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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
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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