Chromatography is a laboratory technique used to separate mixtures by exploiting differences in how compounds partition between a stationary and mobile phase. There are two main types - column chromatography where the stationary phase is within a tube, and planar chromatography like paper or thin layer chromatography where the stationary phase is on a flat surface. Key factors that affect separations include the properties of the stationary and mobile phases used and operating parameters like flow rate. Chromatography is widely applied in fields like pharmaceutical analysis to purify compounds and determine sample composition.

1. PRINCIPLES AND APPLICATION
OF CHROMATOGRAPHY
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2. CHROMATOGRAPHY
 Laboratory technique for the Separation of mixtures
 Chroma -"color" and graphein - "to write”.
 Colour bands - separation of individual compounds
 Measured or analysed.
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3. PURPOSE OF CHROMATOGRAPHY
 Analytical
 Determine Chemical composition of a sample
 Preparative
 Used to purify sufficient quantities of a substance
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4. TSWETT EXPERIMENT
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5. Sample clean up is usually much less of a problem with HPLC than GLC and biological fluids
can often be directly onto an HPLC column. Much sample pretreatment can also be avoided
because aqueous solvents can be used in HPLC.
because of all these advantages, HPLC has already made a significant impact in
pharmaceutical, clinical, forensic and environmental analysis and it is now an ideal
complementary technique to GLC.
2. TYPES OF HPLC TECHNIQUES:
A. Based on modes of chromatography
1. Normal phase mode
2.Reverse phase mode
B. Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Ion pair chromatography
4.Size exclusion(or)Gel permeation chromatography
5. Affinity chromatography
6. Chiral phase chromatography Asheesh Pandey 5

6. C. Based on elution technique
1. Isocratic separation
2. Gradient separation
D. Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
E. Based on the type of analysis
1. Qualitative analysis
2. Quantitative analysis
3. PRINCIPLE:
The principle of separation in normal phase mode and reverse phase mode is
adsorption. When a mixture of components are introduced into a HPLC column, 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 2 components have the same affinity
towards the stationary phase, the components are separated.
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7. High-Performance Liquid Chromatography (HPLC)
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8.  Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
 Eluent - Fluid entering column/ solvent that carries the analyte.
 Eluate - Mobile phase leaving the column.
 Stationary phase - Immobilized phase
 Immobilized on the support particles or on the inner wall of the
column tubing.
 Examples : Silica layer - Thin Layer Chromatography
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CHROMATOGRAPHY TERMS

9.  Mobile phase
Moves in a definite direction. Liquid (LC), Gas (GC).
 The mobile phase moves through the chromatography
column (the stationary phase) where the sample interacts
with the stationary phase and is separated.
 Retention time : Time takes for a particular analyte to
pass through the system (from the column inlet to the
detector) under set conditions.
 Sample (Anylate) :Substance analyzed in
chromatography.
 Solvent : Any substance capable of solubilizing another
substance.
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10.  Chromatogram
 Visual output of the chromatograph.
 Separation - Different peaks or patterns on the
chromatogram correspond to different components of the
separated mixture.
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11. X- axis - Retention time
Y-axis - Signal
Signal is proportional to the concentration of the specific analyte
separated.
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12. HOW TO DESCRIBE A CHROMATOGRAM
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14. Separation of Peaks
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15. Retention
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k = (tr – to)/ to
Where tr = the retention time of the compound, and to = the dead time
Higher values of k mean the analyte will stay in the column longer. The
longer it stays, the more time there is for the peak will widen.

16. Selectivity
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a = kB/kA
the selectivity factor α and is an indication of how well the compounds will
separate. Higher α means larger difference in retention time and more separation

17. Efficiency
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The term that is generally used to describe
column efficiency is “number of theoretical plates” or N
N = L/H
Where: L =column length
H = plate height (both in the same units)

18. N in Practical Terms...
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Units for tr and to….?
Units for W1/2 …..?
N can be measured from the peaks on a chromatogram..
N = 5.54
tr
w1/2( )
2

19. Resolution
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The purpose of chromatography is to separate or resolve compounds. The
separation or distance between two peaks is known as their resolution and is a
function of the 3 factors discussed previously: retention (the time it takes for the
analytes to elute, related to k), selectivity (how different the analytes are from
each other and related to α), and efficiency (how good the column is, related to
N)

20. Resolution
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Rs = ¼ (a-1/a) (k/k+1) N½
The effect on Rs of:
increasing a…?
increasing k…?
increasing N…?
Efficiency
Selectivity Retention

21. Resolution
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Rs = 2 (tR-B – tR-A)/(wb-A + wb-B)
Where: A and B are the two peaks
tR = retention time and
wb = the peak width at the base of
each peak
Rs can also be calculated from actual measurements of peak retention times and
measured peak widths

22. Retention Time: The time from the start of signal detection by the
detector to the peak height of the elution concentration profile of
each different sample.
Curve Width: The width of the concentration profile curve of the
different samples in the chromatogram in units of time.
RESOLUTION (RS) :
Rs = 2(tRB – tRA)/(wB + wA)
Where:
tRB = Retention time of solute B
tRA = Retention time of solute A
wB = Gaussian curve width of solute B
wA = Gaussian curve width of solute A
Plate Number (N):
N = (tR)2/(w/4)2
Plate Height (H):
H = L/N
Where L is the length of the column.Asheesh Pandey 22

23. Resolution
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With a resolution value of 1.0, two peaks that overlap by about 4%. Values
less than 1.0 indicate peaks that overlap, while at a resolution of 1.5, the peaks
are considered fully separated.

24.  Retention factor :
 Rƒ = Distance travelled by a Solute
Distance travelled by a Solvent
 Rƒ = zero, - Solute remains in the stationary phase and
thus it is immobile.
 Rƒ = 1 - Solute has no affinity for the stationary phase
and travels with the solvent front.
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25. Going back to N….
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N = L/H
The value of N is greatly dependent on the value of H.
The value of H depends primarily on four factors:
1) the velocity of the mobile phase,
2) eddy diffusion or multipath diffusion,
3) the diffusion of the compound in the mobile phase
4) the transfer of the compound between the stationary phase
and the mobile phase.

26. H - Theoretical Plate Height
H = A + B/u + (Cs + Cm) u
u = the average linear mobile phase velocity
A is a term expressing multipath diffusion
B/u is the term for longitudinal diffusion
Cs is the mass transfer term in the stationary phase
Cm is the mass transfer term in the mobile phase
H = A + B/u + (Cs + Cm) u
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27. A Multipath
1
2
Flow
Direction
Pathways of two molecules during elution.
Distance traveled by molecule 1 is longer than
that traveled by molecule 2, thus molecule 1 will
take longer to
elute.
The amount of spreading is affected by the nature of
the column material and how well the column is
packed. This factor is generally proportional to the
particle size of the packing material. This factor must
be taken into account for packed columns, but for
capillary columns, this term is not needed since
there are no particles.
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28. B Longitudinal Diffusion
Flow
Flow
Molecules diffuse from areas of high
concentration to areas of low concentration.
Over time….
At low velocities longitudinal diffusion has a negative effect on resolution, but this
effect is negligible at higher velocities. This term is very important in gas
chromatography as diffusion coefficients in gasses are orders of magnitude higher
than in liquids. In liquid chromatography, this term is typically close to zero
relative to the other terms.
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29. Equilibrium between the mobile and stationary phases is never realized
Mass Transfer Terms Cs & Cm
It takes time for analytes to move from the mobile phase into the
stationary phase. Because no equilibrium is reached, some of the
analytes are swept ahead of the of the main band.
It also takes time for molecules to move back out of the stationary phase,
and some of the analyte molecules will be left behind by the rapidly
moving mobile phase.
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30. Mass Transfer Terms Cs & Cm
The faster the mobile phase moves, the less time there is for equilibrium between
the phases and the mass transfer effect on peak broadening is directly related to
mobile phase velocity.
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31. van Deemter Plot
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Linear Velocity, u
PlateHeight,H
Multipath Term, A
Mass Transfer (both), Cu
Longitudinal diffusion, B/u
A + B/u + Cu
H = A + B/u + (Cs + Cm) u

32. PRICNIPLES OF CHROMATOGRAPGHY
 Physical method of separation that distributes components
to separate between two phases moves in a definite
direction.
 Substances are separated based on their differential
distribution between two phases
 Substances will move with the mobile phase at different
rate depending upon their Partition or Distribution co-
efficients.
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33. PRINCIPLES
 The samples are subjected to flow by mobile liquid phase onto
or through the stable stationary phase.
 Separation of fractions of mixture based on their relative
affinity towards the two phases during their travel.
 The fraction with greater affinity to stationary phase travels
slower and shorter while that with less affinity travels faster and
longer.
 The separation is based on Differential partitioning
between the mobile and stationary phases.
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36. FACTORES AFFECTING THE SEPARATION
 Intermolecular interaction between the two phases
 Extent of dispersion of solute molecules over the
stationary phase
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37. CLASSIFICATION OF CHROMATOGRAPHY
 Techniques by Chromatographic bed shape
◦ Column chromatography
◦ Planar chromatography
 Paper chromatography
 Thin layer chromatography
 Techniques by Physical state of mobile phase
◦ Gas chromatography
◦ Liquid chromatography
 Affinity chromatography
◦ Supercritical fluid chromatography
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38. TECHNIQUES BY CHROMATOGRAPHIC BED
SHAPE
A.COLUMN CHROMATOGRAPHY
PRINCIPLES
 Solid materials (Adsorbants) – Ability to hold the molecules
at their surface
 Attractive forces (Vanderwalls & Hydrogen )
 Functional groups (Hydroxyl/ Aromatic)
 Silica
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39.  Stationary bed is within a tube.
 Solvent is driven through the column by applying Positive
pressure.
 Separations - 20 minutes
 Modern flash chromatography :
 Pre-packed plastic cartridges,
 Solvent is pumped through the cartridge.
 Quicker separations
 Less solvent usage.
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40.  Column :
o Diameter - 5 mm to 50 mm
o Height - 5 cm to 1 m with a tap
o Filter (a glass frit or glass wool plug)
 The individual components are retained by the stationary
phase differently and separate from each other while they are
running at different speeds through the column with the eluent.
 During the entire chromatography process the eluent is
collected in a series of fractions. The composition of the eluent
flow can be monitored and each fraction is analyzed for
dissolved compounds, e.g., UV absorption, or fluorescence.
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41. STATIONARY PHASE
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 Silica gel, Alumina. Cellulose

42. SOLVENTS
 Hydroxyl groups - Alcohol
 Carboxyl group - Acetone
 Non polar Compounds – Hexane
Heptane
Toulene
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43.  Flow rate - Separation.
 Pump or compressed gas (e.g. Air, Nitrogen, Argon)
 A faster flow rate of the eluent:
 Minimizes the time required to run a column
 Minimizes diffusion
 Better separation.
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47. B. PLANAR CHROMATOGRAPHY
 Separation technique - Stationary phase is present as or on a
plane.
 Paper – Paper Chromatography
 Layer of solid particles spread on a support such as a glass
plate - Thin layer Chromatography.
 Different compounds in the sample mixture travel different
distances according to how strongly they interact with the
stationary phase as compared to the mobile phase.
 Retention factor (Rf)
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49. PAPER CHROMATOGRAPHY
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51.  This paper is made of cellulose, a polar substance, and the
compounds within the mixture travel farther if they are non-
polar.
 More polar substances bond with the cellulose paper more
quickly, and therefore do not travel as far.
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PRINCIPLE

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54.  b) THIN LAYER CHROMATOGRAPHY
 Widely employed laboratory technique
 Stationary phase - Adsorbent - Silica gel
Alumina
Cellulose
 Widely used in pharmaceutical & food stuff industry
 Advantages :
 Simple, Rapid and Cheap
 Faster runs
 Better separations
 Choice between different adsorbents.
 Better resolution
 Allow for quantification
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55. Used to identify the unknown compounds and to determine
the purity of mixture.
TLC Plate - Aluminium or glass - coated by stationary phase.
Coated material : 0.1-0.3mm in thickness
Fluorescent indicator that will make it florescence during the UV
light exposure.
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56. MOBILE PHASE
 Volatile Organic solvents
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STATIONARY PHASE
Silica gel, Alumina, or Cellulose on a flat, inert substrate.

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59. SPRAYS
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60.  2.TECHNIQUES BY PHYSICAL STATE OF MOBILE
PHASE
A. GAS CHROMATOGRAPHY
 Gas-Liquid chromatography, (GLC)
 Mobile phase – Gas (Helium) Carrier Gas Pressure = 4 kg/cm2
 Stationary phase - Column, which is typically "packed" or "capillary".
 The stationary phase is adhered to the inside of a small-diameter glass
tube (a capillary column) or a solid matrix inside a larger metal tube (a
packed column).
 Partition Coefficient of Volatile analyte between a solid stationary
phase (Silicone) and a mobile gas (Helium).
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61.  Advantages
 High sensitivity,
 High Resolution,
 High speed
 High Accurasy,
 Highly Quantitative

 APPARATUS
 Gas Chromatograph, GC analyzer, Normal syringes and one micro syringe,
Beakers, Sample bottles and Electronic weight.
 CHEMICALS
 Methanol, Isopropyl Alcohol and water
 SAMPLE:
 Gases, Liquid, Solids
 M.Wt: 2-800
 Volatile
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63. APPLICATION
 Quantitative & Qualitative analysis of low polarity compounds
 Analytical chemistry, Biochemistry, Petrochemical,
Environmental monitoring
 Measure picomoles of a substance in a 1 ml liquid sample, or
parts-per-billion concentrations in gaseous samples
 Measuring toxic substances in soil, air or water.
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64. APPLICATION OF GC- MS
 Environmental monitoring : Oraganic Pollutants
 Criminal forensics : Analyze the particles (Fibre) from a human
body in order to help link a criminal to a crime.
 Law enforcement : Detection of illegal narcotics,
 Forensic toxicology : Find drugs and/or poisons in biological
specimens of suspects, victims, or the deceased.
 Sports anti-doping analysis : Test athletes' urine samples
 Security : Explosive detection (September 11 development) systems
have become a part of all US airports.
 Food, beverage and perfume : from spoilage or Adultration -
aromatic compounds, esters, fatty acids, alcohols, aldehydes,
terpenes
 Medicine : Congenital metabolic diseases
In Born error of metabolism
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65.  B. LIQUID CHROMATOGRAPHY
 Mobile phase - Liquid.
 Column or a plane.
 Very small packing particles and a relatively high pressure -
High Performance Liquid Chromatography (HPLC).
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71. LC- MS
 Mass spectra is obtained rapidly
 Small amount of material is required to form the spectra.
 Data collected is highly informative with respect to
molecular structure.
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72. APPLICATION
 Pharmacokinetics : How quickly a drug will be cleared from the
hepatic blood flow and organs of the body.
 Proteomics : Peptide mass fingerprinting
 Drug development: Peptide Mapping, Glycoprotein Mapping,
Natural Products Dereplication, Bioaffinity Screening, In Vivo
Drug Screening, Metabolic Stability Screening, Metabolite
Identification, Impurity Identification, Degradant Identification,
Quantitative Bioanalysis, and Quality Control.
 Fungal toxins
 Pesticides, Herbicides
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73. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC V/S LC TECHNIQUE
 Columns : Small diameter (4.6 mm), stainless steel, glass or
titanium.
 Column packing with very small (3, 5 and 10 μm) particles
 Relatively high inlet pressures and controlled flow of the
mobile phase.
 Detecting very small amounts
 High resolution
 Rapid analysis
 Speed, efficiency, sensitivity and ease of operation
 High degree of versatility
 Easily separate a wide variety of chemical mixtures
 400 atmospheres.
PUMP PRESSURE
 "Ultra High Performance Liquid Chromatography" systems
1000 atmospheres.
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75.  ELUTION : Isocratic and Gradient.
 ISOCRATIC :
 ISO ==> SAME
 - Solvent Composition Stays the Same for the Entire Run
EX: 60:40 Alcohol:Water
 GRADIENT :
 Solvent Composition Changes Throughout the Run
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79. TYPES OF HPLC
 Nature of the stationary phase
 Separation process
 Adsorption chromatography
 Ion-exchange chromatography
 Size exclusion chromatography
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80. APPLICATION
 Protein separation
 Insulin purification
 Plasma fractionation
 Enzyme purification
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81. SIZE EXCLUSION CHROMATOGRAPHY
 Gel filtration or gel permeation chromatography
 Separation - Molecular size of its components.
 Larger molecules are rapidly washed through the column, smaller
molecules penetrate inside the porous of the packing particles and
elute later.
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82. APPLICATIONS
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84.  AFFINITY CHROMATOGRAPHY
 Based on specific & non-covalent binding of the proteins to
other molecules – Ligands ( His-tags, biotin or antigens)
 Physical properties of the analyte.
 Biochemistry in the purification of proteins (Enzymes)
bound to tags.
 After purification, some of these tags are usually removed
and the pure protein is obtained.
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85.  SUPERCRITICAL FLUID CHROMATOGRAPHY
 Used for the analysis and purification of low to moderate molecular weight , thermally labile
molecules.
 Principles are similar to those of (HPLC)
 Mobile phase - High pressure liquid or Super critical Carbon Dioxide.
 Modifiers – Methanol, Ehanol, isopropyl alcohol, acetonitrile and
Chloroform.
 APPLICATION
 Use in industry primarily for separation of Chiral (Asymmetric Carbon atoms) molecules.
• Serine
• Soman
• Glyceraldehyde
• Phosphours (Phosphine)
• Sulfar metal
• Cobalt
• Enkephalins
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86. DETECTOR
 Gas Chromatography or liquid Chromatography
 To visualize components of the mixture being eluted off the
chromatography column.
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87. DETECTORS
 Flame ionization detector
 Aerosol-based detector
 Flame photometric detector ( FPD).
 Atomic-emission detector (AED).
 Mass spectrometer ( MS) detector
 Nitrogen Phosphorus Detector,
 Evaporative Light Scattering Detector (ELD) : LC.
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88. DETECTORS
 UV detectors
 Thermal conductivity Detector, (TCD)
 Fluorescence detector
 Electron Capture Detector, (ECD)
 Photoionization Detector, (PID)
 Refractive index Detector (RI or RID)
 Radio flow Detector
 Chiral Detector
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90. HPLC Instrumentation
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91. In This Section, We Will
Discuss:
 General components of a high performance liquid
chromatograph.
 HPLC solvent delivery systems.
 How automatic injectors work.
 Common HPLC detectors.
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92. HPLC Instrumentation Overview
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Principle Pattern An Example
Detector
Thermostatted
Column Compartment
Autosampler
Binary Pump
Vacuum DegasserSolvent Cabinet
Solvent Reservoirs
Controller

93. Solvent Filters
Solvent Inlet Filer
 Stainless Steel or
glass with 10 micron
porosity.
 Removes particulates
from solvent.
Precolumn Filter
 Used between the injector and
guard column.
 2 to 0.5 micron
 Removes particulates from
sample
and autosampler wear debris.
 Must be well designed to prevent
dispersion.
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Guard
column
Injector
Analytical
Column
Precolumn
Filter
Solvent Inlet Filter

94. Vacuum Degassing
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95. Functions of the Solvent Delivery
System
The solvent delivery system has three basic
functions:
1. Provide accurate and constant flow.
2. Provide accurate mobile phase
compositions.
3. Provide the force necessary to push the
mobile phase through the tightly packed
column.
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96. Multichannel Gradient Valve
 Determines mobile phase composition.
 Largest solvent plug fills first.
 Agilent 1100 and 1200 quaternary pump.
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97. Dual Piston Parallel Pump
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Single
Piston
Delivery
Combined
Delivery
Piston 'A' Advancing Piston B Retracting
Check
Valves
A B
Pumphead
Piston
Rotary
Switching
Valve

98. Dual Piston in Series Pump
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 First piston displaces
solvent at twice the
speed and stroke volume
of the second piston.
 Provides constant flow and the
pressure necessary to get through
column.

99. Ballvalves for Reciprocating
Piston Pumps
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Gold Seal
Sapphire
Insert
Ruby Ball
Spring
Insert

100. Pump Seals and Pistons
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1. Piston
2. Support Rings
3. Seal Keepers
4. Seals
5. Wear
Retainers
2
3
4
5
1

101. Frits and Filters
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Frits, Filters, and Sieves are used to protect
other parts of the LC from pump and seal material.
Purge valve
PTFE Frit

102. Damping Units
 Filled with compressible liquid
separated from the mobile phase by
a membrane.
 Pressure ripples reduced to < 2%
original value.
102
Damping
Unit
Pressure
2%
P/P
Pump
Ripple

103. Gradient Formation
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Low Pressure Gradient High Pressure Gradient

104. Summary
The pump is the most critical piece of
equipment for a
successfully operating HPLC.
Performance parameters for HPLC
pumps:
 Flow Precision
 Flow Range
 Delay Volume
 Pressure Pulse
 Composition Precision
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105. Sample Injectors
Requirements:
Reproducible introduction of the sample
volume into the mobile phase flow.
Two major designs:
Automatic Injectors or Manual Injectors
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106. Manual Injectors
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Front View
Inject
Rear View
Load - Inject
Sample Loop

107. Manual Injectors
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Sample in
Solvent in
Solvent out
Sample Load
Sample Inject
From Pump
To column
Sample in
Solvent in
Solvent out
From Pump
To column

108. Automatic Injectors
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Step 1 Step 2
Step 3

109. Rotor Seals
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Rotor Seal
found within
valve

110. Column Oven
Constant temperature for solvent and column is required to
perform reproducible results.
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111. Common HPLC Detectors
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•UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength
•Mass Spectrometers
•Refractive Index
•Fluorescence
•Light Scattering
•Electrochemical
•Radioactivity
•Conductivity

112. Necessity for More Than One
Detector - Sensitivity
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PAH's extracted from soil;
Sup.LC-PAH 150x4.6mm;
Solv.: H2O/CH3OH= 10:90
Fluorescence
UV-signal
WL
241/394
WL
270/388
WL
248/411
WL
302/420
WL
247/504
Pyrene
C
hrysene
Benzo(e)pyrene
Perylene
Benzo(k)fluoranthene
Benzo(a)pyrene
Benzo(ghi)perylene
Indeno(123-cd)pyrene

113. Necessity for More Than One
Detector - Selectivity
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Flecainide in
Serum
Therapeutic concentration: 1.8mg/l, 20ul injected
UV and fluorescence signal
FL signal
UV signal

114. Necessity for More Than One
Detector - Qualitative Information
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Qualitative Information
Take peak spectrum
(UV)
Chlortoluron
?
44
68
58
96 132 138158
172
215
200
Take peak spectrum
(MS)
104
Mass/Charge
Atrazine
?
Wavelength (nm)
60 80 100 120 140 160 180 200 220

115. HPLC Detector
Characteristics
Detector performance characteristics:
 Sensitivity (LoD, LoQ)
 Selectivity
 Linearity
 Qualitative information
 Reliability
 Ease of use
 Universality
Asheesh Pandey 115

116. LOD
The limit of detection for a detector can be
characterized by its signal to noise ratio (S/N)
for an analyte under a given set of conditions.
Asheesh Pandey 116
Noise
Peak

117. Limit of Detection - Limit of
Quantitation
 Limit of detection (LOD) is a result of the whole chromatography
system, not only the detector performance
 Limit of quantification (LOQ) is a defined limit for a method used for
a specific purpose.
117
Linear range
Slope = sensitivity
MQL
MDL
Response
Amount
Intercept
e.g.,RSD<10%, S/N > 20
e.g., S/N > 3

118. UV-Vis Detectors
Asheesh Pandey 118
b
c
Detector Flow Cell
I0 I
Log I0 = A = abc
I
Principles: The fraction of light transmitted through the detector cell is
related to the solute concentration according to Beer’s Law.
Characteristics: Specific, Concentration Sensitive, good stability,
gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).

119. UV-Vis Detectors - Design Principles
 Single wavelength detection of
multi wavelength detection
possible.
 Wavelength calibration is done
automatically using a holmium
filter.
119
UV Lamp
Grating
Flow cell
Reference diode
Sample
diode
Cut-off filter
Holmium oxide
filter
Slit
Mirror 2
Mirror 1
Variable Wavelength
Detector

120. UV-Vis Detector with Spectral
Capability
 Diode Array UV-Vis Detector allows online measurement of spectra.
 Wavelength range 190 - 950 nm.
 Wavelength Resolution: Up to 1 nm.
 Wavelength calibration with Holmium oxide filter.
120
Diode Array
Grating
Optical
Slit
Detector
Flow Cell
Homium
Filter
Achromatic
Lens
UV
Lamp
Vis
Lamp

121. Fluorescence Detection
Asheesh Pandey 121
Emission
Monochromator
signal&
spectramode
PMTdetector
ReferenceDiode
8µlFlowCell,auto-recognition
Triggerpack
Exitation
Monochromator,
signal&
spectramode
Mirror
Lens
(condensorEX)
Lens(condensorEM)
SlitEM SlitPMT
SlitEX
Diffuser
Xenon
flashLamp,
15W

122. Electrochemical Detectors
 Gold for carbohydrates.
 Platinum for chlorite, sulfate, hydrazine,
etc.
 Carbon for phenols, amines.
 Silver for chloride, bromide, cyanide.
122
Thin-layer design Porous flow-
through design
Wall-jet design

123. HPLC-MSD API- Electrospray
Asheesh Pandey 123

124. Refractive Index Detector DesignThe Refractive Index Detection is
strongly influenced by:
 Pressure changes
 Temperature changes
 Flow pulse
Gradient elution is not possible!
124

125. Conductivity Detectors
Asheesh Pandey 125
ref.capacitor
cell
variable
resistances
fixed
resistor
C
r
Balance
controlA E
F
D
B
~
Schematics Applications
water
soap products
detergents
soft drinks
blood
plating baths
nuclear fuel reprocessing
streams
Ions
Acids
Bases
Salts
in
}