University of Santo Tomas
Faculty of Pharmacy
Department of Biochemistry
High Performance
Liquid
Chromatography
GO | GUTIERREZ | IMBAO | JAMORABON | LABASTIDA
PRINCIPLE
v Highly improved form of
column chromatography
v Solvent is being forced
through a column under high
pressures of up to 400 atm,
making the separation faster
CLASSIFICATIONS OF HPLC
Based on nature of mobile and stationary
phase
vNormal Phase HPLC
Stationary phase polar
Mobile phase non-polar
v Reversed Phase HPLC
Stationary phase non-polar
Mobile phase polar
CLASSIFICATIONS OF HPLC
Based on concentration of solvent
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
Solvent Inlet Filter
INSTRUMENTATION
v Pump adds pressure to force the mobile
phase through the column at a specic ow
rate
normal ow rates are in the 1 to 2-
mL/min range.
v Sample Injection introduces the
required sample volume accurately
without depressurizing the system and
must be able to withstand the high
pressure of the system
INSTRUMENTATION
Types of Injection Systems:
Manual Injection
and
Automatic Injections
INSTRUMENTATION
v Manual Injection
Front View Load - Inject
vuser manually loads the sample using a
syringe
v is also known as Rheodyne or
Valco injectors
INSTRUMENTATION
v Automatic Injection
v also known as autosampler
v user loads vials containing the
sample solution into the auto
sampler tray (100 samples)
INSTRUMENTATION
v Column
heart of the chromatograph, the
columns stationary phase separates the
sample components
made from stainless steel tubings;
glass, polymer sometimes used
Internal diameter: 4-5 mm (usual 4.6 mm)
Length : 10-30 cm (usual 25cm)
Packing particle size: 3 or 5 um
INSTRUMENTATION
Retention is based on the interaction of
the components in the solute and the
stationary phase therefore:
A) Normal Phase polar
substances such as silica
gel
B) Reverse Phase- the typical
stationary phases are
nonpolar hydrocarbons,
waxy liquids, or bonded
hydrocarbons (ex. C18, C8,
etc.)
INSTRUMENTATION
Factors Aecting Eciency of the
Column
v Particle Size
v Flow Rate
v Thickness of the stationary phase
v mobile phase viscosity
v diusion of solute in mobile and
stationary phase
v How well the column is packed
INSTRUMENTATION
v Detectors
vMultiple Wavelength
vVariable Wavelength
vUV-Vis
vDiode Array
vMass Spectrometers
vRefractive Index
vFluorescence
vLight Scattering
vElectrochemical
vRadioactivity
vConductivity
INSTRUMENTATION
v Refractive Index Detector
The refractive index (RI) detector is the
only universal detector in HPLC.
The detection principle involves
measuring of the change in refractive
index of the column euent passing
through the ow-cell. The greater
the RI dierence between sample
and mobile phase, the larger the
imbalance will become
STANDARD
PREPARATION
.25g caeine
1000 ppm standard
Caeine 25mL 60%
MeOH
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
Filter with PTFE
nylon syringe
lter (.2 microliter)
3mL aliquot was
used
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
STANDARD
CURVES
SAMPLE CURVE
CALIBRATION CURVE
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
