Chromatography is a critical technique used to separate and analyze mixtures into their components, applicable in various fields such as forensics and pharmaceuticals. It includes different methods like adsorption, partition, and affinity chromatography, along with various formats such as paper and thin-layer chromatography. The document also discusses practical applications, experimental procedures, and analyses across different types of chromatography, demonstrating their utility in identifying compounds and optimizing separations.

1. Prepared By : Yahia Mohamed Reda
Benha university – faculty of science
Chemistry department

2. What is Chromatography?
Derived from the Greek word Chroma meaning colour,
chromatography provides a way to identify unknown compounds
and separate mixtures

3. What is Chromatography?
Chromatography is a technique for
separating mixtures into their components
in order to analyze, identify, purify,
and/or quantify the mixture or
components.
• Analyze
Separate • Identify
• Purify
Mixture Components
• Quantify

4. Applications of Chromatography
Forensics
Research
Pharmaceutical industry

5. Types of Chromatography…
Thin layer
Paper
HPLC Gas Column

6. •What Are The Branches of
Chromatography
According To Distribution
Between The Mobil & Stationary
Phases ??

7. 1- Adsorption Chromatography
 It Depends On The Ability Of Different Solutes
To be Adsorbed On The Surface Of The
Stationary Phase At Different Strengths
 It Can Be Found In GLC & LSC

8. 2-Partition Chromatography
 It Depends On The Difference in Solubility Of
Different Solutes In The Stationary Liquid Phase
 The Rate Of Separation Depends On The
Equilibrium Of The Solute Between The Mobil
Phase “Liquid “ & Stationary Phase ”liquid”

9. 3 - Ion Exchange Chromatography
 In this type of chromatography, the use of a resin
(the stationary solid phase) is used to covalently
attach anions or cations onto it. Solute ions of the
opposite charge in the mobile liquid phase are
attracted to the resin by electrostatic forces.

10. 4 - Molecular Exclusion
Chromatography
 Also known as gel permeation or gel filtration,
this type of chromatography lacks an attractive
interaction between the stationary phase and solute. The
liquid or gaseous phase passes through a porous gel which
separates the molecules according to its size. The pores are
normally small and exclude the larger solute molecules,
but allows smaller molecules to enter the gel, causing them
to flow through a larger volume. This causes the larger
molecules to pass through the column at a faster rate than
the smaller ones.

12. 5 - Affinity Chromatography
 This is the most selective type of chromatography
employed. It utilizes the specific interaction between one
kind of solute molecule and a second molecule that is
immobilized on a stationary phase. For example, the
immobilized molecule may be an antibody to some specific
protein. When solute containing a mixture of proteins are
passed by this molecule, only the specific protein is reacted
to this antibody, binding it to the stationary phase. This
protein is later extracted by changing the ionic strength or
pH.

14. •What Is The Main Types Of
Chromatography According To
The Kind Of Mobil & Stationary
Phases?

15.  1- Gas-Liquid Chromatography (GLC).
 2- Gas-Solid Chromatography (GSC).
 3- Liquid-Liquid Chromatography (LLC).
 4- Liquid-Solid Chromatography (LSC).

16. •What Are The Techniques
according To Chromatography
Red Shapes?

17. 1- Column Chromatography
 Column chromatography is a separation technique
in which the stationary bed is within a tube. The
particles of the solid stationary phase or the
support coated with a liquid stationary phase may
fill the whole inside volume of the tube (packed
column) or be concentrated on or along the inside
tube wall leaving an open, unrestricted path for the
mobile phase in the middle part of the tube (open
tubular column). Differences in rates of movement
through the medium are calculated to different
retention times of the sample

19. 2- Planar chromatography
 Planar chromatography is a separation technique in which the
stationary phase is present as or on a plane. The plane can be a
paper, serving as such or impregnated by a substance as the
stationary bed (paper chromatography) or a layer of solid particles
spread on a support such as a glass plate (
thin layer chromatography). Different compounds in the sample
mixture travel different distances according to how strongly they
interact with the stationary phase as compared to the mobile
phase. The specific Retention factor (Rf) of each chemical can be
used to aid in the identification of an unknown substance.

20. •Compare Between Paper & Thin
Layer Chromatography ?

21. 1 – Paper Chromatography
•separates dried liquid samples with a liquid
solvent (mobile phase) and a paper strip (stationary
phase)

22. Principles of Paper Chromatography
 Capillary Action – the movement of liquid within the spaces of a porous
material due to the forces of adhesion, cohesion, and surface tension. The
liquid is able to move up the filter paper because its attraction to itself is
stronger than the force of gravity.
 Solubility – the degree to which a material (solute) dissolves into a
solvent. Solutes dissolve into solvents that have similar properties. (Like
dissolves like) This allows different solutes to be separated by different
combinations of solvents.
Separation of components depends on both their solubility in the mobile
phase and their differential affinity to the mobile phase and the stationary
phase.

23. Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture Components
Components Affinity to Stationary Phase Affinity to Mobile Phase
Blue ---------------- Insoluble in Mobile Phase
Black  
Red  
Yellow          

25. Paper Chromatography Experiment
What Color is that Sharpie?

26. Overview of the Experiment
Purpose:
To introduce students to the principles and
terminology of chromatography and demonstrate
separation of the dyes in Sharpie Pens with paper
chromatography.
Time Required:
Prep. time: 10 minutes
Experiment time: 45 minutes
Costs:
Less than $10

27. Materials List
 6 beakers or jars
 6 covers or lids
 Distilled H2O
 Isopropanol
 Graduated cylinder
 6 strips of filter paper
 Different colors of Sharpie
pens
 Pencil
 Ruler
 Scissors
 Tape

28. Preparing the Isopropanol Solutions
• Prepare 15 ml of the following isopropanol solutions
in
appropriately labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%

29. Preparing the Chromatography
Strips
 Cut 6 strips of filter paper
 Draw a line 1 cm above the
bottom edge of the strip with
the pencil
 Label each strip with its
corresponding solution
 Place a spot from each pen on
your starting line

30. Developing the Chromatograms
 Place the strips in the beakers
 Make sure the solution does not
come above your start line
 Keep the beakers covered
 Let strips develop until the
ascending solution front is
about 2 cm from the top of the
strip
 Remove the strips and let them
dry

31. Developing the Chromatograms

32. Developing the Chromatograms

34. Observing the Chromatograms
0% 20% 50% 70% 100%
Concentration of Isopropanol

35. Black Dye
1. Dyes separated – purple and black
2. Not soluble in low concentrations of
isopropanol
3. Partially soluble in concentrations of
isopropanol >20%
0% 20% 50% 70% 100%
Concentration of Isopropanol

36. Blue Dye
1. Dye separated – blue
2. Not very soluble in low
concentrations of isopropanol
3. Completely soluble in high
concentrations of isopropanol
0% 20% 50% 70% 100%
Concentration of Isopropanol

37. Green Dye
1. Dye separated – blue and yellow
2. Blue – Soluble in concentrations
of isopropanol >20%
3. Yellow – Soluble in concentrations
of isopropanol >0%
0% 20% 50% 70% 100%
Concentration of Isopropanol

38. Red Dye
1. Dyes separated – red and yellow
2. Yellow –soluble in low concentrations of isopropanol and
less soluble in high concentrations of isopropanol
3. Red – slightly
soluble in low
concentrations
of isopropanol,
and more
soluble in
concentrations
of isopropanol
>20%
0% 20% 50% 70% 100%
Concentration of Isopropanol

39. Alternative Experiments
 Test different samples:
 Other markers, pens, highlighters
 Flower pigments
 Food Colors
 Test different solvents:
 Other alcohols: methanol, ethanol, propanol,
butanol
 Test different papers:
 Coffee filters
 Paper towels
 Cardstock
 Typing paper

40. Alternative Experiments

41. Alternative Experiments

42. Alternative Experiments

43. 2 - Thin Layer Chromatography
 Sample – marker
 Standard – food dyes
 Stationary phase – chromatography paper
 Mobile phase - water

44. Structures of E numbers…..
E122 pink
E110 yellow
E124 red E133 blue

45. So what will happen?
 Each dye will travel up the paper at different
speeds
 The speed depends on the solubility of the dye
in water and its interaction with the paper
 The dyes are all different molecules with
different characteristics

46. Calculation of results

47. Analysis
Calculation of results
You must now calculate an Rf value for each spot.
Rf = Distance from the start to the middle of a spot
Distance from start to finish point of the water

48. Conclusions – writing up
 One of the key elements of all scientific
experiments is to write up your results
 At the end of this experiment we would like
each person to conclude from the Rf values as
to which E numbers are contained in the
markers

49. Gas Chromatography
•Separates vaporized samples with a carrier gas (mobile
phase) and a column composed of a liquid or of solid beads
(stationary phase)

51. THE CHROMATOGRAPHIC PROCESS - PARTITIONING
(gas or liquid)
MOBILE PHASE
Sample
Sample
out
in
STATIONARY PHASE
(solid or heavy liquid coated onto a solid or support system)

52. GC Methods
Parameter Group Method Compounds
• SDW05.13000’s EPA 502.2 VOC’s
• SDW05.24000’s EPA 551.1 Cl-VOC’s/Pests
• WPP05.04000’s EPA 604 Phenols
• WPP06.03000’s EPA 625 SVOC’s
• SHW06.21000’s SW-846 8141A Org-P Pests

53. Instrumentation
Injection Port - Sample introduction
Manual - Direct Injection
Automated - Autosampler
- Purge and Trap

54. Instrumentation - Oven
Temperature Control
• Isothermal • Gradient
240
200
Temp (deg C)
160
120
80
40
0
0 10 20 30 40 50 60
Time (min)

55. Columns
• Packed
• Capillary

56. Polarity
Non-polar - +
Polar

57. Phases

58. Instrumentation - Detectors
Destructive
• Mass Spectral (CI/EI) [625]
• Flame Ionization (FID) [604]
• Nitrogen-Phosphorus (NPD) [8141A]
• Flame Photometric (FPD) [8141A]
• Electrolytic Conductivity (Hall/ELCD) [502.2]

59. Instrumentation - Detectors
Non-Destructive
• Thermal Conductivity (TCD)
• Electron Capture (ECD) [551.1]
• Photo Ionization (PID) [502.2]

60. Chromatograms - 551.1

61. Chromatograms - 604
Supelco® PTE-5 Supelco® SPB-50

62. m- Cresol

64. Properties of Selected Gas Chromatography
Detectors
Approximate Limit Approximate
Type Comments
of Detection (gs-1) Linear Range
Thermal conductivity Universal detector -measures
10-5-10-6 103-104
(TCD) changes in heat conduction
Universal detector -measures ion
Flame ionization (FID) 10-12 106-107
currents from pyrolysis
Selective detector for compounds
Electron capture (EC or
10 -14
10 -10
2 -3
containing atoms with high
ECD) electron affinities
Flame photometric Selective detector for compounds
10-13 102
(FPD) containing S,P
Selective for N,P containing
Nitrogen-phosphorus 10-8-10-14 105-107
compounds
Universal (some selectivity due to
Photoionisation (PID) 10-8-10-12 105
identity of gas in lamp)
Specific detector for compounds
Hall Detector 10-11 105
which contain halogen, S, or N
variable,
Mass spectrometer (MS) 10-12
depends on MS
Universal
Fourier-transform
10-10 102 Polar molecules
infrared (FTIR)

65. 1- Mass Spectrometry
 Molecular weight can be obtained from a
very small sample.
 It does not involve the absorption or
emission of light.
 A beam of high-energy electrons breaks the
molecule apart.
 The masses of the fragments and their
relative abundance reveal information about
the structure of the molecule.

66. Electron Impact Ionization
 A high-energy electron can dislodge an electron
from a bond, creating a radical cation (a positive
ion with an unpaired e-).
H H
H C C H
H H
H H H H
e- + H C C H H C C+ H
H H H H
H H
H C+ C H
H H

67. Separation of Ions
 Only the cations are deflected by the magnetic
field.
 Amount of deflection depends on m/z.
 The detector signal is proportional to the
number of ions hitting it.
 By varying the magnetic field, ions of all masses
are collected and counted.

68. Mass Spectrometer

69. The Mass Spectrum
Masses are graphed or tabulated according to their
relative abundance.

70. The GC-MS
A mixture of compounds is separated by gas
chromatography, then identified by mass
spectrometry.

71. High Resolution MS
 Masses measured to 1 part in 20,000.
 A molecule with mass of 44 could be C3H8,
C2H4O, CO2, or CN2H4.
 If a more exact mass is 44.029, pick the correct
structure from the table:
 C3H8 C2H4O CO2 CN2H4
 44.06260 44.02620 43.98983 44.03740

72. Molecules with Heteroatoms
 Isotopes: present in their usual abundance.
 Hydrocarbons contain 1.1% C-13, so there will
be a small M+1 peak.
 If Br is present, M+2 is equal to M+.
 If Cl is present, M+2 is one-third of M+.
 If iodine is present, peak at 127, large gap.
 If N is present, M+ will be an odd number.
 If S is present, M+2 will be 4% of M+.

73. Isotopic Abundance
81
Br

74. Mass Spectrum with Sulfur

75. Mass Spectrum with Chlorine

76. Mass Spectrum with Bromine

77. Mass Spectra of Alkanes
More stable carbocations will be more abundant.

78. Mass Spectra of Alkenes
Resonance-stabilized cations favored.

79. Mass Spectra of Alcohols
 Alcohols usually lose a water molecule.
 M+ may not be visible.
=>

80. 2- Flame Ionisation Detector (FID)
Destruction of combustible sample in flame produces measurable current
•Elute is burnt in a mixture of H2 and air
•Electrical conductivity of a gas is
directly proportional to the
concentration of charged particles
within the gas
•It responds to most hydrocarbons
Compounds that give no response
include:
air, water, inert gases, CO, CO2,
CS2, NO, SO2 and H2S
• Very sensitive detector with a
wide linear range
• Excellent detector for
quantitative trace analysis

81. Thermal Conductivity cell (TCD)
(most common in the past)
•Thermal conductivity measures the ability of a substance to transport heat
from a hot region to a cold region
•Differences in the thermal conductivity of gasses are based on the mobility
•The smaller the molecule the higher the mobility and thermal conductivity
•Helium is the carrier gas commonly used
•Simple and universal
•Respond to all analytes
•104 linear response range
•Not sensitive to detect minute quantities
•Mostly used with tubular columns with
>0.053 mm in diameter or packed
columns
•Sensitivity increases with:
Increasing filament current
Decreasing flow rate
Lower detector block temperature

82. Other detectors
Nitrogen-phosphorus detector
Modified Flame Ionization detector. Especially sensitive to N and P, 10 4-
106 greater response than to carbon. Important for the analysis of drugs,
herbicides and pesticides
Sulfur chemiluminescence detector
Sulfur is oxidized to SO during ionization and converted to blue light
emitting SO2 by reaction with ozone. Intensity of emission is proportional to
the mass of S eluted. The response to Sulfur is 107 greater than to carbon
A nitrogen chemiluminescence detector works in analogous
manner, combustion of eluent converts to NO which reacts with O 3 to form
a chemiluminescent product. Response to N is 107 greater than to carbon
Atomic emission detector
Detector can be set to observe almost any element in each analyte as
it emerges through the column.

83. Detectors for HPLC

84. Desirable characteristics of detectors
•High sensitivity
•Negligible baseline noise
•Large linear response range (analyte concentration range over
which detector response is proportional to concentration)
•Insensitive to temperature changes and solvent composition
•Universality or predictable specificity
•Low dead volume
•No sample destruction
•Stability over time
•Reliable
•Inexpensive to produce and continuous operation
•Capable of providing information on solute identity

85. Characteristics of Selected Liquid
Chromatography Detectors
Approximate Limit Approximate
Type Comments
of Detection Linear Range
Ultraviolet and visible Specific for light-absorbing
10-11 g 104
absorption compounds
Universal detector -measures
Differential Refractive
10 -10
-9 -10
g 103
changes in refractive index.
Index Cannot be used with gradients
Electrochemical Specific detector. Compound
10-10-10-11 g 105
Amperometric must be electroactive
Electrochemical
10-8 g/mL 105 Specific detector, but for all ions
Conductometric
Specific detector. Compound
Fluorescence 10-14 g 105
must be fluorescent
Universal detector. Also can be
Mass Spectrometry 10 -10-7 -9
105
used to identify analytes with
great certainty
Used to determine MW’s of
Solution Light polymers as they elute.
10-6 g/mL 105
Scattering Concentration usually far above
limits of detection
Evaporative Light Universal except for volatile
10-9 g 106
Scattering analytes. Not a linear response

86. HPLC Detectors respond to:
Solute property not Bulk property that Direct
exhibited by MP changes with solute
eluted solute detection
UV/VIS (0.1-1 ng)
•Fixed Refractive Mass
•Variable (VWD) Index Spectrometry
•Photodiode array (DAD) (100-1000 ng) (1-1000 pg)
FTIR (1mg) Conductivity ELSD
(500-1000 ng) (100-1000 pg)
Electrochemical (10-1000 pg)
•Amperometric
•Coulobmetric
Fluorescence (1-10 pg)
Requires fluorophore

87. UV-vis Light Absorption Detectors
•Most common HPLC detector
•Many solutes absorb ultraviolet (UV) light
•Most employ the most intense 254 nm
(emission of a mercury lamp)
Light
•Modern HPLC instruments have the source Detector
capability to choose the appropriate wave
length for a given analyte
•Use of photodiode arrays which can record
the entire UV region at once in a fraction of a
second (spectrum of each solute as it is
eluted)
•Full scale absorbance range 0.0005-3 absorbance units
•Linear range 5 orders of magnitude of solute concentration
•Good for gradient elution

88. Refractive Index (RI) Detector
• Light passes through the cell
and is directed to the photocell
by the deflection plate. When
solute with a different RI enters
the cell, the beam is deflected
and the photocell output
changes.
• Respond almost to every solute but sensitivity is lowered by a factor
of 1000 compared to a UV detector
• Sensitive to changes in pressure and temperature
• Unsuitable for gradient elution due to problem of matching sample
and reference while the composition is changing
• Its primary appeal is due to a nearly universal response to all solute
including those with little UV absorption

89. Electrochemical Detector
•Respond to analytes that can be
oxidized or reduced
•Potential is maintained at a
selected value with respect to
Ag/AgCl reference electrode and
current is measured between the
working and counter electrodes.
• Current is proportional to the concentration of
solute over 6 orders of magnitude
• Oxygen free aqueous or polar solvent containing
dissolved electrolyte are required.
•Very sensitive to fluctuations of temperature and the flow rate

90. Electrochemical Detector
Pulsed electrochemical detection of alcohols in an ion exchange column with
0.05M HClO4
Peaks: 1, glycerol; 2,ethylene glycol; 3, propylene glycol 4, methanol; 5,
ethanol; 6, 2-propanol; 7, 1-propanol; 8, 2-butanol; 9, 2-methyl-1-butane; 10,
1-butanol; 11, 3-methyl-1-butanol; 12, 1-pentanol; 13, cyclohexanol; 14,
diethyleneglycol.

91. Evaporative Light-Scattering
Detector (ELSD)
•Responds to any solute which is less volatile than the MP
•Response is related to mass of material (large peak = more
material in contrast to UV detectors peak intensity
is related to how well the solute absorbs UV)
•Response is non linear, so polynomials are used to
construct calibration curve
•Compatible with gradient elution. No peaks associated
with the solvent front, so less interference from
early eluting peaks

92. Evaporative Light-Scattering
Detector (ELSD)

93. Step 1: Nebulization
• Column effluent passes
through nebulizer
needle
• Mixes with nitrogen gas
• Forms dispersion of
droplets

94. Step 2: Mobile Phase Evaporation
• Droplets pass through a
heated zone
• MP evaporates from the
sample particle
• Dried sample particles remain

95. Step 3: Detection
• Sample particles pass through
an optical cell
• Sample particles interrupt
laser beam and scatter light
• Photodiode detects the
scattered light

96. ELSD vs RI

97. ELSD vs UV
• Obtain a more accurate
representation of
sample mass than UV
• See what may be missing
from the UV
chromatogram
• Detects compounds without
chromophores

98. Conclusion: Is ELSD the Ideal Detector?
Advantages Disadvantages
•Universal: Detect any • Destructive
compound less volatile than the
MP • Compatible with isocratic
and gradient elution but NOT
•Very Sensitive: Typical buffers/salts.
detection limits 0.1-1 ng.
•The response is proportional
to the analyte concentration,
and not affected by solvent
properties
•Reliable and easy to use

99. Mass Spectrometer
pump
column
Mass
spectrometer
RT:
10.00 - 15.12
100 847.42 Chromatogram
852.75
80
60
877.43
40 766.40
20
0
1270.60
40
Relative Abundance
35
30 847.41
25
20
2541.21
15
827.45
10
1140.61 2390.83
5 1011.57 1400.61 2169.06
797.08 1625.78 1842.88
0
1000 1500 2000 2500
m/z

100. Important Definitions
 K partition coefficient :- defined as the molar concentration of
analyte in the stationary phase divided by the molar concentration
of the analyte in the mobile phase.
 retention time (tR ) : The time between sample injection and
an analyte peak reaching a detector at the end of the column

101.  Retention factor, k' : is often used to describe the migration
rate of an analyte on a column. You may also find it called
the capacity factor. The retention factor for analyte A is defined as;
k'A = (t R - tM) / tM
 Selectivity factor, a :
which describes the separation of two species (A and B) on the column;
a = k 'B / k 'A
When calculating the selectivity factor, species A elutes faster
than species B. The selectivity factor is always greater
than one.

102. Contact me
Dr.yahia1@yahoo.com
www.facebook.com/yahia.reda