High Pressure Liquid

Chromatography
(HPLC)
HPLC also stands for
“High Performance Liquid Chromatography”
is a form of Modern Liquid Chromatographic Technique that
offers two basic advantages over classical liquid
chromatographic techniques.
1- Very high speed of analysis
2- Many fold increased resolution efficiency.
High speed analysis Very high elution rates, achieved by
pressurized flow of mobile phase solvent using high pressure
pumps.
Increased resolution efficiency using stationary phase of very
fine particle size in the range of 5-40 µm.
History

 Martin and Synge (1941)

 Later on, Cal Giddings, Josef Huber (1960) developed first
form of HPLC particle size 150 µm.
Instrumentation
Solvent Reservoir

 Minimum capacity: 500 cm3 for analytical

labs, while larger reservoirs are used for
preparative work.

 The solvent should be filtered with an inlet

solvent filter to remove any particles.

 Solvent must also be degassed.
Solvent Degassing

Helium purging: helium is
bubbled through the solvent
and removes up to 80% of
dissolved air.
Vacuum degassing: the
solvent is exposed to a
vacuum and the reduced
pressure removes more than
60% of the dissolved air.

Sonication: ultrasonic baths
as a stand-alone technique
only removes up to 30%
dissolved air.
 Pumping System
 Must be capaable of pressure outputs of 500-6000Psi
 Should generate flow rate of 3-100mL/min
 Pulse free slovent flow
 Small Hod up volume
Generally two types of pumps are used
1- Constant Pressure Pump
2- Constant Volume Pump
 Direct Gas-pressure Systems: This
system consists of a cylinder gas
pressure, which is applied directly to
the eluent in a holding coil.
Syringe type Pumps :In these
pumps an electrically driven
lead-screw moves a piston,
which is able to pressurize a
finite volume of solvent, and
thus delivers a pulseless
constant flow of solvent to the
system.
Pneumatic Pumps: These pumps
are operated via gas pressure. A
large area piston drives a small
area piston when acted on by
pressure from a gas line. The gas
pressure is thus amplified in the
ratio of the areas of the forces of
the pistons and a high pressure
liquid at constant  pressure is
Reciprocating Pumps: This pump is
electrically driven by a motor, which
moves back and forth within a
hydraulic chamber. On the backward
stroke the piston sucks in eluent
from the reservoir and due to check
valves the outlet to the separation
Column is closed. During the
forward stroke the eluent is pushed
onto the column and the inlet from
the reservoir is closed.
Injection Port

 Neoprene/Teflon Septum.

 Can be used upto

3000-5000Psi.

 Syringe Injector (Microliter

Syringe) made up of glass/
Stainless steel.
 Columns
 Nature of HPLC column depends
upon nature of sample and
analyte.

 Stainless steel/Glass-Teflon
composite material.

 Inside diameter typically ranges

from 2-50mm, Typical 3-5mm.

 Length: 5-100cm. Typical

10-20cm.
Stationary Phase particles
1-Porous Layer Beads

 Have inert solid core with a thin porous

outer shell of SiO2 Al2O3 or ion exchange
resin.
 Diameter: 20-45 µm
2- Not suitable
Stationary forparticles
Phase large scale preparative
 work.
Available in two sizes:
 (i) 20-40 µm (ii) 5-10 µm
 More widely used than porous layer beads.
 Offer greater resolution.
 Made from SiO2 Al2O3 or ion exchange resin.
 Mobile Phase

Requirements for Mobile Phase of HPLC include:

 Purity
 Reactivity
 Detector Competibility

Eluotropic Series
Detectors

Differential Refractometer
 Works by measuring refractive index
difference between sample analyte and
mobile phase.

 Detection Limit: 0.1 µg

 Can’t be used with gradient elution.

 Photometric Detector
 Focuses UV/Vis radiations
onto a flow cell containing the
column effluent. Extent of
absorption at a particular
wavelength gives qualitative
as well as quantitative
analysis.
 Detection Limit: 1 ng

 Suitable for compounds

containing π-bonding and non-
Fluorescence Detector
 Works only for fluorecent
compounds.

 Response is not linear over wide

range of conc.

 Very high sensitivity with DL:

0.01 ng.

 Can detect trace biological

compounds.
Normal Phase Vs Reverse Phase HPLC
A silica stationary phase is eluted with a
non-polar solvent such as hexane, or a
fairly non-polar solvent mixture such as 2-
propanol in hexanes.
In normal phase chromatography, only
organic solvents are used.
In the normal phase, polar molecules
elute slowly, and non-polar (greasy)
molecules elute quickly.
A non polar stationary phase (often
silica in which the free hydroxyl
groups are end-capped with
something greasy, C18 chains are
common but many many variants
are possible) is eluted with a polar
solvent such as acetonitrile/
methanol, or a fairly polar solvent
mixture (acetonitrile water mixtures
are common, or methanol water
mixtures).
In the reverse phase, polar
molecules elute quickly, and non-
polar (greasy) molecules elute slowly.
 Analysis of HPLC Data

 Qualitative Analysis

 Quantitative Analysis
 Modified Form of HPLC
 FPLC
 Capillary
E
LECTROPHORESIS
1
 Description
A separation technique in which charged species are
separated, based on charge and size, by different rates of
migration in an electric field, is called electrophoresis.

Capillary electrophoresis (CE) is a family of electro-

kinetic separation methods performed in sub-millimeter
diameter capillaries and in micro- and nanofluidic channels.
 History
In 1930, Arnes Tiselius put forward the
concept that electrophoretic separations can
be performed more efficiently if the buffer is
allowed to flow through narrow channels.

In 1960’s Hjerten first time introduced the

use of capillaries in the electrophoretic
separations.

Jorgensen and Lukacs (1980’s) first time

published research papers on capillary
electrophoretic separataions and practically
demonstrated the technique in the Lab.

3
 Also referred as
 High performance capillary electrophoresis (HPCE)

 Capillary zone electrophoresis (CZE)

 Free solution capillary electrophoresis (FSCE)

 Capillary electrophoresis (CE)

 PRINCIPLE
Capillary electrophoresis: principle

▪ Capillary tube is placed between two buffer reservoirs, and an electric

field is applied, separation depends on electrophoretic mobility &
electro-osmosis.

▪ Defined volume of analysate is introduced in to the capillary by

replacing anode buffer reservoir with sample vial.
▪ Electrophoretic separation is measured by detector.
▪ Using narrow bore tubes, CE removes the Joule heating effect, which
decreases band broadening, giving faster separations than gel.
▪ CE uses tubes 20-100 m diameter and 20-100cm in length.
▪ CE is used with/without gel. Longitudinal diffusion is the main source
of band-broadening.
▪ Higher electric fields result in high efficiency and narrow peaks
(analyte migrates faster).
 INSTRUMENTATION

7
 Small amount of
sample is required
(5-30 μm3)

Introduced into
the capillary with
appropriate buffer
at anode end.

8
 Sample Application
Electrokinetic
Injection
High voltage injection
The buffer reservoir is
replaced by the sample
reservoir and the high
voltage is applied.
Buffer reservoir is
placed again and voltage
applied for the separation.
9
Hydrodynamic
Injection
Pressure injection
Anodic end of capillary is
removed from buffer and
placed in air tight sample
solution. With pressure,
sample is pushed into
capillary.
.
 Capillary Tube
The separation is carried in a narrow bore Capillary

50μm – ID.
300 μm – ED.
Length – 50-100cm.
Fused silica capillary tube.
Polyimide coating external.
Packed with the buffer in use.

10
 High voltage is applied (up to 50 kV)
The components migrate at different rate along the length

Although separation is achieved by the electrophoretic

mobility, all the sample components are drawn
towards cathode by electroosmostic flow (EOF).
 The rate of electroosmostic flow (EOF) is greater
than the electrophoretic velocity of the analyte ion.

13
 Positively charged molecule reach the cathode
first (electrophoretic migration + electroosmotic
flow).

12
DETECTION:
oNear to cathode end,
viewing window

oDetected by the
ultraviolet detector, that
transmit signal and
integrated by computer.

oRefractive index

oFluorescence

oCE-MS

13
E-gram
 Troubleshooting :
• Adsorption of protein to the wall of capillary – leading
to smearing of protein – viewed as peak broadening.

Can be avoided by
• Use of neutral coating group to the inner surface of the
capillary.

15
 Advantage over slab type:

Reduce the problem of heating

effect.

Large surface to volume ratio.

Less diffusion of the
separated bands.

16
 Modes of Operation

Capillary zone electrophoresis (CZE):

- Most widely used CE technique, also known as free solution
capillary electrophoresis.
- Separation is based on differences in electrophoretic mobility,
which is directly proportional to charge on molecule, inversely
proportional to size of molecule and viscosity of solution.
- Separation is faster.
- Due to High EOF, the molecules regardless of the charge, they are
moved to cathode.

17
 The fused silica capillaries have silanol groups that become ionized in
the buffer.
 The negatively charged SiO- ions attract positively charged cations,
which form two layers—a stationary and diffuse cation layer.
 In the presence of an applied electric field, the diffuse layer migrates
towards the negatively charged cathode creating an electrophoretic flow
that drags bulk solvent along with it.
 Anions in solution are attracted to the positively charged anode, but get
swept to the cathode as well.
 Cations with the largest charge-to-mass ratios separate out first,
followed by cations with reduced ratios, neutral species, anions with smaller
charge-to-mass ratios, and finally anions with greater ratios.
 The electroosmotic velocity can be adjusted by altering pH, the viscosity
of the solvent, ionic strength, voltage, and the dielectric constant of the
buffer.
 Modes of Operation

Micellar electrokinetic chromatography (MEKC):

- Itis based on solutes partitioning between micelles and the
solvent.
- Micelles have polar negatively charged surfaces and are
naturally attracted to the positively charged anode.
- Because of EOF towards cathode, the micelles are pulled to the
cathode as well, but at a slower rate.

20
 Hydrophobic, neutral molecules will spend the
majority of their time in the micelle and migrate
slowly to cathode and get separated.
 The separation is accomplished by micelles
formation by adding surfactant (e.g. 8-9mmol/L
for SDS)
22
 Modes of Operation

Capillary gel electrophoresis (CGE):

- Separation based on the sieving.

- The capillary is filled with “sieving matrix” or “soluble polymer

network”.
- Low viscosity, self entangling for formation of pore size.

- Variety of polymeric matrices are available for DNA and Protein.

- A commonly used gel apparatus for the separation of proteins is

capillary SDS-PAGE.

18
- .
 • Online detection

• Improved quantification

• Almost complete automation

• Reduced analysis time.

• Wider choice of gel matrices.

Advantage over • Linear polyacrylamide, derivative of
conventional
cellulose, polyvinyl alcohol, polyethyleneoxide,
agarose, dextran, polymethylacrylamide, and
polyacryloylethoxy ethenol.

24
 Modes of Operation

Capillary Isoelectric Focussing (CIEF):

- Comparable to tube IEF, commonly used to separate peptides and
proteins.
- Each molecule has a specific isoelectric point (pI). When the
surrounding pH is equal to this pI, the molecule carries no net charge.
- At a pH below the pI, the molecule is positive, and then negative
when the pH is above the pI.
- Because the charge changes with pH, a pH gradient can be used to
separate molecules in a mixture.
- Typically no EOF is applied (using a coated capillary).
- When the voltage is applied, the ions will migrate to a region where
they become neutral (pH=pI).

25
- The anodic end of the capillary sits in acidic solution

(low pH), while the cathodic end sits in basic solution

(high pH).

- Compounds of equal isoelectric points are “focused”

into sharp segments and remain in their specific zone,

which allows for their distinct detection.
 Modes of Operation

Capillary Isotachophoresis (CITP):

- ITP is the only method to be used in a discontinuous
system.
- The analyte migrates in consecutive zones and each
zone length can be measured to find the quantity of
sample present.

27
 Summary

a. Capillary Isotachophoresis

b. Capillary gel electrophoresis

c.Capillary isoelectric Focussing Electrophoresis

d.Micellar electrokinetic chromatography

28
 Capillary Electrophoresis (CE) versus High
Performance Liquid Chromatography (HPLC)

CE has flat flow, compared to pumped parabolic flow of HPLC.

Flat flow will have narrower peaks & better resolution. CE has
greater peak capacity.

29
 HPLC is more thoroughly developed.

HPLC is more complex than CE.

HPLC has wider variety of column length and packing

Both techniques uses similar modes of detection.

Can be used complementary to one another.

30
31
 The sensitivity has made it as one of the choice
for many biomedical and clinical analyses.

Applications : used to separate

Drugs
Amino Peptide Protein DNA Nucleic / even
acids s s fragment acid metals.
s

32
 Multiple
myeloma
testing (6bands).
Other Haemoglobinopathy
clinical screening.

application
HbA1c
s include
Monitoring chronic
alcoholism (GGT).

33
CAPILLARY ELECTROPHORESIS-
MASS SPECTROMETRY (CE-MS)
CAPILLARY ELECTROPHORESIS-MASS
SPECTROMETRY (CE-MS)

Capillary electrophoresis
(high separation efficiency
in liquid phase)

CE-MS
Mass spectrometry (high
separation efficiency in gas
phase)
INSTRUMENTATION
 CAPILLAR
Y
ELECTROPHORESI
S
 Capillary electrophoresis is an analytical technique that separates ions

based on their electrophoretic mobility with the use of an applied voltage,

1000volts/cm.

 A capillary is present by connectinganode and cathode together.

 The movement of components along the capillary by 2 interactions.

1. Electrophoretic mobility

2. Electroosmotic flow

Electrophoretic mobility(Uep )
 Migration of charged particles ina stationary medium under the influenceof an
applied electric field.

 The positive components move towards the negatively charged cathode.

 electrophoretic mobility is given by the equation:

 Electroosmotic
flow
 The interior wall of capillary contains charged sites that are created by the ionization
of silanol groups on the fused silica.
 The positive component interact with the negatively charged inert surface in the
capillary.

 The EOF alongwith electrophoretic mobility resutls ineffective separation of

components.
 By definition, Movement of the separation buffer through the silica capillary as a
results of the existence of a zeta potential at the solvent/silica interface.
 At very low pH, ionization of silanol groups are very poor results in slow EOF.

 If pH increases, no. of ionized sites increases results in increase of EOF.

 At very high pH, maximum ionization sites and maximum EOF.

 INTERFACING CE WITH MS

 ELECTROSPRAY IONIZATION (ESI)

 Sheath flow interface

 Sheath less interface

 Liquid junction interface

 CONTINUOUS FLOW FAST ATOM BOMBARDMENT (CF-FAB)

 MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI)

 ELECTROSPRAY IONIZATION (ESI)

 It is an evaporative technique.

 Sample introduced through the capillary.

 At the tip of the capillary high voltage will be applied.

 Nitrogen is supplied as nebulizing gas which helps to spray the sample
analyte.
 Desolvation gas is heated nitrogen gas which helps to vaporize the sample.

 The high potential, droplets will be ionized.

 Heated desolvation gas will evaporate the solvent & it will produce the molecular ion.
STRATEGIES FOR COUPING CE TO MS
VIA ESI

Sheath flow interface

 This consists of a Central tube (the CE capillary) surrounded by a second stainless
steel tube-the sheath liquid tube.

 The sheath liquid flows between this tube and the inner CE capillary.
 Between the sheath liquidtube and the third outer tube, or glass tube, flowsthe
nebulizing gas that helps in the nebulizing process.

 For thistype of interface, a sheath liquidisconstantly injected inside the nebulizer

through a coaxial canal, external to the CE capillary.
 The background electrolyte (BGE) and the sheath liquid are forming a junction at the
extremity of the ESI nebulizer, and sprayed in a single process.

 Sheath liquid:

 Commonly used: 1:1 mixture of water-methanol with 0.1% acetic acid or formic
acid.
 functions

 The sheath liquid is connected to the CE outlet electrode, therefore the junction

formed with the BGE enables to maintain the electric field.

 The electrospray process is optimal at flow rates in the μL/min range and because of

the electroosmotic flow, EOF in CE is of the order of 20-200nL/min, there is an obvious

discrepancy between the EOF and the requirements of electrospray. In order to match

the effluent flow to the requirements for electrospray, a make-up liquid is provided by

the sheath liquid.

 In the CE-MS coupling there is a high voltage applied to the inlet side of the capillary
and also a high voltage potential between the sprayer needle and the end- plates near
the MS entrance capillary.

 The potential b/w the sprayer needle and the MS entrance is approx. 3-5 kV.
 If the potential is negative, then positive ions will enter the MS- this is called positive
ion mode.

 If the potential ispositive, then negative ions will enter the MS and thisiscalled
negative ion mode.
Sheath less
interface
 CE capillary is coupled directly to an ESI source with a sheath less interface system.

 The electric contact for ESI is realized by using capillary coated with conductive metal.
 Because no sheath liquid is used, the system has high sensitivity, low flow rates and
minimum background.

 However,theseinterface designs, all have challenges including lowmechanical

robustness, poor reproducibility.
 The latest sheath less interface design features porous ESI emitter through chemical
etching.

 The design effectively provides robust interfacing with massspectrometry and

addresses the reproducibility challenges associated with previous designs.
Liquid junction
interface
 This technique uses a stainless steel tee to mix separation electrolyte from CE
capillary with make up liquid.

 The CE capillary and ESI needle are inserted through opposite sides of the tee and a
narrow gap is maintained.

 The electricalcontact isestablished by makeup liquidsurrounding the junction

between 2 capillaries.

 This system easy to operate.

 However, the sensitivity is reduced and the mixing of 2 liquids could degrade separate.
 CONTINUOUS-FLOW FAST ATOM
BOMBARDMENT

 CE can be coupled to FAB ionization using a continuous flow interface.

 The interface must match the flow rate between the 2 systems.
 The CF-FAB requires a relatively high flow rate but CE need low flow rate for better
separation.

 A make-up flow can be used using a sheath flow or liquid junction.

 Desorption ionization technique.

 Sample and a matrix mixed to form sample – matrix mixture.

 Gas like xenon or argon will be enter the chamber and become radical.

Radical ion react with Xe or Ar, already present in chamber.

Accelerated neutral atoms hit to the sample-matrix mixture.
Free radical cations will be removed by electric field.
Accelerated neutral atoms will be bombarded to the sample-matrix mixture & ionize
the sample.
 COUPLING CE WITH MALDI-MS

 Desorption technique.

 Sample is placed in a matrix.

 Matrix made up of 2,4-dihydroxybenzoic acid and cinnamic acid.

 Matrix liquified at beginning.

 Allow it for solidification.

 Now, sample is entrapped in the matrix.

 Sample : matrix = 1:10000

 Laser hit onto the matrix.

 Transfer of laser energy from matrix to sample.

 Sample particles getting kicked out, i.e.; desorbed from matrix.

 The sample particles become charged now due to the proton transfer to sample.

 Ionized sample-molecular ion.

 Off-linecoupling of CE to MALDI,the CE effluent could be sprayed or added
dropwise on MALDI target plate then dried and analyzed by MS.
 For online coupling, a movingtarget with continuouscontact to CE capillary end
is required.

 The moving target takes analytes into MS where it is desorbed or ionized.

 Musyimi et al. Developed a new technique where rotating ball was used to transfer CE
to MS.
 As the ball rotates the sample is dried beforeit reaches ionization region.

 This technique has high sensitivity since no make-up fluid is used.

 MASS
SPECTROMETRY

PRINCIPL
Ewhich sample is converted to rapidly moving
 MS is an instrumental technique in

positive ions by electron bombardment and charged particles are separated

according to their masses.

 Organic molecules are bombarded with electrons.

 Converted into highly energeticpositively charged ions – molecular ions/parent

 Further break into smaller ions- fragment ions/daughter ions.
 The formed ions are separated by deflection in magnetic field according to their mass
and charge.

 Mass spectrum- relative abundance(%) vs mass/charge ratio.

 Loss of electron from a molecule leads to free radical cation.

 PRINCIPLE OF CE-MS

 In CE-MS combine the high efficiency and high speed of CE with high selectivity
and high sensitivity offered by MS detection.
 Separation first on the basis of an analyte’s charge-to-size ratio and then on the basis
of its mass-to-charge ratio.

 First separating the ionic components of a sample by applying voltageto the

sample.
 The ions will move through the capillary at different rates due to charge and frictional
forces.

 The separated samples is then sprayed into the mass spectrometer which produces a
spectra.

 The spectra is used to identify the individual components of thesample.

 APPLICATION
S
1. Drug analysis and bioanalysis.

 Suitable for analysis of drugs in various matrices.

 In impurity profiling.

 Chiral analysis.

 Determination of drugs.

 Eg: Analysis of Tetrandrine and Fangchinoline which are components of some

Chinese medicines.
2.Analysis of intact proteins and peptides.
 Providing fragmentation data that then be compared against databases to identify
unknown peptide or protein.

 Biopharmaceutical characterization.

 Glycoprotein analysis and Top-down protein analysis.

 Assessment of protein-ligand interactions.

 Metalloprotein characterization.
3.Analysis of amino acids.
 Amino acids have also been analyzed by CE-MS and although the CE separation was
not fully resolved, this was remedied by the MS.

 Eg; separation and quantitative analysis of amino acids in urine.

 A good separation of 27 amino acids , including the isomers L-leucine, L-isoleucine
and L-alloisoleucine, in less than 30 min.
4. Food analysis and foodomics.
 Application of CE-MS in food safety and quality, as well as in other aspects related to
food traceability and bioactivity following classical food analysis as well as novel
foodomics approaches.
5. Metabolomics.
 Metabolomics is a rapidly emerging field of functional genomics research whose aim
is the comprehensive analysis of low molecular weight metabolites in a biological
sample.

 CE-ESI-MS offers a convenient format for the separation of complex mixtures of

cationic, anionic and/or zwitterionic metabolites, as well as their isobaric /isomeric
without complicated sample handling.
6. Separation of isomeric compounds.
 Glucose-6-phosphate and Fructose-6-phosphate, which have the same chemical
formulae and molecular weights, are not be resolved by LC-MS, but can be and
quantitated by CE-MS.

 Separation of Scopolamine and two stereoisomers of Hyoscyamine.

7.Forensic sciences with focus on forensic toxicology.
 REFERENC
E
 GordonA. Ross;Capillary Electrophoresis- Massspectrometry: Practical
implementation and applications; LC.GC Europe- January 2001.

 Rodrigues KT, et al; CE-MS for the analysis of aminoacids; Methods Mol Biol. 2018

 www.biocompare.com

 www.humanmetabolome.com
 Julie Schappler, Victor Gonzalez-Ruiz, Serge Rudaz; CE-MS indrug analysis and
bioanalysis; 16 June 2016.
 Christian W. Klampfi, Markus Himmelsbach; Sheath liquids in CE-MS: Role, Parameters,
and Optimization; 16 June 2016.

 Rob haselberg, Govert W Somsen; CE-MS for the analysis of intact proteins; 16 June
2016.

 Tanize Acunha, Clara Ibanez, Virginia Garcia-Canas; CE-MS infood analysis and
foodomics; 16 June 2016.

Nadia Porpiglia, Elena Giacomazzi, Rossella Gottardo, Franco Tagilaro; CE-MS in

 Akiyoshi Hirayama, Tomoyoshi Soga; CE-MS in Metabolomics; 16 June 2016.
 Venkateswarlu N; A Review on Capillary Electrophoresis- Massspectrometry (CE-
MS);Research & Reviews: Journal of Pharmaceutical Analysis.

 Wikipedia.
Theoretical Plates (N)
 Definition
A theoretical plate in many chromatographic
processes is a hypothetical zone or stage in which
sample analyte develops an equilibrium between two
phases, such as the liquid mobile phase and solid
stationary phase. Such equilibrium stages may also
be referred to as an equilibrium stage, ideal stage, or
a theoretical tray. The performance of many
separation processes depends on having series of
equilibrium stages and is enhanced by providing more
such stages.
 Column Efficiency
Depends upon following parameters:
 Significance
 A theoretical plate is not any physical plate present in the
chromatographic column, rather is a hypothetical equilibrium stage that
is resulted from mathematical calculations of a chromatogram.
 No. of theoretical plates for sample components in a given
chromatographic column represent the column efficiency.
 In a chromatogram, different peaks correspond to different components of the
separated mixture.

 Retention Time, tR is the difference in time between the point of injection and
appearance of peak maxima, expressed in minutes or seconds.

 The value of tR is high, when solute has higher affinity to the stationary phase
and vice versa.
 Columns having a high number of theoretical plates are
considered more efficient in chromatographic separation than
the columns having less number of theoretical plates.
 A more efficient chromatographic column will have a narrow
peak than a less efficient column having lesser number of
theoretical plates at same retention time.
 Resolution of peaks depends upon column efficiency, i.e.
number of theoretical plates.
 Theoretical plates are calculated per meter length of the
column. For GLC, a value of 600/m is sufficient, but for HPLC,
higher values ranging from 40,000 – 70,000/m are
recommended.
Retention Factor or Capacity Factor (k)
•The retention factor, k is equal to
the ratio of retention time of the
analyte on the column to the
retention time of a non-retained
compound.
•The non-retained compound has
no affinity for the stationary phase
and elutes with the solvent front at
a time t0 , which is known as “hold
up
• time”
A highor(k)
“dead time”.
values indicates that sample is highly retained by
the stationary phase. It is suggested that the value of K should
 The selectivity or separation
factor (α) is the ability of a
chromatographic system to
chemically distinguish between
sample components.

 It is usually measured as a ratio

of the retention factor (k) of the
two peaks and can be
visualized as the distance
between the apices of two
peaks.
Resolution (Rs)
 Validation of a chromatographic
procedure is to obtain the optimum
resolution in the minimum time.
 A resolution value of 1.5 or greater
between two peaks will ensure that
sample components are well separated
(separated from the base line)-to a
degree at which area of each peak may
be accurately measured.
 Rs is calculated using the separation of
two peaks in terms of average peak
width at the base (tR2 > tR1) .
Height Equivalent to Theoretical Plate (HETP)
 A theoretical plate is an imaginary or hypothetical unit of a column where
distribution of solute between stationary and mobile phase has attained an
equilibrium. It can also be known as ‘functional unit’ of the column.
 The height of one theoretical plate is referred to as ‘Height Equivalent to
Theoretical Plate’ HETP.
 A theoretical plate can be of any height, which describes the efficiency of
separation.
 If HETP is less, column is more efficient, if HETP is more, column is less efficient.
 HETP = Length of theoretical plate
No. of theoretical plates
 Each plate is the distance over which molecules of one sample component
develops an equilibrium between the stationary and mobile phase in the column.
 HETP is given by Van Deemter Equation.
How peak position and shape controls
theoretical plate number
 In order to optimize saparation efficiency, it is necessary to maximize
the number of theoretical plates, which requires reducing the plate height.

 The plate height is related to the flow rate of the mobile phase, so for a
fixed set of mobile phase, stationary phase and analyte; separation
efficiency can be maximized by optimizing the flow rate.

 Efficiency can be increased by increasing the column length, reducing

the column diameter and decreasing the particle size.

 Moreover, it is needed to have a minimized column dead volume in

order to avoid efficiency losses.
 Formulae for Calculating the Number of
Theoretical Plates
1. Tangent Line Method

N = 16 (tR/W)2

Applied by US Pharmacopiea
(German Pharmacopeia), BP (British Pharmacopeia), and EP (European Pharmacopeia).

2. Half Peak Height Method

Applied by

German Pharmacopeia
British Pharmacopeia
European Pharmacopeia
Japanese Pharmacopeia
3. Area Height Method

This method provides relatively

accurate and reproducible widths,
even for distorted peaks, but results
in somewhat larger N values when
peak overlap is significant.
4. EMG Method (Exponentially Modified Gaussian)
van Deemter Equation
Plate Theory
The theory assumes that the column is divided into a
number of zones called theoretical plates.

At each plate equilibrium of the solute between the

mobile phase and the stationary phase is assumed to
take place.

The partitioning of a solute between the phases

takes plate at each theoretical plate.

Thus, the number theoretical plates in the column is

used as a measure of efficiency of the column to
separate the components from each other
 The number of theoretical plates can be
determined by
N = 16 (tR/W)2

where, N = no. of theoretical plates

tR = retention time
W = base width of the peak
 HETP value can be determined by,

H=HETP
HETP = Height Equivalent to Theoretical Plates
H = L/N
Putting value of N

H=

HαW
H α 1/tR
Plate theory disregards the kinetics of mass transfer;
therefore, it reveals little about the factors influencing
HETP values.

The resulting behavior of the column plate is calculated

on the assumption that the distribution coefficient
remains unaffected by the presence of other solutes and
that the distribution isotherm is linear.

The diffusion of solute in the mobile phase from one

plate to another is also neglected.
 Discrete Continuous
Flow Flow
Model Model

Plate
Theory
Discrete-Flow Model
The assumptions in this model
are

• All the mobile phase moves from one segment to

(a) the next segment at the end of a discrete interval

• The samplemolecules are always inequilibrium

(b) with the mobile and stationary phases
Continuous-Flow Model
The assumptions in this model
are
• The mobile and stationary phases remain
(a) in equilibrium throughout the separation

• The mobile phase flows from one segment to the

(b) next segment at a constant rate

• Perfect mixing takes place in all

segments
(c)
Rate Theory
It was introduced by a Dutch Chemical Engineer,
Van Deemter.

It describes the factors responsible for broadening of

a chromatographic peak as well as its time of
elution.

Van Deemter equation describes the relation of the

height of a theoretical plate H and the average linear
velocity of the mobile phase.
Van Deemter Equation

HETP = height equivalent

to theoretical plate
µ = average linear velocity
of the mobile phase
A = eddy diffusion term
B = longitudinal or
ordinary diffusion term
C = nonequilibrium or
resistance to mass
transfer term

van Deemter H/u curve

 Eddy Diffusion (A)
The term (A) which is referred as Eddy Diffusion or Multiple-path Co-
efficientcorresponds to band broadening caused by dispersion
(multi-pathway) effects.

Eddy Diffusion, A = 2λdp

λ = correction factor for the irregularity of the column packing
dp = average particle diameter.
The particles of the stationary phase, whether irregularly or
spherically shaped, are packed as tightly as possible, and the
solute molecules must pass around them to proceed along the
column.
In this case, the spaces along the column are not uniform.

When a sample migrates down the column, each molecule

“sees”
different paths and each path is of a different length.

Some molecules take the longer paths and others take

the shorter paths.

There are also variations in the velocities of the mobile phase

within these pathways.

The overall result is that some molecules lag behind the center
of
the zone, whereas others move ahead of the zone.
Eddy Diffusion can be minimized by
 Using smaller stationary phase particles

 Selecting well packed columns

 Using particles with a narrow size distribution

Longitudinal Diffusion (B)
The B term represents band broadening by longitudinal
diffusion, the molecular diffusion both in and against the
flow direction:

B = 2γDM
γ = labyrinth factor of the pore channels (0<γ <1)
DM = diffusion coefficient of the analyte in the mobile
phase
This process results when there exists a region of high
concentration and a region of low concentration.

The migration is from the higher to the lower

concentration region in the axial direction of the column.

Diffusion occurs on the molecular level, resulting from

movement of molecules after collision

The diffusion is about 100–1,000-fold faster in gases than

in liquids, therefore B terms shows higher impact in GC
than in LC.
Longitudinal Diffusion can be minimized by
 Very high flow rates of mobile phase

 Using columns of short length

 Using narrow diameter column

Mass transfer under non equilibrium (C
The C terms refers to the mass transfer between stationary and
mobile phase.
The rapid mass transfer depends on the factors originating
from the stationary phase as well as the mobile phase The
term ‘C’ in Van Deemter equation is therefore, the sum of Cs &
CM.
The stationary phase contribution (Cs) to the plate height H, due
to the mass transfer under nonequilibrium condition, is given by,

q = configuration factor
r = a constant dependent upon the relative rate of migration of
a solute & the mobile phase,
d = thickness of the stationary phase
Ds = diffusion coefficient of a solute in the stationary phase.
The mobile phase contribution (CM) to the plate
height H, due to the mass transfer under
nonequilibrium conditions, is given by,

DG = diffusion coefficient in the gas

phase
dp = average particle diameter
ω = obstruction factor for packed bed
the band.
Using smaller stationary phase particles
Using lower mobile phase flow rate
Heating the column that will decrease the over-
adsorption of solute molecules over the surface of
stationary phase.
Van Deemter Plot
ELECTROPHORESI
S
 THEORY OF ELECTROPHORESIS
DEFINITION:
• Electrophoresis is a physical method of analysis which involves
separation of compounds that are capable of acquiring electric charge in
conducting medium.

• Electrophoresismaybe defined as the migration of the charged

particles through a solution under the influence of an external electrical
•field.
Ions that are suspended between two electrodes towards
the
tends to travel electrodes that bears opposite charges.
3

Depending on kind of charge the molecule carry, they move towards either

To cathode

Or to Anode
An ampholyte become positively charged in acidic condition and
migrate to cathode, in alkaline condition they become negatively charge
The rate of migration of an ion in an electrical field
depend on factors:
1.Net charge of molecule 2.Size and shape of
particle 3.Strength of electrical field
4.Properties of supporting medium 5.
Temperature of operation
 CONVENTIONAL
ELECTROPHORESIS
Instrumentation
Two reservoir for the buffer

Power supply and Electrodes

Separation medium
10
At any given PH, exist in a solution an electrically charged
species act either as a cation (+) or anion(-).

Under the influence of an electric field these charged

particles
will migrate either to cathode or anode, depending on the
nature of their charge.
The equipment required for electrophoresis consist basically
of two items,
•Power Pack Unit
Supplies a direct current between electrode in the
electrophoretic separation unit.
•Electrophoretic Separation Unit
Available for running either vertical or horizontal Gel system
or paper or capillary system.
BUFFER
The buffer in electrophoresis has two functions:

Carry applied electrical current

They set the pH at which electrophoresis is carried

out.

Thus they determine;

Type of charge on solute.

Extent of ionization of solute

Electrode towards which the solute will migrate.

The buffer ionic strength will determine the

thickness of the ionic cloud.
SUPPORTING MEDIUM
Supporting medium is a matrix in which the
sample
separation takes place.
Various types of supporting media have been
used for the separation either on slab or
capillary form.

Separation is based on to the charge to mass

ratio of solutes depending on the pore size
of the medium, possibly the molecular size.
 TYPES OF ELECTROPHORESIS

1) Zone Electrophoresis
 Paper Electrophoresis
 Gel Electrophoresis
 Thin Layer Electrophoresis
 Cellulose acetate Electrophoresis
2)Moving Boundary Electrophoresis
a) Capillary Electrophoresis
b) Isotachophoresis
c) Immuno Electrophoresis
d) Isoelectric Focusing
4
 ZONE ELECTROPHORESIS
It involves the migration of the charged particle on the supporting media.
Paper, Cellulose acetate membrane, Starch Gel, Poly acrylamide.
Components separated are distributed into discrete zone on the support media.
Supporting media is saturated with buffer solution, small volume of the sample is
as narrow
appliedband.
On application of voltage at the ends of a strip, components migrate at a rate
determined by its electrophoretic mobility.

ADVANTAGES:
Useful in biochemical investigations.
Small quanity of sample can be analysed.
Cost is low and easy maintenance.

DISADVANTAGES:
Unsuitable for accurate mobility and isoelectric point
determination. 5
Due to the presence of supporting medium, technical complications such as capillary
flow, electro osmosis, adsorption and molecular sieving may affect the separation of
sample components.
 GENERAL METHOD OF OPERATION
Saturation of the medium with the buffer.

Sample application.

Electrophoretic separation. Removal of

the supporting media.

INSTRUMENTATION

Electrophoretic chamber.
Electrodes.
Diffusion barriers.
Supporting/ Stabilizing media.
6
(inert to sample and to any developing reagents).
 PAPER ELECTROPHORESIS

1. Horizontal paper Electrophoresis

2. Vertical paper Electrophoresis
HORIZONTAL PAPER ELECTROPHORESIS

VERTICAL PAPER ELECTROPHORESIS

8
 GEL ELECTROPHORESIS
Separation is brought about through molecular sieving technique, based on the molecular
size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are;
Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
A porous gel acts as a sieve by retarding or, in some cases, by completely
obstructing the movement of macromolecules while allowing smaller molecules to
migrate freely.
GEL ELECTROPHORESIS
Gel Electrophoresis
is caried out in two
methods:

•Vertical starch
Gel electrophoresis

•Horizontal starch
gel electrophoresis

12
 AGAR AND AGAROSE GEL
Agar is a mixture of poly saccharides extracted from sea weeds.
Agarose is a highly purified uncharged polysaccharide derived from agar.
Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L-galactose.
The pores of an agarose gel are large, agarose is used to separate macromolecules such as
nucleic acids, large proteins and protein complexes.

ADVANTAGES:
Easy to prepare and small concentration of agar is required.
Resolution is superior to that of filter paper.
Large quantities of proteins can be separated and recovered.
Adsorption of negatively charged protein molecule is negligible.
It adsorbs proteins relatively less when compared to other medium.
Sharp zones are obtained due to less adsorption.
Recovery of protein is good, good method for preparative purpose.
• DISADVANTAGES
Electro osmosis is high.
•
Resolution is less compared to polyacrylamide gels.
•Different sources and batches of agar tend to give different results and
purification is often necessary.
POLYACRYLAMIDE GEL ELECTROPHORESIS
(PAGE)
•Polyacrylamide gels are garose gels.
•It is thermo stable, transparent, strong and relatively
chemically
inert.
•Gels are uncharged and are prepared ina variety of
pore sizes.
•Proteins are separated on the basis of charge to mas
ratio and molecular a phenomenon called s
size, sieving. Molecular
ADVANTAGES:
• Gels are stable over wide range of pH and
• temperature. Gels of different pore size can be
15
• formed.
Simple and separation speed is good comparatively.
 PAGE-PROCEDURE
The gel of different pore sizes is cast into a column inside a vertical tube, often with
large pore gel at the top and small pore gel at the bottom.

Microgram quantity of the sample is placed over the top of the gel column and
covered
by a buffer solution having such a pH so as to change sample components into
anions.
The foot of the gel column is made to dip in the same buffer in the bottom
reservoir.
Cathode and anode are kept above and below the column to impose an electric
field through the column.
Macromolecular anions move towards the anode down the gel column.
There is no external solvent space, all the migratory particles have to pass through the
gel pores.

Rate of migration depends on the charge to mass ratio.

Different sample components get separated into discrete migratory bands along the
gel
column on the basis of electrophoretic mobility and gel filtration 17
effect.
PAGE SLAB PAGE
PROCEDURE

Polyacrylamide Gel
Electrophoresis (PAGE)
a) The is poured vertically
between twgeol glass plates.
b.) Protein bands are separated
on
th1e8 basis of relativemolecular
 VISUALIZATION

After the electrophoresis is complete, the molecules in the gel can be stained to
make them visible.

Ethidium bromide, silver, or coomassie blue dye may be used for this
process.
If the analyte molecules fluoresce under ultraviolet light, a photograph can be
taken of the gel under ultraviolet lighting conditions. If the molecules to be
separated contain radioac1t9ivity added for visibility, an autoradiogram can be
recorded of the gel.
APPLICATIONS OF ELECTROPHORESIS
1. DNA Sequencing
2. Medical Research
3. Protein research/purification
4. Agricultural testing
5. Separation of organicacid, alkaloids, carbohydrates, amino
acids, alcohols, phenols, nucleic acids, insulin.
6. In food industry
7. It is employed in biochemical and clinical fields i.e. in the study of protein
mixtures such as blood serum, haemoglobins and in the study of antigen-
antibody interactions.
8. Electrophoresis is also used for separation of carbohydrates and vitamins.
9. Quantitative separation of all fractions of cellular entities, antibiotics, RBC,
Enzymes etc is possible.

10.Quantitative separation of all fractions of cellular entities, antibiotics, RBC,

Enzymes etc
is
possible.