chromatography
Chromatographic Process
Chromatographic separations are based on transporting
the liquid carrying the analyte mixture (mobile ph. )
through the porous media (stationary ph.) and the
differences in the interactions of analytes with the
surface of this porous media resulting in different
migration times (retention times) for a mixture
components.
Mobile
phase
Stationary
phase
Analyte
Provides the analyte transport.
Immobile phase.
Mixture of components dispersed in the mobile
phase.
Classification
The nature of the stationary and the mobile phases,
with the mode of the transport through the column, is
the basis for the classification of chromatographic
methods.
HPLC system
Solvent Reservoirs:
Storage of HPLC solvents (mobile phase) and could be
equipped with an online degassing system and special
filters.
pump
Provides the constant and continuous flow of the mobile
phase through the system. Modern pump allow mixing
of different solvents from different reservoirs.
Injector:
Allows the introduction (injection) of the analytes
mixture into the stream of the mobile phase before it
enters the column. Modern injectors are autosamplers.
Column:
It is a device that hold a stationary phase in place, allowing
the mobile phase to carry an injected sample through and
allowing analytes to interact with available surface.
Guard column:
A short column introduced before the analytical
column to increase its life by removing particulate matter
and contaminants.
Detectors:
A device for continuous registration of specific physical
properties of the column effluent
Stationary phase
 It is the column packing material that are the media
producing the separation.
 The properties of this media are important for
successful separations.
Qualitatively
by?
chromatogram
Quantitivly
by ?
Use HPLC to separate the compounds physically
HPLC parameters
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Dead volume.
Retention volume (VR).
Retention time (tR).
Void time (t₀).
Capacity factor (K).
Selectivity (ᾳ)
Efficiency.
Resolution (R).
Peak symmetry factor (s).
Dead volume: Volume of mobile phase inside the column .
 V₀= V column – V particle + V pores.
 =0.65 (µD²L/4) where dead volume equal 65% of empty
column volume
 Retention volume (VR).
Total volume of eluent (in ml) require to elute certain
substance.
VR= tR * F
 Retention time (tR).
Time from injection point to maximum detector response
for corresponding compound.
 Void time (t₀).
Time require for non retaine substance to pass through
the column.
 Capacity factor (K)
Use to describe the migration rate of solutes on columns.
K=(tR -t₀ )/t₀
Recommended to be 2-10
 Selectivity (ᾳ)
Use to describe the separation of band centre
ᾳ =K2/K1 should be > 1.2
 Efficiency.
It is measured in number of theoretical plates.
 N= 16 ( tR/w)².
 H=L/N
*H plate height.
*column length.
 Resolution (R).
R = 2(t R2 –t R1) / (w2 +w1)
Should be 1.5
 Peak symmetry factor (s)
problems
1-On given 200 cm column the sample required 4.80
min. to emerge, an air bubble required 0.5 min. ,and the
time required for the sample pass the detector was
0.40min. Calculate N and platelet height?
2- the adjusted retention distance for a lipid sample on HPLC
chromatogram with a 1.5m column was 59.6 cm. the
recorder was calibrated at 3.60cm/min. the width of the peak
at the base was 12.0 cm calculate N?
The following data apply to a column for a lipid chromatography:
Length of packing
Flow rate
Vm
Vs
24.7 cm
0.313ml/min.
1.37ml
0.164 ml
A chromatogram of a mixture of species A,B,C,D provided the
following data;
Retention time
(min)
Width of peak
base
(w)(min)
Non retained
3.1
-
A
5.4
0.41
B
13.3
1.07
C
14.1
1.16
D
21.6
1.72
Calculate
1- the number of plates from each peak?
2- the plate height for the column?
From the previous data calculate for A,B,C, and D:
- the capacity factor?
Calculate for B and C:
-the resolution?
-the selectivity factor?
Calculate for C and D:
- The resolution?
 Quantitative method.
 Using external standard method (more basic and common) .
 AUC is directly relashion to the concentration.
 It calculated by calibration curve or by matching point.
Procedure:
Stock standard :
-weight 0.1 g of pure paracetamol powder and put it in 100 ml
volumetric flask and complete it with deionized water.
-From stock take 1 ml and put it into 100 ml volumetric flask and
complete it with deionized water and called it solution A.
-From solution A prepare serial dilution 2,4,6,8,10 ml in 25 ml
volumetric flask and complete it with mobile phase.
Mobile phase:
25% acetonitrate +75% acetic acid 1%
Sample preparation:
-From bottle take 1 ml ( contain 100mg) and put it in 100 ml
volumetric flask with deionized water and called it B.
-from B take 1ml and put it in 100ml volumetric flask and complete
it with deionized water and called it C.
- From C take 5 ml put it in 25 volumetric flask and complete it by
mobile phase.
Calculation: