Hplc.pptx

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY [HPLC]
Mr. Mayank David Raiborde
[Department of Forensic Science (Forensic Toxicology)]
mayankraiborde@gmail.com
1
22-12-2015

CHROMATOGRAPHY
 It is define as, it is analytical method in which separation of active constituent in
complex mixture, and the mixture was distributed in two phases i.e. stationary
phase and mobile phase is known as chromatography.
 It is technique is used for separation, purification, Identification and
extraction of compound.
 It is method it can consist of two phases stationary phase and mobile
phase.
 Stationary phase is constant phase or column packaging material.
 Mobile phase is moveable phase.
 The basic principle of chromatography is based on Adsorption and
partition chromatography.
 Adsorption chromatography - The affinity of molecules towards stationary
phase is known as Adsorption chromatography.
 Partition chromatography - The molecule can moves in two phases of liquid
is known as partition chromatography.
 It is important for qualitative and quantitative analysis.
2

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

PRINCIPLES OF LIQUID CHROMATOGRAPHY
4

TYPES OF LIQUID CHROMATOGRAPHY
5

HPLC
6
 HPLC is a High Performance liquid Chromatography.
 High Pressure Liquid Chromatography.
 High Priced Liquid Chromatography.
 It is column chromatography.
 It is Liquid Chromatography.
 It is modified from of gas chromatography, it is applicable for both Volatile as well as
Non volatile compound.
 It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
 It is having a high resolution and separation capacity.
 It is used as qualitative as well as quantitative analysis.
 High performance liquid chromatography (HPLC) is a chromatographic technique used
to separate a mixture of compounds in analytical chemistry and biochemistry with the
purpose of identifying, quantifying or purifying the individual components of the
mixture.

PRINCIPLE
8
 High Performance Liquid Chromatography [HPLC] is principle is based on
adsorption as well as partition chromatography is depending on the nature
of stationary phase, if stationary phase is solid principle is based on adsorption
chromatography and if stationary phase is liquid principle is based on partition
chromatography.
 It is important for determination of volatile and non volatile compounds.
 It is important for determination qualitative and quantitative analysis.
 It is important for determination of Retention Time (the time is required , after
sample injection maximum angle peak reaches to detector)

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

TYPES OF HPLC SEPARATIONS
11
 Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded
stationary phase.
 Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar,
bonded stationary phase.
 Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes
using adsorption onto a pure stationary phase (e.g. alumina or silica)
 Ion Chromatography: Separation of organic and inorganic ions by their partitioning
onto ionic stationary phases bonded to a solid support.
 Size Exclusion Chromatography: Separation of large molecules based in the paths they
take through a “maze” of tunnels in the stationary phase.

HPLC METHODS
12
 Parameter Group Method Compounds
• SDW05.23000’s EPA 555 Cl-PhenoxyAcids
• WPP05.06000’s EPA 605 Benzidines
• WPP05.13000’s EPA 610 PAHs
• SHW06.26000’s SW-846 8316 Acrylics
• SHW06.28000’s SW-846 8330’s Explosives
• SHW07.06000’s SW-846 8325 Benzidines and
N- Pesticides

MODES OF HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Types of Compounds Mode Stationary
Phase
Mobile Phase
Neutrals
Weak Acids
Weak Bases
Reversed
Phase
C18, C8, C4
cyano, amino
Water/Organic
Modifiers
Ionics, Bases, Acids Ion
Pair
C-18, C-8 Water/Organic
Ion-Pair Reagent
Compounds not
soluble in water
Normal
Phase
Silica, Amino,
Cyano, Diol
Organics
Ionics Inorganic Ions Ion
Exchange
Anion or Cation
Exchange
Resin
Aqueous/Buffer
Counter Ion
High Molecular Weight
Compounds
Polymers
Size
Exclusion
Polystyrene
Silica
Gel Filtration-
Aqueous
Gel Permeation- 13
Organic

HPLC CONTAIN
 Stationary Phase - It is non polar and The stationary solid surface is coated with a 2nd liquid (the
Stationary Phase) which is immiscible with mobile phase.
 Mobile Phase -Apolar mobile phase(ACN, MeOH, WATER + BUFFER).
Bonded Phases -
14
-Si-(CH2)3-CN
-Si-(CH2)17-CH3
 C-2 Ethyl Silyl
• C-8 Octyl Silyl
• C-18 Octadecyl Silyl
• CN Cyanopropyl Silyl
-Si-CH2-CH3
-Si-(CH2)7-CH3

BLOCK DIAGRAM OF HPLC
Reservoir
Pump Gradient
controller
Mixing
chamber
Pump
Solvent
Conditioning
column
Reservoir
Injector Precolumn
Analytical
column
Detector
Recorder
Fraction
collector
15

WHY USE HPLC
16
 Simultaneous analysis
 High resolution
 High sensitivity
 Good repeatability
 Moderate analysis condition
 Easy to fractionate and purify
 Not destructive

INSTRUMENTATION OF HPLC
17
 Solvent storage bottle
 Gradient controller and mixing unit
 De-gassing of solvents
 Pump
 Pressure gauge
 Pre-column
 Sample introduction system
 Column
 Detector
 Recorder

23
23
OUTLINE OF LC-2010
System Controller
UV detector
Auto sampler
Column Oven
Pump Unit
Reservior Tray
Degassing Unit
Low pressure
gradient device

24
HPLC SYSTEM
9010 Solvent
Delivery System
9050 Variable
UV/Vis Detector
HPLC Solvent
Reservoirs
HPLC
Column
Rheodyne
Injector
9060 Polychrom
(DiodeArray) Detector
Computer
Workstation

SOLVENT/ MOBILE PHASE RESERVOIRS
 Glass or stainless-steel containers capable of holding up to
1 liter mobile phase (pure organic solvents or aqueous
solutions of salts and buffers)
 Inert to a variety of aqueous and non aqueous mobile
phases.
 Stainless steel should be avoided for use with solvents
containing halide ions.
25

DEGASSING & FILTRATION OF MOBILE PHASE
 In many cases, aqueous solvents & some organic solvents are degassed prior
to use
 Degassing is done to prevent formation of gas bubbles in the pump or
detector ( Mobile phases are degassed by stirring of the mobile phase under
vacuum, sonication or sparing with helium gas)
 The mobile phase are filtered to remove particulate matter that may clog the
system
Tubing
 Should be inert,
 have ability to withstand pressure
 able to carry sufficient volume 26

PUMP
27
 The solvents or mobile phase must be passed through a column at high
pressures at up to 6000 psi(lb/in²) or 414 bar.
 As the particle size of stationary phase is smaller (5 to 10µ) the resistance to the
flow of solvent will be high.
 That is, smaller the particle size of the stationary phase the greater is the
resistance to the flow of solvents.
 Hence high pressure is recommended.

REQUIREMENTS FOR PUMPS
28
 Generation of pressure of about 6000 psi.
 Pulse free output & all materials in the pump should be chemically resistant to
solvents.
 Flow rates ranging from 0.1 to 10 mL/min
 Pumps should be capable of taking the solvent from a single reservoir or more
than one reservoir containing different solvents simultaneously.

Types of pumps
used in HPLC
DISPLACEMENT
PUMPS
RECIPROCATING
PUMPS
PNEUMATIC
PUMPS
29

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
30

DISPLACEMENT PUMPS
31
⚫ 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.

RECIPROCATING PUMPS
 This pump transmits alternative pressure to the solvent via a
flexible diaphragm ,which in turn is hydraulically pumped by a
reciprocating pump.
 Disadvantages
Produces a pulsed flow which is damped because pulses appear as
baseline noise on the chromatograph.
This can be overcome by use of dual pump heads or elliptical
cams to minimize such pulsations. 33

motor and cam
plunger
check valve
pump head
5 - 50µL
out
plunger seal
in
Mobile phase
check valve
34

 Solvent is pumped back and forth by a motor driven piston
 Two ball check valves which open & close which controls the flow
 The piston is in direct contact with the solvent
 Small internal volume 35-400μL
 High output pressure up to 10,000 psi
 Ready adaptability to gradient elution and constant flow rate
35

36
A. Single-piston pump with slow filling cycle
B. Single-piston pump with a rapid filling cycle
C.Adual-piston pump with rapid filling cycles and operate 1800
out of phase.
Schematic representation of solvent flow from

 Advantages
 Have small internal volume of 35-400µL
 Higher output pressures up to 10,000 psi.
 Adaptability to gradient elution.
 Large solvent capacities & constant flow rates.
 Largely independent of column back pressure & solvent viscosity.
37

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.
38

SAMPLE INJECTOR SYSTEM
39
 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
40
⚫ 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.
41

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 .
43
43

44
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.
46

DETECTORS
47
 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
48
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
50
 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

UV-VISIBLE DETECTOR
51
 UV visible detector is widely used as it detects large number of compounds
because most drugs have appropriate structural characteristics for light
absorption.
 These are useful for aromatic compounds and other type of unsaturated systems.
 These are classified as fixed or variable wavelength detectors.
 Fixed wavelength detectors employ filter as a source to provide appropriate
wavelength.
 Most common fixed wavelength detectors are based on 254 nm.
 Variable wavelength detectors are employ a spectrophotometer to provide
dispersion of light and selection of any wavelength in UV visible regions.
 Diffraction gratings are frequently used for wavelength dispersion.

REFRACTIVE INDEX (RI) DETECTOR
 Detection occurs when the light is bent due to samples eluting from the columns, and
this is read as a disparity b/w the two channels.
 It is not much used for analytical applications because of low sensitivity & specificity.
 When a solute is in the sample compartment, refractive index changes will shift the light
beam from the detector.
53

PHOTODIODE ARRAY (PDA)
 A photodiode array (PDA) is a linear array of discrete photodiodes on an integrated
circuit (IC) chip.
 Allows a range of wavelengths to be detected simultaneously. In this regard it can be
thought of as an electronic version of photographic film. Array detectors are especially
useful for recording the full Uv- vis is a absorption spectra of samples that are rapidly
passing through a sample flow cell, such as in an HPLC detector.
 PDAs work on the same principle as simple. Photovoltaic detector
 similar to UV detector, non destructive 190-600 nm for quantization & identification
Spectra is 3D, Response vs time vs WL.
54

FLUORIMETRIC DETECTORS
detector while a
 It is based on the fluorescent radiation emitted by some compounds.
 The excitation source passes through the flow cell to a photo
monochromatic measures the emission wavelengths.
 More sensitive and specific.
 The disadvantage is that most compounds are not fluorescent in nature.
 Fluorescence is a type of luminescence in which the light energy is released in the form of
a photon in nanoseconds to microseconds.
55
S1
T1
S0
Light absorption
Non-radiation transition
Non-radiation transition
Fluorescence
Phosphorescence

RECORDERS AND INTEGRATORS
58
 Recorders are used to record responses obtained from the detectors after amplification, if
necessary.
 They record the baseline & all the peaks obtained with respect tot time.
 Retention time can be found out from this recordings, but area under curve cannot be
determined.
 The Integrators are improved versions of recorders with some data processing
capabilities.
 They can record the individual peaks with retention time, height, width of peaks, peak
area, percentage area, etc.
 Integrators provides more information on peaks than recorders.
 In recent days computers and printers are used for recording and processing the obtained
data & for controlling several operations.

COMPARISION BETWEEN HPTLC AND HPLC
Sr.no. HPTLC HPLC
1 High Performance Thin Layer High
Chromatography
Performance Liquid
Chromatography
2 Column Chromatography
3 Adsorption
Planar Chromatography
Principle based on
Chromatography
Principle is based on Adsorption and
Partition Chromatography
4 Simultaneous method for test as well as
reference material
Not simultaneous method for test as
well as reference material
5 It is simple, rapid, reproducible method It is Tedious method
6 Sample preparation is simple Sample preparation is complex
7 Limited Flexibility Extreme Flexibility
8 Semiautomatic Technique Automatic (Instrumental) Technique
59
9 Determination of SurfaceArea Determination of Retention Time

COMPARISION BETWEEN GC AND HPLC
60
SR.NO. GC HPLC
1 Gas Chromatography High Performance Liquid
Chromatography
2 Less resolution High resolution
3 Limited Flexibility Extreme Flexibility
4 Determination of Volatile
compounds
Determination of Volatile and Non
Volatile Compounds

COMPARISION BETWEEN HPLC AND UPLC
Parameters HPLC Assay UPLC Assay
Column XTerra,C18,50 ×
4.6mm
AQUITY UPLC BEH
C18,50 ×2.1mm
Particle size 4µm particles 1.7µm particles
Flow rate 3.0 ml per min 0.6 ml per min
Injection volume 20 µl 3 µl partial loop fill or
5 µl full loop fill
Total run time 10 min 1.5 min
Theoretical Plate 2000 7500
count 61

62
Parameters HPLC Assay UPLC Assay
Lower limit of
quantization
0.2 µg/ml 0.054µl/ml
Total solvent
consumption
Acetonitrile:10.5ml,
water:21ml
Acetonitrile:0.53ml,
water:0.66ml
Delay volume 720 µl 110 µl
Column temperature 30 °C 65 °C
Maximum back
pressure
35-40 Mpa
less
103.5 Mpa
more
Resolution Less High
Method development
cost
High Low

APPLICATION
 Drug Discovery
 ClinicalAnalysis
 Proteomics
 Forensic Chemistry
 Drug Metabolism study
 Environmental chemistry
 Diagnostic studies
 Cosmetic analysis
 Determination of Green Florescent Protein
 Structural Determination
 PharmaceuticalApplications
 Identification of BileAcid Metabolite
 ClinicalApplications
 Biochemical Genetics
 qualitative and quantitative analysis
 Therapeutic Drug Monitoring
63

REFERENCE
 Knox JH, Done JN, Fell AF et al. High-Performance Liquid Chromatography. Edinburgh:
Edinburgh University Press; 1978.
 Simpson CF. Practical High-Performance Liquid Chromatography. London: Heyden and Son; 1976.
 Pungor E. A Practical Guide to Instrumental Analysis. Boca Raton: CRC Press; 1995.
 Moffat AC, Osselton MD, Widdop B. Clarke’s Analysis of Drugs and Poisons. London: Pharmace
utical Press; 2004.
 Y. Shen and R.D. Smith, Electrophoresis 23 (2002) pp. 3106-3124.
 D.A. Skoog and D.M. West, Fundamentals of Analytical Chemistry, 3rd edit., pp. 643-649.
 D. Sievers, M.E. Swartz and B.J. Murphy, G.I.T. Laboratory Journal (2004) pp.43-45.(2004) pp.
503-504
 L.A. Colon, J.M. Cintron, J.A. Anspach, A.M. Fermier and K.A. Swinney, The Analyst 129
 L.R. Snyder, J.J. Kirkland and J.L. Glajch, Practical HPLC Method development, 2nd edit., pp. 41-
43.
 J.C. Giddings, Dynamics of Chromatography: Part I Principles and Theory (Marcel Dekker, Inc.,
New York: 1965) pp. 25-26, 229-230.
 J.M. Cintron and L.A. Colon, The Analyst 127 (2002) pp. 701-704. A.D. Jerkovich, J.S. Mellors
and J.W. Jorgenson, LCGC 21 (July 2003) pp. 600-611. 64

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