A brief introduction and instrumentation on high-performance liquid chromatography which is widely used in the analytical analysis of different samples in the pharmacy field. In this presentation, there is a small case study in finding out the different mobile and stationary phases used in the HPLC analysis.

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
CONCEPT TO CONCLUSION
Mr.AVINASH.db
-B.Pharmacy
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2. 2

3. HISTORY OF HPLC
Liquid chromatography was initially discovered as an analytical
technique in the early twentieth century and was first used as a
method of separating colored compounds. This is where the name
chromatography {chroma means color, graphy means writing,
was derived.}
A Russian botanist named Mikhail S. Tswett used to separate &
purify mixtures of plant pigments into the pure constituents. He
separated the pigments based on their interaction with a
stationary phase, which is essential to any chromatographic
separation.
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4. CHROMATOGRAPHY
STATIONARY PHASE
MOBILE PHASE
4

5. ADSORPTION PARTITION
COEFFICIENT
LIQUID STATIONARY
PHASE
LIQUID
MOBILE
PHASE
Liq. Mobile
phase
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6. PUMP
SAMPLE
MOBILE PHASE
DETECTOR
SCHEMATIC DIAGRAM OF HPLC
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7. 7

8. DIFFERERNT TYPES TECHNIQUES
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9. BASED ON
SEPERATION
NORMAL
PHASE
REVERSE
PHASE
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10. POLAR
STATIONARY
PHASE
NON-POLAR
MOBILE PHASE
NORMAL
PHASE NON-POLAR
STATIONARY
PHASE
POLAR
MOBILE PHASE
REVERSE
PHASE
Components elute in increasing order of
polarity
Components elute decreasing order of polarit
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11. BASED ON
ELUTION
ISOCRATIC GRADIENT
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12. •A separation in which the
mobile phase composition
remains constant
throughout the procedure
is termed as
•ISOCRATIC ELUSION
ISOCRATIC
ELUTION
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13. •A separation in which
the mobile phase
composition is changed
during the separation
process is described as a
GRADIENT ELUSION
GRADIENT
ELUSION
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14. BASED ON ION
EXCHANGE
ANIONIC
EXCHANGE
CATIONIC
EXCHANGE
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15. Silica has
anionic
residues that
interact
strongly with
cationic species.
Silica has
cationic
residues that
interact
strongly with
anionic species.
CATIONIC
EXCHANGE
ANIONIC
EXCHANGE
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16. TYPES OF
PUMPS USED
IN HPLC
DISPLACEMENT
PUMPS
RECIPROCATING
PUMPS
PNEUMATIC
PUMPS
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17. •The role of this pump is to force mobile phase through the column at a
specific flow rate, expressed in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1to 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).
RECIPROCATING PUMPS17

18. •On the back stroke , the
separation column valve is
closed and the piston pulls
in solvent from the mobile
phase reservoir
•On the forward stroke,
the pump pushes solvent
out of the column from
the reservoir
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19. It consists of large, syringe like
chambers equipped with a
plunger activated by a screw
driven mechanism powered by a
motor.
DISPLACEMENT PUMPS
19

20. In this pumps, the mobile
phase is driven through
the column with the use
of pressure produced
from a gas cylinder.
PNEUMATIC PUMPS
20

21. The injector serves to introduce the liquid sample into the flow stream of
the mobile phase.
Typical sample volumes are 5 to 20 (μL).
INJECTORS
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22. 22

23. •It is Considered as the “heart of the chromatograph” the column’s stationary phase
separates the sample components based on affinity.
•It is usually made of stainless steel to withstand high pressure caused by the pump to
move the mobile phase through the column packing.
•The small particles inside the column are called the “packing material”.
•Guard column is used to remove particular matter and contamination, it protect
the analytical column.
COLUMN23

24. • Columns can be packed with solids
such as silica or alumina; these columns
are called homogeneous columns.
HOMOGENEOUS
COLUMN
• If the stationary phase in the column is
a liquid, the column is called as bonded
column
BONDED COLUMN
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25. Ultraviolet (UV)
•This type of detector responds to substances that absorb light.
•The UV detector is mainly to separate and identify the active
of a mixture.
•UV detectors are the most versatile, having the best sensitivity.
•UV detectors cannot be used for testing substances that are low in
chromophores (colorless or virtually colorless) as they cannot absorb
light at low range.
DETECTORS25

26. Fluorescence
•This is a specific detector that senses only those substances that emit
This detector is popular for trace analysis in environmental science.
• It is very sensitive
Mass Spectrometer
•The mass spectrometer detector coupled with HPLC is called HPLC-
HPLC-MS is the most powerful detector, widely used in pharmaceutical
laboratories and research and development.
•The principal benefit of HPLC-MS is that it is capable of analyzing and
providing molecular identity at minute level.
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27. •Frequently called the data system, the computer not only controls
all the modules of the HPLC instrument but it takes the signal
from the detector and uses it to determine the time of elution
(retention time) of the sample components (qualitative analysis)
and the amount of sample (quantitative analysis).
•The concentration of each detected component is calculated
from the area or height of the corresponding peak and
reported.
Data processing unit (Computer)27

28. PARAMETERS
 DISTRIBUTION CONSTANT
 RETENTION TIME
 VOID TIME
 RETENTION FACTOR
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29. Kc =cS /cM
K , the distribution constant, is the ratio of the affinity of
compound A in the stationary phase and activity of
compound A in the mobile phase
The amount of time between the injection of a
sample and its elution from the column is known as the
retention time.
DISTRIBUTION CONSTANT
RETENTION TIME
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30. VOID TIME
RETENTION FACTOR
The retention factor (k) is a means
of measuring the retention of an
analyte in the chromatographic
column. The retention factor is
equal to the ratio of retention
time of the analyte on the column
to the retention time of a non-
retained compound.
The non-retained compound has no affinity for the stationary
phase and elutes with the solvent front at a time t0, which is known
as void time.
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31. The plate height is related to the flow rate of the mobile
phase
And is given by VAN DEEMTER EQUATION
A is a constant which represents the different possible paths
that can be taken by the analyte through the stationary phase.
B is a constant that describes the longitudinal diffusion that
occurs in the system.
C is a constant that describes the rate of adsorption and
desorption of the analyte to the stationary phase.
VAN DEEMTER EQUATION
υ is the velocity of the mobile phase.
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32. ADVANTAGES OF HPLC:
1. Separations are fast and efficient (high resolution power).
2. It can be applied to the separation and analysis of very complex mixtures.
3. Accurate quantitative measurements.
4. Repetitive and reproducible analysis using the same column.
5. Adsorption, partition, ion exchange and exclusion column
separations are excellently made.
6. HPLC is more versatile than GLC in some respects, because it has the
advantage of not being restricted to volatile and thermally stable solute
and the choice of mobile and stationary phases is much wider in HPLC
7. It providesa means for determination of multiple components in a
single analysis.
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33. Clinical diagnosis of diseases & disorders
In scientific research for discovery
In pharmaceutical labs for analysis
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34. In food industry for quality control
For standards control by Govt.
For separation of similar molecules
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35. DRUGS DOSAGE
FORM
MOBILE
PHASE
STATIONAR
Y PHASE
FLOW RATE DETECTOR RETENTION
TIME
REASON
TENOFOR
VIR-df,
LAMIVUDI
NE,EFAVIR
ENZ
TABLET Methanol:10
mM
phosphate
buffer of pH-
5 in 70:30.
C18
COLUMN(15
0*4.6mm)
with inter
diameter-
5µm
1ml/min PDA detector
with
UV-245nm
L-2.76
T-3.96
E-10.5
Can easily
determine
the drug-
drug
interactions.
SALBUTA
MOL,AMB
ROXOL,G
UAIFENES
IN
TABLET Aecetonitrile:
potassium
dihydrogen
phosphate of
pH-4 in 30:70
C18
column(250*
4.6mm)with
internal
diameter-
5µm
1ml/min PDA detector
with
UV-215nm
S-2.57
A-7.1
G-5.85
Easy for
quantificatio
n n and
estimation.
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36. DRUGSTH
EOPHYLLI
NE
DOSAGE
FORM
MOBILE
PHASE
STATIONA
RY PHASE
FLOW
RATE
DETECTOR RETENTIO
N TIME
REASON
L-
SALBUTAM
OL
SULPHATE,
SYRUP Methanol:1
0mM
tetrabutyla
mmonium
hydrogen
sulphate in
50:50
C18(250*4.
6mm) with
internal
diameter-
5µm.
1.0mL/min UV-214nm S-2.575
T-4.967
Easy for
quantificati
on and
estimation.
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37. Gurdeep R. Chatwal, Sham K. Anand, Instrumental Method Of
Chemical Analysis, Himalaya Publishing House.
Douglas A. Skoog, Instrumental
Analysis.
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38. THANK YOU