HPLC Presentation

1. 1
Raziya Nadeem & Farooq Anwar
Department of Chemistry and Biochemistry
University of Agriculture, Faisalabad

2. Chromatography ?
Chromatography is a broad range of physical
methods used to separate complex mixtures by
their distribution b/w two immiscible phases – one
that is stationary and one that moves
Separate
Components
Mixture
• Analyze
• Identify
• Purify
• Quantify

3. 3
How mixed sample
can be separated?
river bed
direction of flow

4. 4
What is the difference?
Interaction difference
Strong
Weak

5. Classification of Chromatography
 On the basis of Geometry (Planner, Colum)
 Mode of Separation (Adsorption, Partition, Ion Exchange,
Size Exclusion, Affinity)
 Nature of Stationary Phase (GLC,GSC, LSC & LLC)
 Nature of Mobile Phase (Liquid chromatography, Gas
chromatography, SCF chromatography)
5

6. Advancement in Liquid Chromatography
Scientists Year Achievement
Tswett 1903 Introduction of liquid chromatography
N. A. Izmailov 1938 TLC
Martin & Synge 1941 Liquid-Liquid Chromatography
Huber 1967 LLC with small particles
Horvath and Lipsky 1966-67 Instrumentation of HPLC
Wilmington 1971 First Book on HPLC
R.E. Majors 1972-78 Small porous silica particles & modified
st.phases
Hjerten & Liao 1988 Polymer based monolithic column
Nakanishi & Soga 1991 Silica based monolithic column
Knox & Grant 1991 Capillary electrochromatography
Currently HPLC is one of the most widely used technique for Analysis,
purification ,identification, quantification of mixtures of compounds

7. 7
HPLC
 High performance liquid chromatography
 High pressure liquid chromatography
 High price liquid chromatography

8. 8
HPLC Vs Conventional Chromatography
 Simultaneous Qualitative & Quantitative Analysis
 High Resolution
 High Efficiency/Fast Analysis
 High Sensitivity (ppm-ppb)
 High Reproducibility
 High Degree of Selectivity & Specificity
 Automation

9. 9
Components of a mixture are separated through a column
by their partitioning between stationary phase and mobile
phase under pressure
According to distribution or partition coefficient (Kc)
Xm Xs
Kc = [X]s / [X]m
Xs = Concentration of the component in the stationary phase
Xm = Concentration of the component in the mobile phase
Principle of HPLC

10. 10
Separation modes of HPLC
 Normal Phase mode (NP)
 Reversed Phase mode (RP)
 Ion Exchange mode (IC)
 SEC mode ( GPC / GFC )

11.  Mobile phase reservoirs
 Pump(s)
 Injector
 Columns
 Detector
 Data System/Integrator

12. Typical HPLC System

13. A
B
C
D
E
Sample Mixture
Chromatogram
0 5 10 15 20
Time (minutes)
Abundance
A
B
C
D
E
Chromatograph
The HPLC Chromatogram
The time that a peak
appears (it’s retention
time) is diagnostic for a
given compound
The relative size of a
peak (area or height)
is proportional to the
relative abundance of
the compound in the
mixture

14. Mobile Phase Supply System
Individual reservoirs store the
mobile phase components until
they are mixed and used.
Solvent Features
 A range of solvents available
 High Purity
 Degassed
 HPLC Grade (filtered through 0.2 µm)

15. HPLC Pumps
 Pneumatic Type (non reciprocating/constant pressure pumps)
 Syringe Type (Mechanically driven)
 Hydraulic Amplifier Pump
 Reciprocating type (Electrically driven, most common)
minimum flow surges, Dual pistons
 Pressure of 1,000-3,000 psi often required for 1-2 mL/min
 80-90% separation require <1200 psi

16. 16
Isocratic System
pump
injector
column
oven
detector
 Simple system with 1 pump and
1 solvent reservoir.
 If more than 1 solvent is used, solvents
are premixed.
data
processor

17. 17
Low-pressure Gradient System
pump
injector
column
oven
detector
One pump used to
control 4 reservoirs;
mixing is done
before pump.
low pressure
gradient valve
data
processor

18. 18
injector
column
oven
detector
pump
pump
High-pressure Gradient System
pump
mixer
data
processor

19. 19
Elution Mode in HPLC
 Isocratic elution mode
 One mobile phase with a constant composition
 Gradient elution mode
 Multi mobile phases with changing composition

20. 20
Isocratic Elution Mode
Long Time Analysis
Bad Separation
MeOH / H2O = 6 / 4
MeOH / H2O = 8 / 2

21. 21
Gradient Elution Mode
95%
30%
MeOH
concentration

22. Injectors
Introduces the sample into the mobile phase
stream to be carried into the column.
Two major designs:
 Manual Injectors
 Automatic Injectors

23. 23
Manual injector
Auto injector

24. Column-The Heart of HPLC
Guard column
Protects the analytical
column
 Particles
 Interferences
 Prolongs the life of the
analytical column
Analytical column
Performs the separation
Lenghth:10 -30 cm, i.d 3.9 or 4.6mm
 A typical 15-cm long column with 4.6
mm id have 15,000 plates with 3 µm
particles, 9000 with 5 µm and 5000 for
10 µm.
 A 25 cm long column may have
ca.50,000 plates

25. Column Composition
 Solid Support - Backbone for bonded phases.
 High-purity spherical silica (particles 10µm, 5µm or 3µm);
Microporus particles (pore size 60-100 Å)
 Cross linked polymeric particles (polystyrene,
polymethacrylates)
 Monolithic packing [(rod like structure of silica, bimodal pore
macropore(2 µm), mesopore (13nm)]
 Bonded Phases - Functional groups firmly linked (chemically
bound) to the solid support.
 Extremely stable
 Reproducible 25

26. Chromatography Stationary Phases
O O O
| | |
OSiOSiOSiOH
| | |
O O O
| | |
OSiOSiOSiOH
| | |
O O O
bulk (SiO2)x surface
Silica Gel
O O O
| | |
OSiOSiOSiOR
| | |
O O O
| | |
OSiOSiOSiOR
| | |
O O O
bulk (SiO2)x surface
Derivatized Silica Gel
Where R = C18H37
hydrocarbon chain
(octadecylsilyl deriv.
silica or “C18”)
relatively polar surface relatively nonpolar surface
“normal phase” “reversed phase”
DMCS
Silination

27. 27
Normal Phase Mode
Column : polar property
Solvent : non polar property

28. Theoretical Plate Model of Column
Efficiency of a Column
 Theoretical plate number (N)
N = 16 (tR/wb)2
 HETP = L/N

29. 29
Normal Phase HPLC Columns
 Silica gel type : general use
 Cyano type : general use
 Amino type : for sugar analysis
 Diol type : for protein analysis
Silica gel
Si
Si
-Si-CH2CH2CH2CN
-Si-CH2CH2CH2NH2
-Si-CH2CH2CH2OCH(OH)-CH2(OH)
Modified Si

30. 30
What is the interaction?
Silica gel (polar)
Hydrogen bonding
Non-polar

31. Hydrogen bonding
 If the sample has
 -COOH : Carboxyl group
 -NH2 : Amino group
 -OH : Hydroxyl group
Hydrogen bonding becomes strong.
 If the sample has bulky group,
due to steric hindrance
Hydrogen bonding becomes weak.

32. 32
Mobile phase solvents for
normal phase HPLC
 Primary solvents (non-polar)
 Hydrocarbons (Pentane, Hexane, Heptane, Octane)
 Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
 Methylene chloride
 Chloroform
 Carbon tetrachloride
 Secondary solvents
 Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran
(THF), Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-
propaol, ethanol, methanol
 A primary solvent is used as mobile phase. Addition of
secondary solvents is to adjust retention time.

33. 33
Column : Non-polar property
Solvent : Polar property
Reversed Phase mode

34. 34
Reversed Phase HPLC Columns
 C18 (ODS) type
 C8 (octyl) type
 C4 (butyl) type
 Phenyl type
 Cyano type
-Si-C18H37
Si
Non-polar property

35. 35
What is the interaction?
Hydrophobic
interaction
Non-polar
polar solvent

36. 36
Hydrophobicity
 If the sample has more
 CH3CH2CH2--- : Carbon chain
 : Aromatic group
 If the sample has more
 -COOH : Carboxyl group
 -NH2 : Amino group
 -OH : Hydroxyl group
Hydrophobicity
is stronger.
Hydrophobicity
is weaker.

37. 37
Mobile phase solvents for reversed
phase HPLC
 Water (buffer) + Organic solvents
 Methanol (MeOH), acetonitrile
(ACN) or THF are common organic
solvents for r.p HPLC.

38. 38
Effect of stationary phase
C18 (ODS)
Strong
C8
sample
sample
sample
C4
Medium
Weak

39. Normal Phase vs. Reverse Phase HPLC
Skoog and Leary: Principals of Instrumental Analysis, 5th ed. Suanders, 1998

40. 40
Column Temperature Control Devices
•Column temperature control devices are functioning to keep the
column temperature constant.
•The temperature fluctuation of column will influence retention
time reproducibility.

41. 41
Detectors
 Ultraviolet / Visible detector (UV/VIS)
 Photodiode Array detector (PDA)
 Fluorescence detector (RF)
 Conductivity detector (CDD)
 Refractive Index detector (RID)
 Electrochemical detector (ECD)
 Mass spectrometer detector (MS)

42. Important Features of Selected Detectors
Detectors Approx. limit of
detection
Features
UV/Vis. 10-8 g mL-1 (0.01ppm) high sensitivity, insensitive to
changes in temperature and flow rate,
suitable for gradient elution.
Fluorescence 10-11 - 10-12 g mL-1 (0.1 - 1ppt) greater detection sensitivity,
insensitive to changes in temperature
and flow rate, suitable for gradient
elution.
RI 10-5 - 10-6 g mL-1 (10 -1ppm) universal, lack high sensitivity, not
suitable for gradient elution,
temperature and pressure sensitive

43. 43
Sample Preparation for HPLC
 Extraction
 Liquid phase extraction
 Solid phase extraction
 Removal of insoluble material
 Filtration
 Centrifuge (Precipitation)
 Control of concentration
 Concentration
 Dilution
 Derivatization for detection

44. Data Interpretation in HPLC
Qualitative analysis
The retention times of unknown compounds are
compared with those of pure standards
Quantitative analysis
Integration of peak area
base
height
Area = Wb x height
2
Triangulation
Normalization
%Area = Area of peak x 100
Tolal Area

45. 45
Applications of HPLC
•Pharmaceutical /Drugs
•Biomedical and Clinical Analysis
•Food Chemistry
•Forensic Analysis
•Environmental Pollutants
•Inorganic Chemistry
•Industrial Chemicals
•Proteomics

46. Typical Chromatogram of HPLC

47. 47
Separation of Sugars
Peak identification; 1sucrose; 2glucose; 3fructose
Bio-Rad Aminex HPX-87K 300 × 7.8 mm column, mobile phase
was ultra pure H2O at a flow rate of 0.6 mL/min , RI detector

48. Separation of Organic Acids
48
Peak identification; 1tartaric acid; 2ascorbic acid; 3malic acid; 4citric acid
Mobile phase consisted of 5 % methanol in 25 mM potassium dihydrogen
phosphate ; RP-C18 column (250 mm x 4.6mm; 5µ particle size), Detector
diode array detector (DAD)

49. Separation of Phenolic acids
49
Peak Identification: 1. Gallic acid 2. . Chlorogenic acid 3. P- hydroxy-benzoic acid 4. Vanillic acid ,
5. p- coumaric acid 6. Ferulic acid
RP-C18 column (250 mm x 4.6mm, 5µ particle size); Mobile phase consisted of 40 % Trifluoroacetic
acid (0.3 %), 40 % Acetonitrile and 20 % Methanol , detected at 280 nm.

50. Mn(II)
Co(III)
Cu(II)
Cr(III)
Mn(II)
Fe(III)
Co(III)
Ni(II)
Cu(II)
0 5 10 15
min.
MDC
Coupled column chromatographic
separation of metal complexes

51. 1. Cr(III)
2. Mn(II)
3. Fe(III)
4. Co(III)
5. Ni(II)
6. Cu(II)
Conditions: Column
LiChrosorb ODS, 150
x 4.6 mm id, 5 µm,
mobile phase
methanol-water-
1mM sodium acetate
(70:28:02), flow-rate
1.2 ml/min, UV
detection 260 nm.
RP-HPLC separation of PMDTC complexes of
Cr(III), Mn(II), Fe(III), Co(III), Ni(II) and
Cu(II)

52. 0 2 4 6
8
Ion chromatography of F,
CH3COO, Cl, NO3 , Br in
suppressed conductivity mode
RP-HPLC of NO3, Cl, Br, I
with UV detection
Lucy et al. J.Chromatogr. A
1000, 711, 2003
Bhanger, Khuhawar, Arain,
J. Sep. Sci., 25, 462, 2002
Minutes

53. Industrially important compounds
C1: formaldehyde; C2: acetaldehyde; C3: propanal; C4: butanal; C5: pentanal; C6:
hexanal; C7: heptanal; C8: octanal; C9: nonaldehyde; C10: decanal
(Talanta, 66, 2005, 982)
Derivatization with 1,2-benzo-3,4-
dihydrocarbazole-9-ethanol and HPLC
analysis).

56. Separation of Biophosphonate Drugs

58. New Trends in HPLC
FPLC- Fast Protein Liquid Chromatography
•Separation & purification of proteins and other polymeric complex mixtures
•Separation of macromolecules based on size, charge distribution (ion exchange),
hydrophobicity, reverse-phase or biorecognition (as with affinity chromatography)
•FPLC differs from HPLC in that the columns used for FPLC can only be used up to maximum
pressure of 3-4 MPa (435-580 psi).
•Columns used in FPLC are large [mm id] tubes that contain small [µ] particles or gel beads

59. LC-MS
•Powerful technique used for many applications which has very high sensitivity and
selectivity
•In particular used for pharmacokinetic, Proteomics/Metabolomics, Drug development

60. LC-MS-NMR
• A revolution in the separation science
• Power full platform for the analysis complex mixtures
• Determination of molecular weight and composition with in fmol/l concentration
• The exact mass, isotopic pattern and fragmentation characterization
• Intelligently organize data interpretation and visualization

61. HPLC-GC Chromatography
• Isolation of fractions and GC analysis in one process
• high separation, efficiency and high sensitivity
• Analysis of compounds avoiding sample preparation and cleanup
• Cost effective and time reduction
• Analysis of volatile and non-volatile in the HRGC
• Simplified method adjustment and new methods development