|


  





Ida Musfiroh




ë |
 is a form of liquid chromatography used to
separate compounds that are dissolved in solution.

ë ompounds are separated by injecting a sample mixture

onto the column.

ë The different component in the mixture pass through the

column at differentiates due to differences in their
partition behavior between the mobile phase and the
stationary phase.

ë The mobile phase must be degassed to eliminate the

formation of air bubbles.
 ½asic omponents of an |

System
1. Pump System. Mobil phase pressures up to 6000 psi are
necessary to achieve reasonable column elution times (~ minutes).
Typical flow rates are 0.1 to 10 m
minute.
2. Injection System. Used to introduce small samples (0.1 to 500 µ
)
into the carrier stream under high pressure.
3. Reservoirs (Solvents). Multiple solvents are necessary for
performing gradient elution's (i.e. changing the polarity of the mobil
phase during a run).
4. Chromatographic Column.
Column. Typically 10-
10-30 cm in length
containing a packing of 5-
5-10 µm diameter. Many types of columns
are available, depending on the type of liquid chromatography
desired.
5. Detector. Many types are available including UV, IR, refractive
index, fluorescence, conductivity, mass spectrometry, and
electrochemical. Diode array detectors are used when wavelength
scans are desired.
Îhat is HPLC used for ?

1. Separation of mixed components

2. Qualitative analysis  Quantitative analysis
3. reparation of interest components

Separation, analysis andor preparation

of interest components
 Separation and nalysis

C C Separation ½ ½

½ ½ C C C C
C C

Qualitative analysis
What are components A, ½ and  ?
Quantitative analysis
What is the concentration of
components A, ½ and  ?
Results obtained by HPLC

Chromatogram containing three peaks

Qualitative analysis (identification) and
Quantitative analysis (determination)
an be performed using the information contained in the
chromatogram

Chromatography : Method
Chromatogram : Results
Chromatograph : Instrument
 Chromatogram

Sample IN

Mobile phase IN
column
baseline

Sample IN

F E
D
Mobile phase IN
A
½
DE


½
hromatogram
A
 Identification
What is component A?

½
Sample

affeine

omponent A elutes the same time as a caffeine peak.

omponent A is identified as caffeine.

 Determination
What is the concentration of component A?

affeine (1mgml)
5ul injection (5ug)

eak area (or height) is proportional to the concentration

(or amount) of the component.

The concentration of component A(caffeine) is determined by

comparing the peak area with that of the standard caffeine
peak.
 Separation Mechanism
Separation is determined by column (packing material) and
mobile phase (solvent).

Mobile phase elutes components.

acking materials retain components in the column.

Mobile phase (solvent)

Õ Õ Õ Õ
½
½ C
C

olumn
time

>½>A
acking
material
 Separation rinciples in |

eneral Rule of Thumb:
olarity of analytes § polarity of stationary phase 
polarity of mobile phase

To achieve good separation, the analytes should

interact with the stationary phase, but not too
strongly or the retention time will become very long
 ëive modes in HPLC

 mode acking materials Mobile phase Interaction

Normal phase chromatography Silica gel n-|exane Adsorption

Reversed phase chromatography Silica-18(ODS) MeO|Water |ydrophobic
Size exclusion chromatography orous polymer T|F Gel permeation
Ion exchange chromatography Ion exchange gel ½uffer sol. Ion exchange
Affinity chromatography ackings with ligand ½uffer sol. Affinity
Types
of

 HPLC ½asic Instrumentation

Solvent Delivery Detector

Injector
olumn

Separation
Mobile ump
phase

Sample Injection

Data rocessor
 Parameters used in HPLC

1. Retention parameters
2. olumn efficiency parameters
3. eak symmetry parameters
4. ondition for Separation

Retention :
When a component in a sample interacts with the stationary phase in the
column and a delay in elution occurs.

olumn efficiency : Goodness of a column

 2. Parameters used in HPLC

Retention parameters

tR : retention time
(the time between the injection point and the maximum detector response for corresponden
compound)
vR : retention volume (tR x eluent flow rate)
k¶ : capacity factor
t0 : the time required for the component not retained by the column to pass through the column
tR
tR - t0 tR - t0
t0 k¶ =
t0
 2. Parameters used in HPLC

Column efficiency
The number of theoretical plates N is given by:
FW|M method
4 method 5 method

h x 0.044
tR
h x 0.5
h

W4 W5 W 12
N = 16 ( tR  W 4 ) 2 N = 25 ( tR  W 5 ) 2 N = 5.545 ( tR  W 0.5)
2

The height of the theoretical plate | is given by:

|=
N
: olumn length

 2. Parameters used in HPLC

Peak symmetry

S : Symmetry factor ( T : Tailing factor )

h
h x 0.05

f
W 0.05

W 0.05 S = 1 : The peak is completely symmetric.

S= S > 1 : Tailing
2f S < 1 :
eading
 2. Parameters used in HPLC

Degree of separation

tR2
tR1
k¶1 tR2 - tR1
Resolution : Rs = 2 x
W1 + W 2
k¶2
k¶2
Separation factor : =
k¶1

W1 W2
 2. Parameters used in HPLC

Condition for good separation

A larger Rs value means a better separation.

1 - 1 k¶2
Rs = N
4 1 + k¶2

k¶2
: apacity term
1 + k¶2 increases the retention time
- 1
: Selectivity term
increases the time interval between peaks
N : olumn efficiency term
produce narrow peaks
 2. Parameters used in HPLC

Parameters and selectivity

onger retention time

arger

Improved column efficiency

m
 m
  
 Uses of |

ë This technique is used for chemistry and biochemistry
research analyzing complex mixtures, purifying chemical
compounds, developing processes for synthesizing chemical
compounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the purity
of raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product
stability and monitor degradation.

ë In addition, it is used for analyzing air and water pollutants, for

monitoring materials that may jeopardize occupational safety
or health, and for monitoring pesticide levels in the
environment. ëederal and state regulatory agencies use HPLC
to survey food and drug products, for identifying confiscated
narcotics or to check for adherence to label claims.
 |
 
  
ë The function of the injector is to place the sample into
the high-
high-pressure flow in as narrow volume as possible
so that the sample enters the column as a
homogeneous, low- low-volume plug. To minimize spreading
of the injected volume during transport to the column, the
shortest possible length of tubing should be used from
the injector to the column.

ë When an injection is started, an air actuator rotates the

valve: solvent goes directly to the column; and the
injector needle is connected to the syringe. The air
pressure lifts the needle and the vial is moved into
position beneath the needle. Then, the needle is lowered
to the vial.
 |
 columns
ë The column is one of the
most important components
of the |
 chromatograph
because the separation of
the sample components is
achieved when those
components pass through
the column. The |igh
performance liquid
chromatography apparatus
is made out of stainless
steel tubes with a diameter
of 3 to 5mm and a length
ranging from 10 to 30cm.
ë Normally, columns are filled
with silica gel because its
particle shape, surface
properties, and pore structure
help to get a good
separation. Silica is wetted by
nearly every potential mobile
phase, is inert to most
compounds and has a high
surface activity which can be
modified easily with water
and other agents. Silica can
be used to separate a wide
variety of chemical
compounds, and its
chromatographic behavior is
generally predictable and
reproducible.
 
|


 Î|
olumn arameters Instrument arameters

ë olumn Material ë Temperature

ë Deactivation ë Flow
ë Stationary hase ë Signal
ë oating Material ë Sample Sensitivity
ë Detector
 Î|
Sample arameters

ë oncentration
ë Matrix
ë Solvent Effect
ë Sample Effect
 Several column types
(can be classified as )

ë w

ë 

ë 

ë  

 w

ë In this column type, the retention is

governed by the interaction of the polar
parts of the stationary phase and solute.
For retention to occur in normal phase, the
packing must be more polar than the
mobile phase with respect to the sample
 
ë In this column the packing material is relatively
nonpolar and the solvent is polar with respect to
the sample. Retention is the result of the
interaction of the nonpolar components of the
solutes and the nonpolar stationary phase.
Typical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as 18, 8, etc.) and the
solvents are polar aqueous-
aqueous-organic mixtures
such as methanol-
methanol-water or acetonitrile
acetonitrile--water.
 
ë In size exclusion the |
 column is
consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase according to
its molecular size. Small molecules
penetrate into the pores within the packing
while larger molecules only partially
penetrate the pores. The large molecules
elute before the smaller molecules.
  

ë In this column type the sample
components are separated based upon
attractive ionic forces between molecules
carrying charged groups of opposite
charge to those charges on the stationary
phase. Separations are made between a
polar mobile liquid, usually water
containing salts or small amounts of
alcohols, and a stationary phase
containing either acidic or basic fixed sites.
   ! 

ë †¶ values tell us where bands elute relative

to the void volume. These values are
unaffected by such variables as flow rate
and column dimensions. The value tell us
where two peaks elute relative to each
other. This is referred to as the selectivity
factor or separation factor (now and then
as the chemistry factor).
 Types of Liquid Column
Chromatography
(LCC)
ë

 (
iquid
iquid)
ë G
 GS

ë
S (
iquid Solid - ë SF (Supercritical

adsorption) Fluid)

ë SE (Size Exclusion)

 Types of Detectors
ë Absorbance (UV ë Evaporative
ight
with Filters, UV with Scattering Detector
Monochromators) (E
SD)

ë IR Absorbance ë Electrochemical

ë Fluorescence ë Mass-
Mass-
Spectrometric
ë Refractive--Index
Refractive
ë hoto--Diode Array
hoto
"
# $w 
ë EFFIIEN
ë RESO
UTION
ë INERTNESS
ë RETENTION INDEX
ë O
UMN ½
EED
ë A AIT FATOR
 References
ë http:192.215.107.101ebn942techtechfocus1071main.html
ë http:www.chem.usu.edu~sbialklasses565opampsopam
ps.html
ë Skoog, |oller, and Neiman. rinciples of Instrumental
Analysis.. 5th ed. Orlando: |arcourt ½race & o., 1998.
Analysis
ë http:weather.nmsu.edu
ë http:elchem.kaist.ac.krvtchem-
http:elchem.kaist.ac.krvtchem-edseplchplc.htm
ë http:www.chemistry.nmsu.eduInstrumentation
qd_hroma.
html
ë http:weather.nmsu.eduTeaching_MaterialSOI
698Student
_Material|
| 1090|
INJ.|TM
ë http:test
http:test--
equipment.globalspec.com
earnMore
abware_Scientific_In
strumentsAnalytical_Instrumentshromatographs|
_ol
umns
ë http:www.chemistry.adelaide.edu.auexternalsoc
http:www.chemistry.adelaide.edu.auexternalsoc--
relcontentlc
relcontentlc--col.htm