in the
mobile liquid phase are
attracted to the resin by
electrostatic forces
 Ion exchange mechanism
separates analytes based
on their respective
charges
15

MECHANISM
 To optimize binding of all charged molecules, the mobile
phase is generally a low to medium conductivity (i.e., low to
medium salt concentration) solution
 adsorption of the molecules to the solid support is driven by
the ionic interaction between the oppositely charged ionic
groups in the sample molecule and in the functional ligand on
the support
 The strength of the interaction is determined by the number
and location of the charges on the molecule and on the
functional group
 By increasing the salt concentration the molecules with the
weakest ionic interactions start to elute from the column first
 Molecules that have a stronger ionic interaction require a
higher salt concentration and elute later in the gradient
 The binding capacities of ion exchange resins are generally
quite high
16

MOLECULAR EXCLUSION
CHROMATOGRAPHY
 Also known as gel
permeation or gel filtration
chromatography
 Lacks attractive
interaction between solute
and stationary phase
17

 Liquid or gaseous phase passes a porous gel which
separates the molecule according to its size
 The pores are normally small and exclude the
larger solute molecules, but allows the smaller
molecules to enter the gel, causing them to flow
through a larger volume. This causes the larger
molecules to pass through column at faster rate than smaller
ones
 It is generally low resolution technique and thus it is
often reserved for the final “polishing” step of a
purification
18

COLUMN CHROMATOGRAPHY
Stationary phase is
held in a narrow
tube through which
the mobile phase
is forced under
pressure or under
the effect of gravity
19

Factors affecting solutes separation in CC
( Factors affecting column efficiency)
EffectFactor
Decrease of size improves separation (but very small
particles need high pressure).
Particle size of solid stationary
phase (or of support)
Efficiency increases as ratio length / width increases.Column dimensions
Non uniform packing results in irregular movement
of solutes through column & less uniform zone
formation, (i.e. band broadning or tailing).
Uniformity of packing
Increase in column temperature results in speed of
elution but does not improve separation (tailing).
Column temperature
Solvents should be of low viscosity (to give efficient
resolution) & h igh volatility (to get rapid recovery of
the substances).
Eluting solvent
Uniform & low flow rate gives better resolution.Solvent flow rate
Discontinuous flow disturbs resolutionContinuity of flow
Deactivation of adsorbent decreases separation.Condition of adsorbent
Substances of high concentration move slowly.Concentration of solutes 20

ProcedureTechnique
Addition of solvent mixture of fixed composition
during the whole process.
Isocratic elution
Continuous or linear elution: in which there is
continuous change in the composition of the
mobile phase over a period of time (e.g. polarity,
pH or ionic strength).
Gradient elution
Step wise or fractional elution: in which the
change is not continuous i.e. a sudden change in
the composition of the mobile phase is followed
by a period where the mobile phase is held
constant.
Elution techniques
21

PAPER CHROMATOGRAPHY
uses filter paper strips as carrier or
inert support.
The factor governing separation of
mixtures of solutes on filter
paper is the partition between
two immiscible phases.
One is usually water adsorbed on
cellulose fibres in the paper
(stationary phase).
The second is the organic solvent
flows past the sample on the
paper (stationary phase).
22

PAPER CHROMATOGRAPHY
 a small dot or line of sample solution is placed onto a
strip of chromatography paper
 The paper is placed in a jar containing a shallow layer of
solvent and sealed
 As the solvent rises through the paper, it meets the
sample mixture which starts to travel up the paper with
the solvent.
 This paper is made of cellulose, a polar substance,
 and the compounds within the mixture travel farther if
they
are non-polar
More polar substances bond with the cellulose paper
more quickly, and therefore do not travel as far 23

THIN LAYER CHROMATOGRAPHY (TLC)
involves a stationary phase
of a thin layer of
adsorbent like silica gel,
alumina, or cellulose on a
flat, inert substrate
24

HPTLC
 Sophisticated form of thin layer chromatography. It
involves the same theoretical principle of thin layer
chromatography.
 It is also known as planar chromatography or Flat-bed
chromatography.
 Traditional Thin Layer Chromatography & its modern
instrumental quantitative analysis version HPTLC are
very popular for many reasons such as
 visual chromatogram
 simplicity
 multiple sample handling
 low running and maintenance costs, disposable layer
etc.
26

PRINCIPLE
o HPTLC have similar approach and employ the same
physical principles of TLC (adsorption chromatography)
i.e. the principle of separation is adsorption.
o Solvent flows through because of capillary action
o Components move according to their affinities towards the
adsorbent Component with more affinity towards the
stationary phase travels slower
o Component with lesser affinity towards the stationary
phase travels faster
Thus the components are separated on a chromatographic
plate 27

STEPS INVOLVING IN HPTLC
Sample Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of
sample
Chromatography development
Detection of spots
Scanning & documentation
28

Pre washing of pre coated plates
main purpose of the pre-washing is to remove
impurities which include water vapours and other
volatile substances from the atmosphere when they
get exposed in the lab environment.
Silica gel 60F is most widely used sorbent
major disadvantage of this sorbent is that it contain
iron as impurity
This iron is removed by using Methanol : water in the
ratio of 9:1.This is the major advantage of the step of
pre-washing.
29

ACTIVATION OF PLATES
 Freshly opened box of HPTLC plates doesn’t need
activation.
 Plates exposed to high humidity or kept in hand for
long time require activation.
 Plates are placed in oven at 110o-120oc for 30 min
prior to the sample application.
30

PRE-CONDITIONING
• Also called Chamber Saturation
•Un- saturated chamber causes high Rf values
Sample application
• Usual concentration range is 0.1-1µg / µl
Above this causes poor separation
• sample and standard application from
syringe on TLC plates as bands
• Band wise application - better separation
31

POST CHROMATOGRAPHY STEPS
Detection
Photo
documentation
Densitometry
measurements
32

DETECTION
UV CABINET
Detection under UV light is first choice - non
destructive
- Spots of fluorescent compounds can be seen at 254
nm (short wave length) or at 366 nm (long wave
length)
- Spots of non fluorescent compounds can be seen -
fluorescent stationary phase is used - silica gel GF
33

DENSITOMETRY MEASUREMENTS
34
 Measures visible, UV absorbance or
Fluorescence.
 Convert the spot/band into chromatogram
consisting of peaks

Instrumentation of HPTLC consists of following:
Lamp selector
Entrance lens slit
Monochromator entry slit
Grating
Mirror
Slit aperture disc
Mirror
Beam splitter
Reference photo multiplier
Measuring photo multiplier
Photo diode for transmission measurements.
35

DIFFERENCES BETWEEN TLC AND
HPTLC
36

DISPLACEMENT CHROMATOGRAPHY
 The basic principle of displacement chromatography is,
“A molecule with a high affinity for the chromatography
matrix (the displacer) will compete effectively for binding
sites, and thus displace all molecules with lesser
affinities”
 Displacement chromatography has advantages over
elution chromatography
 components are resolved into consecutive zones of pure
substances rather than “peaks”.
 Because the process takes advantage of the
nonlinearity of the isotherms, a larger column feed can
be separated on a given column with the purified
components recovered at significantly higher
concentrations 37

GAS CHROMATOGRAPHY (GC)
 Also called as Gas liquid chromatography (GLC)
 based on a partition equilibrium of analyte between
a solid stationary phase (often a liquid silicone-
based material) and a mobile gas (most often
Helium).
 The stationary phase is adhered to the inside of a
small-diameter glass tube (a capillary column) or a
solid matrix inside a larger metal tube (a packed
column).
38

 high temperatures used in GC make it unsuitable for
high molecular weight biopolymers or proteins (heat
will denature them)
 Well suited for use in the petrochemical, environmental
monitoring, and industrial chemical fields. It is also
used extensively in chemistry research.
 Here the mobile phase is an unreactive gas ( e.g.
Nitrogen) flowing through a tube.
 And the stationary phase is an involatile liquid held on
particles of a solid support.
39

In the animation below the red molecules are more soluble in the
liquid (or less volatile) than are the green molecules.
41

INSTRUMENTATION:
o Mobile Phase: He, Ar, N2, H2
o Flow regulators & flow meters:
o Injection Port: Rubber septum barrier (usually
maintained at a higher temperature than the boiling point
of the least volatile component in the sample mixture)
o Column: (fused silica with a thin coating of stationary
phase on the inner surface)
o Oven: Thermostat controlled forced air oven
o Detector:
o Data System: recorders & integrators
42

COLUMNS:
Columns in GC are two types
1) packed column
2) capillary column
Packed column
(capillary column)
Open tubular column
43

Packed column:
Glass or metals
2-3 m long, 2-4 mm I'd.
Densely packed with packing
materials or solid support coated
with thin layer of stationary liquid
phase
Diatomaceous earth
Size: 60-80 mesh (250-170 m)
or 80-100 mesh (170-149 m)
Better resolution – efficient mass
transfer between gas and SP
Tubing – fused silica, glass, copper,
stainless steel
Open tubular column
44

Liquid phase
Solid support coated
with liquid phase
Porous Adsorbent
Porous Layer Open
Tubular
(PLOT)
Wall-coated Open
Tubular
(WCOT)
Support-coated Open
Tubular
(SCOT)
Types of open tubular column:
45

GC DETECTORS
 TCD (thermal conductivity detector)
 FID (Flame ionization Detector)
 MSD (Mass Selective Detector)
 ECD (Electron Capture Detector)
 NPD (Nitrogen Phosphorus Detector)
 FTIRD (Fourier transformation infrared detector)
 AED (Atomic emission detector)
46

GC-MS
 Gas chromatography-Mass spectroscopy (GC-MS) is
one of the so-called hyphenated analytical technique
 As the name implies, it is actually two techniques that
are combined to form a single method of analyzing
mixtures of chemicals
 Gas chromatography is a technique capable of
separating, detecting and partially characterizing the
organic compounds particularly when present in small
quantity.
 Mass spectroscopy provides some definite structural
information from in small quantity.
47

 Combination of GC-MS provides extremely powerful
tool because it permits direct and effectively
continuous correlation of chromatographic and mass
spectroscopic properties
 The separation and identification of the components
of complex natural and synthetic mixture are
achieved more quickly than any other technique with
less sample
48

PRINCIPLE OF GC-MS
 The sample solution is injected into the GC inlet
where it is vaporized and swept onto a
chromatographic column by the carrier gas (usually
helium).
 The sample flows through the column and the
compounds comprising the mixture of interest are
separated by virtue of their relative interaction with the
coating of the column (stationary phase) and the
carrier gas (mobile phase).
 The latter part of the column passes through a heated
transfer line and ends at the entrance to ion source
where compounds eluting from the column are
converted to ions.
49

GC-MS INSTRUMENT
The insides of the GC-MS, with the column of the gas
chromatograph in the oven on the right. 51

LIQUID CHROMATOGRAPHY
 separation technique in which the mobile phase is a
liquid.
 can be carried out either in a column or a plane.
 Present day it utilizes very small packing particles and a
relatively high pressure is referred as high performance
liquid chromatography (HPLC).
 In the HPLC technique, the sample is forced through a
column that is packed with irregularly or spherically
shaped particles or a porous monolithic layer (stationary
phase) by a liquid (mobile phase) at high pressure.
52

SUPERCRITICAL
FLUIDCHROMATOGRAPHY
 Supercritical fluid chromatography is a separation technique in
which the mobile phase is a fluid above and relatively close to
its critical temperature and pressure
53

AFFINITY CHROMATOGRAPHY
Based on selective non-covalent interaction between an analyte
and specific molecules
Stationary Phase
 Highly specific and selective
 Molecular recognition groups (affinity ligands) covalently
attached to agarose or cellulose beads
 Affinity ligand binds selectively and reversibly the analyte
 Mechanism of retention: interaction with highly specialized
molecular recognition systems which are attached to the
stationary phase
Mobile Phase
 Two distinct roles
– Support the strong binding of the analyte molecule to the
ligand
– Weaken and eliminate the analyte-ligand interaction
54

HPLC
 one of the most powerful tools in analytical chemistry
 has the ability to separate, identify, and quantitate the
compounds that are present in any sample that can be
dissolved in a liquid
 Similar to gas chromatography but uses a liquid mobile
phase
 The stationary phase is usually an inert solid or a liquid
held on the inert solid
 Mobile phase travels through the column forcibly with
the aid of the high pressure pump
 Solutes of the sample separated on column and eluted
with mobile phase
 The technique is applicable to thermally fragile samples,
e.g. amino acids, proteins, nucleic acids, hydrocarbons,
antibiotics, steroids, drugs, inorganic and may organic
substances.
55

.
COLUMNS
 Stainless steel, or heavily walled glass tubing of 10-
30 cm, capable of withstanding 6000 psi, 4 to 10
mm inside dia meter, 0.5 μm particles provide 40-
60000 plates/meter.
 1 - 4.6 mm dia, 3.5 mm particles, 3-5 cm length
columns offer 1, 00,000 Plates/meter. Such
columns have low solvent consumption and speed
up the separations
57

Filter and separate irreversibly bonding
compounds.
The particles have similar composition to that of
the analytical column but slightly larger particle
size to minimize pressure drop.
Column thermostats must be capable of
controlling ± 0.10 C. This is accomplished by
using column heaters or water jackets
GUARD COLUMN:
58

DETECTION METHODS
􀂄 UV – Ultraviolet light--- most popular
– Lamp
– Grating/Lens - Wave length 190-350 nm
– FlowCell
– PhotoDiode - Differential Light Output
􀂄 RI – Refractive Index
– Universal analyte detector
– Solvent must remain the same throughout separation
– VERY temperature sensitive
– Sometimes difficult to stabilize baseline
􀂄 FD – Fluorescence-greater sensitivity, not so popular
– Excitation wavelength generates fluorescence emission at a higher wavelength
– Analytes must have fluorophore group---not very common
– Very sensitive and selective
􀂄 MS – Mass Spectrometry
– Mass to charge ratio (m/z)
– Allows specific compound ID
59

NORMAL-PHASE HPLC
 Adsorption of analytes on the polar, weakly acidic
surface of silica gel
 Stationary Phase.: Silica (pH 2-8), Alumina (pH 2 -
12), Bonded Diol and NH2
 Mobile Phase: Non-polar solvents (Hexane, CHCl3)
 Applications: Non-polar and semi-polar samples;
hexane soluble; positional isomers
 Polar solutes elute later than non-polar lyophilic
ones
60

REVERSED-PHASE HPLC
 Partition of analytes between mobile phase and
stagnant phase inside the pore space + adsorption
on the surface of bonded phase
 Stationary Phase: Hydrophobic surfaces of
moieties bonded on silica (C18, C8, C5, Phenyl,
CN)
 Mobile phase: Methanol or Acetonitrile and Water
 Applications: ~80% of all separations done on RP
HPLC
 organic molecules are separated based on their
degree of hydrophobicity
61

UPLC
 In 2004, further advances in instrumentation and
column technology were made to achieve very
significant increase in:
 RESOLUTION
 SPEED
 SENSITIVITY
 Increase separation EFFICIENCY
 Columns with smaller particles [<1.7um]
 Mobile phase delivery is done at >15,000psi
62

CONTRASTING HPLC AND UPLC
 UPLC gives faster results with better resolution
 UPLC uses less of valuable solvents like
acetonitrile which lowers cost
 The reduction of solvent use is more
environmentally friendly
63

64
INSTRUMENTATION
 Autosampler – Reduced Cycle Time and niglisible
carryove
 Flow cell
 Van guar column – for protecting column
performance
 Tubings – Special for UPLC, reduced volume,
optimized connection
 Mobile Phase Filters – To prevent system damage
 column
 Detectors

65
SCHEMATIC PRESENTATION OF UPLC
Gradient
Controller
Pump
•
Column
Detector
Injector
Mobile Phases

66
TUBING AND FITTING
 Tubing and fittings are carefully designed to
provide leak-free connections, with low dead
volume, and minimal band spreading. For best
performance, these components must be clean,
and designed to work together.

67
MOBILE PHASE FILTERS
 Good practice to always filter your
solvents
 As it prevent system damage
 Filters should be changed periodically
depending on usage and mobile phase

68
DETECTORS
 Evaporative Light Scattering (ELS) Detector
 Fluorescence (FLR) Detector
 Photodiode Array (PDA) Detector
 Tunable UV (TUV) Detector
 Single Quadrapole Detector (SQD)

69
ADVANTAGES OF UPLC
 Decreases run time and increases sensitivity
 Provides the selectivity, sensitivity, and dynamic range of LC analysis
 Maintaining resolution performance.
 Expands scope of Multiresidue Methods
 UPLC’s fast resolving power quickly quantifies related and unrelated
compounds
 Faster analysis through the use of a novel separation material of very
fine particle size
 Operation cost is reduced
 Less solvent consumption
 Reduces process cycle times, so that more product can be produced
with existing resources
 Delivers real-time analysis in step with manufacturing processes
 Assures end-product quality, including final release testing

70
Disadvantages OF UPLC
Due to increased pressure requires more
maintenance and reduces the life of the
columns of this type.

chromatographic techniques

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chromatographic techniques