High Performance Liquid Chromatography HPLC

Description of HPLC

1. 1
Dr. Farooq Anwar(Professor)
Department of Pharmaceutical Chemistry ,
College of Pharmacy, Prince Sattam bin Abdul Aziz University,
Al-Kharj, Saudi Arabia

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
ComponentsMixture
• 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, Super critical fluid chromatography)
5

6. 6
HPLC

High performance liquid chromatography

High pressure liquid chromatography

7. 7
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

8. 8
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 (K
c
)
X
m X
s
K
c = [X]
s / [X]
m
X
s = Concentration of the component in the stationary phase
X
m
= Concentration of the component in the mobile phase
Principle of HPLC

9. Sample Mixture
Chromatogram
0 5 10 15 20
Time (minutes)
A
b
u
n
d
a
n
c
e
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

10. 10
Separation modes of HPLC

Normal Phase mode (NP)

Reversed Phase mode (RP)

11. 11
Normal Phase Mode
ColumnColumn :: polar propertypolar property
SolventSolvent : : non polar non polar
propertyproperty

12. 12
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-CH
2
CH
2
CH
2CN
-Si-CH
2CH
2CH
2NH
2
-Si-CH
2
CH
2
CH
2
OCH(OH)-CH
2(OH)
Modified Si

13. 13
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.

14. 14

Column: Non-polar property
Solvent : Polar property
Reversed Phase modeReversed Phase mode

15. 15
Reversed Phase HPLC
Columns

C18 (ODS) type

C8 (octyl) type

C4 (butyl) type

Phenyl type

Cyano type
-Si-C
18
H
37
Si
Non-polar propertyNon-polar property

16. 16
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.

17. 
Mobile phase reservoirs

Pump(s)

Injector

Columns

Detector

Data System/Integrator

18. Typical HPLC System

19. 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)

20. 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

21. 21
Elution Mode in HPLC

Isocratic elution mode

One mobile phase with a constant composition

Gradient elution mode

Multi mobile phases with changing
composition

22. 22
Isocratic Elution Mode
Long Time AnalysisLong Time Analysis
Bad SeparationBad Separation
MeOH / H
2O = 6 / 4
MeOH / H
2
O = 8 / 2

23. 23
Gradient Elution Mode
95%
30% M
e
O
H

c
o
n
c
e
n
t
r
a
t
i
o
n

24. Injectors

Introduces the sample into the mobile phase
stream to be carried into the column.

Two major designs:
Manual Injectors
Automatic Injectors

25. 25
Manual injector
Auto injector

26. 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

27. 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 27

28. 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 (SiO
2
)
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 (SiO
2
)
x
surface
Derivatized Silica Gel
Where R = C
18
H
37
hydrocarbon chain
(octadecylsilyl deriv.
silica or “C18”)
relatively polar surface relatively nonpolar surface
“normal phase” “reversed phase”
DMCS
Silinatio
n

29. 29
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.

30. 30
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)

31. 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

32. 32
Sample Preparation for HPLC

ExtractionExtraction

Liquid phase extraction

Solid phase extraction

Removal Removal of insoluble material

Filtration

Centrifuge (Precipitation)

Control Control of concentration

Concentration

Dilution

DerivatizationDerivatization for detection

33. Data Interpretation in HPLCData 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

34. 34
Applications of HPLC
•Pharmaceutical /Drugs
•Biomedical and Clinical Analysis
•Food Chemistry
•Forensic Analysis
•Environmental Pollutants
•Inorganic Chemistry
•Industrial Chemicals
•Proteomics

35. Typical Chromatogram of HPLC

36. 36
Separation of Sugars
Peak identification;
1
sucrose;
2
glucose;
3
fructose
Bio-Rad Aminex HPX-87K 300 × 7.8 mm column, mobile
phase was ultra pure H
2O at a flow rate of 0.6 mL/min , RI
detector

37. Separation of Organic Acids
37
Peak identification;
1
tartaric acid;
2
ascorbic acid;
3
malic acid;
4
citric 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)

38. Separation of Phenolic acids
38
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.
 

39. Mn(II)
Co(III)
Cu(II)
C
r
(
I
I
I
)
M
n
(
I
I
)
F
e
(
I
I
I
)
C
o
(
I
I
I
)
N
i
(
I
I
)
C
u
(
I
I
)
0 5 10 15
min.
MDC
Coupled column
chromatographic separation of
metal complexes

40. 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)

41. 0 2 4 6 8
Ion chromatography of F,
CH
3
COO, Cl, NO
3 ,
Br in
suppressed conductivity mode
RP-HPLC of NO
3, 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

42. 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).

45. Separation of Biophosphonate Drugs

47. 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

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

49. 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

50. 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