HPLC Chromatography and its principle Liquid chromatography High Performance Liquid Chromatography ( HPLC ) The components of the high performance liquid chromatograph (HPLC). The separation process. The chromatogram

1. High Performance LiquidHigh Performance Liquid
ChromatographyChromatography
ALAKESH PRADHANALAKESH PRADHAN
COCHIN UNIVERSITY OFCOCHIN UNIVERSITY OF
SCIENCE AND TECHNOLOGYSCIENCE AND TECHNOLOGY
School of IndustrialSchool of Industrial
FisheriesFisheries
M.Sc IInd Sem.M.Sc IInd Sem.

2. Overview:
 Chromatography and its principleChromatography and its principle
 Liquid chromatographyLiquid chromatography
 High Performance Liquid Chromatography ( HPLC )High Performance Liquid Chromatography ( HPLC )
 The components of the high performance liquid chromatograph (HPLC).The components of the high performance liquid chromatograph (HPLC).
 The separation process.The separation process.
 The chromatogram.The chromatogram.

3. Latest instrument

4. BackgroundBackground
Chromatography and its Principle
 Chromatography is a separation technique which is used toChromatography is a separation technique which is used to
separate a mixture of compounds into its individual componentsseparate a mixture of compounds into its individual components
based on certain physical and chemical properties.based on certain physical and chemical properties.

5. Some important terms:Some important terms:
 Mobile phase: The solvent system which carries the mixture to beMobile phase: The solvent system which carries the mixture to be
separated.separated.
 Stationary phase: Immobile surface which is particulate in nature. This is theStationary phase: Immobile surface which is particulate in nature. This is the
region over which the compound gets separated.region over which the compound gets separated.

6. Principle:
 The process involves the interaction of the compounds in the analyte (whichThe process involves the interaction of the compounds in the analyte (which
travels along with a mobile phase) across an immobile surface (stationarytravels along with a mobile phase) across an immobile surface (stationary
phase).phase).
 The compounds bind at specific regions of stationary phase based onThe compounds bind at specific regions of stationary phase based on
certain physical and chemical properties. These bound molecules are thencertain physical and chemical properties. These bound molecules are then
eluted with a suitable buffer and the same are collected with time.eluted with a suitable buffer and the same are collected with time.
These are –These are –
 PolarityPolarity
 ChargeCharge
 Molecular weightMolecular weight
 Present of functional groupPresent of functional group

7. IntroductionIntroduction
 HPLC is a form of liquid chromatography used toHPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.separate compounds that are dissolved in solution.
 HPLC instruments consist of a reservoir of mobileHPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column, and aphase, a pump, an injector, a separation column, and a
detector.detector.
 Compounds are separated by injecting a sample mixtureCompounds are separated by injecting a sample mixture
onto the column.onto the column.
 The different component in the mixture pass through theThe different component in the mixture pass through the
column at differentiates due to differences in theircolumn at differentiates due to differences in their
partition behavior between the mobile phase and thepartition behavior between the mobile phase and the
stationary phase.stationary phase.
 The mobile phase must be degassed to eliminate theThe mobile phase must be degassed to eliminate the
formation of air bubbles.formation of air bubbles.

8. Continued…
What is Liquid Chromatography?
 Liquid chromatography is a separation technique that involves:Liquid chromatography is a separation technique that involves:
•• the placement (injection) of a small volume of liquid samplethe placement (injection) of a small volume of liquid sample
•• into a tube packed with porous particles (stationary phase)into a tube packed with porous particles (stationary phase)
•• where individual components of the sample are transported along thewhere individual components of the sample are transported along the
packed tube (column) by a liquid moved by gravity.packed tube (column) by a liquid moved by gravity.
 The components of the sample are separated from one another by theThe components of the sample are separated from one another by the
column packing that involves various chemical and/or physical interactionscolumn packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.between their molecules and the packing particles.
 The separated components are collected at the exit of this column andThe separated components are collected at the exit of this column and
identified by an external measurement technique , such as aidentified by an external measurement technique , such as a
spectrophotometer that measures the intensity of the color , or by anotherspectrophotometer that measures the intensity of the color , or by another
device that can measure their amount.device that can measure their amount.
 Note:Note: The modern form of liquid chromatography is now referred to asThe modern form of liquid chromatography is now referred to as
““flash chromatographyflash chromatography

9. Principles of Liquid
Chromatography

10. Terminologies for HPLC
 HPLC : High Performance Liquid Chromatography : High Pressure LCHPLC : High Performance Liquid Chromatography : High Pressure LC
 Now, before we go in depth of principle, lets have a basic look at fewNow, before we go in depth of principle, lets have a basic look at few
terms as follows:terms as follows:
 Resolving Power: The extent of separation of the compounds presentResolving Power: The extent of separation of the compounds present
in the mixture across the column.in the mixture across the column.
 Theoretical plates : An imaginary division of the column intoTheoretical plates : An imaginary division of the column into
equilength plates.equilength plates.

11. Principles of HPLC
Principle:Principle:
 The table shows relation between variousThe table shows relation between various
parameters of HPLC.parameters of HPLC.
 Trendline:Trendline:
 Stationary phase have small particulate size andStationary phase have small particulate size and
high surface areas.high surface areas.
 Columns: 20 cm or lessColumns: 20 cm or less
 Mobile phase pumped at high pressures ofMobile phase pumped at high pressures of
200Bar, 3000 psi.200Bar, 3000 psi.
 Flow rates: 1-3 cmFlow rates: 1-3 cm33 per minper min
Column length No. of theoretical plates
per unit area
Resolving power Column length
Particle size Surface area

12. What is HPLC?
 HPLC is a separation technique that involves:HPLC is a separation technique that involves:
••the injection of a small volume of liquid samplethe injection of a small volume of liquid sample
••into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameterinto a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter
called thecalled the stationary phase)stationary phase)
••where individual components of the sample are moved down thewhere individual components of the sample are moved down the
packed tube (packed tube (column) with a liquid (mobile phase) forced throughcolumn) with a liquid (mobile phase) forced through
the column by high pressure delivered by a pump.the column by high pressure delivered by a pump.
 TheseThese components are separated from one another by the columncomponents are separated from one another by the column
packing that involves various chemical and/or physical interactionspacking that involves various chemical and/or physical interactions
between their molecules and the packing particles.between their molecules and the packing particles.
 These separated components are detected at the exit of this tube (These separated components are detected at the exit of this tube (column)column)
by a flow-through device (detector) that measures their amount. Anby a flow-through device (detector) that measures their amount. An
output from this detector is called a “liquid chromatogram”.output from this detector is called a “liquid chromatogram”.
  In principle, LC and HPLC work the same way except the speed ,In principle, LC and HPLC work the same way except the speed ,
 efficiency, sensitivity and ease of operation of HPLC is vastlyefficiency, sensitivity and ease of operation of HPLC is vastly
 superior.superior.

13. HPLC systemHPLC system
Flow chart of HPLC mechanism

14. Varian 9010 Solvent Delivery
System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject

15. Picture of HPLC instrument

16. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEMCOMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
SolventSolvent
Solvent Delivery System (Pump)Solvent Delivery System (Pump)
InjectorInjector
SampleSample
ColumnColumn
DetectorsDetectors
Waste CollectorWaste Collector
Recorder (Data Collection)Recorder (Data Collection)

17. Instrumentation of HPLC
( Describing the 5 major components and their
functions….)
1
2
3
4
5
Solvent
reservoirs
and degassing
Not shown
here
1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer

18. 1. Pump:
•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).

19. Pump Module–typesPump Module–types::
 Isocratic pump - Delivers constant mobile phase composition;Isocratic pump - Delivers constant mobile phase composition;
••solvent must be pre-mixed;solvent must be pre-mixed;
••lowest cost pumplowest cost pump
 Gradient pump - Delivers variable mobile phase composition;Gradient pump - Delivers variable mobile phase composition;
••can be used to mix and deliver an isocratic mobile phase or acan be used to mix and deliver an isocratic mobile phase or a
gradient mobile phasegradient mobile phase
––Binary gradient pumpBinary gradient pump –delivers two solvents–delivers two solvents
––Quaternary gradient pumpQuaternary gradient pump –four solvents–four solvents

21. 2. Injector:
•The injector serves to introduce the liquid sample into the flow stream of
the mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of
the liquid system.
•An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .

22. Sample Injection
……how is a sample actually put into an LC system
Manual InjectorManual Injector::
1.User manually loads sample into the injector using a syringe1.User manually loads sample into the injector using a syringe
2.and then turns the handle to inject sample into the flowing mobile2.and then turns the handle to inject sample into the flowing mobile
phase… which transports the sample into the beginning (head) of thephase… which transports the sample into the beginning (head) of the
column, which is at high pressurecolumn, which is at high pressure
Auto samplerAuto sampler::
1.User loads vials filled with sample solution into the auto sampler tray1.User loads vials filled with sample solution into the auto sampler tray
(100 samples)(100 samples)
2.and the auto sampler automatically2.and the auto sampler automatically
a. measures the appropriate sample volume,a. measures the appropriate sample volume,
b. injects the sample,b. injects the sample,
c. then flushes the injector to be ready for the next sample,c. then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed ……etc., until all sample vials are processed ……
………….for unattended automatic operation.for unattended automatic operation

23. 2323
Manual InjectorsManual Injectors
Front View
Inject
Rear View
Load - Inject
Sample Loop

24. 2424
Automatic InjectorsAutomatic Injectors
Step 1 Step 2
Step 3

25. 3.3. Column:Column:
•• Considered the “heart of the chromatograph” the column’s stationaryConsidered the “heart of the chromatograph” the column’s stationary
phase separates the sample components of interest using various physicalphase separates the sample components of interest using various physical
and chemical parameters.and chemical parameters.
••The small particles inside the column are what cause the highThe small particles inside the column are what cause the high
back pressure at normal flow rates.back pressure at normal flow rates.
••The pump must push hard to move the mobile phase through theThe pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within thecolumn and this resistance causes a high pressure within the
chromatograph.chromatograph.

26. Several Column Types
( can be classified as)
 Normal phaseNormal phase
 Reverse phaseReverse phase
 Size exclusionSize exclusion
 Ion exchangeIon exchange

27. Normal phase
 In this column type, the retention is governed byIn this column type, the retention is governed by
the interaction of the polar parts of the stationarythe interaction of the polar parts of the stationary
phase and solute.phase and solute.
 For retention to occur in normal phase, theFor retention to occur in normal phase, the
packing must be more polar than the mobilepacking must be more polar than the mobile
phase with respect to the samplephase with respect to the sample

28. 28
HO Si
O
O
STATIONARY PHASES
(NORMAL POLARITY)
Silica or alumina possess polar sites that
interact with polar molecules.
Most polar…….Least polar
Components elute in increasing
order of polarity.
Components elute in increasing
order of polarity.
Polar Group
silica

29. Reverse phase
 In this column the packing material is relatively nonpolar and the solvent isIn this column the packing material is relatively nonpolar and the solvent is
polar with respect to the sample. Retention is the result of the interaction ofpolar with respect to the sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the nonpolar stationary phase.the nonpolar components of the solutes and the nonpolar stationary phase.
 Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bondedTypical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-
organic mixtures such as methanol-water or acetonitrile-water.organic mixtures such as methanol-water or acetonitrile-water.
Common Reverse Phase SolventsCommon Reverse Phase Solvents ––
Methanol
Acetonitrile
Tetrahydrofuran
Water
CH3OH
CH3CN
H2O

30. 30
STATIONARY PHASES
(REVERSE POLARITY)
If the polar sites on silica or alumina are capped with non-polar
groups, they interact strongly with non-polar molecules.
Most non-polar…….Least non-polar
Components elute in decreasing
order of polarity.
Components elute in decreasing
order of polarity.
C18 phase
silica
Si
Me
Me
O Si
O
O

31. Size exclusion
 In size exclusion the HPLC column is consisted ofIn size exclusion the HPLC column is consisted of
substances which have controlled pore sizes andsubstances which have controlled pore sizes and
is able to be filtered in an ordinarily phaseis able to be filtered in an ordinarily phase
according to its molecular size.according to its molecular size.
 Small molecules penetrate into the pores withinSmall molecules penetrate into the pores within
the packing while larger molecules only partiallythe packing while larger molecules only partially
penetrate the pores. The large molecules elutepenetrate the pores. The large molecules elute
before the smaller moleculesbefore the smaller molecules..

32. 32
STATIONARY PHASES
(SIZE EXCLUSION)
Size exclusion gels separate on the basis of molecular size.
Individual gel beads have pores of set size, that restrict
entry to molecules of a minium size.
Larger molecules…….Smaller molecules
Large molecules elute fast (restricted path),
while small molecules elute slowly (long path length)
Large molecules elute fast (restricted path),
while small molecules elute slowly (long path length)

33. Ion exchange
 In this column type the sample componentsIn this column type the sample components
are separated based upon attractive ionicare separated based upon attractive ionic
forces between molecules carrying chargedforces between molecules carrying charged
groups of opposite charge to those chargesgroups of opposite charge to those charges
on the stationary phase.on the stationary phase.
 Separations are made between a polar mobileSeparations are made between a polar mobile
liquid, usually water containing salts or smallliquid, usually water containing salts or small
amounts of alcohols, and a stationary phaseamounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.containing either acidic or basic fixed sites.

34. 34
STATIONARY PHASES
(CATION EXCHANGE)
Silica is substituted with anionic residues that interact
strongly with cationic species (+ve charged)
Most +ve…….Least +ve
+ve charged species adhere to the support
and are later eluted with acid (H+
)
+ve charged species adhere to the support
and are later eluted with acid (H+
)
Cations exchange Na+
silica
S
O
O
ONa

35. 35
STATIONARY PHASES
(ANION EXCHANGE)
Silica is substituted with cationic residues that interact
strongly with anionic species (-ve charged)
Most -ve…….Least -ve
-ve charged species adhere to the support
and are later eluted with acid (H+
)
-ve charged species adhere to the support
and are later eluted with acid (H+
)
Anions exchange Cl-
silica
Me N
Me
Me
CH2
Cl

36. HPLC Columns
Within the Column is where separation occurs.Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLCKey Point –Proper choice of column is critical for success in HPLC
Materials of construction for the tubingMaterials of construction for the tubing
 Stainless steel (the most popular; gives high pressure capabilities)Stainless steel (the most popular; gives high pressure capabilities)
 Glass (mostly for biomolecules)Glass (mostly for biomolecules)
 PEEK polymer (biocompatible and chemically inert to most solventsPEEK polymer (biocompatible and chemically inert to most solvents
Packing material:Packing material:
TheThe packing material is prepared from SILICA particle, ALUMINA particlepacking material is prepared from SILICA particle, ALUMINA particle
and ion exchange RESIN.and ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columnsPorous plug of stainless steel or Teflon are used in the end of the columns
to retain the packing material.to retain the packing material.
According to the mode of HPLC , they are available in different size ,According to the mode of HPLC , they are available in different size ,
diameters, pore size or they can have special materials attached ( such asdiameters, pore size or they can have special materials attached ( such as
antigen or antibody ) for immuno affinity chromatography.antigen or antibody ) for immuno affinity chromatography.

37. 3737
Modes of High Performance LiquidModes of High Performance Liquid
ChromatographyChromatography
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-
Organic

38. Types of columns in HPLCTypes of columns in HPLC::
 Guard ColumnGuard Column
 Fast ColumnFast Column
 Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)
 Capillary(i.d. 0.1 -1.0 mm; various lengths)Capillary(i.d. 0.1 -1.0 mm; various lengths)
 Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)
 Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]

39. Guard Column
 These are placed anterior to the separating column. This serves asThese are placed anterior to the separating column. This serves as
protective factor.protective factor.
 They are dependable columns designed to filter or remove :They are dependable columns designed to filter or remove :
Particles that clog the separation columnParticles that clog the separation column
Compounds and ions that could ultimately cause “ Baseline drift ”,Compounds and ions that could ultimately cause “ Baseline drift ”,
decreased resolution, decreased sensitivity and create false peaks.decreased resolution, decreased sensitivity and create false peaks.
These columns must be changed on a regular basis in order to optimizeThese columns must be changed on a regular basis in order to optimize
their protective function.their protective function.

40. Fast Column
 One of the primary reasons for using these column is to obtain improvedOne of the primary reasons for using these column is to obtain improved
sample output ( amount of compound per unit time).sample output ( amount of compound per unit time).
 Fast column are designed to decrease the time of chromatographic analysisFast column are designed to decrease the time of chromatographic analysis
 Here internal diameter is same but length is short and packed with smallerHere internal diameter is same but length is short and packed with smaller
particles , that are 3particles , that are 3 μm diameter.μm diameter.
 Advantages-Advantages-
Increased sensitivityIncreased sensitivity
Decreased analysis timeDecreased analysis time
Decreased mobile phase usageDecreased mobile phase usage
Increase reproducibilityIncrease reproducibility

41. Capillary Column
 It is also known as micro columnsIt is also known as micro columns
 It has a diameter much less than a millimeter and there 3 types:It has a diameter much less than a millimeter and there 3 types:
Open tubularOpen tubular
Partially packedPartially packed
Tightly packedTightly packed
They allow the user to work with nanoliter sample volume , decreasedThey allow the user to work with nanoliter sample volume , decreased
flow rate and decreased solvent usage volume , led to cost effectivenessflow rate and decreased solvent usage volume , led to cost effectiveness

42. Preparatory Column
 Used when objective is to prepare bulk ( milligrams) of sample forUsed when objective is to prepare bulk ( milligrams) of sample for
laboratory preparatory application.laboratory preparatory application.
 It has usually a large column diameter , which is designed to facilitateIt has usually a large column diameter , which is designed to facilitate
large volume injections into the HPLC systemlarge volume injections into the HPLC system

43. 4.4. DetectorDetector::
•• The detector can see (detect) the individual molecules that comeThe detector can see (detect) the individual molecules that come
out (elute) from the column.out (elute) from the column.
••A detector serves to measure the amount of those moleculesA detector serves to measure the amount of those molecules
so that the chemist can quantitatively analyze the sampleso that the chemist can quantitatively analyze the sample
components.components.
••The detector provides an output to a recorder or computerThe detector provides an output to a recorder or computer
that results in the liquid chromatogram(i.e., the graph of thethat results in the liquid chromatogram(i.e., the graph of the
detector response).detector response).

44. HPLC Detectors

45. 4545
Common HPLC Detectors
•UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength
•Mass Spectrometers
•Refractive Index
•Fluorescence
•Light Scattering
•Electrochemical
•Radioactivity
•Conductivity

46. 46
UV-Vis Detectors
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 ).

47. 47
Fluorescence Detection
Emission
Monochromator
signal &
spectra mode
PMT detector
Reference Diode
8 µl Flow Cell, auto-recognition
Trigger pack
Exitation
Monochromator,
signal &
spectra mode
Mirror
Lens
(condensor EX)
Lens (condensor EM)
Slit EM Slit PMT
Slit EX
Diffuser
Xenon
flash Lamp,
15 W

48. 48
Electrochemical Detectors
• Gold for carbohydrates.
• Platinum for chlorite, sulfate, hydrazine, etc.
• Carbon for phenols, amines.
• Silver for chloride, bromide, cyanide.

49. Variable UV/Vis DetectorVariable UV/Vis Detector
ABS AUFS λ RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min
Ready

50. 5.5. ComputerComputer::
•• Frequently called the data system,Frequently called the data system,
The computer not only controls all the modules of theThe computer not only controls all the modules of the
HPLC instrument but it takes the signal from theHPLC instrument but it takes the signal from the
detector and uses it to:detector and uses it to:
1. determine the time of elution (retention time) of the1. determine the time of elution (retention time) of the
sample components (qualitative analysis) andsample components (qualitative analysis) and
2. the amount of sample ( quantitative analysis) .2. the amount of sample ( quantitative analysis) .

51. Varian 9060Varian 9060
PolychromPolychrom
DetectorDetector
UV Spectrum
Chromatogram
Reset
Ready
UV Spectrum
{shows full UV abs
Chromatogram
{shows peaks, Rt}
ABS.
Time
ABS.
Wavelength
UVmax
UVmax
Rt Rt

52. HPLC is optimum for the separation of chemical and biologicalHPLC is optimum for the separation of chemical and biological
compounds that are non-volatile .compounds that are non-volatile .
NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon inNOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in
gasoline, etc.), gas chromatography is a better separation technique .gasoline, etc.), gas chromatography is a better separation technique .
Typical non-volatile compounds are:Typical non-volatile compounds are:
 Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
 Salts like sodium chloride and potassium phosphateSalts like sodium chloride and potassium phosphate
 Proteins like egg white or blood proteinProteins like egg white or blood protein
 Organic chemicals like polymers (e.g. polystyrene, polyethylene)Organic chemicals like polymers (e.g. polystyrene, polyethylene)
 Heavy hydrocarbons like asphalt or motor oilHeavy hydrocarbons like asphalt or motor oil
 Many natural products such as ginseng, herbal medicines, plant extractsMany natural products such as ginseng, herbal medicines, plant extracts
 Thermally unstable compounds such as trinitrotoluene (TNT), enzymesThermally unstable compounds such as trinitrotoluene (TNT), enzymes
What is HPLC used for ?
Separation and analysis of non-volatile or
thermally unstable compounds

53. Separation Technique

54. 54
How can We Analyze the Sample?
For example:
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose
1
2
3
4
5
mAU
time
6

55. 55
Separations
Separation in based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste

56. 56
SeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph

57. 57
SeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time

58. 58
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

59. 59
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

60. 60
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

61. 61
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

62. 62
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

63. 63
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

64. 64
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

65. 65
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

66. 66
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

67. 67
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

68. 68
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

69. 69
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

70. 70
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

71. 71
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

72. 72
The Chromatogram
Injection
to
tR
mAU
time
tR
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportional
to the quantity of analyte.

73. HPLC used for Qualitative
Analysis

74. HPLC used for Quantitative
Analysis

75. HPLC uses
This technique is used for -This technique is used for -
chemistry and biochemistry research analyzingchemistry and biochemistry research analyzing
complex mixturescomplex mixtures
purifying chemical compoundspurifying chemical compounds
developing processes for synthesizing chemicaldeveloping processes for synthesizing chemical
compoundscompounds
isolating natural products, or predicting physicalisolating natural products, or predicting physical
properties.properties.
It is also used in quality control to ensure the purity ofIt is also used in quality control to ensure the purity of
raw materials, to control and improve process yields, toraw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate productquantify assays of final products, or to evaluate product
stability and monitor degradation.stability and monitor degradation.

76. In addition,
it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational
safety or health, and for monitoring pesticide levels in
the environment.

77. 77
HPLC Applications
Chemical
Environmental
Pharmaceuticals
Consumer Products
Clinical
polystyrenes
dyes
phthalates
tetracyclines
corticosteroids
antidepressants
barbiturates
amino acids
vitamins
homocysteine
Bioscience
proteins
peptides
nucleotides
lipids
antioxidants
sugars
polyaromatic hydrocarbons
Inorganic ions
herbicides

78. References
 HPLC instrumentation – Agilent TechnologiesHPLC instrumentation – Agilent Technologies
 Introduction to HPLC – Agilent TechnologiesIntroduction to HPLC – Agilent Technologies
 Principles and Technique of Biochemistry and Moleculer Biology –Principles and Technique of Biochemistry and Moleculer Biology –
Wilson.Keith and Walker. JohnWilson.Keith and Walker. John
 HPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWAREHPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWARE
6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical
Chemistry (Separation Science) at KCLChemistry (Separation Science) at KCL
WEB REFERENCESWEB REFERENCES
 http://192.215.107.101/ebn/942/tech/techfocus/1071main.htmlhttp://192.215.107.101/ebn/942/tech/techfocus/1071main.html
 Skoog, Holler, and Neiman.Skoog, Holler, and Neiman. Principles of Instrumental AnalysisPrinciples of Instrumental Analysis. 5th ed.. 5th ed.
Orlando: Harcourt Brace & Co., 1998.Orlando: Harcourt Brace & Co., 1998.
 http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
 http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.htmlhttp://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
 http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLChttp://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLC
HP1090/HPLCINJ.HTMHP1090/HPLCINJ.HTM
 http://test-http://test-
equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytiequipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analyti
cal_Instruments/Chromatographs/HPLC_Columnscal_Instruments/Chromatographs/HPLC_Columns

79. • THANK YOU…