HPLC Chromatography Mobile Phase & Stationary Phase CLASSIFICATION OF CHROMATOGRAPHY Characteristics of HPLC Purpose Superiority of HPLC TYPES OF HPLC TECHNIQYES Principle PHASING SYSTEM & (normal vs reversed phase) INSTRUMENTATION Flow diagram of HPLC instrument Advantages of HPLC

1. HPLC
PRESENTATION ON

2. Contents:
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC

3. HPLC
➢ High Performance Liquid Chromatography
➢ High Pressure Liquid Chromatography
➢ High Patience Liquid Chromatography
➢ High Priced Liquid Chromatography
➢ High Precision Liquid Chromatography
HPLC machine

4. INTRODUCTION:
Chromatography:
Chromatography is essentially a group of techniques for the separation of the
compounds of mixtures by their continuous distribution between two phases(mobile
phase & stationary phase),one of which is moving past the other.
Mobile Phase:
Mobile-phase is the liquid or gas that flows through a chromatography system,
moving the materials to be separated at different rates over the stationary phase.
Stationary Phase:
Stationary phase is the solid or liquid phase of a chromatography system on
which the materials are to be separated or selectively adsorbed.

5. CLASSIFICATION OF CHROMATOGRAPHY:
Chromatography
On the basis of interaction of
solute to the stationary phase
On the basis of physical
state of mobile phase
Absorption
chromatography
Partition
chromatography
Ion exchange
chromatography
Size exclusion
chromatography
Liquid
chromatography
Gas
chromatography
Super critical fluid
chromatography

6. ❑Liquid chromatography (LC) is one kind of chromatography
in which the mobile phase is a liquid.
❑HPLC is a type of liquid chromatography where sample are
separated from one another by the column packing that
involves various chemical and physical interactions between
their molecules and the packing particles.

7. Characteristics of HPLC:
❑It is column chromatography.
❑It is liquid chromatography.
❑It is modified form of gas chromatography , applicable for both volatile and
non volatile compound.
❑It is mainly divided by two types
1.Normal phase HPLC
2. Reversed phase HPLC.
❑It is having a high resolution and separation capacity.
❑It is used as qualitative as well as quantitative analysis.

8. Purpose:
❑There are three main purpose for HPLC
machine:
A. Identification;
B. Quantification;
C. Purification;
Of the individual components of the mixture.

9. Superiority of HPLC:
➢Simultaneous analysis
➢High resolution
➢High sensitivity
➢Good repeatability
➢Moderate analysis condition
➢Easy to fractionate and purify
➢Not destructive
Industrial HPLC

10. TYPES OF HPLC TECHNIQYES:
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.Ion pair chromatography
4.Size exclusion chromatography
5.Affinity chromatography
6.Chiral chromatography

11. 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 type of analysis
1.Qualitative analysis
2.Quantitative analysis

12. Principle
oHigh Performance Liquid Chromatography [HPLC] is based on principle of adsorption as
well as partition chromatography, depending on the nature of stationary phase, if stationary
phase is solid principle is based on adsorption chromatography and if stationary phase is
liquid principle is based on partition chromatography.
oWhen a mixture of compounds are introduced into a column, they travel according to their
relative affinities to the stationary phase.
oThe component which has more affinity towards the adsorption travels slower.
oThe component which has less affinity towards the stationary phased travels faster, Since no
two component have same affinity towards the stationary phase, the components are
separated.
oIt is important for determination of volatile and non-volatile compounds.
oIt is important for determination qualitative and quantitative analysis.
oIt is important for determination of Retention Time.

13. PHASING SYSTEM
In Chromatography definition, It contains two phases in which one is
mobile phase and another is stationary phase.
➢The mobile phase of chromatography where the sample interacts with the
stationary phase and is separated. Example-Water, Methanol, Benzene etc.
➢The stationary phase of chromatography on which the materials to be
separated are selectively absorbed. Example- Silica , Alumina etc.
➢According to the mode of the separate in HPLC technique is two types
which one is Normal phase and another is Reverse phase.

14. Normal Phase:
In HPLC technique, Normal phase means the stationary phase is polar and the mobile phase
is non polar. In normal phase polar molecules elute slowly, and the non-polar molecules elute
quickly.
Reverse Phase:
In HPLC technique, reverse phase means the stationary phase is non-polar and the mobile
phase is polar. In reverse phase polar molecules elute Quickly, and the non-polar molecules
elute slowly.

15. Point Normal Phase Reverse Phase
Stationary phase Polar (silica gel) Non- polar (C18)
Mobile phase Non – Polar (Organic
solvents)
Polar ( Aqueous /Organic)
Separation movement Non-polar faster Polar faster
Separation based on Different functionally
polarities
Different Hydrocarbon
content

16. INSTRUMENTATION
The main parts of HPLC are:-
1.Solvent Delivery System
2.Pumps
3.Sample Injection system
4.Column
5.Detectors
6.Recorders and integrator

17. Flow diagram of HPLC instrument

18. Flow diagram of HPLC instrument

19. Solvent Reservoirs bottles
#Solvent reservoirs bottles Contains mobile
phase and washing solvents.
#Solvent reservoir bottles are Glass or Stainless-
steel containers capable of holding up to1 liter
mobile phase (pure organic solvents or aqueous
solutions of salts and buffers).
#Inert to a variety of aqueous and non aqueous
mobile-phases.
#Stainless Steel Should be avoided for use with
solvents containing halide ions.
Reservoirs Bottles

20. Degasser :
When solvents are pump under high pressure gas
bubbles are formed which will interfere with the
sample of the steady base line and the sample of the
peak.
➢problems caused by dissolved
air(O2,N2) in mobile-phase
*unstable delivery in pump
*bigger noise and large baseline-
drift in detector cell. Degasser

21. ➢ In order to avoid causing the problems, Mobile phase
should be degassed:
* Vacuum pumping systems
*Distillation system
*A system for heating and stirring the solvents
*Sparging system-bubbles an inert gas of low solubility through the
solvent.

22. HPLC PUMP
❑The role of the pump is force a liquid (called
the mobile phase)Through the liquid
chromatography at a specific flow rate,
expressed in milli liters per min (ml/min).
•Normal flow rates in HPLC are in the 1-to 2-
mi/min range.
•Typical pumps can reach pressures in the
range of 6000-900¬ Psi (400-to 600-bar)
Pump

23. Criteria of HPLC pump:
1)Deliver high volumes (flow rates) of solvent
(to 10 ml/min).
2)Deliver pulse free flow.
3)Deliver high pressure (to 10 600 psi).
4)Be reliable.
5)Deliver precise and accurate flow.
6)Have low pump-head volume.
Pump

24. Functions of HPLC pump
❑ HPLC pump has three basic functions-
1. Provide accurate and constant flow.
2. Provide accurate mobile phase compositions .
3. Provide the force necessary to push the mobile phase
through the tightly packed column.

25. Types of HPLC pump:
1.Reciprocating pump
Reciprocating pump is a positive displacement pump where
certain volume of liquid is collected in enclosed volume and is
discharged using pressure to the
required application. Reciprocating pumps are more suitable
for low volumes of flow at high pressures.
Criteria:
a. Small internal volume.
b. High output pressures.
c. Readily adaptable gradient elution

26. 2. Syringe type pump:
➢Constant flow rate pump.
➢Non-pulsating flow.
➢Low flow rates(1 to 100 ml/min).
➢Isocratic flow only.
➢Refill required when reservoir (~50ml)
expended.

27. 3.Constant pressure pump
In these types of pumps the mobile phase is driven
through the column with the use of pressure from gas
cylinder
• A low – pressure gas source is needed to generate high
liquid pressure
• The valving arrangement allows the rapid refill of the
solvent chamber whose capacity is about 70ml
• This provides continuous phase flow rates.
Constant pressure Pump

28. HPLC pump operating mode
Isocratic system:
A separation in which involved a single solvent or solvent mixture of constant
composition with single pump throughout the run. The main purpose of this system is simple
separation, simplicity, lower cost, simpler instrumentation.
Gradient system:
A separation in which involved two or more solvent composition that are changing this
composition of the mobile phase. The main purpose of this system is to move strongly
retained components of the mixture faster, but having the least retained component well
resolved.
Two types pump are involved-
1. Binary gradient pump
2. Quaternary gradient pump

29. INJECTOR:
❑It is a parts of HPLC machine
which is used to inject the sample
into the flow of mobile phase to
carry the sample.

30. TYPES OF INJECTOR:
Manual injector:
In these injector the sample are injected into the
system manually by a microliter syringe.
Auto injector:
In this system the sample are injected into the flow
lines automatically through self reading.

31. GUARD COLUMN:
❑The column which placed between the
injector and HPLC column is known as
guard column.
❑It prevent the column from impurities,
fibers, particles and air baubles.

32. COLUMN:
▪It is called the heart of HPLC.
▪It contains stationary phase and made by
glass or stainless steel.
▪The main function of column is separation
of compound from the mixture.

33. Column oven
▪Column oven is an oven which use to maintain a
desired temperature into the column because
different temperature is needed to separation of
different compound.
▪Column oven maintain the definite temperature
for proper separation of compound of mixture

34. TYPES OF COLUMN BASED ON POLARITY
▪ Polar column:
The polar column consist of polar stationary phase containing materials(eg.
silica, aluminum)
▪ Nonpolar column:
The nonpolar column consist of nonpolar stationary phase containing
materials(eg. C-8, C-12)

35. MECHANISM OF COLUMN
▪If the column is polar the nonpolar compound elute first and polar
elute last from the mixture of the column.
▪If the column is nonpolar the polar compound elute first and
nonpolar elute last from the mixture of the column.

36. Packing materials
➢The packing material is prepared form 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.
➢Column are packed using high –pressure to
ensure that they are stable during use.
➢Most users purchase pre-packed columns to
use in their liquid chromatography.

37. Parameters
❑Retention Time (RT) :
Retention time is defined as the time taken for the analyte to
travel from the column inlet to the point of detection.(Maximum
peak).
❑Retention Volume : Retention volume is the volume of carrier gas required to
elute 50% of the component from the column. It is the product of retention
time and flow rate.
Retention volume= Retention time flow rate
❑ Separation factor: Separation factor is the ratio of partition coefficient of
the tow components to be separated.

38. HETP
❖HETP is “Height Equivalent to the Theoretical Plate”.
❖It arises from the Plate Theory and is numerically equal to the column length divided by the
number of theoretical plates in the column (and in practice is measured in this way).
❖As the HETP is a function of both the properties of the column and the solute, it will vary from
one column to another and, more importantly, between different solutes eluted from the same
column in the same chromatogram.
❖ 𝐻𝐸𝑇𝑃 =
𝐻
𝑁
Where,
N = the number of theoretical plates.
H = the total bed height.
HETP = the height equivalent to theoretical plate.

39. Detector
➢The detector can detect the individual molecules that elute from the column and convert the data
into an electrical signal.
➢The HPLC detector, located at the end of the column detect the analytes as they elute from the
chromatography.
Common uses detector:
✓Fluorescence
✓UV
✓Refractive index
✓Electrochemical
✓Conductivity
✓Light Scattering
✓Mass spectrometry
Detector(UV)

44. Recorder
➢A recorder, is a device that draws the chromatography
results from a chromatographic process onto chat paper
and provides a visual representation of the separation
that has been achieve.
➢ The time scale of the chart movement normally
ranges from about 1 cm per second to 1 cm per hour
which can also be selected to suit the separation that is
being carried out
Recorder

45. Waste container
❑Waste container is used to collected all waste
products of HPLC.
❑It is composed by glass.
❑It is very important because the solvent may
interact if the container is plastic and if may be
harmful to the environment.
Waste container

46. Washing agent
Washing solvents are agents that used to wash the stationary
phase(column) of HPLC.
❑In order to maintain the accuracy washing agents are used.
❑Column is the heart of HPLC, to remove unwanted material or mobile phase from
column washing agent is used either before or after using HPLC machine or in both.
❑ There could be several test of several different materials , thus maintaining column
affinity and accuracy is must.

47. ❑According to the nature there are two types of washing solvents
1.Polar
2.Non-polar
❑Both types of washing agents are used for a single column to remove both
mobile phase and fragments of separated components adsorbed to the
stationary phase .
❑According to the nature of stationary phase washing agent of similar nature
is used afterward to maintain the nature of column intake.

48. Calibration
Calibration of HPLC is done to check the performance of it’s instrument.
1. Flowrate,
2. Detector and injector linearity,
3. System precision,
4. Column oven temperature,
5. Detector wavelength accuracy.

49. Applications:
❑HPLC is one of the most widely applied analytical separation techniques,
▪In the field of Pharmaceutical:
a) Tablet dissolution of pharmaceutical dosages.
b) Shelf life determinations
c) Identification of counterfeit drug products.
d) Pharmaceutical quality control.

50. ▪Environmental:
a) Phenol in drinking water,
b) Identification of diphenhydramine in sediment samples.
c) Estrogens in coastal waters- The sewage source.
d) Environmentally relevant bacteria.
e) Assessment of TNT toxicity in sediment.

51. ▪Clinical:
a) Analysis of antibiotics
b) Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis
c) Detection of endogenous neuropeptides in brain extracellular fluids
▪Forensics
a) Identification of anabolic steroids in serum, urine, sweat and hair
b) Forensic analysis of textile dyes.
c) Determination of cocaine and metabolites in me conium.

52. ADVANTAGES OF HPLC:
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 mixture
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.

53. 7. both aqueous and non aqueous samples can be analyzed with little or no
sample pre treatment
8. A variety of solvents and column packing are available, providing a high
degree of selectivity for specific analyses.
9. It provides a means for determination of multiple components in a single
analysis.