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What is chromatography
What is HPLC
HPLC Components
Working principle
Applications
Chromatography
Invented by Mikhail Tswett in 1900
Plant pigments
Chromatography
Colors
CaCO3
What is HPLC?
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.
Advantages of HPLC
High performance liquid chromatography is
basically a highly improved form of column
chromatography. Instead of a solvent being
allowed to drip through a column under
gravity, it is forced through under high
pressures of up to 400 atmospheres. That
makes it much faster.
The other major improvement over column
chromatography concerns the detection
methods which can be used. These
methods are highly automated and
extremely sensitive.
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary
phase
Mobile phase
6
Stationary Phases
Polar (Normal Phase):
Silica, alumina
Non-Polar (Reversed Phase)
C18, C8
Diagram of HPLC
Injector
Mixer
Pumps
Column
Detector
Solvents
Waste
Components of HPLC
1.
2.
3.
4.
Solvent Reservoir
Pumps
Sample Injection System
Guard Columns and main
column
5. Detectors
6. Data Processing
7. Waste
Solvent Reservoir
Mobile phase
isocratic elution proportion of the
solvents remains the same throughout
the operation
gradient elution solvent proportion can
be varied during separation
To carry sample into the column
Pumps
To produce an appropriate pressure
to push solvent into the column.
Single pump, binary pump, tertiary
pump, quaternary pump
Syringe :
manual
Autoinjector
Guard column
Placed prior to column
For the safety of column
Particles collect in an HPLC columns inlet
frit or in the first millimeter of the packed
column, can cause degradation of analysis
such as peak tailing and split peaks.
Another source of problems are
compounds that irreversibly bond to the
stationary phase and are often injected
into analytical columns. These compounds
cause permanent damage to columns that
are not protected by a guard column.
Columns
straight, 15 to 250 cm in length; 2 to 5
mm i.d.
packing - silica gel, alumina, Celite
HPLC Detectors
UV/Vis
Diode Array Detector (DAD)
Refractive index
MS
Fluorescence
Data Processing
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)
Separations
Injector
Mixer
Pumps
Column
Detector
Solvents
Waste
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
20
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
21
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
22
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
23
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
24
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
25
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
26
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
27
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
28
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
29
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
30
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
31
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
32
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
33
time
Injector
Mixer
Chromatogram
mAU
Pumps
Start Injection
Column
Detector
Solvents
34
time
The Chromatogram
tR- retention time - determines sample identity
tR
tR
mAU
Injection
35
time
Application of HPLC
1. Pharmaceuticals industry
Application of HPLC
4. Clinical test
- Monitoring of vitamin D levels in the
patients.
5. Food and essence manufacture
- sweetener analysis in the fruit juice
- preservative analysis in sausage.
advantages
1. Needs a small sample with a high
accuracy and precise
2. Non-destructed sample during
operation compared to GC.
Disadvantages
Need a skill to run the instruments
Solvents consuming
Thank you!