This document discusses High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). HPLC uses high pressure to push a mobile phase through a column packed with small particles to separate compounds dissolved in solution based on their affinity to the stationary phase. It has components like pumps, injectors, columns, and detectors. GC vaporizes samples and uses an inert gas as the mobile phase to separate compounds in the gas phase based on their boiling points as they elute from the column. Both techniques are used to qualitatively analyze mixtures by detecting separated components.

1. HPLC & GC
Prepared By :- Dr. Hadeer Adel El-hamdy

2. High Performance Liquid Chromatography
• P can refer to { pressure or price or problem or precision but the
best is P = Performance… }.
• HPLC is a specific form of column & liquid chromatography
used to separate compounds that are dissolved in soln.
• HPLC characterized by use of high pressure why ? To push a
mobile phase solution through a column allowing separation of
mixtures due to using of very small particle diameter of packing
column causing high Back pressure.

3. Principle
• HPLC is a separation technique … injection of small volume of liquid sample into a column
packed with tiny particles ( 3-5 micron ) in diameter called the stationary phase
• Where the individual components of the sample are moved through the packed column with a
liquid mobile phase forced through the column by high pressure delivered by a pump.
• Separation done according to their relative affinities towards the stationary phase.
• The separated components are detected at the exit of tube (column) by flow through device
(detector) that measures their amount
• Output from this detector is called a { liquid chromatogram } Chromatograph

4. COMPONENTS OF HPLC SYSTEM
:-
DiagramfortypicalHPLC
Loopinjector
Pulsedamper
Solvent
proportioning
valve
Pump
Guard
columr
Column
Mobile
phasereservoirs
Detector

5. • 1. Solvent delivery system ( solvent reservoir )
• filtration system
• degasser … remove air bubbles soluble in organic solvents
• Notes
• Mixing unit is used to mix solvents in different proportions.
• The choice of mobile phase is very important determined by
• Polarity of stationary phase & nature of sample components
• Mobile phase is non polar in NP-HPLC & polar usually water or methanol in
RP-HPLC
• Elution technique ( isocratic or gradient elution ).

6. • 2. pump…pumping solvent up to pressure of 4000 psi and at a flow rate 10 ml/min
• 3. sample injection system ( injector)
• Inject solvent through the column
• Volume ( 1-200 ΜL )

7. • 4. Column ….{ Heart Of Chromatograph }
• Usually a stainless steel tube packed , with octadecylsilane (ODS-coated) C18
silica gel.
• The small particles inside the column causing high back pressure at normal rate
flow .

8. • 5. Detector …
• Detect the individual molecules that elute from the column .
• Quantitatively analyze sample components .
• Detector provide an output to recorder or computer software those results in
liquid chromatogram.
• Types :-
• UV-Visible detectors
• Fluorescence detectors
• Refractive index detectors
• Diode assay detector (DAD)

9. •Applications of HPLC
Qualitative Analysis
Purification of Compounds
Identification of
Compounds
Peaks correspond to
individual components
Compound
Impurity
Quantitative Analysis
Authenti
c
Unknown

10. Gas chromatography

11. Gas chromatograph

12. GAS CHROMATOGRAPHY
• Gas chromatography is one of group (analytical separation technique ) used to
• analyze volatile substances in the gas phase .
• M.p gas
• Separation by :-
• Partition between gaseous mobile phase and liquid stat.phase supported by inert
packing (GLC)
• Adsorption between gaseous m.p and solid stat.phase (GSC)
• Depend mainly on temp and boiling point

13. Injection
Detection
Separation
Elution
• In GC, the sample is Vaporized and injected onto the head of a column
• Elution by the flow of an inert gaseous mobile phase,
• the m.p don’t interact with molecules
• of the analyte ( carrier gas ) ; its only function is to transport the analyte
• Through the column
• After separation, the compounds are respectively detecting
• using suitable detectors .
Vaporization

14. Main components of GC
• Carrier gas…..Chemically inert gases ( He, H2&N2) & high pure
• Pressure regulators … reduce pressure of gas & control flow rate
• Injector ….
• It vaporizes and mix the sample with the carrier gas before sample enters the
head of the column .
• The type of column used in the analysis sets the mode of injection
• For packed column ( direct vaporization injector )
• For capillary columns ( split / splitless injector )

15. • Oven ….regulate optimum column temp .
• Column ….. Packed columns & open tubular capillary columns “ most used”

16. Stationary Phase : column is packed with solid the wall of the column
particles coated with liquid stat.phase. is coated with liquid
stat.phase.
M.P : nitrogen helium
Internal diameters : 2-5mm 0.5mm
Packed column Capillary column

17. • GC detectors
1. Flame ionization detector.
2. Electron capture detector .
3. Thermal conductivity detector .
4. Nitrogen phosphorus detector.
GC used mostly for qualitative analysis ….