THIS SLIDES CONTAINS A BRIEF INTRODUCTION ABOUT HPLC ,ITS INSTRUMENTATION AND APPLICATIONS MAINLY IN PHARMACEUTICAL AREAS

1. NEELU MARIAM VARGHESE
INTERNSHIP TRAINEE
HPLC:INSTRUMENTATION AND APPLICATION IN
PHARMACEUTICALS

2. MARTIN SYNGER

3. INTRODUCTION
 HPLC - High Performance Liquid Chromatography
 Powerful tool in analysis, yields high performance and high
speed compared to traditional columns chromatography
because of the forcibly pumped mobile phase
 Defined as:
“A chromatographic technique used to separate
components of mixture for the purpose to identify, quantify
or purify the individual components of the mixture ”
.

4.  Widely used in the field of biochemistry and analytical
chemistry.
 Chromatography : physical method in which separation of
components takes place between two phases-a stationary
phase and a mobile phase.
 Stationary phase : The substance on which adsorption of the
analyte (the substance to be separated during
chromatography) takes place . It can be a solid, a gel, or a
solid liquid combination.
 Mobile phase : solvent which carries the analyte (a liquid or a
gas)

5. PRINCIPLE OF HPLC
 Partitioning of the analytes between the solid phase and
mobile phase.
 Chemical/ physical interactions between sample
molecules and column
 They travel according to their relative affinities towards
the stationary phase.
 The component which has more affinity towards the
adsorbent, travels slower.
 The component which has less affinity towards the
stationary phase travels fast

6. TYPES OF HPLC
 Stationary phase: Polar
eg:SiO2,Al2O3
 Mobile phase : Non-
polar
eg:heptane ,hexane
 Polar molecules elutes
slowly and non polar
molecules elutes
quickly
 Stationary phase: Non-
polar
eg: (Silica bounded with
C-18)
 Mobile phase : Polar
eg:methanol-water
 Polar molecules elutes
quickly and non polar
molecules elutes slowly
NORMAL PHASE HPLC REVERSE PHASE HPLC
NORMAL PHASE
HPLC
REVERSE PHASE
HPLC
STATIONARY PHASE POLAR
eg:SiO2,Al2O3
NON POLAR
eg: (Silica bonded with
C-18)
MOBILE PHASE NON POLAR
eg: heptane ,hexane
POLAR
eg: methanol-water
AFFINITY Polar molecules elutes
slowly and non polar
molecules elutes
quickly
Polar molecules elutes
quickly and non polar
molecules elutes slowly

7. INSTRUMENTATION OF
HPLC
Main components are
 Solvent Reservoir
 Degassing system
 Pump
 Sample Injection Port
 Column
 Detector
 Computer System

8. Schematic representation of HPLC system

9. 1. SOLVENT RESERVOIR
 A reservoir holds the solvent(mobile phase)
 Made up of glass or stainless steel
Requirements
 The reservoir and its attachment to the
pump should be made of materials that
will not contaminate the mobile phase.
 The vessel should have some cap to prevent
particulate matter from contaminating the mobile
phase

10.  Mobile Phase: water solvent/organic solvent/mixture of both
 Acts as a carrier for the sample solution
 Solvent selection depends upon the nature of the compound to
be separated.
 The mobile phase should be of highest purity.
Isocratic elution:
Composition of mobile phase is constant
Gradient elution:
Composition of the mobile phase changes during the
separation process

11. 2. DEGASSING SYSTEM
 Several gases are soluble in organic solvents.
 Dissolved gases in solvents interfere separation
process, steady baseline and the shape of the peak.
 Hence degassing is necessary

12. Degassing Methods
Vacuum filtration:-
Remove air bubbles, but it is not always reliable and
complete.
Helium purging:-
By passing helium through the solvent. This is very
effective but expensive.
Ultrasonification:-
By using Ultrasonicator, which converts ultra high
frequency to mechanical vibrations. This causes the removal of
air bubbles.

13. 3. PUMP
 The role of the pump is to force the mobile phase
through the liquid chromatography at a specific flow
rate, expressed in milliliters per min (mL/min)
 Normal flow rate :: 1-2mL/min range.
 Pump can deliver a constant mobile phase
composition (isocratic) or an increasing mobile phase
composition (gradient).

15. RECIPROCATING PUMP
 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
 Widely used one.
o A small motor driven piston
which moves rapidly back and
forth in a hydraulic chamber

16. DISPLACEMENT PUMP / SYRINGE TYPE PUMP
 Most suitable for small bore
columns
 Pump volume between 250 -
500mL
 Operates by a motorized lead
screw that delivers mobile
phase to the column at a
constant rate
 The rate of solvent delivery is
controlled by changing the
voltage on the motor.

17. 3. PNEUMATIC PUMPS
 The mobile phase is driven through
the column with the use of pressure
from the gas cylinder.
 A low-pressure gas source is needed
to generate high liquid pressures.
 The valving arrangement allows the
rapid refill of the solvent chamber
whose capacity is about 70mL

18. 4. SAMPLE INJECTION PORT
 Introduce the liquid sample into the flow stream of the
mobile phase
 Different types of injector devices :
(i) Septum Injector
(ii) Stop Flow Injector
(iii) Rheodyne Injector

19. i SEPTUM INJECTOR
 Used for injecting the sample through a rubber septum.
 Cannot be commonly used , since the septum has to withstand
high pressures.
ii. STOP FLOW INJECTOR
 Flow of mobile phase is stopped for a while when the sample is
injected
iii. RHEODYNE INJECTOR
 Widely used injector
 Two position : LOAD & INJECT

20.  Typical sample volumes for manual injector are 5-to 20-
microliters(μL)

21. • An auto sampler is the automatic version for when the user has
many samples to analyze or when manual injection is not practical.
• It can continuously inject variable volume a of 1 μL – 1 mL
AUTOSAMPLER

22. 5.COLUMN
 Heart of the chromatogram
 The column stationary phase separates the sample
components of interest using various physical and
chemical parameter
 It consist of
Guard Column
Analytical Column

23. GUARD COLUMN
 Increase the life of analytical
column
 It contains a packing
chemically identical to that in
analytical column.
 Mainly used to remove the
impurities from the solvent
and thus prevents
contamination of the
analytical column
 The success or failure of
analysis depends upon choice of
column.
 Actual separation is carried out
here.
 Stainless –steel tube
 size – length -25 to 100 cm
 Internal diameter – 2 to 4.6 mm
ANALYTICAL COLUMN

24. MATERIALS OF CONSTRUCTION FOR THE TUBING
 PACKING MATERIAL
Stainless steel ( the most popular, gives high pressure capabilities)
 Glass (mostly for biomolecules)
PEEK (poly ether ether ketone) polymer (biocompatibility and
chemically inert to most solvents).
The packing material is prepared from SILICA particle, ALUMINA
particle and ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of
the columns to retain the packing material.

25. 6. DETECTOR
 Detects the individual molecules that elute from the
column and convert the data into an electrical
signal.
 Measure the amount of those molecules.
 Detector is selected based on the analyte or the
sample under detection

26. The detectors used in HPLC should have following ideal properties:
 High sensitivity.
 Good stability and reproducibility.
 A linear response to solute.
 Negligible base line noise.
 Should be inexpensive.
 Capable of providing information on the identity of the solute.
 A short response time independent of flow-rate.
 High reliability and ease of operation.
 The detector should be non-destructive.
 Responses independent of mobile phase composition.
 A temperature range from room temperature to at least 400º C
IDEAL PROPERTIES OF A DETECTOR

27. COMMONLY USED DETECTORS IN HPLC
1.Ultraviolet(UV)/Visible Detector
 Measures the ability of solutes to absorb light at a
particular wavelength(s) in the ultraviolet (UV) or visible
(Vis) wavelength range.
 Separate and identify the principal active components of
a mixture.
 Versatile, have the best sensitivity and linearity.
 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.

28.  Cost-effective and widely used
 Useful for aromatic compounds and other type
unsaturated systems.
Classified as :
 Fixed detectors :
employ filter as a source to provide appropriate
wavelength.
 Variable detectors :
employ a spectrophotometer to provide dispersion of
light and selection of any wavelength

29.  Diffraction gratings are frequently used for wavelength
dispersion

30. 2.Flourescence Detector
 Ideal detector for those solutes that exhibit molecular
fluorescence
 The excitation source passes through the flow cell to a
photo detector while a monochromatic measures the
emission wavelengths.
 Their sensitivity depends on the fluorescence
properties of the components in the elute.
 The light source is usually a broad spectrum deuterium
or xenon flash lamp

31. Schematic representation of flourescence detector

32. 3.Photo Diode Array (PDA) Detector:
 Linear array of discrete photodiodes on an integrated circuit (IC)
chip.
 Light from the broad emission source such as a deuterium lamp
is collimated by an achromatic lens system so that the total light
passes through the detector cell onto a holographic grating.
 The array may contain many hundreds of diodes and the output
from each diode is stored on a hard disc.

33. 4.Refractive index Detector
 Measures the overall ability of the mobile phase and its
solutes to refract or bend light.
 The amount of defection is proportional to the concentration
of the solute in the mobile phase.
 Useful for detecting those compounds that are non-ionic, do
not absorb ultraviolet light and do not fluorescent
e.g. sugar, alcohol, fatty acid and polymers.

34. 5. Electrochemical Detector
 It is based on the measurement of the current resulting
from an oxidation/ reduction reaction of the analyte at a
suitable electrode.
 The level of current is directly proportional to the analyte
concentration.
 Three electrodes are employed which are:
Working electrode
Auxiliary electrode
Reference electrode

35. 6. Computer(Data Processing System)
 Frequently called the data system
 The concentration of each detected component is
calculated from the area or height of the corresponding
peak and reported.

36. MANGIFERIN
SOLVENTS:
ACETONITRILE
O-PHOSPHORIC ACID

37. APPLICATIONS OF HPLC

38. HPLC IN PHARMACEUTICALS
 To control the drug stability
 Quantity of drug determination from pharmaceutical dosage
forms
eg:Paracetamol determination in panadol tablet
 Quantity of drug determination from biological fluids
eg: Blood glucose level
 Therapeutic drug monitoring
 Purity Checking
 Toxicology eg:Paracetamol Poisoning

40.  HPLC is useful in compound identification and
pharmaceutical purification testing, as it is estimated that
over 60% of existing molecules have been uniquely
identified by HPLC testing.
1.IN DRUG ANALYSIS
 Most important analytical method for identification and
quantification of drugs.
 Either in their active pharmaceutical ingredient or in their
formulations during the process of their discovery,
development and manufacturing.
Discovery Development Manufacturing

41. Example1 : HPLC analysis of the novel antipsychotic drug
quetiapine in human plasma
R. Mandrioli a, S. Fanali b, A. Ferranti a, M.A. Raggi
*Carried out on a C8 (150/4.6 mm i.d., 5 mm) reversed-phase column
*Mixture of acetonitrile,methanol phenoll
and pH 1.9 phosphate buffer as the
mobile phase;
*Triprolidine was used as the internal
standard.
Example2 : HPLC analysis of raloxifene hydrochloride and its
application to drug quality control studies
Jurij Trontelj ∗, Tomaˇz Vovk, Marija Bogataj, Aleˇs Mrhar
* Raloxifene hydrochloride is a selective estrogen receptor modulator
and is currently being used for prevention of osteoporosis in
postmenopausal women

42.  HPLC method for detection of raloxifene hydrochloride was
developed and validated using an ultraviolet (UV) and
coulometric detectors.
 The mobile phase consisted of methanol, acetonitrile, water
(28:32:30%, v/v/v) and ammonium acetate (5 mM) and was
delivered isocratically with a flow rate of 0.5 mL min−1
Chromatograms obtained with an UV detector (a) and coulometric detector (b).
The main peaks correspond to 1 mg L−1 raloxifene hydrochloride.

43.  2.Quantity of drug determination from pharmaceutical dosage
forms
Example 1 :A RP-HPLC method for simultaneous estimation of paracetamol and
aceclofenac in tablets
R. Gopinath, S. Rajan, S. N. Meyyanathan*, N. Krishnaveni and B. Suresh
*A simple, selective, rapid, precise and economical reverse phase HPLC method has
been developed for the simultaneous estimation of paracetamol and aceclofenac from
pharmaceutical dosage forms.
*The method was carried out on a Hichrom C18 (25 cm×4.6 mm i.d., 5 µ) column with a
mobile phase consisting of acetonitrile: 20 mM phosphate buffer (pH 5.0) (60:40 v/v) at
a flow rate of 0.8 ml/min.
*Detection was carried out at 265 nm.
*Etoricoxib was used as an internal standard.
*The retention time of paracetamol, aceclofenac and etoricoxib was 4.75, 6.44 and 8.83
min, respectively.

44. Example 2 :.RP-HPLC Estimation of Risperidone in Tablet
Dosage Forms
S L Baldania, K K Bhatt, R S Mehta
*A Phenomenex Gemini C-18, 5 μm column having 250×4.6 mm i.d. in
isocratic mode
*Mobile phase containing methanol: acetonitrile: 50 mM potassium
dihydrogen orthophosphate (80:10:10 v/v) was used.
*The flow rate was 1.3 ml/min and effluents were monitored at 234 nm.
*Clozapine was used as an internal standard.

45.  The retention time of risperidone and clozapine were 2.5
min and 3.3 min, respectively.
 Risperidone (RIS) is
psychotropic agent
3.Quantity of drug determination from biological fluids
Example: Rapid determination of metformin in human
plasma using ion-pair HPLC
A. Zarghi a,*, S.M. Foroutan b , A. Shafaati a , A. Khoddam

46.  The separation was performed on an analytical 150/4.6 mm i.d.
µbondapak C18 column.
 The wavelength was set at 235 nm.
 The mobile phase was 40% acetonitrile, 0.01 M sodium dodecyl
sulphate, 0.01 M sodium dihydrogen phosphate, and distilled water
to 100%, adjusted to pH 5.1 at a flow rate of 1.5 ml/min.
 Metformin is an antidiabetic agent which
used in the treatment of non-insulin
-dependent diabetes
 phenytoin is the internal standard