Chromatography

• Chromatography is a separation
technique in which sample A = polar B = non-polar

components distributes themselves

between two immiscible phases to Mobile phase
hexane
varying degrees. Consequently, they Non-Polar
move at different rates and then
separated. Sample mixture
A+B

Steps of Chromatographic analysis….

Stationary phase =
1- Separation Silica gel or CaCO3
2- qualitative or quantitative analysis Polar

Always Remember the general rule;

“Like dissolves like” …. Or ….
“Like attracts like”
 1 2 3 4 5

Elution order of compounds: 1-B, 2-A

Stationary phase = Silica gel (Polar)

Normal phase-Liquid
Mobile phase = Hexane (non-polar) Chromatography
S. Phase is more polar than M. phase NP-LC
Example: What is the elution order of
the compounds A, B and C from the Mobile phase
Methanol + Water
charcoal column using methanol + water
Polar
as a mobile phase.

Stationary phase
= Charcoal
Non-Polar
A B C

Reversed phase-Liquid
Chromatography
Remember, “Like attracts like” RP-LC

B (non-polar) will be delayed because it is more strongly attracted to

the stationary phase. C (polar) will move faster because it is more
soluble in the mobile phase.

Elution order; 1 = C, 2 = A, 3=B

Plate Theory
The column can be visualized as being divided into a number of imaginary sections
called plates.
N = number of theoretical plates.
H = Height of theoretical plate. (a) (b)
L = Column length in cm

If peaks are not well separated…

increase column efficiency by ….

1- increasing column length (L). … 

(not practical …)
2- decreasing the particle size ….
(i.e., decreasing the H).
The smaller particle size results in an increased back pressure. Therefore, MP will not
move down the column by gravity. ….  Pumps should be used to push the MP
throughout the column!! ….    HPLC
High Performance Liquid Chromatography (HPLC)

Why HPLC is so Popular?

Sensitive
Accurate
Separation of non-volatile components, and heat unstable
compounds.
Non-destructive

Sample Detector
Pump Column

Injector
MP Readout

https://www.youtube.com/watch?v=MLoitPJQH3g
A chromatogram is essentially the output of a
chromatography run. It is an electronic file or hardcopy
containing the information generated during the
chromatography run.

mV
30 B
tr=3.5
W= 0.5 • tr = Retention time: is the time
A
tr=2.0 between the sample injection and the
20 W= 0.4
detector response of the compound.
Peak
Area • to = Column dead time or solvent
Peak
10 Height peak (Time required for an unretained
solute).
to= 1.2
Wb = Peak width at base line
PH= Peak Height
PA = Peak Area
0 1 2 3 4
Retention time, (min)
Common Relationships in Chromatography
•Capacity Factor, k', is often used to describe the migration rate of an analyte on a
column. Ideally, the retention factor for an analyte is between 1 –5.
• Column Selectivity (α): Selectivity represents the separation power of particular
adsorbent to the mixture of this particular components.
• Resolution (Rs): Resolution is the parameter describing the separation power.
If Rs = 1 then components are completely separated.
If R < 1, then components are overlapped.
If R = 1.5, Excellent baseline resolution is achieved

 tr 
2
t R  t0
N  16   k'
 Wb  t0

t 2  t o k '2 2 [t B  t A ]
  Always,  1 R
t1  to k '1 WA  WB
Example: Calculate NA, NB, , average N, k’A, k’B, α and R for
the compounds A and B in the previous chromatogram.

2
 tr 
2
 2 
NA  16    16    400 NB  784 Naverage  592
 Wb   0.4 

t A  t0 2  1.2 3.5  1.2

kA'    0.67 kB '   1.92
t0 1.2 1.2

tB  to 3.5  1.2 kB ' 1.92

   2.87 or    2.87
tA  t o 2  1.2 kA' 0.67

2 [t B  t A ] 2 [3.5  2.0]
R   3.33
WA  WB 0.4  0.5
 Qualitative and Quantitative Analysis
1) Qualitative Analysis by HPLC Ground water sample /extract

What compounds are present??

4.25

1- Determine the retention time (tr) by injecting

pure analyte. 5.23
4.67
2- Compare retention times ….

Example: The chromatograms for a ground water

extract and for the pure fungicide
4.25
epoxyconazole.
Pure epoxyconazole

By comparing the retention times, … we can

conclude that the ground water contains the
fungicide epoxyconazole.
 C X CStaandard
Quantitative Analysis 
For quantitative analysis, use either peak height or peak area. AX AS tan dard
The simplest way of calculation is to use single external standard.
Example: The chromatograms below are for a sample containing unknown amounts of
A and B and for a standard containing known amounts of A and B. Calculate the
concentrations of A and B in the unknown sample.
mV B, mV A
30 tr=3.5 30 tr=2.0
B,
A tr=3.5
tr=2.0 Standard:
20
CA= 4 ppm 20 Unknown
CB = 4 ppm sample
10 10
to= 1.2 to= 1.2

0 1 2 3 4 0 1 2 3 4
Retention time, (min) Retention time, (min)

CS 4
Cx  x Ax ....  CA  x 28  6.2 ppm
AS 18
4
CB  x 22  3.1ppm
28
 Classification of chromatographic techniques
(according to mobile phase)

Chromatography

Liquid Chromatography (LC) Gas Chromatography (GC)

-Thermally unstable compounds -Thermally stable compounds
- Non-volatile compounds -Volatile or can be volatilized

 The general principle of chromatography and the mathematical

relationships found for LC are applicable to GC (with only minor
modifications related to compressibility of the gas).
 Only 10-20% of the known compounds can be analyzed by GC.
 All forms of GC are more efficient in many respects than LC.
 Instrumental components of GC

He
Or
N2
…

The carrier gas (inert gas) serves as the mobile phase that elutes the
components of a mixture from a column containing the stationary phase. In
contrast to most other types of chromatography, the mobile phase dose not
interact with molecules of the analytes.

https://www.youtube.com/watch?v=iX25exzwKhI
https://www.youtube.com/watch?v=939N9JFQXYY
 Gas Chromatography Columns: Packed and Capillary

a) wall-coated open tubular (capillary) column (WCOT)

b) packed column
 Packed
PackedGCGC Capillary
Capillary GCGC column
column
Packed GC Capillary GC column
Packed GC
column
column
column
column

Packed
Capillary
Gas Chromatography Columns

1. Packed GC Columns
- 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
2. Capillary columns
- 10-50 m length, internal diameter of a few mm

Column temperature

• Column temperature must be controlled.

• The optimum column temperature is dependant upon the boiling
point of the sample.
• As a rule of thumb, a temperature slightly above the average
boiling point of the sample results in an elution time of 2 - 30
minutes.
• If a sample has a wide boiling range, then temperature
programming can be useful. The column temperature is increased
as separation proceeds.
How to solve the general elution problem in GC? …. Use temperature
 Gas Chromatography (GC) Detectors
• After the components of a mixture are
separated using gas chromatography, they
must be detected as they exit the GC column.
The requirements of a GC detector depends
on the separation application. For example,
one analysis might require a detector that is
selective for chlorine-containing molecules,
another analysis might require a detector that
is non-destructive so that the analyte can be
recovered for further spectroscopic analysis.
 Thermal Conductivity Detector (TCD)

 Thermal conductivities of He, H2 are much larger than organics.

Organics cause temperature rise in filament
 When a compound elutes, the thermal conductivity of the gaseous
mixture of carrier gas and compound gas is lowered, and the filament
becomes hotter.

Advantages:
• Responds to all compounds
• Wide dynamic range (105)
• Nondestructive
• Simple construction

Disadvantage: He
or
low H2

sensitivity
 Flame-Ionization Detector (FID)

• An FID consists of a
hydrogen/air flame and a + +-- +
- +-
collector plate. The effluent +
…. mV CH + O  CHO+ +
from the GC column passes e-
-
through the flame, which
breaks down organic
molecules and produces ions. Air H2
The ions produce an electrical
signal. The FID is extremely
sensitive with a large dynamic
range. Its only disadvantage is
that it destroys the He

compounds
 Flame-Ionization Detector (FID)

• The FID is a mass sensitive detector. The more CH-

groups a molecule contains, the larger is the detector
response. Molecules without CH-groups do not deliver
a signal. The FID is therefore not universal, as e.g.
H2O, NOx, CS2, CCl4, etc. are not or poorly detected.

• Sensitive (10-13 g/s), especially for hydrocarbons …

• Destructive (-)
 Electron Capture Detector (ECD)
• The ECD uses a radioactive Beta emitter (electrons) to ionize some
of the carrier gas and produce a current between a pair of
electrodes. When organic molecules that contain electronegative
functional groups, such as halogens, phosphorous, and nitro groups
pass by the detector, they capture some of the electrons and reduce
the current measured between the electrodes.

Radioactive + + + + + Electrode
-emitter Insulator
• The ECD is as sensitive
- - - -
as the FID but has a - Electrode
limited dynamic range
and finds its greatest
application in analysis N2

of halogenated
compounds.
Electron Capture Detector

 63Ni (β-emitter) is used to ionize the carrier gas.

 Organic molecules capture electrons and decrease current
 N2 (Carrier gas) +  (e-) = N2+ + 2e-
 The N2+ and e- establish a “base line”
 halogenated compounds X (F, Cl and Br) collect
electrons
 The “base line” due to the e- will decrease and this
decrease the signal. Insecticides, pesticides, and
fluorocarbons
Characteristics
• Simple and reliable
• Sensitive (10-15 g/s) to electronegative groups
• Non-destructive
 ECD vs. FID

Note the different selectivity