Chromatographic techniques

Principle and Applications of
Chromatographic Techniques
TEJASVI BHATIA

ORIGIN
Chromatography (from Greek word) “Chroma” ( colour) and
“Grafein" (to write) collective term-: laboratory techniques for the
separation of mixtures.
Russian botanist Mikhail Semyonoyich Tsvet invented the first
chromatography technique in 1900 during his research on
chlorophyll.
The method was described on December 30, 1901 at the 11th
Congress of Naturalists and Doctors in St. Petersburg.
Chromatography basically involves the separation of mixtures
due to differences in the distribution coefficient of sample
components between 2 different phases.

Principle of Chromatography
When a gas / vapour comes in contact with an absorbent,
certain amount of the gas gets absorbed on the solid surface.
The phenomenon takes place according to the laws of
Freaundlich (x/m =kC1/n) or Longmuir (x/m=k1C+k2C)
where x is the mass of the gas or vapour sorbent in mass of the sorbent and
C is the vapour concentration in the gas phase and k, k1 & k2 are constants.
Similarly if the vapour, gas and a compound comes in contact
with a liquid , a fixed amount of compound gets dissolved in the
liquid.This phenomenon is known as Henry’s Law of partition
x/m= kC.

Chromatography terms
 Chromatogram: The visual output of the
chromatograph. In the case of an optimal
separation, different peaks or patterns on the
chromatogram correspond to different components
of the separated mixture.
 A chromatograph- Equipment that enables a
sophisticated separation (Gas chromatographic or
liquid chromatographic separation)

Mobile phase- The mobile phase consists of the sample
being separated/analyzed and the solvent that moves the
sample through the column.
In case of HPLC the solvent is mobile phase which carry the
analytes. The mobile phase moves through the
chromatographic column (the stationary phase) where the
sample interacts with the stationary phase and is separated.
Phase which moves in a definite direction.
In case of GLC the mobile phase is gas.
 Stationary phase- The substance which is covalently bonded to
the support particles or to the inside wall of the column tubing
in chromatography procedure. e.g. silica layer in thin layer
chromatography.
 The effluent - The mobile phase leaving the column.

 Sample- The matter analysed in chromatography. It may consist of
a single component or mixture of components.
 Solute- Refers to the analytes present in sample during partition
chromatography.
 Analyte- Substance that is to be separated during
chromatography.
 Solvent- Refers to any substance capable of solubilizing other
substance.

 Analytical chromatography- To determine the existence/
presence and the concentration of analyte(s) in a sample.
 Preparative chromatography- Preparative chromatography is
used to isolate and purify the compounds and collect the
sufficient quantities of a substance for further use, rather than
analysis.
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile.
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >Cyclohexane.

It is a characteristic of particular compound. Time taken for a
particular analyte to pass through the system (from the column
inlet to the detector) under set conditions.
Retention time (RT)

It is a quantitative indication of how far a
particular compound travels in a particular
solvent.
The retention factor( Rf) = distance the solute (D1)
moves / the distance traveled by the solvent front
(D2)
Rf = D1 / D2
where
D1 = distance that color traveled, measured from
center of the band of color to the point where the
color was applied.
D2 = total distance that solvent traveled.
Retention Factor (Rf)

Important Parameters
Capacity factor (k')
It is a measurement of the retention time of a sample
molecule, relative to column dead volume (V0)
k' =
V1-Vo
Vo
Where:
k' = Capacity Factor of the column
Vo =Void volume ( or dead volume) of the column
(volume at which an unretained component elutes)
V1 = Retention volume of peak 1

Capacity factor (k') depends upon
 Solvent polarity
 Composition
 Purity
 Temperature
 Column Chemistry
 Sample

Column Efficiency (N)
The Column Efficiency (N) (also called theoretical plate count ) is a measure of the
band spreading of a peak .
Smaller the band spread higher the number of theoretical plates which
indicates good column and column efficiency
Or
Larger the value of N is for a column, better separation of two compounds.
- N is independent of solute retention
- N is dependent on the length of the column
Poor column efficiency due to:
 Age and history of the column.
 Extra column band broadening (such as due to malfunctioning injector or
improper tubing ID)
 Inappropriate detector setting .
 Change in flow rate and solvent viscosity.

Methods of measuring column efficiency (N)
5 Sigma 4 Sigma
Tangent 3 Sigma
½ Height 2 Sigma (inflection)
N =  Vr
W
( )
2
W  Method
W1 4 inflection(2)
Wh 5.54 ½ Peak height
W3 9 3
W4 16 4
W5 25 5
Wtan 16 Tangent
Wasymmetry 10 Asymmetry-based
 = constant dependent on high
where peak width measured
Vr= retention time
W= peak width

Resolution is distance between the peak centre of the
two components peaks divided by the average base
width of the peaks.
Resolution (Rs)
Rs = V2 - V1
½(W1+W2
)

Factors affecting the Resolution
 Increase column length
 Decrease column diameter
 Decrease flow-rate
 Uniformly packed Column
 Use uniform stationary phase (packing material)
 Sample size
 Select proper stationary phase
 Select proper mobile phase
 Use proper pressure
 Use gradient elution

Why Do Bands Spread?
a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion

a) Eddy diffusion – a process that leads to peak broadening due to
the presence of multiple flow paths through a packed column.
As solute molecules travel through the column, some arrive at
the end sooner then others simply due to the different path
traveled around the support particles in the column that result in
different travel distances.

A solute in the center of the channel moves more quickly than
solute at the edges, it will tend to reach the end of the channel
first leading to band-broadening
The degree of band-broadening due to eddy diffusion
and mobile phase mass transfer depends mainly on:
1) Size of the packing material
2) Diffusion rate of the solute
b.) Mobile phase mass transfer –caused by the presence of different
flow profile within channels or between particles of the column.

C.) Stagnant mobile phase mass transfer – band-broadening due to
differences in rate of diffusion of the solute molecules between the mobile
phase outside the pores of the support (flowing mobile phase) to the mobile
phase within the pores of the support (stagnant mobile phase).
Since a solute does not travel down the column
when it is in the stagnant mobile phase, it spends
a longer time in the column than solute that
remains in the flowing mobile phase.
Stagnant mobile phase mass transfer depends on:
1) Size, shape and pore structure of the packing material
2) Diffusion and retention of the solute
3) Flow-rate of the solute through the column

d.) Stationary phase mass transfer – band-broadening due to the movement of
solute between the stagnant phase and the stationary phase.
Since different solute molecules spend different lengths of time in the
stationary phase, they also spend different amounts of time on the
column, giving rise to band-broadening.
Stationary phase mass transfer depends on:
1) the retention and diffusion of the solute
2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and
the stationary phase

e.) Longitudinal diffusion – band-broadening due to the diffusion of the
solute along the length of the column in the flowing mobile phase.
Longitudinal diffusion depends on:
1) Diffusion of the solute
2) Flow-rate of the solute through
the column

The baseline is any part of the chromatogram where only mobile phase is
emerging from the column.
The peak maximum is the highest point of the peak.
The injection point is that point in time/position time when/where the
sample is placed on the column.
The dead point is the position of the peak-maximum of an unretained solute.
The dead time (to) is the time elapsed between the injection point and the
dead point.
The dead volume (Vo) is the volume of mobile phase passed through the
column between the injection point and the dead point.
Thus, Vo = Qto where Q is the flow rate in ml/min.
Terms of Chromatography

The retention time (tr) is the time elapsed between the injection point and the
peak maximum. Each solute has a characteristic retention time.
The retention volume (Vr) is the volume of mobile phase passed through the
column between the injection point and the peak Maximum.
Thus, Vr = Qtr where Q is the flow rate in ml/min. (Each solute
will also have a characteristic retention volume.)
The corrected retention time (t'r) is the time elapsed between the dead point
and the peak maximum.
The corrected retention volume (V'r) is the volume of mobile phase passed
through the column between the dead point and the peak
maximum. It will also be the retention volume minus the dead
volume. (Thus,V'r =Vr -Vo = Q(tr - to) where Q is the flow rate in
ml/min.)

The peak height (h) is the distance between the peak maximum and the base
line of the peak.
The peak width (w) is the distance between each side of a peak measure at
0.6065 of the peak height (ca 0.607h).
The peak width at half height (w0.5) is the distance between each side of a
peak measured at half the peak height.The peak width measured at half
height has no significance with respect to chromatography theory.
The peak width at the base (wB) is the distance between the intersections of
the tangents drawn to the sides of the peak and the peak base geometrically
produced.The peak width at the base is equivalent to four standard
deviations (4s) of the Gaussian curve and thus also has significance when
dealing with chromatography theory.

1. Liquid/Solid Chromatography (adsorption chromatography)
Liquid-solid: adsorption on solid which is generally polar (cellulose, poly
amides) or (silica gel, alumina, magnesium silicates).
2. Liquid/Liquid Chromatography (partition chromatography)
Liquid-liquid partition between 2 bulk phases (immiscible solvents)
3. Ion Exchange Chromatography Specific interactions with ionic species
(change relative strengths of acid or base)
4. Gel Permeation Chromatography (exclusion chromatography)
5. Affinity Chromatography.
Types of Chromatography

Liquid Solid Chromatography- The separation mechanism
in LSC is based on the competition of the components of the mixture sample for
the active sites on an absorbent such as Silica Gel. (Paper, TLC)
Liquid-Liquid Chromatography- The stationary solid
surface is coated with a liquid (the Stationary Phase) which is immiscible in
the solvent (Mobile) phase. Partitioning of the sample between 2 phases
delays or retains some components more than others to effect separation.

Ion-Exchange Chromatography
In Ion-exchange Chromatography the separation is based on
the competition of different ionic compounds of the sample
for the active sites on the ion-exchange resin (anion or cation
resins) (column-packing).
Low salt High salt

Ion Exchange Chromatography
Applied to
 Biochemical drugs, their metabolites,
amino acid, proteins, and peptide
 Food preservatives
 Vitamin
 Mixtures of sugars
 Pharmaceutical preparation
 Purification of proteins

Gel-Permeation Chromatography is a mechanical
sorting of molecules based on the size of the
molecules in solution.
Small molecules are retained longer time than large
molecules.
Gel-Permeation Chromatography

Separation mechanism is sieving not partitioning.
Technique applicable for the separation of high-molecular weight
species (proteins and polymer).
Solute and solvent molecules can diffuse into pores / trapped and
removed from the flow of the mobile phase.
 Stationary phase are porous silica or polymer particles
polystyrene, polyacrylamide) (5-10 mm) is used.
Large molecules not retained in the pores and elute first.
Small molecules permeate into pores strongly retained, eluted last
Gel Permeation Size Exclusion

Advantages
 Short & well-defined separation times.
 Narrow bands spread & good sensitivity.
 Solutes do not interact with the stationary phase.
Disadvantages
 Only limited number of compounds can be separated
because the time scale of the chromatogram is short.
 Inapplicability to samples of similar size, such as
isomers.
 At least 10% difference in molecular weight is required
for reasonable resolution.

It is generally a low resolution chromatography
technique and thus it is often reserved for the final,
"polishing" step of a purification.
It is also useful for determining the tertiary structure
and quaternary structure of purified proteins.
Application

Affinity chromatography
 Interaction of the solute with an immobilized affinity ligand.
 A washing step, where non-bonded solutes are rinsed off.
 In elution step, where the selectively attached molecules are
recovered by elution with a competing substrate, or by applying
conditions which alter the conformation of the attached
macromolecule.

Applications
 In the field of biochemistry for the separation of bio
macromolecules from biological compounds.
For example, S. Loukas and colleagues studied the existence of one type of suspected
opiate receptor in the brain (1994). They separated the (opioid binding protein by using an
opioid receptor antagonist that was specific to the (-opioid binding protein. These studies
may one day lead to a better understanding of how drugs such as opium affect our brains.
 Affinity chromatography can be used to determine
dissociation constants of ligands and molecules.

 Paper chromatography.
 Thin layer chromatography.
 Gas liquid chromatography.
 High performance liquid chromatography.
 Gas chromatography- Mass spectroscopy.
Common Chromatography

Paper Chromatography
Paper chromatography is a technique that
involves placing a small dot of sample solution
onto a strip of chromatography paper.
Different compounds in the sample mixture travel
different distances according to how strongly they
interact with the paper.
The paper is placed in a jar containing a shallow
layer of solvent and sealed. As the solvent rises
through the paper it meets the sample mixture
which starts to travel up the paper with the solvent.
This allows the calculation of Rf value and can be
compared to standard compounds to aid in the
identification of an unknown substance

Thin layer chromatography
Thin layer chromatography (TLC) is a widely-employed
laboratory technique and is similar to paper
chromatography.
However, instead of using a stationary phase of paper, it
involves a stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert substrate.
Compared to paper, it has the advantage of faster runs,
better separations, and the choice between different
adsorbents.
Different compounds in the sample mixture travel
different distances according to the interaction with the
adsorbent. This allows the calculation of an Rf value and
can be compared to standard compounds to aid in the
identification of an unknown substance.

Gas Liquid Chromatography
 The concept of gas-liquid was first elucidated in 1941 by Martin and
Synge, who were also responsible for the development of liquid-liquid
partition chromatography .
 Volatile Organic compounds can be separated due to differences in their
participating behavior between the mobile phase (gas) and the
stationary phase in the column.
 Inert gases - Ar, Ne, N2 , He can be used as mobile phase.
 O2 is usually avoided since it will oxidize the solid phase.
 The lighter gases He and H2 require faster analysis
 The mobile phase does not interact with molecules of the analyte; its
only function is to transport the analyte through the column.

Capillary columns – fused silica particles in the packed column require
chemical modification (below). The stationary phase surface (silica) is a
hydroylated surface. This caused problems with nonpolar stationary phases.
When polar or mildly polar species partition into the s.p. they stand a good
chance of being trapped, causing a excessive band broadening beyond what
is expected from van Deemter considerations.
Packed columns – uniform silica particles (150-250 μm) required to
ensure uniform path lengths (the “A” term in the van Deemter eqn.)
Surfaces are chemically modified (see below). The columns themselves
were either glass or stainless steel.
Packed Column Capillary Column
Types of columns

GC Injector & Syringe –
It is important to rapidly vaporize the sample. Slow vaporization
increases band broadening, by increasing the sample “plug”.
Injection port temperature is usually held 500C higher than the
BP of the least volatile compound.
InjectorInjector Port

Detectors
 Flame Ionization Detector (FID).
 Electron Capture Detector (ECD)
 Nitrogen-Phosphorous Detector (NPD)
 Flame Photometric Detector (FPD)
 Photoionization Detector (PID)
 Discharge ionization detector (DID)
 Hall electrolytic conductivity detector (ELCD)
 Mass selective detector (MSD)
 Pulsed discharge ionization detector (PDD)
 Thermal energy analyzer (TEA)

Flame Ionization Detector
(FID)
Sensitive towards hydrocarbons-
 Analyte is burned in H2/air, which
produces CH, CHO+& radicals of the
compound
 Specific for organic carbon, insensitive to
inorganics, CO2, SO2 etc.
 Response to specific organic depends on
the number of organic carbons.

Sensitive to electron withdrawing groups
especially towards organics containing –F, -Cl, -
Br, -I & -CN, NO2 ,
Nickel-63 source emits energetic electrons collides
with N2 (introduced as make-up gas or can be
used as carrier gas) producing more electrons:
Ni-63 => ee-
+ N2 => 2e- + N2
The result is a constant current that is detected by
the electron collector (anode).
Electron Capture Detector (ECD)

The nitrogen phosphorous detector
also called the thermo ionic detector
 Very sensitive to nitrogen and
phosphorous compounds.
 It is based on the flame ionization
detector but differs in that it contains a
rubidium or cesium silicate (glass) bead
situated in a heater coil, a little
distance from the hydrogen flame.
 The sensitivity of the detector to
phosphorous is about 10-12 ppm and
for nitrogen about 10-11 ppm
NPD
Nitrogen-Phosphorous Detector

Nitrogen-Phosphorus Detector (NPD)
Advantages:
 Useful for environmental testing
 Detection of organophosphate pesticides
 Useful for drug analysis determination of amine-containing or basic drugs
 Like FID, does not detect common mobile phase impurities or carrier gases
 Limit of detection: NPD is 500x better than FID in detecting nitrogen- and
 Phosphorus- containing compounds
 NPD more sensitive to other heterocompounds, such as sulfur-, halogen-
,and arsenic- containing molecules
Disadvantages:
 Destructive detector

 Analyte ionized by UV radiation.
 Electrodes collect ion current.
 Compound must absorb UV
radiation (usually 254 nm) to be
detected.
 Application
• BTX compounds
• Styrene
• Trichloro ethane
• Isobutane
• Cyclohexane
• Polyvinyl
Photo ionization Detector (PID)

Flame Photometric Detector
 Highly specific for S and P containing
compounds.
 Sensitive detector with mass flow
dependent response behavior.
 Well suited for capillary GC.
 Quadratic dependence of response for S.

1  HCH
2  HCH
3 βHCH
4 HCH
5 Aldrin
6 -Endo
7 pp- DDE
8 op-DDT
9 pp-DDD
10 pp-DDT
Spiked water containing
120ppb OC mixture
60ppb OC mixture
30 ppb OC mixture
15ppb OC mixture
GC Chromatogr. of pesticides in Water
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10

Uses of Gas Chromatography
 Determination of volatile compounds
(gases & liquids).
 Determination of partition coefficients
and absorption isotherms.
 Isolating pure components from complex
mixtures.

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Once called High Pressure Liquid Chromatography

HPLC
In HPLC pressure (well above the atmosphere ) are required to
operate high efficiency columns which is packed with small diameter
particles at a flow rate of few ml/min. A solvent or solvent mixture is
pressurized through a pump and delivered a pulse free flow to the
column to separate the compounds.
Most widely used analytical separations technique.
Gives the sensitivity upto parts per trillion (ppt) level.
Used for accurate quantitative determination.
Suitable for nonvolatile species or thermally fragile ones.

Types of HPLC
A Partition chromatography (liquid mobile phase/liquid stationary phase)
solid support: silica or silica-based
treated similarly to GLC supports
1. Normal phase chromatography
nonpolar mobile/polar stationary: nonpolar solutes elute first
2. Reversed phase chromatography
polar mobile/nonpolar stationary: polar solutes elute first
e.g.,water mobile/hydrocarbon stationary
3. Chiral chromatography
stationary phase is chiral
can separate enantiomers

B. Adsorption chromatography (liquid mobile phase/solid stationary phase)
oldest type stationary phase: silica, alumina
C. Ion chromatography
separate and determine different ions using ion-exchange resins
resins contain “loosely” held ions these ions will exchange with solute
ions that bind more tightly to resin
D. Size exclusion chromatography
packing: approx. 10 m silica or polymer particles forming a network of
pores of uniform size “very small” molecules get trapped in pores and
are last to be eluted “very large” “molecules” are too big to fit in pores
and get eluted first “intermediate sized” molecules undergo fractionation
very useful technique for polymers

Mobile phase
Mobile phase are liquids which carries analytes
to the column for separation
Types of elution
a) Isocratic- A solvent or premixed two or more solvents are
delivered through pump through whole analysis.
b) Gradient- Two or more solvents are steadily changed
during the analysis.

Normal and Reverse phase
Normal phase (NP) - The stationary phase is strongly polar in
nature (e.g. silica gel) and the mobile phase is non polar (such
as n-hexane or tetra hydro furan)
Reverse phase (RP) - The stationary phase is non polar
(hydrophobic ) in nature while the mobile phase is polar liquid
(such as mixture of water and methanol or acetonitrile.

 Stainless steel
 2-30 cm length
 4 -10 mm internal diameter
 0.45-10 µ particle size
 High Speed Isocratic / Gradient separation
 Variation in solvent changes
 Elution at different pH
Analytical Column
HPLC Columns

Types of Detector
 UV/Visible
 Photo diode array
 Fluorescence
 Conductivity
 Refractive index
 Electrochemical
 Light scattering

UV / Visible
 Detect UV & visible region
 Measure according to Bear Lamberts law
 Photocell measures absorbance of light
 Modern UV detector has filter for
repetitive and quantitative analysis
 Advantages
• high sensitivity
• small sample volume required
• linearity over wide concentration
ranges
• can be used with gradient detection
Disadvantage
Not work for the compounds that do not
absorb light at this wavelength region.

HPLC Chromatogram of PAHs Std.
and blood sample
1
2
3
4
5
6
7
8
9
10
11
12
13

Fluorescence
 For compounds having natural
fluorescing capability
 Fluorescence observed by
photoelectric detector
 Mercury or Xenon source are used
with grating monochromator to
isolate fluorescent radiation

Conductivity
 Measure conductivity of column effluent
 Concentration of the sample
proportional to change in conductivity
 Best use in ion-exchange
chromatography
 Cell instability
Advantages
• extremely high sensitivity
• high selectivity
Disadvantage
• may not give linear response over
wide range of concentrations.

Refractive Index
 Measure displacement of beam with respect
to photosensitive surface of detector.
Advantages
• Universal respond to nearly all solutes
• Reliable
• Unaffected by flow rate
• Low sensitive to dirt and air bubbles.
Disadvantages
• Expensive
• Highly temperature sensitive
• Moderate sensitivity
• Cannot be used with gradient elution flow
cell

Electrochemical
 Based on reduction or oxidation of the eluting
compound at a suitable electrode and
measure the resulting current
Advantages
• High sensitivity
• Easy to use
Disadvantages
• Mobile phase must be made conductive
• Mobile phase must be purified from
oxygen, metal contamination & halides

Advantages of HPLC
 Higher resolution and speed of analysis can be performed.
 HPLC columns can be reused without repacking or regeneration.
 Greater reproducibility.
 Easy operation of instrument and data analysis.
 Adaptability to large-scale for analytical / preparative
procedures.
 Stationary supports with very small particle sizes and large
surface areas.

Fast Protein Liquid Chromatography
 It is a form of column chromatography used to separate or
purify proteins from complex mixtures. It is very commonly used
in biochemistry and enzymology. Columns used with an FPLC
can separate macromolecules based on size, charge distribution,
hydrophobicity, or biorecognition (as with affinity
chromatography)
 Applications of fast protein liquid chromatography in the
separation of plasma proteins in urine and cerebrospinal fluid.
 Chromatographic separation of the proteins takes 1 h for urine
specimens and 45 min for CSF.

Application of HPLC
For the separation, identification and quantification-
 Carbohydrates, Nucleosides (purines and pyrimidines)
 Amino acids, proteins, Vitamins
Pharmaceutical products
 Fatty acids, fats ,Aflatoxins ,Antioxidants
Pollutants like PAHs, PCBs, pesticides, phenols, phthalates
Carotenoids, chlorophylls, cocaine,
 Alcohol in blood,
Explosive materials
The Police, F.B.I., and other detectives use chromatography
when trying to solve a crime.
A variety of other organic substances.

Mass Spectrometry
MS is an analytical tool that is used to identify unknown
compounds, quantify known material and elucidate the structure
and physical properties of ions. It require less than picogram
amt. (10-12) of material
MS can separate charged atoms or molecules according to their
mass-to-charge ratio ( m/z ).

Working of MS
 Sample is introduced under high vacuum to convert into gas
phase.
 The compound under investigation is bombarded with a
beam of electron which produce ionic molecule or fragments
of the original species.
 Ions (+&- ions) are repelled out of the ion source and
accelerated towards the analyser region.
 Analyser can detect either positive or negative ions.

Components of MS
 Inlet system
 Ionization source
 Electrostatic accelerating device
 Ion separator (magnetic field)
 Ion collector (detector)
 Vacuum pump

Ionization Sources
 Electron Ionization
 Chemical ionization (isobutane, methane,
ammonia)
 Fast atomic bombardment
 Liquid secondary ionization
 MALDI
 Electro spray ionization
 SELDI

Detector
Detector monitors ion current, amplifies it and then
transmits signal to data system.
Types of detectors used
Field- Electromagnetic field/ Electric field
Quadrupole and ion trap- radio frequency& direct
current
 TOF
Photomultiplier tube
Electron multiplier
Micro channel plate

5.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
STDPAHmix261006 Scan EI+
TIC
7.51e4
16.20
15.41
7.31
3.33
3.73
7.44
8.81
11.43
30.92
25.73
21.28
21.43
31.69
1
2
3
4
5
6
7
8
9 10
5.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
Sam45-261006 Scan EI+
TIC
1.91e5
16.24
8.08
3.04 15.4113.8512.46
10.33
22.69
18.40
18.05
18.53
21.43
20.38
26.74
25.77
23.15
31.71
30.94
28.78

5.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
Sam45-261006 Scan EI+
TIC
1.91e5
16.24
8.08
3.04 15.4113.8512.46
10.33
22.69
18.40
18.05
18.53
21.43
20.38
26.74
25.77
23.15
31.71
30.94
28.78
Acenaphthene
Pyrene
Fluoranthene
B(j) fluoranthene
B(ghi)Pyrelene
GC-MS chromatogram after
matching with library (NIST)

 Reaction monitoring- enzyme activity, chemical modification , protein digestion
 Amino acid sequencing
 Oligonucleotide sequencing
 Protein structure determination
 Environmental Monitoring and Cleanup
GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. There are some compounds for
which GC-MS is not sufficiently sensitive, including certain pesticides and herbicides, but for most organic analysis of
environmental samples, including many major classes of pesticides, it is very sensitive and effective.
 Criminal Forensics
GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. The analysis of free debris using
GC-MS is well established, and there is even an established American Society for Testing Materials (ASTM) standard for fire
debris analysis.
 Food, Beverage and Perfume Analysis
For the analysis of these compounds which include esters, fatty acids, alcohols and terpenes etc. It is also used to detect and
measure contaminants from spoilage or adulteration which may be harmful and which is often controlled by governmental
agencies, for example pesticide.
 Medicine
In combination with isotopic labeling of metabolic compounds, the GC-MS is used for determining.
 Law Enforcement
GC-MS is increasingly used for detection of illegal narcotic, and may eventually supplant drug-sniffing dogs.
 Security
Explosive detections, many of them based on GC-MS.
Uses of MASS

Chiral chromatography
Counter current chromatography
Lectin chromatography
Hydroxyapatite chromatography
Some specific chromatography

Chiral chromatography
 This involves the separation of stereoisomers.
 In the case of enantiomers, these have no chemical or
physical differences apart from being three dimensional
mirror images.
 Conventional chromatography or other separation processes
are incapable of separating them.
 To enable chiral separations to take place, either the mobile
phase or the stationary phase must themselves be made
chiral, giving differing affinities between the analytes.
 Chiral chromatography HPLC columns (with a chiral
stationary phase) in both normal and reversed phase are
commercially available.

Countercurrent chromatography
This is a type of liquid-liquid chromatography,
where both the stationary and mobile phases
are liquids. It involves mixing a solution of
liquids, allowing them to settle into layers and
then separating the layers.

Lectin affinity chromatography
This is a form of affinity chromatography where
lectin are used to separate components within the
sample. Lectin , such as concanavalin A, are proteins
which can bind specific carbohydrate (sugar)
molecules. The most common application is to
separate protein based on their glycan groups.

Future of Chromatography
National safety/ Defense Forensic science
R&D
Health-Drugs
 Environmental - Food & beverages and
pollutant chemicals
Material Characterization

SPECTROSCOPY
Spectral Distribution of Radiant Energy
X-Ray UV Visible IR Mic row ave
200nm 400nm 800nm
WAVELENGTH(nm)
Wave Number (cycles/cm)
Spectroscopy was originally the study of the interaction between
radiation and matter as a function of wavelength λ.
Spectrophotometer can be used to determine the entity of
anunknown substance, or the concentration of a number of known
substances. The type of source / filters used typically determines
the type of the spectrophotometer.

DISPERSION OF POLYCHROMATIC LIGHT WITH A PRISM
Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Prism - spray out the spectrum and choose the certain wavelength
(l) that you want by slit.

2.Fluorometer - measures the intensity of fluorescent light emitted by a
sample exposed to UV light under specific conditions.
Emit fluorescent light
as energy decreases
Ground state
Sample
90C
Detector
UV Light Source
Monochromator Monochromator
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Energy






'
'
'
'
'
n->
n
 n->'
Electron's molecular energy levels
Fluorometer

BEER LAMBERT LAW
Glas s cell filled with
co ncen tration of solution (C)
II
Light
0
As the cell thickness increases, the intensity of I (transmitted intensity of light )
decreases.
Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits

R- Transmittance
R = I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 - Log%T
Log is proportional to C (concentration of solution) and is
also proportional to L (length of light path
through the solution).
I
I0
I
I0
I0
I
1
T
I
I0

A  CL = KCL by definition and it is called the Beer
Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of solute
per liter of solution
A = ECL
E = Molar Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution

CELLS
UV Spectrophotometer - Quartz (crystalline silica)
Visible Spectrophotometer - Glass
IR Spectrophotometer - NaCl
LIGHT SOURCES
UV Spectrophotometer
1.Hydrogen Gas Lamp
2.Mercury Lamp
 Visible Spectrophotometer
1.Tungsten Lamp
 IR Spectrophotometer
1.Carborundum (SIC)

CHROMOPHORIC STRUCTURE
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261

Type of spectroscopy
Spectroscopy depends on the physical quantity
measured. Normally, the quantity that is measured
is an intensity, either of energy absorbed or
produced.
 Optical Spectroscopy or Ultraviolet-visible Spectroscopy
(involves interactions of matter with electromagnetic
radiation light).
 Electron spectroscopy is an analytical technique to study the
electronic structure and its dynamics in atoms and molecules.
In general an excitation source such as x rays, electrons, will
eject an electron from an inner-shell orbital of an atom.

 Mass spectrometry is an analytical technique that
measures the mass to charge ratio of ions. It is most
generally used to find the composition of a physical
sample by generating a mass spectrum representing
the masses of sample components. The mass
spectrum is measured by a mass spectrometer.
 Dielectric spectroscopy (sometimes called impedence
spectroscopy) measures the dielectric properties of a
medium as a function of frequency. It is based on the
interaction of an external field with the electric dipole
moments of the sample.

Most spectroscopic methods are differentiated as either
Atomic or molecular
 Absorption spectroscopy (Uses the range of the
electromagnetic spectra in which a substance absorbs.)
This includes {Atomic Absorption spectroscopy and various
molecular techniques (infra red spectroscopy, Nuclear
magnetic resonance spectroscopy (NMR) }
 Emission spectroscopy {Uses the range of electromagnetic
spectra in which a substance radiates (emits)}. Techniques
include spectrofluorimeter.
 light scattering spectroscopy -measures the amount of light
that a substance scatters at certain wavelengths, incident
angles, and polarization angles. The scattering process is much
faster than the absorption/emission process. e.g. (Raman
spectroscopy)

Atomic absorption
spectrophotometer
 Atomic absorption spectroscopy is a quantitative method of analysis that is
applicable to metals and Metalloids .
 An atomic absorption spectrophotometer consists of a light source, a sample
compartment and a detector.
 In this method, light from a source is directed through the sample to a
detector. The greater the amount of sample present, the greater the
absorbance produced by the sample.
 The source of light is a lamp whose cathode is composed of the element being
measured.
 Each element requires a different lamp.
 One of the most common means of introducing the sample into the flame is
by preparing a solution of the sample in a suitable solvent, frequently water.
 The flame gases flowing into the burner create a suction that pulls the liquid
into the small tube from the sample container. This liquid is transferred to the
flame where the ions are atomized. These atoms absorb light from the
source.

Atomic Absorption spectroscopy(AAS)
Basic components-
 Nebulizer- create a sample mist
 Burner - Slot-shaped which
gives a longer path length
flame
 Lamps
 Graphite furnace- Solid or
slurry samples, greater
sensitivity
 Recorder

A hollow cathode lamp for Aluminum (Al)
The lamp is housed inside
the lamp compartment of
the instrument. The sample compartment is really the
flame since it is in the flame that the
atoms absorb radiation from the
source.

 Quantitative analysis can be
achieved by measuring the
absorbance of a series of
solutions of known
concentration.
 A calibration curve and the
equation for the line can be used
to determine an unknown
concentration based on its
absorbance.

Atomic Fluorescence
spectroscopy “Atomic fluorescence spectroscopy (AFS) is the optical
emission from gas-phase atoms that have been excited
to higher energy levels by absorption of radiation.”
 “AFS is useful to study the electronic structure of atoms
and to make quantitative measurements of sample
concentrations.”

 The atomic emission spectrum of an element is the set of frequencies of
the electromagnetic waves emitted by atoms of that element. Each atom's
atomic emission spectrum is unique and can be used to determine if that
element is part of an unknown compound.
 The emission spectrum characteristics of some elements are plainly visible
to the naked eye when these elements are heated. For example, when
platinum wire is dipped into a strontium nitrate solution and then inserted
into a flame, the strontium atoms emit a red color. Similarly, when copper
is inserted into a flame, the flame becomes light blue.These definite
characteristics allow elements to be identified by their atomic emission
spectrum. Not all lights emitted by the spectrum are viewable to the naked
eye, it also includes ultra violet rays and infra red lighting.
 The fact that only certain colors appear in an element's atomic emission
spectrum means that only certain frequencies of light are emitted. Each of
these frequencies are related to energy by the formula:
E=h ν
herein E is energy, h isPlank’s constant and ν is the frequency.
Atomic emission spectroscopy

 UV SPECTROMETER : Protein,Amino Acids (aromatic),Pantothenic Acid,Glucose
Determination,Enzyme Activity (Hexokinase).
FLUOROMETER :Thiamin (365 nm, 435 nm),Riboflavin,Vitamin A,Vitamin C.
 VISIBLE SPECTROPHOTOMETER :Niacin,Pyridoxine,Vitamin B12,Metal
Determination (Fe),Fat-quality Determination (TBA),Enzyme Activity (glucose
oxidase).
APPLICATIONS-

Chromatography
Generic Protocol
1. Prepare Column (±)
2. Apply Sample
3. Wash
4. Elute
5. Analyze Fractions
Equipment
1. HPLC/FPLC
2. GLC
3. TLC
4. Paper Chromatography

Theory of Chromatography
1.)Typical response obtained by chromatography (i.e., a chromatogram):
chromatogram - concentration versus elution time
Wh
Wb
Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
Inject

(A) A difference in the retention of solutes (i.e., a difference in their time or
volume of elution)
(B) A sufficiently narrow width of the solute peaks (i.e, good efficiency for the
separation system)
A similar plot can be made in terms of elution volume instead of elution time. If volumes are
used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as
the retention volume (VR) and the amount of mobile phase that it takes to elute a non-retained
component is referred to as the void volume (VM).
Peak width & peak position
determine separation of peaks
The separation of solutes in chromatography depends on two factors:

Retention time:
A solute’s retention time or retention volume in chromatography is directly related to the
strength of the solute’s interaction with the mobile and stationary phases.
Retention on a given column pertain to the particulars of that system:
- size of the column
- flow rate of the mobile phase
Capacity factor (k’): more universal measure of retention, determined from tR or VR.
k’ = (tR –tM)/tM
or
k’ = (VR –VM)/VM
capacity factor is useful for comparing results obtained on different systems since it is
independent on column length and flow-rate.

The value of the capacity factor is useful in understanding the retention mechanisms for
a solute, since the fundamental definition of k’ is:
k’ is directly related to the strength of the interaction between a solute with the
stationary and mobile phases.
Moles Astationary phase and moles Amobile phase represents the amount of solute present in
each phase at equilibrium.
Equilibrium is achieved or approached at the center of a chromatographic peak.
k’ =
moles Astationary phase
moles Amobile phase
When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum

A simple example relating k’ to the interactions of a solute in a column is
illustrated for partition chromatography:
A (mobile phase) A (stationary phase)

KD
where: KD = equilibrium constant for the distribution of A between the
mobile phase and stationary phase
Assuming local equilibrium at the center of the chromatographic peak:
k’ =
[A]stationary phase Volumestationary phase
[A]mobile phase Volumemobile phase
k’ = KD
Volumestationary phase
Volumemobile phase
As KD increases, interaction of the solute with the stationary phase
becomes more favorable and the solute’s retention (k’) increases

k’ = KD
Volumestationary phase
Volumemobile phase
Separation between two solutes requires different KD’s for their
interactions with the mobile and stationary phases
since
peak separation also represents different changes in free energy
DG = -RT ln KD

3.) Efficiency:
Efficiency is related experimentally to a solute’s peak width.
- an efficient system will produce narrow peaks
- narrow peaks  smaller difference in interactions in order to separate two
solutes
Efficiency is related theoretically to the various kinetic processes that are
involved in solute retention and transport in the column
- determine the width or standard deviation () of peaks
Wh
Estimate  from peak widths,
assuming Gaussian shaped peak:
Wb = 4
Wh = 2.354
Dependent on the amount of time that a solute spends in the column (k’ or tR)

Efficiencies of columns with different lengths
H = L/N
where: L = column length
N = number of theoretical plates for the column
Note: H simply gives the length of the column that corresponds to one theoretical plate
H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,
etc.) to the kinetic processes that give rise to peak broadening:
Plate height (H)

Number of theoretical plates (N): compare efficiencies of a system for solutes that have
different retention times
N = (tR/s)2
or for a Gaussian shaped peak
N = 16 (tR/Wb)2
N = 5.54 (tR/Wh)2

Resolution (RS) – resolution between two peaks is a second measure of how well
two peaks are separated:
RS =
where:
tr1, Wb1 = retention time and baseline width for the
first eluting peak
tr2, Wb2 = retention time and baseline width for the
second eluting peak
tr2 – tr1
(Wb2 + Wb1)/2
Rs is preferred over  since both
retention (tr) and column efficiency
(Wb) are considered in defining
peak separation.
Rs $ 1.5 represents baseline
resolution, or complete separation
of two neighboring solutes  ideal
case.
Rs $ 1.0 considered adequate for
most separations.

Van Deemter equation: relates flow-rate or linear velocity to H:
H = A + B/ + C
where:
 = linear velocity (flow-rate x Vm/L)
H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer
One use of plate height (H) is to relate these kinetic process to band broadening to a
parameter of the chromatographic system (e.g., flow-rate).
This relationship is used to predict what the resulting effect would be of varying this
parameter on the overall efficiency of the chromatographic system.
Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2 peak width (Wh)
H = L/N

 optimum
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of
the mobile phase
Optimum linear velocity (opt) - where H has a minimum value and the point of maximum
column efficiency:
opt = rB/C
opt is easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds

Separation factor (a) – parameter used to describe how well two solutes are
separated by a chromatographic system:
 = k’2/k’1 k’ = (tR –tM)/tM
where:
k’1 = the capacity factor of the first solute
k’2 = the capacity factor of the second solute,
with k’2 $ k’1
A value of  $1.1 is usually indicative of a good separation
Does not consider the effect of column efficiency or peak widths, only retention.

Chromatographic techniques

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