Hello Everyone, Here I am attaching a PPT on the topic of HPLC

1. PRINCIPLE , WORKING &
APPLICATION OF HPLC

2. By :-
SHABIR KHAN
ASST. PROFESSOR
MICROBIOLOGY

3. P
igh
erformance
Liquid
Chromatography

4.  High performance liquid chromatography
 A form of liquid chromatography used to separate
compounds that are dissolved in solution .
 HPLC instruments consists of a reservoir of mobile
phase , a pump, and injector , a column and a detector
.
 Compounds are separated by injecting a mixture onto
the column .
 Separation is based on partition of compounds towards
stationary and mobile phase .

5.  Chromatography is an analytical method used to
separate compounds physically prior to measurement.
 Satisfies the purpose of separation and quantification of
target.
 HISTORY OF CHROMATOGRAPHY:-
 M. Tswett- a Russian botanist

6.  Stationary phase
 Mobile phase
 Chromatogram
 Retention time

7. Physical method in which separation of components takes place between
two phases:
1. Stationary phase
2. Mobile phase
Stationary phase : the substance on which absorption of the analyte (the
substance to be separated during chromatography ) take place .
It can be a solid , a gel , or a solid liquid combination.
Mobil phase : solvent which carries the analyte (a liquid or a gas )
HPLC is a type of liquid chromatography where the sample is forced
through a column that is packed with a stationary phase composed of
irregularly or spherically shaped particles , a porous monolithic layer , or a
porous membrane by a liquid (mobile phase ) at high pressure.

8.  Separation is not efficient
 More time consumption
 Wastage of solvent
Column chromatography
column: diameter – 5cm
length – 1 meter
Packing material : silica
Particle size : 100 micrometer

9. High performance High pressure
HPLC
PERFORMANCE PRESSURE
HIGH
ACCURACY
HIGH
SENSITIVITY
HIGH
PRESSURE

10. • To understand the principle of HPLC , we must first
look at the principle behind liquid chromatography .
• 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.

11.  The main principle of separation is adsorption .
 When a mixture of components are introduced into the
column .
 Various chemical and/or physical interaction take place
between the sample molecules and the particles of the
column packing.
 They travel according to their relative affinities toward
the stationary phase.
 The component which has more affinity towards the
absorbent , travel slower.
 the component which has less affinity toward the
stationary phase travels faster.

12.  Since no two components have the same affinity toward
the stationary phase , the components are separated .
 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 in diameter
called the stationary phase).
 Where individual components of the sample are moved
down the packed tube (column) with a liquid (mobile
phase) forced through the column by high pressure
delivered by a pump.

13.  These components are separated from one another by
the column packing that involves various chemical and
physical interactions between their molecules and the
packing particles.
 These separated components are detected at the exit of
this tube (column) by a flow- through device (detector)
that measures their amount .
 The output from the detector is called a liquid
chromatogram .
 In principle , LC and HPLC work the same way except
the speed , efficiency, sensitivity and ease of operation
of HPLC is vastly superior.

14. (A) Based on modes of chromatography
1). NORMAL PHASE MODE
2). REVERSE PHASE MODE
(B) Based on principle of separation
1). ADSORPTION CHROMATOGRAPHY
2). ION EXCHANGE CHROMATOGRAPHY
3). PARTITION CHROMATOGRAPHY
4). SIZE EXCLUSION

15. (C) Based on elution technique
1. ISOCRATIC SEPARATION
2. GRADIENT SEPARATION
(D) Based on the scale of operation
1. ANALYTICAL HPLC
2. PREPARATIVE HPLC
(E) Based on the types of analysis
1. QUALITATIVE ANALYSIS
2. QUANTITATIVE ANALYSIS

16. (A) 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)
 Mobile phase is polar (hydrophilic).
 Polar –polar bonds and non polar- non polar bonds are more
affinity than polar –non polar bonds.
 Reverse phase chromatography is more commonly used as drugs
are usually hydrophilic.

17. (1) Adsorption chromatography:-
 In the absorption chromatography solute molecules
bond directly to the surface of the stationary phase
 The component which has more affinity toward
mobile phase elutes first & component which has less
affinity toward stationary phase elutes later.
 No two component have same affinity toward mobile
phase &stationary phase.

18. ABSORPTION CHROMATOGRAPHY

19. (2) ION EXCHANGE CHROMATOGRAPHY
 Ion exchange chromatography is a process that allows
the separation of ions and polar molecules based on
their charge.
 It can be use for almost any kind of charged molecules
including large proteins ,small nucleotides and amino
acid .
 Retention is based on the attraction between solute ions
and charged sites bound to the stationary phase . Ions
of the same charge are excluded .
 The use of resin (the stationary solid phase ) is used to
covalently attach an ions or cations onto it.
 Solute ions of the opposite charge in the mobile liquid
phase are attracted to the resin by electrostatic forces.

21.  It is a from 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
that imparts a certain hydrophobacity so that
the ion pair can be retained on a reversed-
phase column.

22.  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.

23. (C) BASED ON ELUTION TECHNIQUE
1) Isocratic elution:-
 A separation in which the mobile phase composition
remains constant through out the procedure is termed
isocratic elution.
 In isocratic elution , peak width increases with
retention time linearly with the number of theoretical
plates.
 This leads to the disadvantage that late-eluting peaks
get very flat and broad.
 Best for simple separation
 Often used in quality control application that support
and are in close proximity to a manufacturing process.

24. (2) gradient elution
 A separation in which the mobile phase composition
is changed during the separation process is described
as a gradient elution .
 Gradient elution is decreases the retention of the
later-eluting components so that they elute faster ,
giving narrower peaks.
 This also improves the peak shape and the peak
height.
 Best for the analysis of complex samplers
 Often used in method development for unknown
mixtures.
 Linear gradients are most popular

25. (D) BASED ON SCALE OF OPERATION
(1) Analytical HPLC:-
 No recovery of individual components of substance.
(2) Preparative HPLC :-
 Individual components of substance can be
recovered.

26. (E) BASED ON TYPE OF ANALYSIS :-
(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) Quantitative analysis :
 Determining the amounts and proportions of its
chemical constituents.
 Quantity of the impurity and individual components
can be assessed

27.  Solvent
 Solvent delivery system (pump)
 Injector
 Sample
 Column
 Detectors
 Waste collector
 Recorder (data collection)

31.  The role of the pump is to force a liquid (called the
mobile phase) through the liquid chromatograph at
specific flow rate.
 Expressed in milliliters per min.(ml/min.)
 Normal flow rate in HPLC are in the 1to2 ml/min
range.
 Typical pumps can reach pressures in the range of 6000
to 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.

33.  Isocratic pump- delivers constant mobile phase composition:
 solvent must be pre-mixed
 lowest cost pump
 Gradient pump:- delivers variable mobile phase composition:
 Can be used to mix and deliver an isocratic mobile phase or a
gradient mobile phase

34.  The injector serves to introduce the liquid sample into
the flow stream of the mobile phase .
 Typical sample volumes are 5 to 20 micro liters (μl).
 The injector must also be able to withstand the high
pressures of the liquid system.
 An auto sampler is the automatic version for when the
user has many samples to analyze or when manual
injection is not practical.

38.  Considered the ‘heart of the chromatograph ‘ the
columns stationary phase separates the sample
components of interest using various physical and
chemical parameters .
 The small particles inside the column are what cause
the high back pressure at normal flow rates.
 The pump must push hard to move the mobile phase
through the columns and this resistance cause a high
pressure within the chromatograph.

39.  Normal phase
 Reverse phase
 Size exclusion
 Ion exchange

40.  Guard column
 Fast column
 Preparative(i.d. >4.6 mm : lengths 50-250 mm)
 Capillary (i.d. 0.1-1.0mm ;various lengths)
 Nano (i.d. <0.1 mm or sometimes stated as <100μm)
 Analytical (internal diameter (i.d.) 1.0-4.6 mm ; lengths
15- 250 mm)

41.  One of the primary reasons for using these column is to
obtain improved sample output (amount of compound per
unit time )
 Fast column are designed to decrease the time of
chromatographic analysis
 Here internal diameter same but length is short and packed
with smaller particles , that’s are 3 μm diameter.
Advantages:
 Increased sensitivity
 Decreased analysis time
 Decreased mobile phase usage
 Increase reproducibility

42.  It is also known as micro columns.
 It has a diameter much less than a millimeter and there
3 types:
(1) Open tubular
(2) Partially packed
(3) Tightly packed
 They allow the user to work with nanoliter sample
volume , decreased flow rate and decreased solvent
usage volume, led to cost effectiveness.

43.  Used when objective is to prepare bulk (milligrams) of
sample for laboratory application.
 It has usually a large column diameter , which is
designed to facilitate large volume injections into the
HPLC system.

44.  The detector can see (detect) the individual molecules
that come out (elute) from the column .
 A detector serves to measure the amount of those
molecules so that the chemist can qualitatively analyze
the sample components.
 The detector provides an output to a recorder or
computer that results in liquid chromatogram (i.e. the
graph of the detector response).

46.  UV-VIS
-Diode array
-Multiple wavelength
-variable wavelength
 Mass spectrometers
 Refractive index
 Fluorescence
 Light scattering
 Electrochemical
 Radioactivity
 conductivity

47.  Frequently called the data system .
 The computer not only controls all the modules of the
HPLC instrument but a it takes the signal from the
detector and uses it to:
1. Determine the time of elution (retention time ) of the
sample components (qualitative analysis ) and
2. The amount of sample (quantitative analysis ).

56.  This technique is used for :
 Chemistry and Biochemistry research analyzing
complex mixtures.
 Purifying chemical compounds.
 Developing processes for synthesizing chemical
compounds.
 Isolating natural products , or predicting physical
properties.
 Its also used in quality control to ensure the purity of
raw materials etc.

57.  Lesser time consumption
 Less solvent
 Efficient separation
 High sensitivity & resolution
 Small sample size (ppm/ppb)

58.  Pharmaceuticals
 Bio-chemicals
 Food products
 Industrial chemicals
 forensic chemistry
 Environmental science.