2. CONTENTS
• INTRODUCTION
• BASIC TERMINOLOGIES
• DIFFERENT TYPES OF CHROMATOGRAPHY
• DIFFERENT TYPES OF LIQUID CHROMATOGRAPHY
• DEFINITION OF HPLC
• BASIC TERMS USED IN HPLC
• INSTRUMENTATION
• COMPONENTS USED IN HPLC
• PARAMETERS USED
5. INTRODUCTION TO CHROMATOGRAPHY
• Chromatography is used to separate mixtures of substances into their
components. All forms of chromatography work on the same principle.
• They all have a stationary phase (a solid, or a liquid supported on a solid)
and a mobile phase (a liquid or a gas). The mobile phase flows through
the stationary phase and carries the components of the mixture with it.
Different components travel at different rates.
• The purpose of preparative chromatography is to separate the
components of a mixture for more advanced use (and is thus a form
of purification). Analytical chromatography is done normally with smaller
amounts of material and is for measuring the relative proportions of
analytes in a mixture. The two are not mutually exclusive
6. BASIC CHROMATOGRAPHIC TERMS
• Chromatogram - A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks
or patterns on the chromatogram correspond to different components of the separated mixture.
• Chromatograph - A chromatograph is equipment that enables a sophisticated separation, e.g. gas chromatographic or liquid
chromatographic separation.
• Eluent - The eluent or eluent is the "carrier" portion of the mobile phase. It moves the analytes through the chromatograph.
In liquid chromatography, the eluent is the liquid solvent
• Eluate - The eluate is the analyte material that emerges from the chromatograph. It specifically includes both the analytes and
solutes passing through the column, while the eluent is only the carrier.
• Mobile Phase
• Stationary Phase
• Sample
• Retention Time
• Detector
7. ADVANTAGES OF HPLC
1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column separations are excellently made.
7. HPLC is more versatile than GLC in some respects, because it has the advantage of not being restricted to volatile and
thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC
8. 8. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment
9. A variety of solvents and column packing are available, providing a high degree of selectivity for specific analyses.
10. It provides a means for determination of multiple components in a single analysis.
10. DEFINITION• High-performance liquid chromatography is
a technique in analytical chemistry used to
separate, identify, and quantify each
component in a mixture. It relies on pumps to
pass a pressurized liquid solvent containing
the sample mixture through a column filled
with a solid adsorbent material. Each
component in the sample interacts slightly
differently with the adsorbent material,
causing different flow rates for the
different components and leading to the
separation of the components as they flow
out the column.
• HPLC has been used for manufacturing (e.g.
during the production process of
pharmaceutical and biological products), legal
(e.g. detecting performance enhancement
drugs in urine), research (e.g. separating the
components of a complex biological sample, or
of similar synthetic chemicals from each
other), and medical (e.g. detecting vitamin D
levels in blood serum) purposes.
11. WHAT IS HPLC ???
• It is the most widely used analytical techniques among other
chromatographic techniques
• It basically utilizes a liquid mobile phase and a packed column
• It’s popularity when compared to other techniques is more because
a) Its Sensitivity
b) Suitability for separating Non Volatile Species & Thermally fragile
ones
c) The most important thing is that it is used because it is ideally suited
for identification and separation of Amino acids,
proteins,carbohydrates,pigments.pesticides,steroids and many other
inorganic substances
12. PRINCIPLE
To understand the principle of HPLC , we must first look at the principle behind liquid
chromatography
Liquid chromatography is a separation technique that involves:
the placement (injection) of a small volume of liquid sample
into a tube packed with porous particles (stationary phase)
where individual components of the sample are transported along the packed tube (column) by
a liquid moved by gravity.
The main principle of separation is adsorption .
13. • When a mixture of components are introduced into the column . various chemical
and/or physical interactions take place between the sample molecules and the
particles of the column packing .
• They travel according to their relative affinities towards the stationary phase. The
component which has more affinity towards the adsorbent, travels slower.
• The component which has less affinity towards the stationary phase travels faster.
• Since no two components have the same affinity towards the stationary phase, the
components are separated
15. BASIC TERMINOLOGIES IN HPLC
• BASELINE - is the name given to that part of a chromatogram that represents any time period during
which only mobile phase is passing through the detector
• PEAK WIDTH - represent retention dimensions (time or volume) parallel to the baseline. If the
baseline is not parallel to the axis representing time or volume, then the peak-widths are to be drawn
parallel to this axis
• PEAK FRONTING - Peak fronting is the name given to asymmetric peaks having a wider front half of the peak
compared to the back half.
• PEAK TAILING – It is defined as the distance from the front slope divided by twice the distance from
the centre line of the peak to the front slope
19. COMPONENTS
OF HPLC UNIT
1. SOLVENT
2. SOLVENT DELIVERY PUMP
3. SAMPLE
4. INJECTOR
5. COLUMN
6. DETECTOR
7. DATA SYSTEM
8. WASTE COLLECTOR
20. DIFFERENT TYPES OF HPLC
Types of HPLC
I -There are following variants of HPLC, depending upon the phase system (stationary) in
the process :
1. Normal Phase HPLC:
This method separates analytes on the basis of polarity. NP-HPLC uses polar
stationary phase and non-polar mobile phase. Therefore, the stationary phase is
usually silica and typical mobile phases are hexane, methylene chloride, chloroform,
diethyl ether, and mixtures of these. Polar samples are thus retained on the polar
surface of the column packing longer than less polar materials. The technique is non
advantageous in pharmaceutical applications since most of the drug substances are
polar in nature and takes longer time to be eluted from the column
2. Reverse Phase HPLC:
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a
polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the
principle of hydrophobic interactions hence the more nonpolar the material is, the
longer it will be retained & the polar compound gets eluted first. This method is widely
used compared to normal phase HPLC
21. II.BASED ON PRINCIPLE OF SEPERATION
1.Absorption Chromatography
In the Absorption Chromatography solute molecules bond directly to the surface of the stationary phase
the component which has more affinity towards mobile phase elutes first & the component which has
less affinity towards stationary phase elutes later.
No two components have same affinity towards mobile phase & stationary phase.
22. • 2. Ion-exchange chromatography
• Ion exchange chromatography is a process that allows the separation of ions and polar molecules
based on their charge.
• It can be used for almost any kind of charged molecule including large proteins, small nucleotides and
amino acids.
• Retention is based on the attraction between solute ions and charged sites bound to the stationary
phase. Ions of the same charge are excluded.
23. 3.Ion-pair chromatography
• It is a form of chromatography in which ions in solution can be "paired" or
neutralized and separated as an ion pair on a reversed-phase column.
• Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that
imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-
phase column.
4. Gel permeation chromatography
• This type of chromatography lacks an attractive interaction between the stationary
phase and solute.
• The liquid or gaseous phase passes through a porous gel which separates the
molecules according to its size.
24. • 5.Affinity Chromatography
• This is the most selective type of chromatography employed. It utilizes the specific interaction
between one kind of solute molecule and a second molecule that is immobilized on a stationary phase.
For example, the immobilized molecule may be an antibody to some specific protein. When solute
containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this
antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic
strength or pH.
25. • 6.Chiral chromatography
• It involves the separation of stereoisomers. In the case of enantiomers,
these have no chemical or physical differences apart from being three-
dimensional mirror images. Conventional chromatography or other
separation processes are incapable of separating them. To enable chiral
separations to take place, either the mobile phase or the stationary phase
must themselves be made chiral, giving differing affinities between the
analytes.
26. III. BASED ON ELUTION TECHNIQUE
1.Isocratic elution
A separation in which the mobile phase composition
remains constant throughout the procedure is termed
isocratic elution
In isocratic elution, peak width increases with retention
time linearly with the number of theoretical plates.
This leads to the disadvantage that late-eluting peaks
get very flat and broad.
Best for simple separations
Often used in quality control applications that support
and are in close proximity to a manufacturing process
27. 2. Gradient elution
A separation in which the mobile phase composition is changed during the separation
process is described as a gradient elution
Gradient elution decreases the retention of the later-eluting components so that they
elute faster, giving narrower peaks . This also improves the peak shape and the peak
height
• Best for the analysis of complex samples
• Often used in method development for unknown mixtures
• Linear gradients are most popular
28. IV.BASED ON TYPE OF ANALYSIS
1.Qualitative analysis
Analysis of a substance in order to ascertain the nature of its chemical constituents
We can separate individual components but cannot assess the quantity in this analysis
2.Quantitaive analysis
Determining the amounts and proportions of its chemical constituents .
Quantity of the impurity and individual components can be assessed
29. SOLVENT DELIVERY SYSTEM(MOBILE
PHASE)
• The mobile phase in HPLC refers to the solvent being continuously applied to the column or stationary phase
• The mobile phase acts as a carrier to the sample solution
• A sample solution is injected into the mobile phase of an assay through the injector port
• As a sample solution flows through a column with the mobile phase,the components of that solution migrate according to the non-
covalent interaction of the compound with the column
30. •The chemical interaction of the mobile phase and sample , with the column , determine the degree of migration and
separation of components contained in the sample
•The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi. this
is because as the particle size of stationary phase is around 5-10µ, so the resistance to the flow of solvent is high.
31. REQUIREMENTS OF AN IDEAL MOBILE PHASE
1. Polarity – It is an important criterion for mobile phase selection. The solvent system chosen should
have neither high polarity nor should have low polarity, the polarity of the solvent should be
moderate. In normal phase chromatography the solvent system used is non polar while in reverse
phase it is a polar solvent. One other criterion is that the polarity of the solvent is also decided on the
functional group of the analyte to be separated, in better words solvent can be selected by matching
the relative polarity of the solvent to that of the sample component or a solvent to match the most
polar functional group in the sample. E.g. - Alcohols for hydroxyl group and ketones, acetates for
carbonyl groups
2. The solvent system should be able to carry loosely held substances or weakly soluble substances and
carry out the process of separation efficiently
3. Mobile phase used must be compatible to the detector that is used for the specific HPLC process E.g.
UV detector limits the number of solvents that can be used. It usually uses solvents like
hexane,chloroform,methanol,water etc. as it is compatible as well as possess low viscosity. As
viscosity is a major consideration and requirement for the development of high column efficiencies.
4. Grade of solvents should be selected for HPLC which is reasonably constant in physical and chemical
characteristics E.g. Tetrahydrofuran is susceptible to peroxide formation so to deal with that anti
oxidant preservatives are used but in HPLC preservative free tetrahydrofuran is used. High grade
solvents or analytical grade solvents are used
32. PUMPS USED IN HPLC
•The role of the pump is to force a liquid (called the mobile phase) through the liquid
chromatograph at a specific flow rate, expressed in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a constant mobile phase
composition (isocratic) or an increasing mobile phase composition (gradient).
33. TYPES OF HPLC PUMPS
• There are several types of pumps used for HPLC analysis, most commonly used are reciprocating piston pump, syringe pump and
constant pressure pump
• Reciprocating piston pumps:
• Consists of a small motor driven piston which moves rapidly back and forth in a hydraulic chamber that may vary from 35-400µL
in volume
• On the back stroke , the separation column valve is closed , and the piston pulls in solvent from the mobile phase reservoir
• On the forward stroke, the pump pushes solvent out of the column from the reservoir
• A wide range of flow rates can be attained by altering the piston stroke volume during each cycle , or by altering the stroke
frequency.
34.
35. SYRINGE TYPE PUMPS
• These are most suitable for small bore columns
because this pump delivers only a finite
volume of mobile phase before it has to be
refilled. These pumps have a volume between
250 to 500mL
• The pump operates by a motorized lead screw
that delivers mobile phase to the column at a
constant rate .The rate of solvent delivery is
controlled by changing the voltage on the
motor.
36. CONSTANT PRESSURE PUMP
• In these types of pumps , the mobile phase is driven through the column with the use of pressure from the gas cylinder
• .A low-pressure gas source is needed to generate high liquid pressures
• The valving arrangement allows the rapid refill of the solvent chamber whose capacity is about 70mL
• This provides continuous phase flow rates
37. INJECTOR
• The injector serves to introduce the liquid sample into the flow
stream of the mobile phase for analysis.
• It is equipped with six port valves so that a sample can be
injected into the flow path at continuous pressure
• For a manual injector, the knob is manually operated to deliver
the sample to the column
• The knob is set to LOAD position for sample injection using a
syringe , the sample is injected into the sample loop , which is
separated from the flow path
• The knob is turned to INJECT position and the eluent travels
through the loop from the pump and delivers the sample to the
column
38. • Typical sample volumes for manual injector are 5-to 20-
microliters (μL).
• The injector must also be able to withstand the high pressures of
the liquid system.
• An autos ampler is the automatic version for when the user has
many samples to analyze or when manual injection is not
practical. It can continuously Inject variable volume a of 1 μL –
1 mL
39. DESIRABLE FEATURES OF AN INJECTOR
• The smallest possible contribution to peak broadening
• Should be convenient to use
• Able to operate at high pressure
• Chemically inert with the eluent and the sample
• Should be reproducible
40. DIFFERENT TYPES OF INJECTORS USED IN HPLC
I SYRINGE INJECTION
This is the earliest and simplest technique, here the syringes are used to inject the sample and are designed to withstand high pressure. Fixed
volume is introduced by making use of fixed volume injector. The sample devices are six pored rotary valves. The sample is loaded by means of
loading syringe. Excess sample is used to ensure that the storage line is completely filled. This line may either be an external loop with adequate
volume is to be changed or an internal cavity or fixed value grooved at the surface of the rotor or drilled into the column The different types of
Syringe Injections are :-
1] Rotor Fill
2] Sample Injection
Contd….
41. Contd…
II STOP FLOW INJECTION
A variable volume is introduced by making use of an injection valve. Variable volume injectors contain a needle port, which can be closed or sealed
at high pressure to insert the syringe. This too is a syringe injection but here the solvent flow is stopped momentarily. A fitting at the column head
is removed and sample is injected directly onto the head of the column packing at the atmospheric pressure ,then the fitting is replaced and the
system is again pressurized. Then solvent line switching on causes the sample to be injected into the column.
III SOLVENT FLOWING INJECTION
Here the variable volume is introduced by means of sampling valves or loops, the sampling valve or loop which contains the desired amount of the
sample to be injected ,is introduced onto the pressurised column through septum and its contents are discharged into the eluent stream. Septum
injection ports are used, a syringe is used to inject the sample through an inert septum directly into the mobile phase tubing to sample tubing. The
sample is injected to the sample line, the valve is rotated and the mobile phase washes the sample into the main line. This type of injectors are used
for injecting samples volume more than 10 Alit is now a days used in all automatic systems as it does not affect the column efficiency
42.
43.
44. COLUMNS
• Considered the “most important part of the chromatograph” the column’s stationary phase
separates the sample components of interest using various physical and chemical
parameters.
• It is usually made of stainless steel to withstand high pressure caused by the pump to move
the mobile phase through the column packing other material include PEEK and glass
• The small particles inside the column are called the “packing” what cause the high back
pressure at normal flow rates.
• Column packing is usually silica gel because of its particle shape , surface properties , and
pore structure give us a good separation
45. Other material used include alumina, a polystyrene-divinyl
benzene
synthetic or an ion-exchange resin
– Pellicular particle: original, Spherical, nonporous beads,
proteins and large biomolecules separation (dp: 5 μm)
– Porous particle: common used, dp: 3 ~ 10 μm. Narrow size
distribution, porous microparticle coated with thin organic
film
The dimensions of the analytical column are usually
-straight, Length(5 ~ 25 cm), diameter of column(3 ~ 5 mm),
diameter of particle(35 μm). Number (40 k ~ 70 k plates/m)
46. DIFFERENT PARTS
OF COLUMN
• It comprises usually of : -
1. Cap nut with compression
2. Guard Column
3. Split collets
4. Cartridge
5. Housing with outer thread
47. TYPES OF COLUMNS USED IN HPLC
1. Guard Column
A guard column is placed between injector and HPLC column to protect the HPLC column from damage and loss of efficiency caused
by particulate matter and strong adsorbent in sample or solvent. In HPLC guard columns serves to saturate the mobile phase with the
stationary phase from analytical column is minimized. The composition of the guard column should be similar to that of the analytical
column- the particle size is usually large, to minimize the pressure drop. It is mainly used to remove the impurities from the solvent
and thus prevent contamination of the analytical column
2. Analytical Column
Column chromatography columns are constructed from smooth bore stainless steel tubing, or heavy walled glass tubing. Column length ranges
from 10-100 cm and internal diameter of 2-6 mm for analytical column and ID of 6mm and more for preparative columns
Columns packing have a particle size of 3-5um. Recently micro columns are available with internal diameter of 1-4.6mm, particle size of 3-5um &
length of 3-7.5cm which works with high speed and gives high resolution and minimal solvent consumption
48. DIFFERENT TYPES OF ANALYTICAL COLUMNS
1. Standard Columns
HPLC are done on columns with an internal diameter of usually of 4-5 mm .
The provide a good compromise between efficiency, sample capacity and
the amount of packing and solvent required. Column packing materials are
uniformly sized and mechanically stable.
2. Narrow bore columns
Bore size or the internal diameter of the column is decreased, then there
is an increase in the signal of the sample component. When bore size is
decreased the volume of solvent required is decreased. Mobile phase of
high purity is used. In these columns there is better homogeneity in
packing density and smaller temp gradient across the column
49. 3. Radial Columns
In the radial column, the packing takes the form of a cylinder and the flow of
mobile phase passes from outside of the cylinder, through the packing to the
inside of the cylinder, the separation taking place n the way through. The packing
is supported between two cylindrical frits and the gap between represents the
bed height or column length. The outer frit is the column inlet and consequently
the sample initially has a large area of stationary phase with which to interact.
The sample is injected into a radial column that disperses the sample over the
total peripheral area of the external frit. This type of column has a low
resolution but high loading capacity and is not suitable for separating complex
mixtures. It can be used effectively with gradient elution
50. CAPILLARY COLUMNS
• HPLC led to smaller analytical columns called as micro-columns, capillary columns which have
diameter less than a millimeter.
• Sample used – is in nanolitre volumes, decreased flow rate, decreased solvent volume usage which
leads to cost effectiveness.
• Disadvantage:- since it is miniatured flow rate is difficult to produce & gradient elution is not
efficient.
51. • MICROBORE and SMALLBORE columns are also used for analytical and small volumes assay.
• Diameter of small-bore columns is 1-2mm.
• The instrument must also be modified to accommodate these smaller capacity columns.
FAST COLUMNS:
This column also have the same internal diameter but much shorter length than most other columns &
packed with particles of 3µm in diameter.
Increased sensitivity, decreased analysis time, decreased mobile phase usage & increased reproducibility.
52. TYPES OF COLUMN PACKING MATERIALS IN
HPLC
1.Pellicular Or Superficially Porous Material – It consists of a core hat,
surrounded by a crust or a layer of material that is chromatographically active.
The core is usually a non porous spherical glass heads or polymer beads and the
outer layer consists of porous particles. The particle size ranges from 30-40 um.
Limitations of this is that there is reduction or decrease in the surface area
available in the column
Eg Perisorb A – contains silica , Perisorb RP – contains silanized silica
Perisorb AN – quaternary ammonium
2. Bonded phase packing
The bonded phase HPLC packings consists of a silica support with an organic
moiety bonded to it through a silicon carbon covalent bonding system. This
bonding is obtained by chemical bonding reactions and it is used in HPLC –
partition chromatography
E.g. RP-18,RP-2,RP-8,C8,C22
53. 3. Micro particulate Packings
This packing material is totally porous packing material, and the particle
size ranges from 3-10um . Usually silica, alumina or an ion exchange resins
are used. Mainly silica particles are used with small particle size and
spherical shapes. The limitation is that silica is not chemically stable , it
reacts with many compounds and also dissolves away in the mobile phase.
Silica also requires to use buffer solutions to maintain the pH and prevent
the stationary phase from degrading
Eg Adsorb sphere – silica, RSIL – silica ,IBM - silica
54. DETECTOR
• The detector can detect the individual molecules
that elute from the column and convert the data
into an electrical signal
• •A detector serves to measure the amount of those
molecules
• •The detector provides an output to a recorder or
computer that results in the liquid chromatogram
• Detector is selected based on the analyte or the
sample under detection
55. HOW A DETECTOR SHALL BE !!!
• A detector can be compared to a gate watchman who verifies the visitors
before permitting them entry inside a building. The chromatographic
detector is capable of establishing both the identity and concentration of
eluting components in the mobile phase stream. A broad range of
detectors is available to meet different sample requirements. Specific
detectors respond to a particular compound only and the response is
independent of mobile phase composition. On the other hand the response
of bulk property detectors is dependent on collective changes in
composition of sample and mobile phase.
• The desirable features of a detector are:
• Sensitivity towards solute over mobile phase
• Low cell volumes to minimize memory effects
• Low detector noise
• Low detection limits
• Large linear dynamic range
56. DIFFERENT TYPES OF DETECTORS USED IN
HPLC
• ULTRAVIOLET [UV]
a. Fixed Wavelength Detector
b. Variable Wavelength Detector
c. Diode Array
• Fluorescence
• Electrical Conductivity
• Refractive Index
• Electro Chemical
• Light Scattering
• Mass Spectrometric
57. DIFFERENT TYPES OF DETECTORS USED IN
HPLC
Ultraviolet (UV)
• This type of detector responds to substances that absorb light.
• The UV detector is mainly to separate and identify the principal active components of a
mixture.
• UV detectors are the most versatile, having the best sensitivity and linearity.
• UV detectors cannot be used for testing substances that are low in chromophores (colorless
or virtually colorless) as they cannot absorb light at low range.
• They are cost-effective and popular and are widely used in industry
58. UV VISIBLE DETECTORS
UV Visible detectors most widely used in HPLC. They
work on the principle of beer’s law i.e. they measure
the ability of sample to absorb light at one or more
wavelengths.
UV Visible detectors are used to detect solutes that
specifically
absorb UV Visible radiations
EXAMPLE: Alkenes, aromatic compounds and
compounds having multiple
bonds between carbon, Oxygen, nitrogen & sulphur.
60. UV Visible absorption detector divided into two types:
1. FIXED WAVELENGTH DETECTORS:
They measure the sample absorbance at fixed wavelengths like
245, 250, 280,334 & 436nm. Desired wavelength is obtained by allowing the
radiations emitted by the mercury Vapour lamp to pass through an optical filter
These detectors are operated at a wavelength of 254 nm as most
of the drug substances absorb light at this wavelength.
EXAMPLE: Low pressure mercury vapour lamp
61. VARIABLE UV DETECTORS:
They measure the sample absorbance between wavelengths
190- 600 nm.
They consist of either deuterium or tungsten lamp
as the radiation source, a diffraction grating for wavelength
selection and a photomultiplier detector for detection.
62. In variable UV detectors the concentrated light falls on the
refraction grating.
Which splits The light into its individual components allowing
selection of specific wavelength to be Passed through the flow cell.
63. FLUORESCENCE
DETECTOR
• This is a specific detector that senses only those
substances that emit light. This detector is
popular for trace analysis in environmental
science.
• As it is very sensitive, its response is only linear
over a relatively limited concentration range. As
there are not many elements that fluoresce ,
samples must be synthesized to make them
detectable.
64. Some organic & inorganic compounds (like polycyclic aromatic
hydrocarbons, alkaloids, quinolines) exhibit natural fluorescence and
are detected by using fluorescence detectors .which measure the
fluorescent radiations.
Detection of non-fluorescent compounds is achieved by reacting
them with appropriate fluorogenic solvents.
EXAMPLE: Fluorescent derivatives of amines, amino acids, phenols are obtained by treating
them with fluoro genic reagent, dansyl chloride
In these detectors radiations emitted from a xenon or deuterium
lamp are focused on the flow cell containing the column effluent
through a filter.
66. The fluorescent radiations emitted by the sample are measured
at 90 C to the incident beam
The desired radiation is picked up by the second filter and detected
by using a photomultiplier detector
ADVANTAGES:
1. Increased sensitivity and specificity.
2. Independent of changes in flow rates, pressure, temperature etc..,
67. DISADVANTAGES:
1. Only fluorescence compounds or those that can be derivatised
can be detected
2. The solvents used should be pure & devoid of any
fluorescencing impurities
70. PHOTO DIODE ARRAY DETECTOR:
Photo diode array detector modified from of UV Visible
absorbance detector and operates between 190-600 micro meter
In this, a poly chromatic light is made to pass through the flow
cell and the emerging radiations obtained are diffracted by grating
to fall on an array of photodiodes.
Each photodiode receives a different wave length bond which is
scanned by a microprocessor and the resulting spectrum is
obtained as a three dimensional plot of response time vs
wavelength On a visual display unit.
72. ADVANTAGES:
It simulantaneously records the absorbance at several wavelength.
It is possible to monitor a broad range of wavelength with out
interrupting the flow in the column
It acts like a perfect detector since almost all organic compounds
absorb light in the UV Visible region.
73. DISADVANTAGES:
Real time data display. With all the information available, it can
display only a maximum of two wavelengths at a time on a strip
chart recorder or up to 4 -8 small chromatograms at a time on
computer display screen. Hence very expensive variable UV
detector
Array chatter is a problem which limits sensitivity and goes worse
when the system ages.
75. Electro chemical detector measure the current associated with the
oxidation/ reduction of solute at a suitable electrode.
The mobile phase used for detection should exhibit conductivity.
Inorganic salts or mixtures of water & miscible organic solvents
are generally used as Conducting mobile phase. Compounds
such as aldehydes, ketones, aromatic amines, Heterocyclic nitrogen
compounds, phenols etc.., are detected.
76. APPLICATIONS:
It is highly sensitive and is often measured for biogenic substances
such as catecholamine.
77. REFRACTIVE INDEX (RI) DETECTION
• The refractive index (RI) detector uses a
monochromator and is one of the least
sensitive LC detectors.
• This detector is extremely useful for
detecting those compounds that are non-
ionic, do not absorb ultraviolet light and do
not fluoresce.
• e.g. sugar, alcohol, fatty acid and polymers.
78. RI detector has lower sensitivity compared to UV detector,
and that's the main reason why RI is not as commonly used as UV.
However there are some advantages over UV detector.
It is suitable for detecting all components. For an example,
samples which do not have UV absorption,
such as sugar, alcohol, or inorganic ions obviously cannot be measured by a UV detector.
In contrast, change in reflective index occurs for all analyte,
thus a RI detector can be used to measure all analyte.
It is applicable for the use with solvent that has UV absorbance.
A UV detector cannot be used with solvent which has UV absorbance.
Sometimes the organic solvent used for GPC analysis absorbs UV, and thus UV detector cannot be used.
It provides a direct relationship between the intensity and analyte concentration.
The amount of UV absorbed depends on each analyte,
thus the intensity of UV detector peak does not provide information on the analyte concentration.
While intensity observed by a RI detector is comparable to the concentration of analyte.
Because of those advantages, RI is often used for the detection of sugars and for SEC analysis.
79. MASS SPECTROMETRY DETECTOR
• The mass spectrometry detector coupled with HPLC is called HPLC-
MS. HPLC-MS is the most powerful detector, widely used in
pharmaceutical laboratories and research and development.
• The principal benefit of HPLC-MS is that it is capable of analyzing and
providing molecular identity of a wide range of components
81. STAGE 1: IONISATION
The atom is ionized by knocking one or more electrons off the to give
a positive ion. This is true even for things which you would normally expect
to form negative ions (chlorine, for example) . Mass spectrometer always
work with positive ions
STAGE 2: ACCELERATION
The ions are accelerated so that they all have the same kinetic
energy.
82. STAGE 3: DEFLECTON
The ions are then deflected by a magnetic field according to their
masses the lighter they are , the more they are deflected.
The amount of deflection also depends on the number of positive
charges on the ion-in other words. On how many electrons were
knocked off in the first stage. The more the ion is charged, the
more it gets deflected.
STAGE 4: DETECTION
The beam of ions passing through the machine is detected
electrically
83. ADVANTAGES:
• It gives extremely superb and high level of efficiency.
• Reasonably minute detection limits
85. These detectors used for the determination of protein and polymer
molecules based on their ability to scatter incident light
proportionally to their sizes and concentration.
HPLC effluent passes through a nebulizer to produce a uniform
mist of analyte and solvent within a flow of either pure nitrogen or
air.
Misty is made to enter into a temperature modulated drift tube where
in the solvent gets evaporated giving rise to fine uniform gaseous
suspension of analyte particulate matters
A laser beam is passed through these analyte particles they
scatter the light.
Scattered light is monitored or deflected at 90 to the path of laser
beam.
86. APPLICATIONS:
These detectors find best application in analysis of fatty acids,carbohydrates
& Polymers with little UV absorption.
Used to tackle and monitor non-volatile compounds as soon as they get
eluted from the HPLC columns
87.
88. DETECTORS PROPERTIES ADVANTAGE DISADVANTAGE
1. UV-VISIBLE ABSORPTION
DETECTORS
A wide variety of
compounds can be
detected that absorb light
from 190- 900 nm
They offer best, most
economical detection
for wide ranges of
concentrations.
Only those samples can be
analysed which show UV
absorption.
2. DIODE ARRAY DETECTORS 1.The diode array detector
modifies the position of the
refracting grating by
placing it after the flow cell.
2. The flow cell is irradiated
with a polychromatic
UV/visible light source.
3. Several hundred
photodiodes can be used,
each one monitoring a
narrow wavelength range
(1nm ).
4. A wide variety of
compounds can be
detected that absorb light
from 190- 900 nm
It simultaneously
records the absorbance
at several wavelength.
With all the information
available, it can display
only a maximum of two
wavelengths at a time on a
strip chart recorder or up
to 4 to 8 small
chromatograms at a time
on computer display
screen. Hence very
expensive variable UV
detector.
89. 3. FLOURESCENCE DETECTOR A compound can be
detected by
fluorescence if it
meets two criteria:
1. It must absorb in
the ultraviolet region
2. It should be
fluorescent in nature.
1.Compounds that
meet both the criteria
can generally be
detected 2 to 10
times more sensitively
by a fluorometer than
by a UV detector.
2. They are more
sensitive and more
specific than a UV
detector
Only those compounds
that can be converted into
fluorescent derivatives can
be detected.
4. REFRACTIVE INDEX
DETECTOR
Compounds which
show greater
difference in RI when
compared to mobile
phase.
The ability to cover
the entire refractive
index range from
1.000 to 1.750 RI with
a single, easily
balanced cell.
Relatively low sensitivity
and a general disability to
easily remove and clean or
replace the cell when
filming or clogging occurs.
90. 5. MASS SPECTROMETERS It gives extremely
superb and high level
of efficiency
Sample has to be
converted into vapour
form
6. EVAPORATE LIGHT
SCATTERING DETECTOR
These detectors are
used for the
determination of
protein and polymer
molecules
7. CONDUCTIVITY DETECTOR The solutions should
show conductivity.
1.This detector is
mainly used to
measure inorganic
ions and small
organic substances
including organic
acids and amines.
2. It is highly sensitive
It is very susceptible to
effect of temperature
variation ( a change of 1˚ C
in the solution
temperature causes
roughly 2% change in
electric conductivity.
91. DATA PROCESSING UNIT
(COMPUTER)
• •Frequently called the data system, the
computer not only controls all the modules of
the HPLC instrument but it takes the signal
from the detector and uses it to determine the
time of elution (retention time) of the sample
components (qualitative analysis) and the
amount of sample (quantitative analysis).
• The concentration of each detected component
is calculated from the area or height of the
corresponding peak and reported.
92. WASTE
COLLECTOR
• It is used in HPLC to collect waste
fluids from the pumps after
performing the analysis
• They are of 2 types
a) Automated
b) Manual
93. ULTRA PERFORMANCE LIQUID
CHROMATOGRAPHY
• It is an advanced technology in column technology and chromatography
instrumentation
• It utilizes very small packaging size particles and allows significant increases in LC
speed reproducibility and sensitivity
• It is found to better when compared to HPLC because
a) It gives faster results with better resolution
b) It uses less of valuable solvents like Acetonitrile
c) The reduction of solvent results in the method being Eco Friendly
94.
95. PARAMETERS USED IN
HPLC
1.Retention time (RT)
• In a chromatogram, different peaks correspond
to different components of the separated mixture.
• Time elapsed between sample introduction and
maximum of response, it is the characteristic
time it takes for a particular analyte to pass
through the system
• Time taken for the analyte to travel from the
column inlet to the point of detection(maximum
peak)
96. RETENTION TIME
• For assay the main peak(main component) is considered
• For purity test all the peaks are considered
• Area of peak A= height × base/2
• Peak area gives us the concentration of the analyte
97. 2.Retention volume:
Retention volume is the volume of carrier gas required to elute 50% of the
component from the column. It is the product of retention time and flow rate.
Retention volume = Retention time × flow rate
3.Seperation factor:
Separation factor is the ratio of partition coefficient of the two components to
be separated.
S=Ka/Kb=(tb-to)/(ta-to)
Where : -
to = Retention time of unretained substance
Ka, Kb = Partition coefficients of a, b
ta, tb = Retention time of substance a, b
98. 4. Resolution:
Resolution is the measure of extent of separation of 2 components and the base
line separation achieved.
Rs = 2 (Rt1-Rt2) / w1+w2
5. Height Equivalent to a Theoretical Plate (HETP):
A theoretical plate is an imaginary or hypothetical unit of a column where
distribution of solute between stationary phase and mobile phase has attained
equilibrium. It can also be called as a functional unit of the column.
A theoretical plate can be of any height, which describes the efficiency of separation. If
HETP is less, the column is more efficient. If HETP is more, the column is less efficient.
.
99. 6. Efficiency:
Efficiency of a column is expressed by the theoretical plates.
n = 16 Rt2/ w2
Where n = no of theoretical plates
Rt= retention time
w = peak width at base
Rt and w are measured in common units (cm or mm , min or sec ). No of theoretical plates
Is high, the column is said to be highly efficient. For GLC, a value of 600/meter is
sufficient. But in HPLC, high values like 40,000 to 70,000/ meter are recommended.
100. APPLICATIONS OF HPLC
HPLC is one of the most widely applied analytical separation techniques.
Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• Pharmaceutical quality control.
101. ENVIRONMENTAL
• Phenols in Drinking Water.
• Identification of diphenhydramine in sediment samples.
• Biomonitoring of PAH pollution in high-altitude mountain lakes through the
analysis of fish bile.
• Toxicity of tetracycline's and tetracycline degradation products to environmentally
relevant bacteria.
• Assessment of TNT toxicity in sediment..
102. FORENSICS
• A mobile HPLC apparatus at dance parties - on-site identification and
quantification of the drug Ecstasy.
• Identification of anabolic steroids in serum, urine, sweat and hair.
• Forensic analysis of textile dyes.
• Determination of cocaine and metabolites in meconium.
• Simultaneous quantification of psychotherapeutic drugs in human plasma.
103. CLINICAL
• Quantification of DEET in Human Urine.
• Analysis of antibiotics.
• Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.
• Detection of endogenous neuropeptides in brain extracellular fluids.
104. FOOD AND FLAVOR
• Ensuring soft drink consistency and quality.
• Sugar analysis in fruit juices.
• Trace analysis of military high explosives in agricultural crops.
Stability of aspartame in the presence of glucose and vanillin
