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Prof. Dr. Amani S. Awaad
Professor of PharmacognosyPharmacognosy Department,
College of Pharmacy Salman Bin Abdulaziz
University,
Al-Kharj. KSA.
Email: [email protected]
Pharmacognosy- 1
PHG 222
Chromatography
High Performance Liquid Chromatography
After studying this topic student should be
able to :
Define HPLC
Describe HPLC principle
Explain major components of HPLC and their function
Explain application of HPLC
Explain factors affected function of HPLC
Describe advantages & disadvantages of HPLC
• Originally referred to as High-Pressure Liquid Chromatography
• Now more commonly called High
Performance Liquid Chromatography
• HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time, utilizing very small particles, small column diameters, and very high fluid pressures.
What is HPLC?
Introduction
In HPLC chromatography:
the mixture is dissolved in a solvent (mobile
phase) and then forced to flow through a
chromatographic column under a high pressure.
In the column, the mixture is resolved into its
components.
(HPLC) is an advanced form of liquid
chromatography used to separate the
components of a mixture (Analytes).
The separation occurs because each component inthe mixture interacts differently with the stationaryphase. Molecules that interact strongly with thestationary phase (yellow component) will moveslowly through the column, while the moleculesthat interact less strongly (blue component) willmove rapidly through the column.
The start to the detect
- Why high pressure?
In HPLC the stationary phase has two
characters:
- Has small particles size (5- 10 µm).
-And packed under high pressure.
Thus pressure from 1000 to 5000 psi, pound
per square inch (68 to 340 atm.) is applied
to overcome the obstructive effect of the
fine particles.
Reduction of the particle size of the
stationary phase leads to:
- Leaving less space for the mobile phase to
pass through.
- Decrease the flow rate of the liquid mobile
phase.
• HPLC instrument includes:
• A- Reservoir for solvents (mobile phase)
• B- High pressure pump
• C- Sample inlet device
• D- Column
• E- Detector
• F- Recorder
Instrumentation of HPLC
-Mobile phase is usually organic oraqueous or mixture of both.
- Mobile phase is placed in bottles ofglass.
Instrumentation of HPLC
High Performance Liquid Chromatography
A- Reservoir for solvents (mobile phase)
Characters of mobile phase:
1- Pure
2 - Low viscosity
3-Chemically inert
4- Low price
5- Compatible with detector
6- Solubility of the sample
Mobile phase
Solvent A Solvent B
Water Organic solvent
Miscible with water, such as acetonitrile,
methanol, or isopropanol.
Instrumentation of HPLC
High Performance Liquid Chromatography
A- Reservoir for solvents (mobile phase)
Elution Techniques (Programinig)
1- Isocratic elution:
The mobile phase composition
remains constant throughout the
separation procedure.
2- Gradient elution:
The mobile phase composition is
changed during the separation
process.
Gradient elution is divided into
two types:
A- Continuous (linear)
B- Discontinuous (stepwise)
Stepwise (discontinuous)
Linear (continuous)
Time
% of B
10%
20%
30%
40%
50%
60%
70%
0 5` 10` 15` 20` 25` 30`
Isocratic elution
Gradient elution
Instrumentation of HPLC
High Performance Liquid Chromatography
A- Reservoir for solvents (mobile phase)
Elution Techniques (Programing)
Advantages of gradient elution technique
1- Shortening the time of analysis.
2- Reduces tailing, gives sharp peak.
3- Increases the sensitivity of analysis.
4- Decreases the retention of the later-eluting
components so that they elute faster.
PH of mobile phase
The pH of the solvent (water) may be adjusted using
phosphate or perchlorate or trifloroacetate acid or
sulphate buffer.
The selectivity of HPLC is affected by :
1- Type of mobile phase, organic or aqueous.
2- The composition of the mobile phase, whether one
solvent or more.
3- The pH of the mobile phase.
Incomplete
separation
No
separation
for 2 and 3
Best
separation
30% MeCN
70% Water
45% MeOH
55% Water
Mobile Phase Composition Effect on Selectivity
Fast Slow and better separation
Methanol and water give slow and better separation while use of actonitrile
and water give fast and bad separation
High Performance Liquid ChromatographyInstrumentation of HPLCElution Techniques (Programing)
A- Reservoir for solvents (mobile phase)
High Performance Liquid Chromatography
Instrumentation of HPLCElution Techniques (Programing)
A- Reservoir for solvents (mobile phase)
Some solvents used in
HPLC and their polar PolaritySolvents
10.2
7.2
6.9
5.8
5.1
5.1
4.8
4.3
4.0
3.9
Water
Dimethyl
sulfoxide
Ethylene glycol
Acetonitrile
Methanol
Acetone
Dioxane
Ethanol
Tetrahydrofuran
I-propanol
N.B. Chlorinated solvents
do not used in HPLC to
prevent rusting of
stainless parts of the
instruments
High Performance Liquid Chromatography
Instrumentation of HPLCElution Techniques (Programing)A- Reservoir for solvents (mobile phase)Treatment of mobile phase
C- Pre-saturation with the stationary phase in case of
liquid liquid chromatography.
A- Filtration before entering the column.
B- Degassing using degasser.
To get red of dissolved gases and
bubbles which disturb the detector
1- Heating with stirring
2- Applying vacuum,
3- Passing nitrogen or helium
4- Ultrasound
High Performance Liquid Chromatography
B- High pressure pump
• Function of the pump:
• Pump is used for forcing the mobile phase through the column
There are two types of pump:
1- Constant pressure pump
It is free from pulsation resulting in smooth baseline
2- Constant flow pump
It is able to give constant flow rate of mobile phase.
1- The pump should be capable of delivering accurateand pulse free flow rate (e.g. 5 ml/min).
2- The pump should be capable of delivering highvolume of solvent.
3- The pump should be capable of delivering highpressure up to 5000 psi.
Instrumentation of HPLC
High Performance Liquid Chromatography
C- Sample inlet device
(Injection port)1- Manual injection
2-Automated injection
The injection port consists of
A- The injection valve.
B- The sample loop.
Manual injection
1- The sample is typically
dissolved in the mobile phase.
2- It is drawn into a syringe and
injected into the loop via
injection valve.
Instrumentation of HPLC
Type of injection
1- Manual injection
2-Automated injection
High Performance Liquid Chromatography
D- ColumnInstrumentation of HPLC
Column in HPLC is either
1- Analytical, 1-6 mm i.d.
2- Preparative up to 3 cm i.d.
Made from: Stainless. Shape: Straight.Length: Variable
Other types of columns used in HPC
Guard column:
1- Protect the analytical column
2- Organization of separation in HPLC
High Performance Liquid Chromatography
E- Detectors (Brain of HPLC)Instrumentation of HPLC
1 -UV absorption detector
2 - Refractive index detector
3- Mass spectrometer detector
4- Fluorescence detector
5- Photodiode array detector
6-Infra Red detector
Characters of detectorsTypes of detectors
1- High sensitivity
2- Low noise (straight base line)
3-Wide range of response to
different compounds
4- Unaffected by temperature or
mobile phase
5- Non destructive to the
compounds
6- Provides qualitative and
quantitative information about
the detected sample
High Performance Liquid Chromatography
E- Detectors (Brain of HPLC)Instrumentation of HPLC
Types of detectors
1 -UV absorption detector
2 - Refractive index detector
-Not used in case of gradient elution
- Less sensitive
-It measures the difference in RI between pure
mobile phase and the column eluate (mobile
phase + solute).
It is the most sensitive, sensitive to ng of
compound.
- The most widely used, it measure the UV
absorption of the solute
3- Mass spectrometer detector
It is used with capillary column in analytical
HPLC to give information about nature of the
material by giving the mass spectrum of the
material.
High Performance Liquid Chromatography
E- Detectors (Brain of HPLC)Instrumentation of HPLC
Types of detectors
4- Fluorescence detector
- More sensitive than UV detector (1000 fold as UV)
-It is used with compounds which are naturally
fluorescent. Or compound which
-can be converted to fluorescent
- derivative.
5- Photodiode array detector
- It is series of detectors each is responsible
for receiving a different wavelength.
6-Infra Red detector
-Used for substances whose boiling point is
higher than that of the mobile phase
-- It is more sensitive than refractive index
detector
High Performance Liquid Chromatography
F- Data ProcessingInstrumentation of HPLC
Using specific software that is connected to
HPLC machine
Receive the information from HPLC
machine and present it as a graph
The graph describes about qualitative data
(Retention time) and quantitative data (area
under curve)
1- Analytical type: which is used
a- In identification and assay ofthe components in a mixture .
b- To know the number of components in a mixture (screening).
2- Preparative or semipreparative type: used in isolation and purification.
Analytical Preparative
Dimensions of the
column
Analytical, 1-6
mm i.d
Preparative up
to 3 cm i.d.
Flow rate of
mobile phase
(pump)
should has flow
rates that range
from 1 to 10
ml/min.
flow rates in
excess of 100
ml/min.
Injected volume of
the sample
flow rates in
excess of 100
ml/min.
from 1 ml to 5
ml or more.
Size of the loop of
injection
port
HPLC can be divided into two
main types according to the uses:
The difference between analytical and
preparative HPLC
High Performance Liquid Chromatography
Types of HPLC according to the uses
High Performance Liquid Chromatography
The factors which influence the
HPLC performance
1. Internal diameter of column
- the smaller in diameter, the higher in sensitivity
2. Pump pressure
- the higher in pressure, the higher in separation
3. Sample size
4. The polarity sample, solvent and column
5. Temperature
- the higher in temperature, the higher in separation
High Performance Liquid Chromatography
Advantages & Disadvantages of HLPC
1- High speed
2- High resolution
3- High sensitivity
4- Re-usable column
5- No destruction of the components
6- The instrumentation are automatic,
computerized
7- Sample is recovered completely
8- Quantitative work is more easily and most
sensitive
I-Advantages
II-Disadvantages
Need a skill to run the instruments
Solvents consuming
High Performance Liquid Chromatography
4. Clinical test- Monitoring of hepatic chirosis
patient through aquaporin 2 in the urine.
5. Food and essence manufacture- sweetener analysis in the fruit juice- preservative analysis in sausage
1.Pharmaceuticals industry
-To control the drug stability-Quantity of drug determination from
pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet
-Quantity of drug determination from biological fluids, ex: blood glucose level
2. Analysis of natural contamination- Phenol & Mercury from sea water
3. Forensic test- Determination of steroid in blood, urine & sweat.- Detection of psychotropic drug in plasma
Applications of HPLC
Chemical
Environmental
Pharmaceuticals
Consumer
Products
Clinical
polystyr
enes
dyes
phthalat
es
tetracyclines
corticosteroids
antidepressants
barbiturates
amino acids
vitamins
homocysteine
Bioscience
proteins
peptides
nucleotides
lipids
antioxidants
sugars
polyaromatic hydrocarbons
Inorganic ions
herbicides
29
I need a quantitative
separation of
carbohydrates in some
of our products
as soon as possible. I’ll get
on it!
I’ll need a separation
technique. in my lab,
HPLC & GC
Which should I use?
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose
Zorbax NH2 (4.6 x 250 mm)
70/30 Acetonitrile/Water
1 mL/min
Detect=Refractive Index
30
Separations Separation in based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
31
Separations Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
32
Separations Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
33
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
34
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
35
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
36
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
37
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
38
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
39
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
40
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
41
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
42
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
43
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
44
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
45
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
46
Separations Injector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time
47
The Chromatogram
Injection
to
tR
mAU
time
tR
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportional
to the quantity of analyte.