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Faculty of Pharmacy
Department of Biochemistry
High Performance
Liquid
Chromatography
vIsocratic Elution
-single solvent system
v Gradient Elution
-multiple solvent system
DEFINITION OF
TERMS
v Retention Time
v Retention Volume
v Flow Rate
vResolution
v Peak Area
INSTRUMENTATION
INSTRUMENTATION
v Solvent Delivery Systems
a. Solvent Reservoir 3-4 inert containers
for holding the eluted solvents
b. Inlet Filter type of lter that is usually
stainless steel or glass which removes particles
from solvent.
c. Degassing Facilities not always
available, are used to remove dissolved gases
or remove bubbles from solvents
Manual Injection
and
Automatic Injections
INSTRUMENTATION
v Manual Injection
Diluted to 100,
200, 300, 400.
500, 600, 700, C1V1=C2V2
800, and 900 ppm
SAMPLE PREPARATION
10 mL Caeine
Sample
Dilute to mark
using 60%
methanol in 50mL
volumetric ask
CONCENTRATION:
32.25977733 ppm
CALIBRATION CURVE
CONCENTRATION:
44.24038238 ppm
THEORETICAL
VALUES
v Retention Time: 2 to 3 minutes
v Wavelength: 260 nm
v Concentration of Energy Drink
Used: 320 ppm