Analytical Techniques 11%

Analytical methods are defined as the set of techniques that allow us to determine the quality and quantity
of any material and chemical state. An analytical technique is a method that is used to determine the
concentration of a chemical compound or chemical element visioning.

High Performance Liquid Chromatography (HPLC)

HPLC is a technique used to separate, identify, and quantify each component in a mixture.
Principle; It is based on the principle of the difference in the relative affinities of different molecules for the
mobile phase and the stationary phase used in the separation.

Basic components of HPLC

1. Solvent (Mobile Phase): Usually a mixture of polar and non-polar liquid components in two solvent
reservoir which holds 1000 mL of solvents.

2. High Pressure Pump: It drives the solvent and sample through the column at a specific flow rate.

3. Sample Injector System: It introduces the liquid sample into the mobile phase.

Column (stationary phase): It is packed with SiO2 or Al2O3 and separates the sample into individual
components.

5. Detector: It detects the individual molecule that comes out from the column. It also measure the amount
of molecules in the sample.
6. Data Collection Device: Computer collects all the signal from the detector and uses it to determine the
time of elution and amount of sample.
Procedure for the Analysis of Sample by HPLC
 1. Small volume of the liquid sample is injected into the injector.
2. Sample is carried by the mobile phase into the column with the help of high pressure pump.
3. The components in the sample gets separated due to their different affinities for mobile and
stationary phase
4. The separated components are detected at the exit of the column with the UV absorption detector.
The output from this detector is called Liquid Chromatogram

Applications of HPLC

1. For the separation of chemical and biological compounds.

 Proteins like egg white or blood proteins.
 Many natural products such as ginseng, herbal medicines, plant extracts etc..
2. For the identification of individual components in the sample. Different components in the sample
get separated due to difference in their affinities for stationary phase and mobile phase.
3. For the measurement of the amount of compound in a sample by measuring its peak height and
peak area.
4. For the preparation of pure compounds by collecting the chromatographic peaks at the exit of the
detector.
5. For the determination of trace compounds in pharmaceutical, biochemistry and environmental
studies.

Mass Spectrometry
Mass spectrometry is an instrument used to measure the masses of atoms and molecules. The first
spectrometer was invented by Aston.
Mass spectroscopy is an analytical technique in which:
 The sample of an organic compound is converted into gaseous positive ions, with or
without fragmentation.
 The mass to charge ratio ( m/z) and relative abundance of the ions are then analyzed.

Purpose: To identify the composition of the compound and elucidate its structure.

Principle of Mass Spectrometry; The mass spectrometry is based on the fact that, when charged
particles move in a magnetic field, they move in curved paths. Heavy ions are deflected the least, light ions
the most.

 The molecules are bombarded with an energetic electron to produce positively charged ions such as
molecular ions and the fragmented ions.
 Each kind of the molecular ion and the fragmented ion has certain mass to charge ratio ( i.e m/z
ratio).
 Example: fragmentation of ammonia
 + + + + 15
Ion N NH NH2 NH3 N isotope in
Third fragment Second fragment First fragment molecular ion or NH3
+
parent ion (M +1) peak
+
( M peak)
m/z ratio 14 15 16 17 18
Relative 2.2 7.5 80 100 0.4
Intensity (Base peak)

Procedure of Mass Spectroscopy

Step 1: Organic molecules are vaporized and bombarded with a beam of high energy electrons. The
energetic electron knock out most loosely bound electrons to produce positively charged ions.

ABC+ molecular ion

Electron beam
ABC AB+ C+
+ BC+
Step 2. The positive ions are accelerated towards aA
negatively charged plate by passing through a magnetic
field, which deflects the ions. Lighter ions are deflected more than heavier ions.
 Step 3: Each
daughter ion has a particular m/e ratio. For most ions produced in the fragmentation of the molecule, the
charge is +1. therefore, m/e represents the mass of the ions.

H H H
NH 3  e - 
-2 e
[NH 3 ]  [NH 2 ]  [NH]  [NH]
m/e  17 m/e  16 m/e  15 m/e  16

Step 4. A signal is obtained corresponding to each m/e value. The intensity of a signal is proportional to the
number of ions giving rise to that signal.

NH 3 (1 7 )

100 NH 2 (1 6 )
R e la tiv e in t e n s ity

80

60
N H (15)
40
N (1 4 )
20

m /e
M a s s s p e c tru m o f N H 3 m o le c u le

Important parts of Mass Spectrometry

1. Ion Source: Ions are produced in mass spectrometry by bombardment of molecules by electrons having
energy of about 70ev.

AB  e - 

e
AB  2e  (most probable)


e
ABn   (n  1)e (least probable)


e
A   B  e


e
A   B  2e
2. Mass Analyzer: The fragmented ions are differentiated on the basis of their m/e ratio by the mass
analyzer.
3. Detector: The ions separated by the analyzer are detected and measured in the detector.

Interpretation of mass spectrum

 The peak with the highest m/z ratio is the peak of the molecular ion and found usually towards the
extreme right of the spectrum.

 The peak of the molecular ions ( parent peak) is usually with the higher intensity and is taken as the
base peak (100% ).

 For example in the case of NH3, the molecular ion NH3+ has the peak with highest intensity and is
taken as the base peak.

 However, the parent peak is not always the base peak in some of the compounds whose molecular
ions are unstable and tend to undergoes further fragmentation.

 Example:

 The molecular mass of the molecular ion as analyzed by the mass spectroscopy gives the molecular
mass of the compound.

 The mass spectrum of a particular compound is unique and no two compounds will have the same
spectrum. The mass spectrum of a compound depends on its mode of fragmentation .

Some of the features of parent peak.

 The molecular ion peak ( parent peak ) in aromatic compounds is comparatively much intense due to
delocalized pi- electron system.

 Unsaturated compounds ( with double or triple bonds) give more intense peak than those saturated
or the cyclic compounds.

 The intensity of the peak given by the saturated compounds is more than the corresponding
branched chain with same number of carbon atoms. For example the intensity of molecular ion of n-
pentane is intense than that of neo-pentane.
 For example; the intensity of molecular ion of n- pentane is intense than that of neopentane.

Question 1. Observe m/z value of neo- pentane and write its fragmentation mode.

Questio 2. Now write the fragmentation for n- pentane.

Mass spectrometry of Chloro and Bromo Compound

 In the case of chloro and bromo compounds, isotope peaks along with the molecular ion peak (
parent peak) are also formed .
 In the case of bromo compounds, the isotopic peaks, M+ and (M+ + 2 ) with intensity ratio 1:1 are
formed. The mass spectrum of ethyl bromide:
 In the case of chlorocompound the isotope peaks, M+ and (M+ + 2) peaks have intensities ratio 1:3
 For example, the mass spectrum of ethyl chloride
  The compounds of fluorine and iodine do not show M + + 2 peaks as they do not have their
isotopes.
 If the mass spectrum does not contain molecular ion peak, the compound is either highly branched
or is a tertiary alcohol.
 For straight chain alkane, fragment peaks are formed 14 units apart and the base peak is due to
C3H7+ ion (43). It also gives additional peak of M+ 1 peak due to isotope of carbon.
 For example a compound forms peaks at m/z values of 100, 85, 71, 57 and 43 ( 100%)

Question 3. Compound forms peaks at m/z values of 100, 85, 71, 57 and 43 ( 100%). Deduce the molecular
formula of the compound and its fragmentation pattern.
Solution; The successive peaks are at 14 units apart, so it a straight chain hydrocarbon.The highest m/z
value is 100 which is the molecular mass of the parent ion which corresponds to the molecular mass of the
sample. The hydro carbon ( sample) with molecular mass 100 is that of a heptane -
CH3CH2CH2CH2CH2CH2CH3 - (C7H16)
Fragmentation pattern :
C6H16 + e– → C6 H16+ (100)
(heptane) ( molecular ion)
 The mass
spectrum the compound can be drawn accordingly.

Applications of Mass Spectroscopy

 Identification of substance. Mass spectrum is highly characteristic of a substance. It can be

used to identify the substance.
 Identification of substance. Mass spectrum is highly characteristic of a substance. It can be
used to identify the substance.
 Determination of molecular structure: Through study of various fragments, molecular
structure can be determined.
 Infrared Spectroscopy (IR spectroscopy)
IR is one of the important analytical technique used to identify the given sample of organic compound. It
is useful in identifying the different functional groups present in the compound. In this technique, the
compounds are irradiated with infrared radiations of electromagnetic spectrum. The sample absorbs
certain energy of the IR radiation to get excited from lower to higher vibrational level. The rest of the
energy get transmitted which is detected and recorded in the form of spectrum. The energy of the IR
radiation is expressed in the form of wave number as they are directly related to each other.

��
= = ῡ, ( ῡ − wave number), wave number is expressed usually in cm-1

The infrared region of the spectrum lies within wave number of 4000 to 400 cm–1. IR spectrum is theplot of

percentage of transmittance or the absorbance of energy against the wave number.

Principle of IR spectrometry.

When the infrared radiation is passed through the sample of an organic compound, certain part of the
radiation is absorbed and the rest get transmitted. The % of transmittance of the radiation of certain

wave number is recorded in the form of peaks in the spectrum. If the % of absorbance of the radiation of
certain wave number by the sample is more, its transmittance will be low and vice versa. The absorption of
radiation causes the molecules to vibrate at higher energy level. The absorption of radiation takes place
only when the particular frequency of the IR radiation matches with the frequency of the particular
molecular vibration of the sample. Different atoms in the molecules vibrate at different frequencies and
therefore absorb different frequencies of IR radiation.

Molecular vibration:
The atoms bonded by a covalent bond in a molecule are not held rigidly. The molecules are visualized to
be consisting of a balls (atoms) of varying size tied by string (bond) of varying strength. The atoms vibrate
along such covalent bond with certain frequencies. When the frequency of such vibration matches with
frequencies of IR radiation, absorption takes place. The molecular vibrations are of different types:

A.Stretching: in such vibration, the distance between the two bonded atoms increases or decrease but the
atoms remain in the same bond axis. The stretching vibrations are of two types:

1. Symmetric stretching : in this type, the atoms, with respect to a particular atom in the molecule
stretch in the same direction (either approach or depart).

2. Asymmetric stretching : In this type, one atom approaches the central atom while the other
departs from it.
B. Bending : During bending the atoms move out of bond axis

Bending are of four types.

The bending vibration require less energy than stretching vibration and the therefore occur at lower
wave number region in the spectrum i.e in the finger print region of the spectrum.

The molecular vibration results into the change in dipole moment of the molecule. If the net change in
the dipole moment of the molecule is zero, the molecule is said to be IR inactive and does not give
absorption band. Only those molecules whose change in dipole moment is greater than zero are said to
be IR active.

Example : in CO2 molecule

 The IR spectrum consists of two regions:

Functional group region : This region of the IR spectrum lies within 4000 to 1500 cm-1

Each type of functional group of the molecules has its own absorption bands depending on its energy of
vibration. By careful observation of the absorption bands, the functional groups can be identified. The
range of wave numbers between which the functional group show the absorption bands are given below.

Finger print region :

This is the region in which the absorption takes place below 1500cm-1 and is due to bending
vibrations in the molecule. Some of the stretching vibrations of C- O, C-C and C-N are also shown in
this region. No two compounds have the same spectra in the finger print region and thus can be
useful in identifying the compound. Two compounds may have the same spectra in the functional
group region but will have different spectra inthefinger print region.
Absorption bands of some of the functional groups:

Spectrum of some of the compounds

a. Alcohol : R-OH The alcohol in the IR spectrum give strong and slightly broader absorption band
in between 3000 to 3500 cm-1 which is due to O-H group. It also gives strong and slightly sharp band,
near 3000cm-1 which is due to C-H stretching in the molecule. But it does not show the band of C=O
( carbonyl group) stretching near 1700cm-1 in the spectrum.
b. Aldehydes : R-CHO

The spectra of aldehyde show strong and sharp absorption band of C=O (carbonyl group) stretching
between 1800 – 1600 cm-1 and C- H stretching. But it do not show the absorption band of O- H stretching.
C. Carboxylic acid : R- COOH

the spectrum of carboxylic acid shows absorption band of O-H, C-H and C= O stretching. The
absorption band of O-H and C-H stretching overlap each other to give a very broad trough like band.

d. Amides: R - CONH 2 The spectrum of amides will give three types of absorption bands which are
N- H stretching C-H stretching and C=O stretching. The absorption band of N-H stretching lie
somewhere close to that of O-H stretching bands. But the N-H will not be broader as in the case of
OH. It will also have spikes.
 e. Amines : Amines will have similar type of spectrum as that of the amides except that the C=O
bands will be absent. Tertiary amines will not have N-H stretching band due to absence of N- H
bond in it.

Questions : 1 An aromatic compound with molecular formula C 6 H 7 O has the following IR spectrum. Identify the
compound.

Since the spectrum contains broad and strong absorption band near 3500 cm-1 which specifically is that of O- H
stretching, the compound is can be C 6H 5OH (phenol).

2. Identify the compound from the list that would give the spectrum of the type given below.

Acetone, propionic acid, n- propyl alcohol, ethyl formate.

Ans : the spectrum shows broad peak near 3500 cm-1 which is of O-H stretching and strong –sharp peak near

1700cm-1 which is of C=O stretching. Since the compound contains both O-H and C= O, it is propionic
acid.
 3. IR spectrum of acetone gives two peaks of C-H vibrations – at 1360 cm-1 and at 3100 cm-1. Identify the
vibration mode.

The absorption at 1360 cm-1 is due to C-H bending and at 3100cm-1 is due to C-H stretching.

4. Indicate the expected location in the functional group region of the IR spectrum of the following
compounds.

i. CH 3 CH 2OH

ii. CH 3 OCH 3

Ans : For CH 3CH 2 OH. The broad and strong peak due to O-H stretching : near 3500cm-1 ( 3200 – 3450) Due to C-
H stretching : near 3000cm-1

For CH 3OCH 3

Due to C-H stretching of methyl group : near 3000cm-1

5. How will you distinguish between acetone and the ethanol by IR spectrum? Distinction between intra
molecular and intermolecular hydrogen bonding :

Compounds containing -OH and -COOH groups form aggregate due to intermolecular hydrogen bonding. Such
compounds in aggregate form show stretching absorption bands/ peaks at lower frequencies or wave numbers.
However when such molecules are dissolved in non- polar solvents such as CCl 4 , the molecules separate from

the aggregate and show absorption bands at higher frequencies or wave number ( in usual manner). So in the case

of intermolecular hydrogen bonding, the compounds show shift in the absorption bands when solvents are
changed. In the case of compounds such as o-nitro phenol which shows intra molecular hydrogen bonding,
there will not be shift in the their absorption bands when different solvents are used.
 Nuclear Magnetic Resonance (NMR) spectroscopy

Nuclear Magnetic Resonance spectroscopy is one of the important technique used in the field of scientific
research for determining the components, purity and the molecular structure of a given substance. It is
based on the fact that the atomic nuclei posses charge and spin around their own axis due to which they
develop magnetic moment. The atomic nucleus therefore behaves as a small bar magnet associated with
certain magnetic moment. Only those atoms with odd atomic number of mass

number such as 1H, 13C , etc. can generate magnetic field during spinning. If hydrogen nucleus is
taken, then it is known as HNMR or simply proton NMR or PMR. HNMR is useful in studying the
number of hydrogen atoms and their environment in the molecules.

Principle : When protons behaving as a small bar magnet are placed in an external magnetic field, the
magnetic field of the protons either align with or oppose the external field.

The protons which align with the external magnetic have lower energy and the ones which oppose the
external magnetic field have higher energy. The lower and higher energy states of the proton are known as α
– spin state and β - spin state respectively. When such nucleus is irradiated with electromagnetic radiations
( radio wave frequency), the proton absorbs energy of the radiation equal to energy difference between the
two states (ΔE) and get excited to higher energy state with reversed spin. This is called ‘spin flip’ or
flipping of magnetic proton. The proton then comes back to lower energy state by releasing the absorbed
energy which is recorded in the spectrum. When the energy of the electromagnetic radiation matches with
that of the spin flip energy of the proton, the absorption takes place and is known as resonance.
Higher the strength of applied magnetic field higher will be the frequency of the energy required to flip the
proton.

Equivalent and non-equivalent protons:

In the proton NMR of the organic compounds, proton signals are obtained at different frequencies of the
electromagnetic radiations. This is because the protons in the molecule are in different electronic
environment.

Those protons in the same electronic environment are called equivalent protons and those in the different
electronic environment are called non- equivalent protons. The equivalent protons in the molecule absorb
 at same frequency of the electromagnetic radiation. The non-equivalent protons absorb at different
frequencies and hence give rise to more than one signal in HNMR spectrum.

For example: in methane molecule all the four protons are in same chemical environment and
therefore give one signal in the NMR spectrum.

Shielding and Deshielding Effect : In organic molecule the hydrogen nuclei (protons) are surrounded by
electrons. These electrons generate small magnetic field which oppose the applied magnetic field (B 0 ) due to
which the effective magnetic field (Be) experienced by the nucleus will be less than the applied field. This
effect is known as shielding effect as the electrons shield the nucleus from the influence of external magnetic
field. Since the shielded proton experiences less external magnetic field, the energy gap between the two spin
states is also low and require lass frequency ( energy) for resonance (to flip form lower energy spin state to
higher energy spin state). Higher the electron density around proton greater will be the shielding and the signals
are up field in the NMR spectrum.

If the electron density around the nucleus decreases, the effective magnetic field experienced by the
nucleus is more than that of external magnetic field. This is called deshielding. The deshielded

protons require higher frequency (energy) for resonance. The presence of highly electronegative atom pull
the surrounding electrons away from protons and causes desheilding and the signals are

downfield in the NMR spectrum.

Chemical shift: Different sets of protons in a molecules,due to shielding or deshielding give signals at
different resonance frequencies and hence give peaks at different position in the spectrum. The difference in
the absorption of the protons in the molecule - as a result of shielding and deshielding by the surrounding
electrons - with respect to tetra methyl saline (TMS) taken as reference standard signal is called chemical
shift. The equivalent protons in the molecule have same chemical shift those in the different chemical
environment have different chemical shift.

The chemical shift is measured in two scales :

Delta scale (δ) , Tau scale (τ)

In the above spectrum‘b’ protons have higher chemical shift than that of ‘a’ protons. The ‘a’ protons
therefore are more shielded than ‘b’ protons.
 Example of Chemical shifts:

The chemical shift for methylene (CH 2 ) protons occur at 3.4 ∂ and that of methyl (CH 3 ) protons
occur at 1.7∂ with respect to TMS. CH 3 protons are more shielded than CH 2 protons.

Chemical shift values of protons in different compound:

Spin – spin coupling :In the HNMR spectrum, each of the peaks obtained are observed to be
further split into smaller peaks. The splitting of peaks takes place due to interaction of the
magnetic field of the protons with the magnetic field of the neighbouring non – equivalent
protons. Such interaction between the magnetic fields of neighbouring non-equivalent
protons which results into splitting of peaks is known as spin-spin coupling.The number of
split up peaks in the spectrum is determined by using (n+1) rule, in which ‘n’ is the number of

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 neighbouring protons.The splitting will take place only between the neighbouring non-
equivalent protons. If the neighbouring protons are equivalents, splitting will not be observed.
Example :

Splitting pattern using (n+ 1) rule.

CH 3 CH 2 Cl : the protons of CH 3 groups have two non- equivalent neighbouring protons on

CH 2 group, so its signal splits into (1 + 2) = 3 smaller peaks called triplet. Similarly the protons
of CH 2 group have three neighbouring non-equivalent protons on CH 3 group, so its signal
splits into (1+ 3) =

4 smaller peaks called quartet.

NMR spectrum of ethyl alcohol : three signals ( triplet,quartet, singlet)

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 The triplet is given by the three methyl protons which are coupled with
two methylene protons to give upfield triplet.The quartet is given by the two methylene protons
which are coupled with three methyl protons to produce downfield quartet.The singlet is
produced due to one OH proton which is not coupled with any other protons.

Questions : How many NMR signals do the following compounds give? Indicate the splitting
pattern of each of the signals.

A CH 3 OCH 3 : One signal (singlet)

b. CH 3 OCH 2 CH 3 : three signals (singlet, quartet, triplet)

 How will you distinguish between the following pairs?

CH 3 COCH 3 CH3CH 2 CHO

 Gives only one signal  Gives three signals

CH 3 COCH 3 CH 3 COOCH 3
 Gives only one signal  Gives two signals

a compound with molecular formula C 2 H 4 O has NMR spectrum as given below. Identify
the compound.

The two signals with different chemical shifts indicate that there two sets of protons
in the molecule. The duplet produced upfield (lower chemical shift) indicates that there is
one proton attached to the adjacent carbon and the quartet produced downfield indicates that
there are three protons attached to the neighbouring carbon. The quartet produced with
higher chemical shift also indicates that the electronegative oxygen atom is attached to
carbon with one proton. So the formula of the compound is CH 3 — CHO ( acetaldehyde

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