2. Introduction
High-performance liquid chromatography (HPLC) is a form
of liquid chromatography to separate compounds that are dissolved
in solution.
HPLC involves a solid stationary phase, normally packed inside a
stainless-steel column, and a liquid mobile phase.
The different components in the mixture pass through the column at
different rates due to differences in their partitioning brhavior
between the mobile liquid phase and the stationary phase
Separation of the components of a solution results from the
difference in the relative distribution ratios of the solutes between
the two phases.
3. Components of HPLC
Stationary phase
Components
of HPLC
Mobile phase
Injector
Detectors
Pumping system
Chromatographic
column
4.
5. Components of HPLC
Pumping system
Computer- or microprocessor-controlled pumping systems are
capable of accurately delivering a mobile phase of either constant
(isocratic elution) or varying (gradient elution) composition,
according to a defined programme.
In the case of gradient elution, solvent mixing can be achieved on
either the low- or high-pressure side of the pump(s).
6. Injector
The sample solution is usually introduced into the flowing mobile phase at
or near the head of the column using an injection system based on an
injection valve design which can operate at high pressure.
Chromatographic column
Columns are usually made of polished stainless steel, are between 50 and
300 mm long
internal diameter of between 2 and 5 mm.
They are commonly filled with a stationary phase with a particle size of 3–
10 μm.
un modified Silica alumina or porous graphite (normal phase)
Modified silica C8 and C18 (reverse phase)
7.
8. Stationary phase
They are usually solids with polar compounds stick to the
column wall.
HPLC systems consisting of polar stationary phases and non-polar
mobile phases are described as normal-phase
chromatography.
Those with non-polar stationary phases and polar mobile
phases are called reversed-phase chromatography.
9. Detectors
• Detectors provide flow cells that give energy to show result.
Ultraviolet/visible (UV/vis) absorption spectrophotometers
evaporative light-scattering detectors (ELSD)
charged aerosol detectors (CAD)
mass spectrometers (MS) or other special detectors may be used.
Mobile phase
The mobile phase, or solvent, in HPLC is usually a mixture of polar
and non-polar liquid components whose respective concentrations are
varied depending on the composition of the sample.
As the solvent is passed through a very narrow column, any
contaminants could at worst plug the column.
10. Working Principle
The sample mixture to be separated and analyzed is
introduced, in a discrete small volume (typically microliters),
into the stream of mobile phase.
The components of sample in the mobile phase moves with
different velocity due to its interaction with the stationary
phase.
The smaller particle size of the stationary phase provides less
binding of the sample in mobile phase with the stationary
phase and also increases the flow rate of the sample to be
eluted out more quickly.
11. To enhance the fast
flow rate pump is
used
Then, outside the
column they are sent
into a detector, where
individual compounds
are detected and
recorded in a computer
The detector is wired to the
computer data station, that
records the electrical signal to
generate the chromatogram to
display and to identify and
quantitate the concentration of
the sample constituents.
Preparatory
chromatography
The recordings (preferably in
the form of quantitative peaks)
are compared with those of
standard compound's HPLC
values and the individual
compounds are identified
12.
13. • The time at which a specific analyte elutes (emerges from the
column) is called its retention time.
14.
15. Normal phase HPLC
• The column is filled with tiny silica (polar)particles, and the solvent
is non-polar – hexane.
• Polar compounds in the mixture being passed through the column
will stick longer to the polar silica than non-polar compounds will.
• The non-polar ones will therefore pass more quickly through the
column.
16. Reverse Phase Chromatography
• In this case, the column size is the same, but the silica is modified to make
it non-polar by attaching long hydrocarbon chains to its surface - typically
with either 8 or 18 carbon atoms in them (Stationary phase).
• A polar solvent is used - for example, a mixture of water and an alcohol
such as methano (Mobile phase).
• In this case, there will be a strong attraction between the polar solvent
and polar molecules in the mixture being passed through the column.
There won't be as much attraction between the hydrocarbon chains
attached to the silica (the stationary phase) and the polar molecules in the
solution.
• Non-polar compounds in the mixture will tend to form attractions with the
hydrocarbon groups because of van der Waals dispersion forces.
• That means that now it is the polar molecules that will travel through the
column more quickly. Reversed phase HPLC is the most commonly used
form of HPLC .
18. Pharmaceutical applications
• Identification of active ingredients of dosage forms
• Pharmaceutical quality control
Environmental applications
• Detection of phenolic compounds in Drinking Water
• Identification of diphenhydramine in sedimented samples
• Bio-monitoring of pollutant
Forensics
• Quantification of the drug in biological samples.
• • Determination of cocaine and metabolites in blood
19. Clinical
• Quantification of ions in human urine Analysis of antibiotics in
blood plasma.
Food and Flavor
• Ensuring the quality of soft drink and drinking water.
• Analysis of beer.
• Sugar analysis in fruit juices.
21. INTRODUCTION
• Gas chromatography (GC), also called gas-liquid
chromatography
• Common type of chromatography used in analytical chemistry
for separating and analyzing volatile organic compounds
without decomposition.
USES
• Testing the purity of a particular substance
• separating the different components of a mixture (the relative
amounts of such components can also be determined)
• help in identifying a compound.
22. • Gaseous compounds interact with the walls of
the column(narrow tube) which is coated with a
stationary phase and located in an oven where
the temperature of the gas can be controlled.
• Each compound elutes at a different time,
known as the retention time of the compound.
The comparison of retention times is what gives
GC its analytical usefulness.
• GC separates the components of a mixture
primarily based on boiling point (or vapor
pressure) differences.
23. HISTORY
• Archer John Porter Martin was awarded the Nobel Prize for his
work in:
• developing liquid–liquid (1941) and
• paper (1944) chromatography,
• laid the foundation for the development of gas chromatography
and he later produced liquid-gas chromatography (1950).
24. GC ANALYSIS
• A known volume of gaseous or liquid analyte is injected into
the "entrance" (head) of the column, usually using a
microsyringe .
• As the carrier gas sweeps the analyte molecules through the
column, this motion is inhibited by the adsorption of the
analyte molecules either onto the column walls or onto packing
materials in the column.
• The rate at which the molecules progress along the column
depends on the strength of adsorption, which in turn depends
on the type of molecule and on the stationary phase materials.
• The components of the analyte mixture are separated at the end
of the column at different retention times.
26. INSTRUMENTAL
COMPONENTS
• Inlet/Sample injector
provides the means to introduce a sample into a continuous
flow of carrier gas. The inlet is a piece of hardware attached to
the column head.
The most common injection method:
A microsyringe is used to inject sample through a rubber
septum into a flash vapouriser port at the head of the column.
The temperature of the sample port is usually about 50°C
higher than the boiling point of the least volatile component
of the sample.
27. The injector can be used in one
of two modes;
• split or splitless.
• The injector contains a
heated chamber containing a
glass liner into which the
sample is injected through
the septum. The carrier gas
enters the chamber and can
leave by three routes (when
the injector is in split mode).
• The sample vapourises to
form a mixture of carrier gas,
vapourised solvent and
solutes. A proportion of this
mixture passes onto the
column, but most exits
through the split outlet.
28. Carrier gas
• Chemically inert. Commonly used gases include nitrogen,
helium, argon, and carbon dioxide.
• The choice of carrier gas is often dependant upon the type of
detector.
• The carrier gas system also contains a molecular sieve to
remove water and other impurities.
29. Detectors
• Non-selective detector responds to all
compounds except the carrier gas,
• Selective detector responds to a range of
compounds with a common physical or
chemical property and a specific detector
responds to a single chemical compound.
• Concentration dependant detectors-concentration
of solute in the detector.
• Mass flow dependant detectors -rate at
which solute molecules enter the detector.
30. Commonly Used Detectors
• flame ionization detector (FID)
• thermal conductivity detector (TCD).
Both are sensitive to a wide range of
components and concentrations.
TCDs-universal and can be used to detect
any component other than the carrier gas.
FIDs are sensitive to hydrocarbons.
31. METHODS
• The method used depends on the collection of conditions
used.
Conditions:
• inlet temperature,
• detector temperature,
• column temperature
• carrier gas
• carrier gas flow rates,
• the column's stationary phase, diameter and length,
• Inlet type and flow rates,
• sample size and injection technique.
32. Carrier gas selection and flow rates
• Typical carrier gases include helium, nitrogen,
argon, hydrogen and air.
• The purity of the carrier gas
• The carrier gas linear velocity.
The higher the linear velocity the faster the
analysis, but the lower the separation between
analytes.
Polarity of the stationary phase
• Polar compounds interact strongly with a polar
stationary phase, hence have a longer retention
time than non-polar columns.
33. Inlet types and flow rates
• The choice of inlet type depends on if the sample is in
liquid, gas, adsorbed, or solid form.
Sample size and injection technique
Sample injection
• The amount injected should not overload the column.
• The width of the injected plug should be small.
34. Column selection
• depends on the sample measured.
• polarity of the mixture must closely match the polarity of the
column stationary phase to increase separation while reducing
run time.
• The separation and run time also depends on the film
thickness (of the stationary phase), the column diameter and
the column length.
35. • Column temperature
• The temperature is precisely controlled electronically.
• The rate at which a sample passes through the column is
directly proportional to the temperature of the column
36. DATA REDUCTION AND
ANALYSIS
Qualitative analysis
• Data- in the form of graph. detector response (y-axis) is
plotted against retention time (x-axis), which is called a
chromatogram.
• spectrum of peaks of a sample- analytes eluting at different
times.
• Retention time-identify analytes if the method conditions are
constant.
38. Quantitative analysis
• area under a peak is proportional to the
amount of analyte present in the
chromatogram.
• Calculation of the peak area-concentration
of an analyte in the original
sample can be determined.
41. • Mass spectrometry has been described as the smallest scale in
the world, not because of the mass spectrometer’s size but
because of the size of what it weighs – molecules.
• In a typical MS procedure, a sample, which may be solid, liquid,
or gas, is ionized. The ions are separated according to their mass-to-
charge ratio. The ions are detected by a mechanism capable of
detecting charged particles. Signal processing results are
displayed as spectra of the relative abundance of ions as a
function of the mass-to-charge ratio. The atoms or molecules can
be identified by correlating known masses to the identified
masses or through a characteristic fragmentation pattern.
• Due to ionization sources such as electrospray ionization and
matrix-assisted laser desorption/ ionization (MALDI), mass
spectrometry has become an irreplaceable tool in the biological
sciences.
42. Principle
• Four basic components are, for the most part, standard in all
mass spectrometers .
1. Sample inlet
2. Ionization chamber
3. Mass analyzer
4. Ion detector
44. Sequence
• Stage 1: Ionisation
The vaporised sample passes into the ionisation chamber.The
electrically heated metal coil gives off electrons which are
attracted to the electron trap which is a positively charged plate.
The particles in the sample (atoms or molecules) are therefore
bombarded with a stream of electrons, and some of the collisions
are energetic enough to knock one or more electrons out of the
sample particles to make positive ions
45.
46. • Most of the positive ions formed will carry a charge of +1
because it is much more difficult to remove further electrons
from an already positive ion.
47. Acceleration
• The positive ions are repelled away from the very positive
ionisation chamber and pass through three slits, the final one of
which is at 0 volts. The middle slit carries some intermediate
voltage. All the ions are accelerated into a finely focused beam.
48.
49. Deflection
Different ions are deflected by the magnetic field by
different amounts.
1.the mass of the ion.
Lighter ions are deflected more than heavier ones.
2. The charge on the ion.
Ions with 2 (or more) positive charges are deflected more
than ones with only 1 positive charge
50.
51. Mass/charge ratio is given the symbol m/z (or sometimes m/e).
For example, if an ion had a mass of 28 and a charge of 1+, its
mass/charge ratio would be 28. An ion with a mass of 56 and a
charge of 2+ would also have a mass/charge ratio of 28
52. • In the diagram, ion stream A is most deflected it will contain
ions with the smallest mass/charge ratio. Ion stream C is the least
deflected - it contains ions with the greatest mass/charge ratio.
53. Detection
• When an ion hits the metal box, its charge is neutralised by an
electron jumping from the metal on to the ion (right hand
diagram). That leaves a space amongst the electrons in the
metal, and the electrons in the wire shuffle along to fill it.
• A flow of electrons in the wire is detected as an electric current
which can be amplified and recorded. The more ions arriving,
the greater the current.
54.
55. If you vary the magnetic field, you can bring each ion stream in
turn on to the detector to produce a current which is proportional
to the number of ions arriving.
The mass of each ion being detected is related to the size of the
magnetic field used to bring it on to the detector
The machine can be calibrated to record current against m/z
directly.
57. Sample introduction techniques
• Sample must be introduced so that the vacuum inside instrument
remains unchanged.
• Sample placed on
probe/plate
• Inserted into the ionization
chamber
Direct
insertion
• Capillary or capillary
column. Sample – gas or
solution
• Small quantities of sample
Direct
infusion
58. Ionization methods
Protonation
Ionization
methods
Deprotonation
Cationization
Transfer into
gas phase
Electron
Capture
Electron
ejection
59. Protonation
M + H MH+
H2N-RGASSRR-OH + H+ MH+
Protonation is a method of ionization by
which a proton is added to a molecule,
producing a net positive charge of 1+ for every
proton added. Positive charges tend to reside
on the more basic residues of the molecule,
such as amines, to form stable cations.
Peptides are often ionized via protonation.
Protonation can be achieved via matrix-assisted
laser desorption/-ionization (MALDI),
electrospray ionization (ESI) and atmospheric
pressure chemical ionization (APCI)
Deprotonation
M - H+ [M – H] --
Deprotonation is an ionization method by
which the net negative charge of 1- is achieved
through the removal of a proton from a
molecule. This mechanism of ionization is
very useful for acidic species including
phenols, carboxylic acids, and sulfonic acids.
60. Cationization
M + Cation+ → MCation+
Cationization is a method of ionization that
produces a charged complex by non-covalently
adding a positively charged ion to
a neutral molecule. While protonation could
fall under this same definition, cationization is
distinct for its addition of a cation adduct
other than a proton (e.g. alkali, ammonium).
Moreover, it is known to be useful with
molecules unstable to protonation. The
binding of cations other than protons to a
molecule is naturally less covalent, therefore,
the charge remains localized on the cation.
This minimizes delocalization of the charge
and fragmentation of the molecule.
Cationization is commonly achieved via
MALDI, ESI, and APCI. Carbohydrates are
excellent candidates for this ionization
mechanism, with Na+a common cation
adduct.
61. Electron ejection
-e--
M M+
electron ejection achieves ionization
through the ejection of an electron to
produce a 1+ net positive charge,
often forming radical cations.
Observed most commonly with
electron ionization (EI) sources,
electron ejection is usually performed
on relatively nonpolar compounds
with low molecular weights and it is
also known to generate significant
fragment ions.
62. Electron capture
+e--
M M--
With the electron captureionization
method, a net negative charge of 1- is
achieved with the absorption or
capture of an electron. It is a
mechanism of ion-ization primarily
observed for molecules with a high
electron affinity, such as halogenated
compounds.
Transfer of charged molecule to gas
phase
M+ solution M+ gas
The transfer of compounds already
charged in solution is normally
achieved through the desorption or
ejection of the charged species from
the condensed phase into the gas
phase. This transfer is commonly
achieved via MALDI or ESI.
63. Ionization sources
• Electron ionization (EI) limited chemists and biochemists to
small molecules well below the mass range of common bio-organic
compounds. This limitation motivated scientists to
develop the new generation of ionization techniques
• These techniques have revolutionized biomolecular analyses,
especially for large molecules. Among them, ESI and MALDI
have clearly evolved to be the methods of choice when it
comes to biomolecular analysis.
64. Ionization Source Acronym Event
Electrospray ionization ESI Evaporation of charged
droplets
Nanoelectrospray ionization nanoESI Evaporation of charged
droplets
Atmospheric pressure chemical
ionization
APCI Corona discharge and proton
transfer
Matrix-assisted laser desorption
ionziation
MALDI Photon absorption/proton
transfer
Desorption/ ionization on silicon DIOS Photon absorption/proton
transfer
Fast atom/ion bombardment FAB Ion desorption/ proton
transfer
Electron ionization EI Electron beam/electron
Chemical ionization CI Proton transfer
65. Ionization methods, advantages and disadvantagesods, advantages and
disadvantages
Ionization
Method
Advantages Disadvantages
Protonation
(positive
ions)
•many compounds will
accept a proton to become
ionized
•many ionization sources
such as ESI, APCI, FAB, CI
and MALDI will generate
these species
•some compounds are not
stable to protonation (i.e.
carbohydrates) or cannot
accept a proton easily (i.e.
hydrocarbons)
Cationization
(positive
ions)
•many compounds will
accept a cation, such as
Na+ or K+ to become
ionized
•many ionization sources
such as ESI, APCI, FAB and
MALDI will generate these
species
•tandem mass spectrometry
experiments on cationized
molecules will often
generate limited or no
fragmentation information
66. Deprotonation
(negative ions)
•most useful for compounds that
are somewhat acidic
•many ionization sources such as
ESI, APCI, FAB and MALDI will
generate these species
•compound specific
Transfer of
charged
molecule to gas
phase
(positive or
negative ions)
•useful when the compound is
already charged
•many ionization sources such as
ESI, APCI, FAB and MALDI will
generate these species
•only for precharged ions
Electron
ejection
(positive ions)
•observed with electron ionization
and can provide molecular mass as
well as fragmentation information
•often generates too much
fragmentation
•it can be unclear whether the
highest mass ion is the molecular
ion or a fragment
Electron
capture
(negative ions)
•observed with electron ionization
and can provide molecular mass as
well as fragmentation information
•often generates too much
fragmentation
•it can be unclear whether the
highest mass ion is the molecular
ion or a fragment
67. Electrospray Ionization MS
• Most commonly used for biomolecular MS
• Strong electric field is applied to a liquid passing through a capillary
tube with a weak flux under atmospheric pressure. Multiply charged
ions are obtained from proteins.
• Research of non-covalent complexes with the advantages of speed,
sensitivity, specificity and low sample consumption.
• Conjugates retain their structural integrity upon the transition from
solution state into gas phase
• Evidence for complex formation and accurate determination of their
binding stoichiometries
68.
69. Matrix-Assisted Laser Desorption/Ionization
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
was first introduced in 1988 by Tanaka, Karas, and Hillenkamp. It has since
become a widespread analytical tool for peptides, proteins, and most other
biomolecules (oligonucleotides, carbohydrates, natural products, and lipids).
The efficient and directed energy transfer during a matrix-assisted laser-induced
desorption event provides high ion yields of the intact analyte, and
allows for the measurement of compounds with sub-picomole sensitivity.
In addition, the utility of MALDI for the analysis of heterogeneous samples
makes it very attractive for the mass analysis of complex biological samples
such as proteolytic digests.
70. In MALDI analysis, the analyte is first co-crystallized with a large molar
excess of a matrix compound, usually a UV-absorbing weak organic
acid. Irradiation of this analyte-matrix mixture by a laser results in the
vaporization of the matrix, which carries the analyte with it. The matrix
plays a key role in this technique. The co-crystallized sample molecules
also vaporize, but without having to directly absorb energy from the
laser. Molecules sensitive to the laser light are therefore protected from
direct UV laser excitation.
Once in the gas phase, the desorbed charged molecules are then
directed electrostatically from the MALDI ionization source into the
mass analyzer. Time-of-flight (TOF) mass analyzers are often used to
separate the ions according to their mass-to-charge ratio (m/z). The
pulsed nature of MALDI is directly applicable to TOF analyzers since
the ion’s initial time-of-flight can be started with each pulse of the laser
and completed when the ion reaches the detector
71.
72.
73. MATRIX
The matrix consists of crystallized molecules, of
which the three most commonly used are 3,5-
dimethoxy-4-hydroxycinnamic acid (sinapinic acid
), α-cyano-4-hydroxycinnamic acid (alpha-cyano
or alpha-matrix) and 2,5-dihydroxybenzoic acid
(DHB). A solution of one of these molecules is
made, often in a mixture of highly purified water
and an organic solvent (normally acetonitrile
(ACN) or ethanol ).Trifluoroacetic acid (TFA) may
also be added. A good example of a matrix-solution
would be 20 mg/mL sinapinic acid in
ACN:water:TFA (50:50:0.1).
74.
75.
76.
77.
78. p = m/z
p1 = (Mr + z1)/z1
p is a peak in the mass spectrum
m is the total mass of an ion
z is the total charge
Mr is the average mass of protein
