Spectroscopic Techniques: Introduction of UV-Vis & IR spectrophotometer, Principle,

Mechanism, Instrumental Set-up, Practical: few Spectral data of molecules with

interpretation

Tapeesh Bharti | Team Leader-QC | Jubilant Biosys Ltd.

 Classical (old-fashioned) analytical chemistry is
based on techniques such as:

Titration Gravimetric Electrochemical methods

• volume of a standard reagent reacting • measurements based on mass.
with the sample is measured Simple examples are:
• Acid-base titrations: e.g. monitor the • pH measurement.
color of a solution containing a • Mass lost on heating of a solid
• pH-sensitive indicator as an acid (or gives the amount of water of
base) is added. crystallization. • Ion-selective electrodes.
• Complexation titrations: e.g. monitor • Mass of precipitate formed during
the pH of a solution whilst reagent EDTA a reaction can be measured.
(ethylenediaminetetraacetic acid) • For instance, adding excess silver
(HOOCCH2)2NCH2CH2N(CH2COOH)2,
is added. EDTA reacts in a 1:1 molar nitrate solution to determine the
ratio with almost all metal cations (except concentration of chloride ions
alkali metals), enabling the metal cation present.
concentration to be determined.

Modern analytical chemistry is largely based on instrumental techniques. In this lecture course, we shall discuss about
Molecular spectroscopy techniques & chroMatographic techniques.
Schematic diagram of Absorption Spectrometer

Wavelengths below ~200 nm cannot

easily be studied for instrumental
reasons that
the sample cell window absorbs
radiation at these wavelengths.
Ultra-Violet Spectrometer
Due to simultaneous vibrational and rotational transitions UV spectra normally
consist of fairly broad peaks. The absorbing groups in a molecule are called
chromophores. Two isolated chromophores in a molecule give roughly
independent absorptions for each one. e.g. CH3CH2–CNS: λmax= 245 nm
and SNC–CH2CH2CH2–CNS: λmax= 247 nm.

In organic chemistry, π-conjugated systems (when multiple bonds are

separated by a single bond) tend to give particularly informative spectra.
Overlap of adjacent π orbitals results in a decrease in the energy gap between
the occupied π orbital and the unoccupied π* antibonding orbital. This results
in an increase in absorption wavelength (even into the visible region
for greatly conjugated systems e.g. organic dyes), and normally an
increase in the intensity as well.

General rule: increased conjugation increases λmax and ε.

Aromatic systems exhibit conjugation, but tend to give complex spectra,

frequently with more than one absorption band. Conjugation with lone pairs (n-
π conjugation) can also result in spectral transitions, though these are much
weaker than those originating from overlap of π orbitals.
Uses of Ultra-Violet Spectrometer
• Sample is usually liquid.
• Can follow changes in color/composition quite rapidly (timescale may be
down to ~1 s). Can measure concentration of any colored compound, or
any compound that absorbs in the UV region.

• Beer-Lambert law is useful, though for accurate work absorbance will

be measured on solutions of known concentration for calibration purposes.
Reasonably straightforward to do the measurement “on-line”. Can study
the process fluid through a glass window, or using a fiber optic cable. In
practice, a technique called attenuated total reflectance is likely to be used
if the sample absorbs strongly.

• This technique is limited:

It doesn’t work if the sample doesn’t absorb in the UV / visible region!
It’s not good if the sample contains several species that absorb in the UV /
visible region: the absorption bands are broad and so overlap too much.
FT-Infrared Spectrometer
FT-Infrared Spectrometer
FT-Infrared Basic
Spectral Data
 Some characteristic infrared absorption frequencies
BOND COMPOUND TYPE FREQUENCY RANGE, cm-1
C-H alkanes 2850-2960 and 1350-1470
alkenes 3020-3080 (m) and
RCH=CH2 910-920 and 990-1000
R2C=CH2 880-900
cis-RCH=CHR 675-730 (v)
trans-RCH=CHR 965-975
aromatic rings 3000-3100 (m) and
monosubst. 690-710 and 730-770
ortho-disubst. 735-770
meta-disubst. 690-710 and 750-810 (m)
para-disubst. 810-840 (m)
alkynes 3300
O-H alcohols or phenols 3200-3640 (b)
C=C alkenes 1640-1680 (v)
aromatic rings 1500 and 1600 (v)
C≡C alkynes 2100-2260 (v)
C-O primary alcohols 1050 (b)
secondary alcohols 1100 (b) IR_frequecy_table
tertiary alcohols 1150 (b)
phenols 1230 (b)
alkyl ethers 1060-1150
aryl ethers 1200-1275(b) and 1020-1075 (m)

all abs. strong unless marked: m, moderate; v, variable; b, broad

IR spectra of ALKANES
C—H bond “saturated”
(sp3) 2850-2960 cm-1
+ 1350-1470 cm-1

-CH2- + 1430-1470
-CH3 + “ and 1375
-CH(CH3)2 + “ and 1370, 1385
-C(CH3)3 + “ and 1370(s), 1395 (m)
 n-pentane

2850-2960 cm-1
3000 cm-1
sat’d C-H

1470 &1375 cm-1

CH3CH2CH2CH2CH3
n-hexane

CH3CH2CH2CH2CH2CH3
2-methylbutane (isopentane)
2,3-dimethylbutane
cyclohexane

no 1375 cm-1
no –CH3
IR of ALKENES
=C—H bond, “unsaturated” vinyl
(sp2) 3020-3080 cm-1
+ 675-1000
RCH=CH2 + 910-920 & 990-1000
R2C=CH2 + 880-900
cis-RCH=CHR + 675-730 (v)
trans-RCH=CHR + 965-975

C=C bond 1640-1680 cm-1 (v)

 1-decene

unsat’d
C-H

3020-3080
cm-1
910-920 &
C=C 1640-1680 990-1000
RCH=CH2
4-methyl-1-pentene

910-920 &
990-1000
RCH=CH2
2-methyl-1-butene

880-900
R2C=CH2
2,3-dimethyl-1-butene

880-900
R2C=CH2
IR spectra BENZENEs
=C—H bond, “unsaturated” “aryl”
(sp2) 3000-3100 cm-1
+ 690-840
mono-substituted + 690-710, 730-770
ortho-disubstituted + 735-770
meta-disubstituted + 690-710, 750-810(m)
para-disubstituted + 810-840(m)

C=C bond 1500, 1600 cm-1

 ethylbenzene

3000-3100
cm-1
Unsat’d C-
H
1500 & 1600
690-710, 730-
Benzene ring 770 mono-
o-xylene

735-770
ortho
p-xylene

810-840(m)
para
m-xylene

meta

690-710, 750-
810(m)
styrene

no sat’d C-H

1640
C=C 910-920 & 990-
1000
mono
RCH=CH2
2-phenylpropene

Sat’d C-H

880-900
mono
R2C=CH2
p-methylstyrene

para
IR spectra ALCOHOLS & ETHERS

C—O bond 1050-1275 (b) cm-1

1o ROH 1050
2o ROH 1100
3o ROH 1150
ethers 1060-1150

O—H bond 3200-3640 (b) 

 1-butanol

3200-3640 (b) O-H

C-O 1o

CH3CH2CH2CH2-OH
 2-butanol

O-H
C-O 2o
 tert-butyl alcohol

O-H
C-O 3o
 methyl n-propyl ether

no O--H

C-O ether
2-butanone

 C=O
~1700 (s)
 C9H12

1500 & 1600

benzene

C-H unsat’d & sat’d mono

C9H12 – C6H5 = -C3H7

isopropylbenzene
n-propylbenzene?
n-propylbenzene
isopropylbenzene

isopropyl split 1370 + 1385

 C8H6

C-H unsat’d 1500, 1600

benzene

3300 C8H6 – C6H5 = C2H

C-H mono

phenylacetylene
Chromatographic Techniques: Introduction of LC and GC, Principle, Mechanism,
Types, Instrumental Set-up, Difference between NP and RP, Difference between
Isocratic and Gradient modes.

Tapeesh Bharti | Team Leader-QC | Jubilant Biosys Ltd.

 Chromatographic Techniques

Techniques discussed so far are limited in one important respect that they can only easily analyze pure
compounds, or simple mixtures at best.

What do we do if the sample is a complicated mixture?

1. Chromatography is the key separation technique used by analytical chemists when the sample is a mixture.
After calibration, chromatography can be used for structural identification and quantitative measurement as well
as simply being a separation technique.

2. Chromatography is also a chemical engineering unit operation for purification of high value chemicals,
particularly in the pharmaceutical and biotechnology industries.

3. Chromatography is based on the physical separation of individual chemical components in a sample:

a) The sample is present in a mobile or carrier phase: may be gas, liquid, or even supercritical fluid.
b) The sample is separated into components due to differences in affinity for a stationary phase.
Chromatographic Techniques
HPLC instrumentation
 Liquid Chromatographic Technique (LC)
Modern instruments often use the acronym HPLC (for “high-performance” or “high-pressure” LC).

In this case the sample is in the liquid phase (by dissolving into solution if it’s not already a liquid). The stationary phase is
a solid packed into a column; it can be a liquid-coated solid. Different detector systems are used (e.g. UV, fluorescence,
refractometry). A variety of different separation mechanisms are used.

Liquid-Solid separations are based on the intermolecular interactions between sample molecules and the solid phase.
For instance, these may be “polar interactions” or “hydrogen bonding interactions”.

1. The “normal” case is that the solid has hydroxyl groups at the surface and so has an affinity for polar groups. Less
polar molecules will pass through the column faster than polar molecules if the surface of the solid likes polar
species.
2. In “reverse phase” chromatography, the stationary phase is made hydrophobic (e.g. silica with n-alkyl chains
covalently bound to its surface). In this case, hydrophobic compounds will have longer retention times than
hydrophilic ones.

The retention times are critically affected by the polarity of the solvent (carrier phase).

Mixtures that don’t separate when using one solvent as carrier phase may separate easily using a solvent of different
polarity. This technique is widely used in synthetic organic chemistry labs as a bench-scale purification technique.
 Retention Order
NP-HPLC RP-HPLC
Difference between RP and NP Concept; Polar
to Non-Polar
Different types of Chromatographic
Techniques and their Detection
 Different Chromatographic Technique (LC)
A. Size-Exclusion chromatography is based on the molecular size of the compounds present.

1. The stationary phase consists of solid beads containing small pores.

2. Large compounds can’t enter inside the beads and thus will elute first.
3. Smaller compounds enter the beads and will have longer retention times.

B. Ion-exchange chromatography operates on the basis of selective exchange of ions in the sample with those in
the stationary phase.
1. The column consists of a polymer matrix bearing certain ionic functional groups: e.g. M–SO3–H+ for the case of
cation exchange (anion exchange columns also exist).
2. Molecules capable of ion-exchange will be retained at these sites :e.g. if they contain cations or acidic hydrogen in
this example.
3. Molecules retained on the column can be subsequently collected by changing the properties of the mobile phase:
e.g. by changing the pH so that the carrier liquid will displace sample molecules attached to the column.

C. Affinity chromatography uses immobilized biochemical that have a specific affinity to the compound of interest.
Gas Chromatographic Technique (GC)
 Gas Chromatographic Technique (GC)
1. For GC, the carrier phase is an inert gas (e.g. He, Ar, N2).

2. The sample needs to be vaporized if it’s not already a gas, and injected as a pulse into the carrier
stream. The stationary phase is usually a column containing the stationary phase on a fused silica
support. The column is usually very narrow (say 2 mm diameter) and may be 1-10 m long; physically
it will look like a coiled loop. There are many types of silica and modified silica so different
separations can be achieved.

3. Separation is based on the components having different retention times on the column:
a) affected by boiling points of the substances to be separated.
b) affected by selective adsorption of a component onto the stationary phase.

4. The column is located in an oven, the temperature of which can be controlled.

5. For good separations in a reasonable length of time, it’s common for the temperature of the oven to
be increased over the course of the experiment.
 Commonly Detectors used in GC
Flame ionization detectors (FID):
1. These are widely used for analysis of organic compounds.
2. Gas at the column exit is mixed with hydrogen and air and burnt. Any organic compounds present produce ions and
electrons in the flame making it capable of conducting electricity. The FID measures the current response to an electric
potential at the burner tip.
3. The FID response has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but
it destroys the sample.
4. For hydrocarbons, the FID peak areas are proportional to the number of carbon atoms present in that component of the
sample.

Thermal conductivity detectors (TCD):

1. These compare the thermal conductivity of the gas at the column exit with a reference flow of carrier gas (usually He).
Any change is due to the presence of sample compounds.
2. Disadvantage: TCDs are slightly less sensitive than FIDs and have slightly lower resolution (as they have a larger dead
volume).
3. Advantage: TCDs can be used to detect any compound (i.e. not just hydrocarbons) and the sample isn’t destroyed.
4. Because the thermal conductivity of organic compounds tend to be similar to each other, TCD peak areas for
hydrocarbons are roughly proportional to the concentration of that component.