hplc.pptx

 HPLC stands for “High Performance Liquid Chromatography”.
 High performance liquid chromatography is a powerful tool in
analysis, it yields high performance and high speed compared to
traditional column chromatography because of the forcibly
pumped mobile phase
Chromatography:
• Separation of mixture of samples into individual samples.
• It is a separation technique which separation take Between two
phases 1.Stationary phase 2.Mobile phase
1.Stationary phase : The substance on which adsorption of analyte
take place.
2.Mobile phase: Solvent which carries the analyte.
1

Liquid chromatography:
2
 It is a chromatographic technique in which mobile phase is a liquid.
 The main principle involved in liquid chromatography is adsorption.
PRINCIPLE
When a mixture of samples introduced into column various chemical
and physical interactions takes place between column material and
components present in the sample travel according to their relative
affinities towards the stationary phase . the component which has more
affinity towards the stationary phase ,travels slower. The components
which having less affinity towards stationary phase travels faster.
More over it work very efficiently compare toother chromatographic
techniques. In this high pressure pump is used to transfer the sample to
stationary phase. so it is also called as high pressure liquid
chromatography.

 The principle of HPLC are based on Van Demeterequation
which relates the efficiency of the chromatographic column to
the particle size of the column, molecular diffusion and
thickness of stationary phase.
 The Van Deemter Equation is given as
H or HETP = A + B/u + C υ
Where,
A= Represents eddy diffusion,
B= Represents molecular diffusion,
C =Represents rate of mass transfer,
υ =Represents flow rate.
3

TYPES OF CHROMATOGRAPHY
 Based on modes of chromatography
1. Normal phase mode
2.Reverse phase mode
 Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Partition chromatography
4. Size exclusion
 Based on elution technique
1. Isocratic separation
2. Gradient separation
 Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
 Based on the type of analysis
1. Qualitative analysis
2. Quantitative analysis
3

1.BASED ON MODE OF SEPARATION
5
 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.
 Reverse Phase HPLC :The stationary phase is non polar
(hydrophobic) in nature, such as C18, C8, Cyano
columns used. While the mobile phase is a polar liquid,
such as mixtures of water and methanol or acetonitrile.

2.BASED ON PRINCIPLE OF SEPRATION
i)Adsorption chromatography:
⚫ In which stationary phase is an
adsorbent.
⚫ The compounds separated based
on their affinity towards stationa
-ry phase.
⚫ More affinity-slow elution
⚫ Less affinity-fast elution
ii)Partition chromatography:
⚫ A process of separation of solutes utilizing the
partition of the solutes between two liquid phases
⚫ Namely the original solvent and the film of solvent on the
adsorption column.
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iii)Size-Exclusion HPLC:
8
⚫ In which the stationary phase is a gel having a closely
controlled pore size.
⚫ Molecules are separated based on molecular size, shape
smaller molecules being temporarily retained in the pores and
mainly Agarose , Dextran used as mobile phase.
iv)Ion-Exchange HPLC:
⚫ Ion-exchange chromatography separates molecules based on
their respective charged groups.
⚫ Ion-exchange chromatography retains analyte molecules on
the column based on coulombic (ionic) interactions.
⚫ Essentially, molecules undergo electrostatic interactions with
opposite charges on the stationary phase matrix.

3.BASED ON ELUTION TECHNIQUE
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During the chromatographic experiment, a pump can deliver a
constant mobile phase composition(isocratic) or an increasing
mobile phase composition (gradient).
i) Isocratic Elution
⚫ Delivers constant mobile phase composition;
⚫ Solvent must be pre-mixed;
⚫ Lowest cost pump;
⚫ Best for simple preparation.
ii) Gradient Elution
⚫ Delivers variable mobile phase composition;
⚫ In this mobile phase is programmed to change in composition
during elution time;
⚫ Best for complex preparations.

4.BASED ON SCALE OF OPERATION
i)Analytical HPLC:
No recovery of individual components of substance.
ii)Preparative HPLC:
Individual components of substance can be recovered.
5.BASED ON TYPE OF ANALYSIS
i)Qualitative Analysis:
Determine the quality of sample.
ii)Quantitative Analysis:
Determine the quantity(concentration) of sample.
10

INSTRUMENTATION OF HPLC
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 Solvent storage bottle
 Gradient controller and mixing unit
 De-gassing of solvents
 Pump
 Pressure gauge
 Pre-column
 Sample introduction system
 Column
 Detector
 Recorder

HPLC INSTRUMENTATION OVER-VIEW
12

I. Solvent reservoir & Mixing unit & degassing
system
14
⚫ The appropriate solvents [mobile phase] from the reservoirs
are allowed to enter the mixing chamber where a
homogenous mixture is obtained.
⚫ Several gases are soluble in organic solvents.
⚫ When solvents are pumped under high pressure, gas
bubbles are formed which will interfere with the separation
process, steady base line and the shape of the peak.
⚫ Hence degassing of solvent is important. This can be done by
-Vacuum filtration,
-Helium purging,
-Ultrasonication.

II. High pressure pump
⚫ The role of the pump is to force a liquid (called the mobile
phase)through the liquid chromatograph at a specific flow
rate, expressedin 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-
9000psi (400-to 600bar).
⚫ There are several types of pumps used for HPLC most
commonly used are:-
1.Reciprocating Piston Pump,
2.Syringe Pump,
3.Constant Pressure Pump.
15

HPLC Pump
Constant flow rate (Mechanical) Pump Constant Pressure Pump (Pneumatic)
Manner in which they operate
syringe
(Displace
ment
pump)
Reciprocating pump
Single
piston
reciprocati
ng pump
Double piston
reciprocating
pump
Reciprocating
diaphragm
pump
Direct pressure pump Amplifier pump
16

1.Reciprocating pump:
-The piston is moved in and out of a solvent chamber by an -
eccentric cam or gear.
-The forward-stroke closes the inlet-check value while the
outlet valve opens and the respective mobile phase is duly
pumped into the column.
-Consequently, the return-stroke-closes the outlet valve and it
-
refills the chamber.
Advantages:
*The internal-volume can be
made very small from 10-100 μl,
*The flow-rate can be monitored either
by changing the length of the piston
or by varying the speed of the motor.
*It has an easy access to the valves and
seals.
17

DISPLACEMENT PUMPS
18
⚫ It consists of large, syringe like chambers equipped with a plunger
activated by a screw driven mechanism powered by a stepping
motor.
⚫ So it is also called as Screw Driven Syringe Type Pump.
⚫ Advantages:- It produces a flow that tends to be independent of
viscosity & back pressure.
⚫ Disadvantages:- It has a limited solvent capacity(~250) &
considerably inconvenient when solvents must be changed.

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3.Constant Pressure Pump :
-A constant-pressure pump acts by applying a constant pressure
to the mobile-phase.
-The flow rate through the column is determined by the flow
resistance of the column.

PNEUMATIC PUMPS
 In this pumps, the mobile phase is driven through the column with the use of
pressure produced from a gas cylinder.
 It has limited capacity of solvent
 Due to solvent viscosity back pressure may develop.
21

III. Injector:
 The injector serves to introduce the liquid sample into the
flow stream of the mobile phase.
 Typical sample volumes are 5 to 20μL.
 The injector must also be able to withstand the high pressures
of the liquid system.
Types of injectors :
A.Manual injectors:
User manually loads sample into the injector using a syringe
and then turns the handle to inject sample into the flowing
mobile
B. Auto sampler injector:
User loads vials filled with sample solution into the auto sampler
tray(100 samples)
-Measures the appropriate sample volume,
-Injects the sample,
18

SAMPLE INJECTOR SYSTEM
23
 Several injector devices are available either for manual or auto
injection of the sample.
(i) Septum Injector
(ii) Stop Flow Injector
(iii) Rheodyne Injector

i. SEPTUM INJECTOR
24
⚫ These are used for injecting the sample through a rubber septum.
⚫ This kind of injectors cannot be commonly used , since the septum
has to withstand high pressures.
ii. Stop Flow
 In this type the flow of mobile phase is stopped for a while & the
sample is injected through a valve.

III. RHEODYNE INJECTOR
 It is the most popular injector and is widely used.
 This has a fixed volume of loop, for holding sample until its injected into the
column, like 20µL, 50µL or more.
 Through an injector the sample is introduced into the column.
 The injector is positioned just before the inlet of the column.
25

SELECTION VALVE
BY USING THE SELECTION VALVE
WE CAN SELECT WHETHER THE
PURPOSE IS FOR ANALYTICAL
PURPOSE OR PREPARATIVE
PURPOSE.
INJECT POSITION
In this position the loaded
sample is injected into the
column by the forceful flow
of the solvent into the sample
loop by which the sample is
introduced into the column.
LOAD POSITION
In this position the sample is
loaded into the sample loop .
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43

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44
HPLC AUTO INJECTORS
Inside of SIL-20AC

COLUMN
1. Precolumn
1. It contains a packing chemically identical to that in analytical column.
2.Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytical
column, it can protect analytical column.
3. It is also called as guard column or protective column.
4. it is having large particle size.
5. It is having short length of 2 to 10 cm, so does not affect separation.
2. Analytical column
1. The success or failure of analysis depends upon choice of column.
2. Actual separation is carried out here.
3. Stainless –steel tube
4. size – length -25 to 100 cm
5. Internal diameter – 2 to 4.6 mm
6. Column is filled with small particles 5 – 10 micron. The solid support can be silica gel, alumina.
7. The separation is result of different components adhering to or diffusion into the packing particles when
the mobile phase is forced through column.
45

8.C8 and C18 columns are considered as examples of reversed phase liquid
chromatography (RP).
9.The stationary phase here is seen as a thin film of non-polar liquid phase that has
been designed to be chemically similar to an inert material (Silica gel particles).
10.The non-polar layer is chemically linked to the silica particles surface by
reaction with the polar silanol groups on the stationary phase surface and so
rendering them less polar or non-polar.
11. The difference between the two columns will be in the length of the carbon-
chain attached to the silica surface.
12.Accordingly C8 hplc columns have packing material composed of silica
particles attached to C8 carbon units.
13. C18 will, of course, have packing materials coated with C18 hydrophobic
units.
14.Categorically both are reversed phase but C18 columns will definitely be
more "hydrophobic rather than the C8 columns.
30

DETECTORS
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 Absorbance (UV/Vis and PDA)
 Refractive index (detects the change in turbidity)
 Fluorescence (if the analyte is fluorescent)
 Electrochemical (measures current flowing through a pair of electrodes, on
which a potential difference is imposed, due to oxidation or reduction of
solute)
 Conductivity (for ions)
 Light scattering
 Mass spectrometry (HPLC-MS)

SELECTION OF DETECTORS
Detectors
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Type of compounds can be detected
UV-Vis & PDA
RF
CDD
Compounds with chromophores, such as
aromatic rings or multiple alternating
double bonds.
Fluorescent compounds, usually with
fused rings or highly conjugated planar
system.
Charged compounds, such as inorganic
ions and organic acid.
ECD For easily oxidized compounds like quinones or amines.
RID & ELSD
For compounds that do not show characteristics usable by
the other detectors, e.g. polymers, saccharides.

TYPES OF HPLC DETECTORS
UV-Vis Works w/all molecules Non-specific; complex
samples; absorption
wavelength
DAD Works for all wavelengths High LOD
Fluorescence Very specific; low LOD Not everything fluoresces
IR Works w/all molecules Many solvents IR active
Refractive Index Works w/nearly all
molecules
Temperature sensitive;
high LOD
Scattering Uniform response;
5ng/25mL LOD
Non-specific; interference
from solvent
Electrochemical Commercially available Non-specific; high LOD
Mass Spec Low LOD; analyte
identification
Ability to ionize analyte
49
Name Advantage Disadvantage

IDEAL DETECTOR PROPERTIES
34
 High Sensitivity
 Universality or predictable specificity
 Large linear response range
 Low dead volume
 Non-Destructive
 Insensitive to temperature & mobile phase
 Continuous operation
 Reliable and easy to use
 No single detector fits all these criteria

19
Manual injectors
Auto sampler injector

IV. Column
 The column [stationary phase] separates the sample components of
interest using various physical and chemical parameters.
 The columns used for HPLC are generally made up of stainless steel .
so they can withstand up to high pressure [8000psi].
 Straight columns of 20-50 cm in length & 1-4mm in diameter are
used .
 Particle size should be for porous particles 20-40μm. For porous
micro particles it should be 3-10 μm.
 porous plugs of stainless steel are used in ends of column to retain
the packing material.
Stationary phases used in column:
-Alumina
-Silica
-Polystyrene
-Polyvinyl acetate beads 20

TYPES OF COLUMNS IN HPLC:
A. Guard Column
B. Fast Column
C. Preparative(i.d. > 4.6 mm; lengths50-250mm)
D. Capillary (i.d. 0.1 -1.0 mm; various lengths)
A. Guard column: Guard columns, set between the injector and an
analytical column, are used to protect analytical columns from
chemical impurities in samples. We have two types of guard
columns.
Cartridge type which can be changed easily by hand, and
Packed type, which are packed in stainless cartridge like
analytical columns. 21

.
B. Fast column:
38
One of the primary reasons for using these column is to obtain
improved sample output ( amount of compound per unit time).
Fast column are designed to decrease the time of
chromatographic analysis.
C. Capillary column:
They allow the user to work with nano liter sample volume ,
decreased flow rate and decreased solvent usage volume , led to
cost effectiveness.
D. Preparative column:
It Used when objective is to prepare bulk ( milligrams) of
sample for laboratory preparatory application.
It has usually a large column diameter , which is designed to
facilitate large volume injections into the HPLC system.

COLUMN EFFICIENCY IN SEPARATION PROCESS
A. Theoretical plates:
⚫ Chromatographic column contains a large number of separate
layers, called ‘Theoretical Plates’.
⚫ Separate equilibrations of the sample between the stationary and
mobile phase occur in these "plates".
⚫ The analyte moves down the column by transfer of equilibrated
mobile phase from one plate to the next.
⚫ It is important to remember that the plates do not really exist ; they
are a figment of the imagination that helps us understand the
processes at work in the column.
B. HETP [Height Equivalent to a Theoretical plate]:
⚫ A theoretical plate is an imaginary unit of a column, where
distribution of solute between , stationary phase and mobile phase,
has attained equilibrium.
39

⚫ It also called as functional unit of the column.
⚫ It can beof any height ,which describes the efficiency of
separation
HETP = L /N
Where
HETP = LENTH OF COLUMN /NUMBER OF THEORETICAL PLATES
⚫ If HETP is less , the column is more efficient .
⚫ If HETP is more , the column is les s efficient .
C. RESOLUTION:
⚫The most important thing in HPLC
is to obtain the optimum resolution
in the minimum time
⚫ Resolution is the ability to separate
two signals i.e., separation of
two constituents.
40

D. PEAK ASYMMETRY
⚫ In the ideal world all chromatographic peaks would be
symmetrical (or Gaussian) .
⚫ However, due to the effects of instrument ,adsorptive effects of
the stationary phase and the quality of the column packing
peaks may often show tailing & fronting behavior.
Fronting : Deformation at the beginning of the peak. It is due to
saturation of stationary phase with higher quantity of
components.
Tailing: Deformation at the end of the peak. It is due to
similarity of polarity for a component towards stationary phase.
41

FACTORS AFFECTING EFFICIENCY OF COLUMN
42

V.DETECTORS:
43
1. UV- Visible Dectectors.
2. Photo Diode Array Detectors.
3. Refractive Index Detectors.
4. Fluorescence Detectors.
5. Conductivity Detectors.
6. Mass Spectrometer.
7. Evaporative Light Scattering Detectors.

Refractive index detector
Fluorescence detectors
44

ADVANTAGES
 It is simple, rapid , reproducible.
 High sensitivity.
 High performance.
 Rapid process and hence time saving.
 It is having a high resolution and separation capacity.
 Accuracy and Precision.
 Stationary phase was chemically innert.
 Wide varities of stationary phase.
 Mobile phase was chemically innert.
 Less requirement of mobile phase in developing chamber.
 Early recovery of separated component.
 Easy visualization of separated components.
 It is having Good reproducibility and repeatability.
 It is analytical technique is important for validation of product, quality control
studies of product.
 It is important for qualitative and quantitative analysis.
 It is used for both analytical and preparative purpose.
10

46
HPLC DISADVANTAGES :
 Co-elution.
 Cost.
 Complexity.

APPLICATIONS
47
 Purification Of Samples.
 Identification Of Compounds.
 Determination Of Impurities.
 Determine the Concentration Of Drug.
 Biopharmaceutical & Pharmacokinetic Studies.
 Drug Stability Studies.
 Qualitative & Quantitative Analysis.
 Environmental Applications[ analysing air & water
pollutants.
 Separation Of Mixture Of Samples.
Ex: Carbohydrates

QUANTITATIVE ANAYSIS
 To measure the concentration of each compound in a sample.
 There are 2 main ways to interpret a chromatogram.
 Determination of the peak height of a chromatographic peak
as measured from the baseline.
 Detection of the peak area.
48

QUALITATIVE ANALYSIS
 The identification of individual compounds in the sample.
 The most common parameter for compound identification is its
Retention Time [Is the amount of time a compound spends on
the column after injection]
 Depending on the detector used, compound. identification is
also based on the chemical structure, molecular weight or some
other molecular parameter.
49

REFERENCES
50
 Principles of instrumental analysis- Doglas A
skoog
 Instrumental methods of chemical analysis-
Gurudeep R.chatwal, sham K. anand
 Textbook of chemical analysis-
Francis Rouessac and Annick Rouessac 2nd edition
john wiley & sons ltd
 Text book of modern analytical chemistry-
David harvey

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