Pharmaceutical Analysis & Q.C.

II
Presented by:
Group 3
Shariful Alam
Masud Rana
Piash Kar
Sumaiya Binta Fashiuddin
Batch:19th
Section: A
Dept. of Pharmacy
Southeast University

Presentation On:
High Performance Liquid
Chromatography
(Hplc)
Introduction
Different Phase in HPLC
Configuration of HPLC
Detector of HPLC

High Performance Liquid

Chromatography
High performance (or high pressure) liquid
chromatography (HPLC) is a separation
technique.
HPLC is an abbreviation for High Performance
Liquid Chromatography(It has also been
referred to as High Pressure LC).
HPLC has been around for about 35 years and
is the largest separations technique used

The history of HPLC

Since 60-70

Since 80

since2006

Types of HPLC
Adsorption
Normal Phase polar bed, non polar
Reverse Phase non-polar bed w/ polar mobile
* most common
Ion Exchange
Stationary bed ionically charged surface,
opposite to sample ions
Use with ionic or ionizable samples
Mobile Phase aqueous buffer
Size Exclusion

Masud Rana
ID: 2012000300019

Stationary phases On HPLC

unmodified silica, alumina or porous graphite, used in normal-phase
chromatography,
Common bonded phases/ Reverse Phase

octyl

C8

octadecyl

C18

phenyl

C6H5

cyanopropyl

CN

aminopropyl

NH2

diol

Si-(CH2)3-OCH(OH)-CH2-OH

Mobile Phase / Eluent

Normal Chromatography:
Hexane,dichloromethane,isopropanol,methanol
According to increase streangth
Reverse Phase Chromatography:
water, methanol,acetonitrile,terahydrofuran
According to increase polarity

CONTINUE.
Isocratic elution
Eluent composition remains constant
Single solvent or single solvent mixture
Gradient elution:
Eluent composition (and strength) changed
Increases separation efficiency
Decreases retention time
Peak shape is improved (Less tailing)

CONSIDERATION

Purity
Low viscosity
Detector compatibility
Chemical inertness
Solubility of sample
Price

Piash Kar
ID: 2012000300016

Configuration of HPLC

Configuration of HPLC
Pumping unit
It is necessary to pump the eluent at a constant flow
rate and pressure.
Conventional, analytical HPLC pumps are the most
common type, but semi-micro and a preparative
pumps are also used depending
on the range of the eluent flow
rate required.

Configuration of HPLC
Two mixing methods are available;

Low-pressure
The eluent to be absorbed is switched via electromagnetic
valves. One pump is used for mixing. System price is
lower. Up to four eluents can be mixed.

High-pressure
Two pumps are used. The eluents are mixed after
pumping. The response of the gradient is superior because
of the small volume from the mixing unit to the column.
System price is higher due to the increase in the number
of pumps.

Configuration of HPLC
Sample-injection unit
A sample is injected into the flow path for analysis.
This is accomplished via a manual injector or an auto
sampler.
Each type is equipped with six-port valves, so that a
sample can be injected into the
flow path at continuous pressure.
For the manual injector, the knob
is manually operated to deliver
the sample to the column.

Configuration of HPLC
Sample-injection unit

Configuration of HPLC
Separation unit
A column is used for separate the sample.
The column oven is used to maintain a constant
column temperature. If the column temperature were
allowed to vary during qualitative or quantitative
analysis, the elution time of the components would
change, so that an accurate
analysis could not be
performed. Temperature
between 25-50C is often
selected.

Configuration of HPLC
Detection unit
The components eluted from the column are detected,
and the detection data are converted into an electrical
signal. The detector is selected to suit the sample.
Major types of detectors
UV detector
UV-VIS detector
Diode array detector (DAD)
Fluorescence (FL) detector

Configuration of HPLC
Data processing unit
The concentration of each detected component is
calculated from the area or height of the corresponding
peak, and reported.
The data system is performed by a PC.

Sumaiya Binta Fashiuddin

ID: 2012000300015

HPLC Bulk Property Detectors

1.Refractive index detector(IR)
2. UV/UV-VIS detector
3.Diode array detector(DAD)
4. Fluorescence detection
5. Electrochemical detector (ECD)

Refractive Index Detector

Refractive Index Detector

Plus:
1) Measures a bulk property
2) Nearly Universal (different RI than mobile phase)
3) Comparable response for different analytes
4) Detects species with no chromophores
Minus:
1) Temperature dependent
2) Poor sensitivity (LOD 100 ng)
3) No gradient elution

Diode array detector (DAD)

Diode array detector (DAD)

DAD has multiple photodiode arrays to obtain
information over a wide range of wavelengths
at one time,
DAD can be used to identify components by a
comparison of the spectrum.
as well as quantitative analysis with a
specified chromatogram.

Conventional UV-Vis Absorption

Detector

UV-Vis Absorption Detector

light from the lamp is shone onto the diffraction
grating,
dispersed according to wavelength.
For example, when the measurement is performed
with a
wavelength of 280 nm, the angle of the diffraction
grating is adjusted
so that 280 nm light can shine on the flow cell.

Fluorescence Detector

Fluorescence Detector

Fluorescence Detector
Fluorescence detection is suitable for
trace analysis because of generally
having high sensitivity .
many components that originally emit
fluorescence . amino acids, etc.
can be detected as fluorescent
substances, after reaction with a
fluorescence reagent .

Electrochemical Detector

Electrochemical Detector
An ECD is used to measure components
displaying oxidation-reduction reactions.
detects electric currents generated by these
reactions
ECD has high selectivity
often used for measurement of biogenic
substances such as catechol amine.

The End