CONTAINS ALL THE INSTRUMENTATIONS & NECESSARY DETAILS CAN BE REFERRED BY PHARMACEUTICAL ANALYSIS STUDENTS

1. Presented by:
AKHIL ARUN ELANGO
IVth B. pharm

2.  The definition of chromatography is a
process of separating out different parts of
chemical mixtures onto an absorbent
material that can then be individually
analyzed because different parts are caught
on the material at different rates.

4. Liquid chromatography is a separation technique that
involves:
 the placement (injection) of a small volume of liquid
sample
 into a tube packed with porous particles (stationary
phase)
 where individual components of the sample are
transported along the packed tube (column) by a
liquid moved by gravity
 When a mixture of components are introduced into
the column .Various chemical and physical
interactions take place between the sample molecules
and the particles of the column packing

7.  HPLC is a separation technique that involves:
 the injection of a small volume of liquid sample
into a tube packed with tiny particles (3 to 5
micron (μm) in diameter called the stationary
phase)
 where individual components of the sample are
moved down the packedtube (column) with a
liquid (mobile phase) forced through the column
by high pressure delivered by a pump

8.  These components are separated from one
another by the column packing.
 These separated components are detected at
the exit of this tube(column) by a flow-
through device (detector) that measures their
amount.

9.  I.BASED ON MODE OF SEPARATION
 1.Normal phase chromatography -
stationary phase is polar(hydrophilic) and
mobile phase is non-polar (hydrophobic).
 2.Reverse phase chromatography-
 stationary phase is non-polar(hydrophobic)
and mobile phase is polar (hydrophilic).

10. II.BASED ON PRINCIPLE OF SEPARATION
1.Absorption Chromatography
2. Ion-exchange chromatography
3.Ion-pair chromatography
4 .Gel permeation chromatography
5.Affinity Chromatography
6.Chiral chromatography

11. •In the Absorption Chromatography solute
molecules bond directly to the surface of the
stationary phase
•the component which has more affinity
towards mobile phase elutes first & the
component which has less affinity towards
stationary phase elutes later.

13. •Ion exchange chromatography is a process that
allows the separation of ions and polar molecules
based on their charge.
•Retention is based on the attraction between
solute ions and charged sites bound to the
stationary phase.
•The use of a resin (the stationary solid phase) is
used to covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile
liquid phase are attracted to the resin by
electrostatic forces.

15. •It is a form of chromatography in which ions in
solution can be "paired“ or neutralized and
separated as an ion pair on a reversed-phase
column.
•Ion-pairing agents are usually ionic
compounds that contain a hydrocarbon
chain.

16. •This type of chromatography lacks an
attractive interaction between the stationary
phase and solute.
•The liquid or gaseous phase passes through a
porous gel which separates the molecules
according to its size

18. •This is the most selective type of
chromatography employed. It utilizes the
specific interaction between one kind of
solute molecule and a second molecule that is
immobilized on a stationary phase

19. It involves the separation of stereoisomers .
In the case of enantiomers, these have no
chemical or physical differences apart from
being three-dimensional mirror images.
Conventional chromatography or other
separation processes are incapable of
separating them.

20.  1.Isocratic elution
A separation in which the mobile phase
composition remains constant throughout
the procedure is termed isocratic elution
 2. Gradient elution
A separation in which the mobile phase
composition is changed during the separation
process .

21. 1.Analytical HPLC
 No recovery of individual components of
substance
2.Preparative HPLC
 Individual components of substance can be
recovered

22.  1.Qualitative analysis
*Analysis of a substance in order to ascertain the nature
of its chemical constituents.
*We can separate individual components but cannot
assess the quantity in this analysis
 2.Quantitaive analysis
Determining the amounts and proportions of its
chemical constituents .
Quantity of the impurity and individual components can
be assessed

23.  Solvent delivery system [RESERVOIR]
 Gradient controller and mixing unit
 De-gassing of solvents
 Pump
 Pressure gauge
 Pre-column
 Sample introduction system
 Column
 Detector
 Recorder

28. The mobile phase in HPLC refers to the solvent being
continuously applied to the column or stationary phase
• The mobile phase acts as a carrier to the sample
solution.
• A sample solution is injected into the mobile phase of
an assay through the injector port
• As a sample solution flows through a column with the
mobile phase, the components of that solution
migrate according to the non-covalent interaction of
the compound with the column

30.  The solvents or mobile phase must be passed
through a column at high pressures at up to
6000 psi(lb/in²) or 414bar.
 As the particle size of stationary phase is smaller
(5 to10μ) the resistance to the flow of solvent
will be high.
 That is, smaller the particle size of the stationary
phase the greater is the resistance to the flow of
solvents.
 Hence high pressure is recommended

32.  It consists of large, syringe like chambers
equipped with a plunger activated by a screw
driven mechanism powered by a stepping
motor.
 So it is also called as Screw Driven SyringeType
Pump
 Advantages:- It produces a flow that tends to be
independent of viscosity & back pressure.
 Disadvantages:- It has a limited solvent
capacity(~250) & considerably inconvenient
when solvents must be changed.

34. This pump transmits alternative pressure to the
solvent via a flexible diaphragm ,which in turn is
hydraulically pumped by a reciprocating pump.
Disadavantages:
 Produces a pulsed flow which is damped
because pulses appear as baseline noise on the
chromatograph.
 This can be overcome by use of dual pump
heads or elliptical cams to minimize such
pulsations.

36.  In this pumps, the mobile phase is driven
through the column with the use of pressure
produced from a gas cylinder.
It has limited capacity of solvent .

38.  Isocratic solvents- mobile phase is prepared
by using pure solvent or mixture of solvents
which has same eluting power or polarity.
 Gradient solvents- in this the polarity of the
solvent is gradually increased & hence the
solvent composition has to be changed.

42.  Several gases are soluble in organic solvents,
when high pressure is pumped, the formation
of gas bubbles increases which interferes with
the separation process, steady baseline &
shape of the peak.
 TYPES:
*Vacuum filtration
*Helium purging
*Ultrasonication

43. (i)Vacuum filtration:
De-gassing is accomplanished by applying a partial
vacuum to the solvent container.
But it is not always reliable & complete.
(ii) Helium Purging:
Done by passing Helium through the solvent.
This is very effective but Helium is expensive.
(iii) Ultrasonication:
Done by using ultrasonicator which converts ultra
high frequency to mechanical vibrations.

44.  Several injector devices are available either
for manual or auto injection of the sample.
TYPES:
(i) Septum Injector
(ii) Stop Flow Injector
(iii) Rheodyne Injector

45.  These are used for injecting the sample
through a rubber septum.
 This kind of injectors cannot be commonly
used, since the septum has to withstand high
pressures.

47.  In this type the flow of mobile phase is
stopped for a
 while & the sample is injected through a valve

48.  It is the most popular injector and is widely
used.
 This has a fixed volume of loop, for holding
sample until its injected into the column, like
20μL, 50μL or more.
 Through an injector the sample is introduced
into the column.
 The injector is positioned just before the
inlet of the column.

51. *Considered the “heart of the chromatograph”
the column’s stationary phase separates the
sample components of interest using various
physical and chemical parameters.
 The small particles inside the column are called
the “packing” what cause the high back pressure
at normal flow rates.
 Column packing is usually silica gel because of
its particle shape , surface properties , and pore
structure give us a good separation.

52.  Guard Column
 DerivatizingColumn
 Capillary Column
 Fast column
 AnalyticalColumn
 Preparatory Column

53.  Guard columns are placed anterior to the separating
column.
 This protects and prolongs the life & usefulness of the
separating column.
 They are dependable columns designed to filter or
remove:-
 particles that clog the separating column,
 compounds and ions that could ultimately cause
‘baseline drift’, decreased resolution, decreased
sensitivity and create false peaks.

54.  Derivatization involves a chemical reaction
between an analyte and a reagent to change the
chemical and physical properties of an analyte.
 The four main uses of derivatization in HPLC are:
 Improve detectability,
 Change the molecular structure or polarity of
analyte for better chromatography,
 Change the matrix for better separation,
 Stabilize a sensitive analyte.

55.  Disadvantages: It becomes a complex
procedure and so it acts as a source of error
to analysis and increases the total analysis
time.
 Advantages: Although derivatization has
drawbacks, it may still be required to solve a
specific separation or detection problem

56.  HPLC led to smaller analytical columns called as
micro-columns, capillary columns which have
diameter less than a millimeter.
 Sample used – is in nanolitre volumes,
decreased flow rate, decreased solvent volume
usage which leads to cost effectiveness.
 Disadvantage:- since it is miniatured flow rate is
difficult to produce & gradient elution is not
efficient.

57.  This is the most important part of HPLC
which decides the efficiency of separation.
 Length- 5 to 25 cm ,Internal Diameter 3 to
5mm.
 Particle size of packing material is 3 to 5μm.
 LC columns achieve separation by different
 intermolecular forces b/w the solute & the
stationary phase and those b/w the solute &
mobile phase

58.  Length – 10 to 15 cm, Int. diameter – 4.6mm
 Packed with particles having 5μm as
diameter.
 Columns of this time generate 10,000 plates
per column.
 It consists of back pressure regulator and
fraction collector.
 This back pressure regulator is placed
posterior to the HPLC detector.

59. DETECTORS

60.  The detector can detect the individual molecules
that elute from the column and convert the data
into an electrical signal.
•A detector serves to measure the amount of
those molecules
•The detector provides an output to a recorder or
computer that results in the liquid
chromatogram
•Detector is selected based on the analyte or the
sample under detection

61.  Absorbance (UV/Vis or PDA)
 Refractive index (detects the change in turbidity)
 Fluorescence (if the analyte is fluorescent)
 Electrochemical (measures current flowing
through a pair of electrodes, on which a
potential difference is imposed, due to oxidation
or reduction of solute)
 Conductivity (for ions)
 Light scattering
 Mass spectrometry (HPLC-MS)

63.  The UV/Vis source usually comes from a
monochromator so the wavelength can be
selected, or scanned.
 If wavelength scanning is desired, the flow is
stopped long enough for the scan to take place.
 Fixed wavelength-measures at single
wavelength usually 254nm.
 Variable wavelength-measures at single
wavelength at a time but can detect over a wide
range of wavelengths simultaneously.

65.  Advantage:
• Sensitivity is high
• Relative robust to temperature and flow rate
change
• Compatible with gradient elution
 Disadvantage:
• Only compounds with UV or visible absorption
could be detected.
 Additional Functions
• Dual Wavelength mode
•WavelengthTime Program mode
•Wavelength Scan mode

66.  This is a recent detector which is similar to UV
detector which operates from 190-600nm.
 Radiations of all wavelength fall on the
detector simultaneously.
 The resulting spectra is a three dimensional
plot of ResponseVsTimeVsWavelength.
 ADVANTAGE:The wavelength need not be
selected but detector detects the responses
of all compounds.

68.  Detection occurs when the light is bent due
to samples eluting from the columns, and this
is read as a disparity b/w the two channels.
 It is not much used for analytical applications
because of low sensitivity & specificity.
 When a solute is in the sample compartment,
refractive index changes will shift the light
beam from the detector

70.  It is based on the fluorescent radiation
emitted by some compounds.
 The excitation source passes through the
flow cell to a photodetector while a
monochromator measures the emission
wavelengths.
 More sensitive and specific.
 The disadvantage is that most compounds
are not fluorescent in nature.

72.  Amperometric detectors works based on the
reducing and oxidizing property of the sample
when a potential is applied.
 The diffusion current recorded is directly
proportional to the concentration of the
compound recorded.
 DISADVANTAGE: This detector is applicable
only when the functional groups present in the
sample can be either oxidized or reduced.
 ADVANTAGE: Highly sensitive detector.

74.  Recorders are used to record responses
obtained from the detectors after
amplification ,if necessary.
 They record the baseline & all the peaks
obtained with respect to time.
 Retention time can be found out from this
 Recordings ,but area under curve cannot be
determined.

75.  The mass spectrometry detector coupled
with HPLC is called HPLCMS.
 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 of a wide range of components

76.  Pharmaceutical:
•Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical
products.
• Identification of counterfeit drug products.
• Pharmaceutical quality control.

77.  Environmental
•Phenols in DrinkingWater.
•Identification of diphenhydramine in sediment
samples.
•Biomonitering of pollution in high-altitude
mountain lakes through the analysis of fish bile.
•Estrogens in coastal waters -The sewage source.
•Toxicity of tetracyclines and tetracycline
degradation products to environmentally
relevant bacteria.

78.  Clinical
 •Quantification of DEET in Human Urine.
 •Analysis of antibiotics.
 •Increased urinary excretion of aquaporin 2 in
patients with liver
 cirrhosis.
 •Detection of endogenous neuropeptides in
brain extracellular fluids

79.  1. Separations fast and efficient (high resolution power)
 2. Continuous monitoring of the column effluent
 3. It can be applied to the separation and analysis of very
complex
 mixtures
 4. Accurate quantitative measurements.
 5. Repetitive and reproducible analysis using the same
column.
 6. Adsorption, partition, ion exchange and exclusion column
 separations are excellently made.
 7. 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.

81.  Gurdeep R. Chatwal, Instrumental Method Of
ChemicalAnalysis, Himalaya Publishing [p.
2.624 to 2.638]
 Dr. S Ravi Shankar,Textbook Of
PharmaceuticalAnalysis, 2005, p. 18-1 to 18-
11
 Skoog , InstrumentalAnalysis