High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC) head points:
HPLC Advantages Vs GC
Instrumentation
HPLC System
Separations
Mobile Phase Reservoirs
Degasser
Aim of Gradient system
High/Low pressure gradient system
HPLC Pump Criteria
HPLC Pumps: Types
Reciprocating Pumps
Sample introduction
Manual Injector
Auto Injector
HPLC Modes
The Mobile Phase
Hydrophobic interaction
Common reverse phase solvents
Detectors

https://www.linkedin.com/in/preeti-choudhary-266414182/
https://www.instagram.com/chaudharypreeti1997/
https://www.facebook.com/profile.php?id=100013419194533
https://twitter.com/preetic27018281

Please like, share, comment and follow.
stay connected

If any query then contact:
[email protected]

Thanking-You
Preeti Choudhary

1. Presented by Preeti Choudhary
M.Sc. Applied Physics
[email protected]

2. High Performance Liquid Chromatography
HPLCis characterized by the use of high
pressure to push a mobile phasesolution
through a column of stationary phase
allowing separation of complex mixtures with
high resolution.

3. From High Pressure Liquid Chromatography to High
Performance Liquid Chromatography
Higher degree of separation!
Refinement of packing material (3 to 10 µm)
Reduction of analysis time!
Delivery of eluent by pump
Demand for special equipment that can
withstand high pressures
The arrival ofhigh performance liquid chromatography!
3

4. High-Performance Liquid Chromatography
MobilePhase:Liquid
StationaryPhase SeparationMechanism
-Solid Adsorption
-LiquidLayer Partition
-Ionexchangeresin Ionexchange
-Microporousbeads SizeExclusion
-ChemicallymodifiedresinAffinity

5. HPLC Advantages vs GC
Notlimitedbysamplevolatilityorthermalstability
Twointeractingphases–mobilephaseandstationary
phase
Roomtemperatureanalysis
Easeofsamplerecovery

6. Instrumentation
Solvent Reservoirs
Pump
Sample Injector
Column
Detector
Data System

9. Separations
9
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

10. Separations
10
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph

11. Separations
11
Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time

12. Separations
12
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

13. Separations
13
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

14. Separations
14
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

15. Separations
15
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

16. Separations
16
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

17. Separations
17
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

18. Separations
18
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

19. Separations
19
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

20. Separations
20
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

21. Separations
21
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

22. Separations
22
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

23. Separations
23
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

24. Separations
24
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

25. Separations
25
Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

26. Mobile Phase Reservoirs
Inertcontainerwithinerttubingsleadingtothe
pumparerequired.
Reservoirfilters(2-10mm)areplacedatreservoir
endofsolventdeliverylines
Reservoirsareelevatedabovepumps.
filter

27. Degasser
Problems caused by dissolved air in the eluent
Unstable delivery by pump
More noise and large baseline drift in detector cell
Inordertoavoidtheseproblems,theeluentmust
bedegassed.Itisdoneby
-Vacuumfiltration
-Spargingwithinertgas(N
2orHe)
-Ultrasonicundervacuum
27

28. Isocraticelution:Aseparationthatemploysasingle
solventorsolventmixtureofconstantcomposition.
Gradientelution:Heretwoormoresolventsystems
thatdiffersignificantlyinpolarityareemployed.After
elutionisbegun;theratioofthesolventsisvariedina
programmedway,sometimescontinuouslyand
sometimesinaseriesofsteps.Separationefficiencyis
greatlyenhancedbygradientelution.

29. Aim of Gradient System
In isocratic mode
29
Long analysis time!!
Poor
separation!!
CH
3OH / H
2O = 6 / 4
CH
3OH / H
2O = 8 / 2
(Column: ODS type)

30. Aim of Gradient System
If the eluent composition is changed gradually during
analysis...
30
95%
30%
Concentration of methanol in eluent

31. High-/ Low-Pressure Gradient System
31
High-pressure gradient
Mixer
Low-pressure
gradient unit
Low-pressure gradient
Mixer

32. Varian 9010 Solvent Delivery System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H
2O}{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

34. HPLC Pump Criteria
Constructedofmaterialsinerttowardsolventstobe
used
Deliverhighvolumes(flowrates)ofsolvent(upto10
mL/min)
Deliverpreciseandaccurateflow(<0.5%variation)
Deliverhighpressure(to6000psi)
Deliverpulsefreeflow
Havelowpump-headvolume
Bereliable

35. HPLC Pumps: Types
Reciprocating pumps
Syringe pumps
Constant pressure pumps

36. Reciprocating Pumps
Mostwidelyused
Consistsofasmallcylindricalchamberthatisfilled
andthenemptiedbybackandforthmotionofa
piston
Thepumpingmotionproducesapulsedflowthat
mustbesubsequentlydamped
Advantages:
-theyhavesmallinternalvolume
-highoutputpressure(upto10,000psi)
-readyadaptabilitytogradientelution
-constantflowrates

38. Schematic Diagram of Reciprocating Pump
38
Motor and cam
Plunger
Plunger seal
Check
valves
Pump head
10 -100µL

39. Sample Introduction
Valve-typeinjectors
reproducibleinjectionvolumes
variableloopsize
easytouse,reliable

40. Manual Injector:
Operating Principle of Sample Injection
40
LOAD INJECT
To column
From pump
To column
From pump
Loop
Loop

41. Front view Back view
Injection

42. Auto Injectors
Continuousinjectionsoperatorfree
Comparableprecisionandaccuracytomanual
injection
Muchmoreexpensiveinitially
Muchmoreconvenient.Upto100samplesand
standardswithmicroprocessorcontrol

43. Auto sampler

44. Liquid-chromatographiccolumnsrangeinlengthfrom
10to30cm.
Theinsidediameterofliquidcolumnsisoften4to10
mm;
Theparticlesizeofpackingare10,5or3m.
Themostcommoncolumncurrentlyinuseisonethatis
25cminlength,4.6mminsidediameter,andpacked
with5mparticles.
Columnsofthistypecontain40,000to60,000
plates/meter.
Analytical Columns

45. Guard Columns
Aguardcolumnisintroducedbeforetheanalyticalcolumnto
increasethelifeoftheanalyticalcolumnbyremovingnotonly
particulatematterandcontaminantsfromthesolventsbut
alsosamplecomponentsthatbindirreversiblytothe
stationaryphase.
Theguardcolumnservestosaturatethemobilephasewith
thestationaryphasesothatlossesofthissolventfromthe
analyticalcolumnareminimized.
Thecompositionoftheguard-columnpackingissimilarto
thatoftheanalyticalcolumn;theparticlesizeisusuallylarger.
Whentheguardcolumnhasbecomecontaminated,itis
repackedordiscardedandreplacedwithanewone.

46. Inside the column…..

47. PARTITION CHROMATOGRAPHY
Partitionchromatographycanbesubdividedinto
(i)liquid-liquidchromatographyand
(ii)bonded-phasechromatography.
Withliquid-liquid,aliquidstationaryphaseisretained
onthesurfaceofthepackingbyphysicaladsorption.
Withbonded-phase,thestationaryphaseisbonded
chemicallytothesupportsurfaces.

48. PARTITION CHROMATOGRAPHY
Earlypartitionchromatographywastheliquid-liquid
type;nowthebonded-phasemethodhasbecome
predominatebecauseofcertaindisadvantagesofliquid-
liquidsystems.
Oneofthesedisadvantagesisthelossofstationary
phasebydissolutioninthemobilephase,which
requiresperiodicrecoatingofthesupportparticles.
Furthermore,stationary-phasesolubilityproblems
prohibittheuseofliquid-phasepackingsforgradient
elution.

49. Bonded-PhaseChromatography
Thesupportsforthemajorityofbonded-phasepackingsfor
partitionchromatographyarepreparedfromrigidsilica,or
silica-based,compositions.
Thesesolidsareformedasuniform,porous,mechanically
sturdyparticlescommonlyhavingdiametersof3,5,or10m.
Thesurfaceoffullyhydrolyzedsilicaismadeupof
chemicallyreactivesilanolgroups.Themostusefulbonded-
phasecoatingsaresiloxanesformedbyreactionofthe
hydrolyzedsurfacewithanorganochlorosilane.

50. Organochlorosilane siloxane

51. Chromatography Stationary Phases
relatively polarsurface
bulk (SiO
2)
x surface
relatively nonpolarsurface
Silica Gel Derivatized Silica Gel
Where R =
generally
C
18H
37or
C
8H
17
hydrocarbon
chain
“normal phase” “reversed phase”

52. Bonded Phases
C-2 Ethyl Silyl -Si-CH
2-CH
3
•CN Cyanopropyl Silyl -Si-(CH
2)
3-CN
Polar
•C-18OctadecylSilyl -Si-(CH
2)
17-CH
3
•C-8 Octyl Silyl -Si-(CH
2)
7-CH
3

53. 53
Effect of Chain Length of Stationary
Phase
C
18(ODS)
Strong
C
8
C
4
Medium
Weak

55. HPLC -Modes
Normal Phase
Polar stationary phase and non-polar solvent.
highly polar stationary phases such as water or triethyleneglycol
supported on silica or alumina particles; a relatively nonpolar
solvent such as hexane or propyletherthen served as the mobile
phase.
Reverse Phase
Non-polar stationary phase and a polar solvent.
the stationary phase is nonpolar, often a hydrocarbon, and the mobile
phase is relatively polar (such as water, methanol, or acetonitrile).

56. Innormal-phasechromatography,theleastpolar
componentiselutedfirstbecauseitisthemostsoluble
inthemobilephase;increasingthepolarityofthe
mobilephasehastheeffectofdecreasingtheelution
time.
Incontrast,inthereversed-phasemethod,themost
polarcomponentappearsfirst,andincreasingthe
mobilephasepolarityincreasestheelutiontime.

57. 57
Relationship Between Retention
Time and Polarity
C
18(ODS)
CH
3
Strong
Weak
OH

59. Normal vs. Reversed Phase Chromatography

60. The Mobile Phase
Normal chromatography
Hexane ; dichloromethane; isopropanol; methanol
Increasing strength
Reverse phase chromatography
water ; methanol; acetonitrile; tetrahydrofuran (THF)
Increasing strength

61. 61
Hydrophobic Interaction
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
Network of hydrogen bonds
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
Nonpolar solute
If a nonpolar
substance is added...
…the network is broken and...
H
2O
H
2O H
2O
H
2O
H
2O H
2O
H
2O
Nonpolar solute
Nonpolar stationary phase
…the nonpolar substance
is pushed to a nonpolar
location.

62. 62
Difference in Solute Retention Strengths for Water
and Water-Soluble Organic Solvents
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
H
2O
Tightly packed network
CH
3OH
Nonpolar solute
Nonpolar solute
Nonpolar stationary phase
Loose network
CH
3OH
CH
3OH
CH
3OH
CH
3OH
CH
3OH
CH
3OH

63. Common Reverse Phase Solvents
Methanol CH
3OH
• Acetonitrile CH
3CN
• Tetrahydrofuran
• Water
H
2O

64. HPLC CHROMATOGRAM -choosing the ‘right’
mobile phase (having ‘right’ polarity)

65. Detectors
Unlikegaschromatography,liquidchromatographyhasno
detectorsthatareasuniversallyapplicableandasreliableas
theflameionizationandthermalconductivitydetectors.A
majorchallengeinthedevelopmentofliquid
chromatographyhasbeenindetectorimprovement.
TypesofDetectors:
Bulkpropertydetectorsrespondtoamobile-phasebulk
property,suchasrefractiveindex,dielectricconstant,or
density.
Solutepropertydetectorsrespondtosomepropertyof
solutes,suchasUVabsorbance,fluorescence,ordiffusion
current,thatisnotpossessedbythemobilephase.

66. HPLC Detector Characteristics
Detector performance characteristics:
Sensitivity (LoD, LoQ)
Selectivity
Linearity
Qualitative information
Reliability
Ease of use
Universality
66

67. 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.
67
Noise
Peak

68. 68
Representative HPLC Detectors
UV-VIS absorbance detector
Photodiode array-type UV-VIS absorbance
detector
Fluorescence detector
Refractive index detector
Evaporative light scattering detector
Electrical conductivity detector
Electrochemical detector
Mass spectrometer

69. Absorbance Detectors
TheseareZ-shaped,flow-throughcellforabsorbance
measurementsoneluentsfromachromatographic
column.Manyabsorbancedetectorsaredouble-beam
devicesinwhichonebeampassesthroughtheeluentcell
andtheotherisreferencebeam.

70. UltravioletAbsorbanceDetectorswithFilters
ThesimplestUVabsorptiondetectorsarefilter
photometerswithamercurylampasthesource.Most
commonlytheintenselineat254nmisisolatedbyfilters.
Deuteriumortungstenfilamentsourceswith
interferencefiltersalsoprovideasimplemeansof
detectingabsorbingspecies
UVAbsorbanceDetectorwithMonochromator
Therearedetectorsthatconsistofascanning
spectrophotometerwithgratingoptics.Somearelimited
touvradiation;othersencompassbothuvandvisible
radiation.Themostpowerfuluvspectrophotometric
detectorsarediode-arrayinstruments.

71. 71
Data Obtained with a Photodiode
Array Detector
Retention
time
Absorbance
Chromatogram
Spectrum

72. Online Spectra -UV-Vis Detector
72
Wavelength
Time
Absorbance
Spectra

73. FluorescenceDetectors
Fluorescenceisobservedbyaphotoelectricdetector
locatedat90degtotheexcitationbeam.
Thesimplestdetectorsemployamercuryexcitation
sourceandoneormorefilterstoisolateabandof
emittedradiation.
MoresophisticatedinstrumentsarebaseduponaXenon
sourceandemployagratingmonochromatortoisolate
thefluorescentradiation.
Aninherentadvantageoffluorescencemethodsistheir
highsensitivity,whichistypicallygreaterthanmost
absorbanceprocedures.

74. 74
Optical System of Fluorescence Detector
Xenon lamp
Excitation
grating
Excitation
light
Fluorescence
Fluorescence
grating
Sample cell
Photomultiplier
tube

75. Refractive-IndexDetectors
Refractive-indexdetectorshavethesignificant
advantageofrespondingtonearlyallsolutes.Thatis
theyaregeneraldetectorsanalogoustoflameor
thermalconductivitydetectorsingaschromatography.
Theyarereliableandunaffectedbyflowrate.
Theyare,however,highlytemperaturesensitiveand
mustbemaintainedataconstanttemperaturetoafew
thousandthsofadegreecentigrade.
Theyarenotassensitiveasmostothertypesof
detectorsandgradientelutionisnotpossible.

76. 76
Differential Refractive Index
Detector (Deflection-Type)
Light
Sample
cell
Reference
cell
Light-receiving
unit

77. ElectrochemicalDetectors:Thesedevicesare
baseduponamperometry,polarography,coulometry,
andconductometry.Theyappeartoofferadvantages,
inmanyinstances,ofhighsensitivity,simplicity,
convenience,andwidespreadapplicability.

78. 78
Electrochemical Detector
RHO
HO
RO
O
+ 2H
+
2e
-
Electrode

79. 79
Cell Structure of Electrochemical
Detector (Amperometric Type)
Working electrode
(glassy carbon)
Electrode couple
Reference electrode
(Ag/AgCl)
Eluent

80. HPLC Applications
80
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

82. Application of HPLC
1. Pharmaceuticals industry
To control the drug stability
Quantity of drug determination from pharmaceutical dosage
forms, ex. Paracetamol determination in panadol tablet
Quantity of drug determination from biological fluids, ex:
blood glucose level
2. Analysis of natural contamination
-Phenol & Mercury from sea water
3. Forensic test
-Determination of steroid in blood, urine & sweat.
-Detection of psychotropic drug in plasma

83. Application of HPLC
4. Clinical test
-Monitoring of hepatic chirosis patient through
aquaporin 2 in the urine.
5. Food and essence manufacture
-sweetener analysis in the fruit juice
-preservative analysis in sausage.