A collection of notes for the

subject

‘MODERN PHARMACEUTICAL
ANALYSIS (MPA)’

~ per t “

As ae

*harmac
Fiat ihtas y =

CONTENTS

Content ©. | Page no.

UV-Vis Spectroscopy 2

IR Spectroscopy 3-14

NMR Spectroscopy 15-28

Mass Spectrometry 29-45

Affinity Chromatography 46-48

Electrophoresis 49-61

X-Ray Crystallography 62-67

Immunological Assay 68-70

Note: | have not been able to include some chapters/topics prescribed in the syllabus due to lack
of time. Fortunately, the excluded topics are the ones whose reference materials are easily
obtained from relevant sources.

UV-VISIBLE SPECTROSCOPY

CHOICE OF SOLVENTS:

The choice of the solvent to be used in ultraviolet spectroscopy is quite important. The first cnterion
for a good solvent is that it should not absorb ultraviolet radiation in the same region as the sub-
stance whose spectrum is being determined. Usually solvents that do not contain conjugated sys-

tems are most suitable for this purpose, although they vary as to the shortest wavelength at which
they remain transparent to ultraviolet radiation. Table 7.1 lists some common ultraviolet spec-
troscopy solvents and their cutoff points, or minimum regions of transparency.

Of the solvents listed in Table 7.1, water, 95% ethanol, and hexane are most commonly used.
Each is transparent in the regions of the ultraviolet spectrum where interesting absorption peaks
from sample molecules are likely to occur.

A second critenon for a good solvent is its effect on the fine structure of an absorption band.
Figure 7.5 illustrates the effects of polar and nonpolar solvents on an absorption bund. A nonpolar
solvent does not hydrogen bond with the solute, and the spectrum of the solute closely approx-
imates the spectrum that would be produced in the gaseous state, where fine structure is often
observed. In a polar solvent, the hydrogen bonding forms a solute—solvent complex, and the fine
structure may disappeur.

TABLE 7.1
SOLVENT CUTOFFS

Wavelength (nm)

FIGURE 7.5 Ulteaviolet spectra of phenol in ethanol and in isooctane. (From Coggeshall, N. D., and
E. M. Lang. J. Am. Chem. Soc., 70 (1948): 3288. Reprinted by permission.)

IR SPECTROSCOPY

THEORY OF IR SPECTROSCOPY:

* Almost any coenpound having covalent bonds. whether organic or inorpanic, absorbs various frequencies of EMR. rachation in

the infrared region of the EMR

The infrared rewion of EMR may be divided mito the fcllowing sections

Table 1. Subxikvren of TR

Wavelength O) rangein je =| Wave number (0) range to cm!

Al wavelensths below 25m the radiahon has sufficient enersy

to cause changes mm the vibratoeal eoerpy bevels of the

molecule and these are accompansed by changers m the

eee tes es ee aL

The mam region of interest for analyheal purposes a froe

waveleapties 2.5 to 25 uta i.e weverumbers 4000 to 400cn”

a ——— Frequency (5}—_——_———_ ——________-
high ¢——_——_______—_—_—E_ne—r_gy _ — low

X-RAY utrravioceT | p | ~ | Frequency

ieee

| 1 25 um <—> 15 am
200 nn ¢————_ 400 nv <————_) 600 nm

BLUE RED

short Viavelength (a) ———qquqeum—_»

Pig, 1. A portion of FATK spectrum showing relationship of vilrational infrared to other types of radiation.

* Mobeciies conten bonds of specie enatial Orentetion and enerey

Po dee eR ee eae ME eRe Bey URGE ae Tepe et es Melieo gmag ig ce)

=
ia

>

* A Very approximate model compares the vibration to that of a harmosic osciliaior, sock as an ideal sprite

aewyyyyeevvy~a.R

in the enrine-Gall syetern depicted above, if the sorine conmeéctene the two balls is struck with @ force, vibrations are produced

Re Re E ea el RG Slee ee BP Sa) By leslesi abae a ata

Lf the spring has a force cometant, K and masses mm, and m, al the ends, then the theoretical wibration frequency, vt piven
a

Bene
4d ewe e, rn eee Ate Re eae Miro Bism yp eels

egois Gee he RT Bet TT Reese) eager

I ie Pta B s

= nu 655 x1660x 10″ ke

Varro theses values in the above formulas. v can be calculated and found to be 1 iSem”

Practically, the observed C—O band for methanol ts af 1034cm*, which ts apprcammataly close to the calculated valee above

Therefor, if force Constant rs known, spproximate Iraqeency of band can be calculated

PE Di Pee ER Be Re BT er eee Eee see Te Blak yw iho

For absorption of energy to occur from tbe incidert enfrared rediation and vibrational transitions fo occur, if es eseeritial that «

chanee in dipole moment oOocurs Curse the Vibration

See een eee eee ae ee en TTT ee ee Ba

Vibration between two atoms i diatomic molecules, will mot neeult in a chase of electocal symmetry or depole mocnent of the

eee OR Se ele aT BateS lee sree de Bilis eB gal

* Otbers mury sequire a dtpole moment when they whrate. E.@ metCHh, ahas npo deipol,e, but when one of the C-iH eee

stretches. the molecule will develop a temporary dipole

© Since every type of bond has a different natural frequency of vibration, and since two of the sume type of bonds an two different

compounds are mn two alaghtly different environments, no two structure have oxactly the same Pitse os B hspe Rt) ss)

* ‘The vibrational modes or transitions are characteristic of the proups in a molecule and are useful in the identification of a

compound, particularly in establishing the structure of an tenknown compound

* Thus, the infrared spectrum cin be used foe molecules neuch as a fingerprint can be used for humans

« For identification of a pare compound, the spectrin of the anknown substance a compared with the spectsa of 4 imited sia

eee ro Bi Re eee oe Me BSR SB Bel ee sitte bthee By Bees gl

MODES OF VIBRATION: (see Ravi Sankar’s book )

SAMPLE HANDLING:

PREPARATION OF SAMPLES FOR INFRARED SPECTROSCOPY

To determine the infrared spectrum of a compound, one must place the compound in a sample
holder, or cell. In infrared spectroscopy this immediately poses a problem. Glass and plastics absorb
strongly throughout the infrared region of the spectrum. Cells must be constructed of ionic
substances—typically sodium chloride or potassium bromide, Potassium bromide plates are more
expensive than sodium chloride plates and have the advantage of usefulness in the range of 4000 to
400 cm~*. Sodium chloride plates are used widely because of their relatively low cost. The practical
range for their use in spectroscopy extends from 4000 to 650 cm™’. Sodium chloride begins to ab-
sorb at 650 cm™’, and any bands with frequencies less than this value will not be observed. Since
few important bands appear below 650 cm™’, sodium chloride plates are in most common use for
routine infrared spectroscopy.

Liquids. A drop of a liquid organic compound is placed between a pair of polished sodium chloride
of potassium bromide plates, referred to as salt plates. When the plates are squeezed gently, a thin
liquid film forms between them. A spectrum determined by this method is referred to as a neat
spectrum since no solvent is used. Salt plates break easily and are water soluble. Organic com-
pounds analyzed by this technique must be free of water. The pair of plates is inserted into a holder
which fits into the spectrometer,

Solids. There are at least three common methods for preparing a solid sample for spectroscopy.
The first method involves mixing the finely ground solid sample with powdered potassium bro-
mide and pressing the mixture under high pressure. Under pressure, the potassium bromide melts
and seals the compound into a matrix. The result is a KBr pellet which can be inserted into a
holder in the spectrometer. The main disadvantage of this method is that potassium bromide ab-
sorbs water, which may interfere with the spectrum that is obtained. If a good pellet is prepared,
the spectrum obtained will have no interfering bands since potassium bromide is transparent down
to 400 cm™!,

The second method, a Nujol mull, involves grinding the compound with mineral oi! (Nujol) to
create a suspension of the finely ground sample dispersed in the mineral oil. The thick suspension
is placed between salt plates. The main disadvantage of this method is that the mineral oil ob-
scures bands that may be present in the analyzed compound. Nujol bands appear at 2924, 1462,
and 1377 cm™ (p. 30).

The third common method used with solids is to dissolve the organic compound in a solvent,
most commonly carbon tetrachloride (CCl,), Again, as was the case with mineral oil, some regions
of the spectrum are obscured by bands in the solvent. Although it is possible to cancel out the sol-
vent from the spectrum by computer or instrumental techniques, the region around 785 cm™! is
often obscured by the strong C—C] stretch that occurs there.

THE INFRARED SPECTROMETER

The instrument that determines the absorption spectrum for a compound is called an infrared
spectrometer or, more preciscly, a spectrophotometer. Two types of infrared spectrometers
are in common use in the organic laboratory: dispersive and Fourier transform (FT) instru-
ments. Both of these types of instruments provide spectra of compounds in the common range
of 4000 to 400 cm. Although the two provide nearly identical spectra for a given compound,
FT infrared spectrometers provide the infrared spectrum much more rapidly than the dispersive
instruments.

Dispersive Infrared Spectrometers

The following are the essential components of an infra-red spectrophotometer.

1. Light source.

Monochromator and optical materials

Sample holder

Detector and

Instrument for recording the response (Recorder)

1. Infra Red Radiation Sources:

The infra red radiation sources are the hot bodies, continuously emitting the radiations,
which approximate a black body radiator in their emission properties.

(a) Incandescent lamp : A closed wound nichrome coil can be raised to incandescence by
resistive heating. A black oxide film formed on the coil give acceptable emmisivity.
In this the temperature can be reached up 1100°. The nichrome coil does not require
water cooling. It requires little or no maintenance and gives long service. This

source is recommended where reliability is essential. Though this source is simple
and rugged, it is less intense than some other infra-red radiation sources.

A rhodium wire heater sealed in a ceramic cylinder has also been used as a source of
infra red radiations.

(b) Nernst glower ; In IR spectroscopy, Nernst glower is the most commonly used source
of radiation. It is constructed by fusing a mixture of oxides of metals like zirconium,
yttrium and thorium. They are moulded in the form of hollow tubes or rods about
1-3 mm in diameter and 2-5 cm in length. The ends of the rods are cemented to short
ceramic tubes for mounting and short platinum leads are provided for power
connections.

mm wf

Nernst glowers are fragile. They have negative coefficient of resistance and they are
preheated as to be conductive. Thus they are provided with auxiliary heaters. In order
to prevent overheating they are provided with ballast, but they should also be protected
from draft and at the same time ventilation is needed to remove surplus heat.
The energy output of Nernat glower is predominantly concentrated between 1-10
with relatively low energy beyond 10 p. Radiation intensity is approximately thrice
that of nichrome and globar sources, except in the near infra red region,
The main advantages of Nernst glower is that it emits infra red radiations over a
wide wavelength range and the intensity of radiation remains steady and constant
over a long period of time. Secondly, it can be used in air as it is not oxidised.

(c) Globar Source : It is a rod of sintered silicon carbide 6-8 mm in diameter and 50 mm

in length. It is self starting and is electrically heated. The operating temperature is
about 1300°. It has a positive coefficient of resistance and can conveniently be

controlled with a variable transformer. It is often enclosed in a water cooled brass
tube, with a slot provided for the emission of radiations. It emits maximum radiation
at 5200 em=!. In comparison with Nernst glower the Globar is a less intense source
below 10 p. The two sources are comparable to about 15 p, and the Globar is superior

beyond about 15 p.

(d) Mercury Arc : In the very far infra red region. i.e. beyond 50 p (200 cm-‘), black body
type sources lose effectiveness as their radiations decreases with the fourth power of
wavelength. Mercury arc gives intense radiation in this region. It is enclosed in a

quartz jacket to reduce loss. The output from mercury arc is similar to that of black
body sources, but additional radiation is emitted from a plasma which enhances the
long wavelength output.

(e) Tungsten Filament Lamp : This source is useful for near infra red region only.

2. Monochromators:

The radiation source emits radiations of various frequencies. As the sample in IR
spectroscopy absorbs only at certain frequencies, it is therefore, necessary to select desired
frequencies from the radiation source and reject the radiations of other frequencies. This
selection is achieved by means of monochromators. The monochromators are of two types :
(i) Prism monochromators and (ii) Grating monochromators.

(i) Prism Monochromators : These are favoured because of greater range and simplicity.

Neither glass nor quartz is sufficiently transparent to infra red radiations and therefore other

materials like halogen salts are used in prism monochromators as they are transparent to
infra red radiations.

Quartz prisms are used only in the near infra red region (0.8 – 3 1). It is absorbed strongly
heyond 4

The great bulk of analytical work in the infra-red region is done using crystalline sodium

chloride as the prism material. It has high dispersion in the region between 5-15 p and

adequate upto 2.5 1 crystalline potassium bromide and cesium bromide are satisfactory for far

infra red region (15 p to 40 p). In the near infra red region (1-5 1), lithium fluoride is used as

prism material.

All the commonly used prism materials except quartz are water soluble and are easily

scratched. These materials must be protected from moisture either by using dessicants or by

placing in a sealed housing which is evacuated.

In the infra red spectrometers the focusing of the radiations is achieved by using concave

mirrors rather than prisms. These mirrors can be prepared from various materials like

metals or glass coated aluminium. The main advantage of these materials is that it has no

chromatic abbration and are also sturdy. Besides concave mirrors plane reflecting mirrors

are also used.

The prism monochromator may be a single pass monochromator or a double pass
monochromator as shown in figures 24.4 and 24.5 respectively.

Exit sit

Source

Fig. 24.4: Single pass monochromator

/ 1 Littrow mirror

_ ss
en e e

<— ~ Se Exit slit
}

Plane mitror Atntrance sit

Fig. 24.5 : Double pass monochromator

Single pass monochromator:

The sample is kept at or near the focus of the beam, ; just before the entran ce slit A to the
monochromator. The radiation from the source after passing through the sample and the slit
strikes the off-axis parabolic Littrow mirror 3. This renders the radiation ‘parallel and is
transmitted to the prism ‘C’. The dispersed radiation after reflecting from a plane mirror ‘D’
eretaurnes through the prirsma ise cond time a: nd focuses into the e exit exit slistl it of the monochromator and

Double pass monochromator:

more resolution of radiation as compared to mono pass monochromator.
In both mono and double pass monochromators. : sodi um chloride (rock salt) pri

employed for the entire region from 4000 – 650 cm-! (2.5 to 15.4 ya).
Prisms of lithium fluoride and calcium fluoride give more

the significant stretching vibrations are located. . enero SSD ROO weaN

(ii) Grating monochromators 1 The gratii ng is essentially a series of lel
= cut out into a plane surface. It is usually coastructed from glass or sidaite which nee

th aluminium, In order to minimise greater amounts of scattered radiations and the
unwanted radiations of other spectral orders, th
radiation into a single order. . the gratings are blaze to concentrate the

A grating is generally used in combination with a small prism which acts as order sorter.

Sometimes filters transparent over a limited wavelength range are incorporated with
gratings.

Grating monochromators have certain advantages over prism monochromators as :

(i) The grating construction material is not attacked by moisture and is not subjected to
etching where on the salt prisms are affected by moisture and can be subjected to etching.

(ii) Secondly grating monochromators can be used over considerable wavelength range and

(iii) Grating monochromators are sturdy and long lasting.

3. Sampling: (see previous pages)

4. Detectors:

There are two types of detectors used in infra red spectrophotometry and they are

(a) thermal detectors and (b) photo-detectors.

(a) Thermal detectors : When the infra radiations falls on these detectors, they cause

heating which gives rise to a potential difference which is measured. This potential

difference depends upon the amount of radiation. The thermal detectors commonly

used are thermocouples, bolometers and thermisters and Golay cell or Golay detector.

(i) Thermocouple : It is the most commonly used detector in infra red

spectrophotometry, Thermocouples are basically the dissimilar strips of metals
joined together at one end. Thermocouples are constructed in various ways. In
one of the thermecouple detectors two fine wires of metals which have different

thermoelectrical properties are welded with blackened gold foil, and which
absorbs the radiations. One welded joint (cold junction) is kept at constant

temperature and the other welded joint (hot junction) is exposed to radiations.

This exposure of hot junction causes a rise in its temperature Thus, as the two

junctions are at different temperatures, it causes a potential difference which is

proportional to degree of heating of hot junction (or amount of radiations

falling on the hot junction).

(ii) Bolometers : They are constructed from metals or semiconductors. In this large

change of electrical resistance depends on temperature. When the radiations

fall on bolometer, its temperature changes which cause change in the resistance
of the conductor. This change in resistance depends upon the amount of
radiations falling on the bolometer.

Bolometer is made in one arm of the wheatstone bridge and a similar strip of
metal is used as balancing arm of the bridge, which is not exposed to infra red
radiations. When no infra red radiations fall on the bolometer, the bridge
remains balanced. As the radiations fall on the bolometer, the bridge becomes
unbalanced due to change in electrical resistance and thus the electrical
current flows through galvanometer G. The amount of current flowing through
galvanometer is a measure of the intensity of the radiations falling on the
detector. The response time for bolometer is 4 m sec. The schematic
representation is miven below.

Fig. 24.7

Both thermocouples or bolometers are fitted in steel housing having potassium
bromide or cesium iodide window and it is evacuated, which decreases the
noise and increases sensitivity.

(iii) Thermisters : These functions similar to bolometers. They are the resisters
made by fusing several metallic oxides. These shows a negative thermal

coefficient of electrical resistance.

(iv) Golay cell or Golay detector: Golay cell is now-a-days used in several
commercial spectrophotometers. It consists of a small metal cylinder, one end
of which closed by blackened metal plate and the other with a metallised
diaphragm. A light beam falls on the diaphragm which reflects to phototube.
The cylinder is filled with non-absorbing gas like xenon. When the radiations
fall on blackened metal plate it is heated, which causes the expansion of gas
which in turn affects the diaphragm (motion of the diaphragm). This causes the
change in the output of cell received by the phototube, which can be modulated
according to the power of the falling radiations on Golay cell.
Thermocouples and Golay detectors possesses similar sensitivity in the mid
infra red region.

(b) Photon detectors : Photon detectors are widely used in near infra red region. They
consist of suitable semiconductors like lead sulphide, lead telluride or germanium
which are non-conducting at lower energy state. When the radiations fall on these

in

they are raised to higher level which can conduct and produce a signal which is

proportional to the amount of radiation. In these there is a drop of electrical resistance

and if small voltage is applied there is a large increase in current which can be

amplified and indicated on a meter or recorder.
5. Recorder:

In infra red recording spectrophotometers as the sample absorbs some energy, the sample

beam and reference beam differs in their radiant energies. Then detector system generates

the signal which is normally amplified and goes to servometer. The servometer which is

connected to attenuator comb blocks the part of reference beam till energies of reference and
sample beams are equal and thus beam balance is achieved (i.e. optical null). The attenuator
comb is tied mechanically to the pen of the recorder and paper driver. They are synchronised
with the automatic rotation of wavelength mirror. The transmittance of the sample is recorded

as a function of wavelength.

—_=>=>—

Mirror Y
fi

FIGURE 2.3 A schematic diagram of a dispersive infrared spectrometer.

FT-IR:

Instrumentation

Several Fourier transform spectrometers are
available commercially. Their prices lic in the
$35,000 to $120,000 range (including the
dedicated computer for performing the Fou-
rier transformation).

Drive Mechanism. A requirement for satis-
factory interferograms (and thus satisfactory
Spectra) is that the speed of the moving
mirror be relatively constant and its position
exactly known at any instant. The planarity
of the mirror must also remain constant
during its entire sweep of 10 cm or more.

In the far-infrared region, where the wave-
lengths are in the micrometer range, dis-
placement of the mirror by a fraction of a
wavelength, and accurate measurement of its
Position, can be accomplished by means of
a motor-driven micrometer screw. A more
precise and sophisticated mechanism is re-
quired the mid- and near-{nirared regions,
however. Here, the mirror mount is gencrally
floated on air cushions held within close-
fitting stainless steel sleeves (sce Figure 8-22).
The mount is driven by an clectromagnetic
coil similar to the voice coil in a loudspeaker:
a slowly increasing current in the coil drives
the mirror at constant velocity. After reaching
its terminus, the mirror is returned rapidly to
the starting point for the next sweep by a
rapid reversal of the current. The length of
travel vanes from 2 to about 18 cm; the scan
rates range from 0.05 cm/s to 4 cm/s.

Two additional features of the mirror
system are necessary for successful opcration
in the infrared regions. The first of these is
a means ‘of sampling the interferogram at
precisely spaced retardation intervals. The
second is,a method for determining exactly
the zero-fetardation point to permit signal
averaging. If this point is not known pre-

cisely, the signals from repetitive sweeps
would not be fully in phasc; averaging would
tend to degrade rather than improve_ the
signal. :

The problem of precise signal sampling

and signal averaging ts accomplished in mod-
em instruments by using three interferom-

‘ @ters rather than one, with a single mirror
mount holding the three movable mirrors.

1?

Beam Splitters. Beam splitters are con-
structed of transparent materials with re-
fractive indices such that approximately 50%

of the radiation is refleAc witdelyd u.se d
materia] for the far-inrefgirona irs ea tdhi n
film of Mylar sandwiched between two plates
ofa low refractive-index solid. Thin films of
germanium or silicon deposited on cesium
iodide or bromide, sodium chloride, or potas-
sium bromide are satisfactory for the mid-
infrared region. A film of iron(III) oxide is
deposited on calcium fluoride for work in the
near-infrared.

Searces and Detectors. The sources for
Fourier transform infrared instruments are
similar to those discussed carlicr in this chap-
ter. Generally, pyroelectric detectors must be
employed because their response times are
shorter than those of the other infrared
detectors. °
DoabDlesiegn. -InsBtruementas fmor t he

far-infrared region are often of single-beam
design. Most instruments for the higher-
frequency range are double-baefatemr :ex;it –
ing from the interferometer, the beam is
alternated between a sample compartment
and a reference comparbty mmeeansn otf a
moving mirror. The beams are then recom-
bined and pass on to the detector.

neem | |
Iniarfarogram:

Movin, g | ] 4 the signal the
Mirror ue computer racelves.

Detector

NA
oy run

FACTORS:

Factors Influencing Vibrational Frequency :

From the discussion above we know that the probable frequency of absorption can be
calculated by the Hook’s law. However, it has been observed that the calculated value of
frequency of absorption is not exactly equal to the experimental value. There are many factors
which are responsible for shifts in vibrational frequencies.

(a) The frequency shift may occur due to the effect of molecule in the immediate
neighbourhood of bond,

(b) Change in force constant of bond due to electronic structure and

(c) Due to different states of the same substance e.g. solid, liquid or gas (vapour),

The energy of vibration and thus the wavelength of its absorption peak is influenced by
other vibrations in a molecule. The influence and extent of coupling of vibrations plays
significant role.

APPLICATIONS:

(see book)

?

CHAPTER-2

NMR SPECTROSCOPY

NMR spectroscopy ts the study of spin changes at the nuclear level when a radiofrequency
energy is absorbed in the presence of magnetic field.

Quantum numbers and their role in NMR:

Many atomic nuclei have a property called spin: the nuclei behave as if they were spinning. In fact,
any atomic nucicus that possesses either odd mass, odd atomic number, or both has a quantized spin
angular momentum and a magnetic moment. The more common nuclei that possess spin include {H,
iH, ‘&c, ‘SN, ZO, and ‘3F. Notice that the nuclei of the ordinary (most abundant) isotopes of carbon
and oxygen, ‘ZC and ‘§O, are not included among those with the spin property. However, the nucleus
of the ordinary hydrogen atom, the proton, does have spin. For cach nucleus with spin, the number of
allowed spin states it may adopt is quantized and is determined by its nuclear spin quantum number, /.
For each nucleus, the number / is a physical constant, and there are 2/ + | allowed spin states with in-
tegral differences ranging from +/ to —/. The individual spin states fit into the sequence

+/,([/—1),…,(-/+1),-l Equation 3.1

For instance, a proton (hydrogen nucleus) has the spin quantum number / = and has two allowed spin
states [2(/) + 1 = 2) for its nucleus: ~! and +4. For the chlorine nucleus, / = and there are four allowed
spin states [2(3) + 1 = 4]: -t, —}, +}, and +2. Table 3.1 gives the spin quantum numbers of several nuclei.

TABLE 3.1
SPIN QUANTUM NUMBERS OF SOME COMMON NUCLEI

Element in UH ic UMN: = 90– Yo. Fe a
Nuclear spin ‘ t 5 t 1 3

quantum number + I 0 ; j 0 : :

Number of
spin states 2 3 0 2 3 0 6 2 2 4

In the absence of an applied magnetic field, all the spin states of a given nucleus are of equivalent
energy (degenerate), and in a collection of atoms, all of the spin states should be almost equally
populated, with the same number of atoms having each of the allowed spins.

ABSORPTION OF ENERGY

The nuclear magnetic resonance phenomenon occurs when nuclei aligned with an applied field are
imuced to absorb energy and change their spin orientation with respect to the applied ficld. Figure
3.5 illustrates this process for a hydrogen nuclcus.

The energy absorption is a quantized process, and the encrgy absorbed must equal the energy
difference between the two states involved,

Fanccrtrod @ (E~1 ante ~ Fo case) @ AY Equation 3.2

In practice, this energy difference is a function of the strength of the applied magnetic field, Bp, as
illustrated in Figure 3.6,

| — | |

i. ae -j

FIGURE 3.3 The spin sates of a peoton in the No field Applied field
abscace and in the presence of an applied magnetic
field Energies Alignenents

‘The stronger the applied magnetic field, the greater the energy difference between the possible
spin states:

AE =f (Bo) Equation 3.3

The magnitude of the energy-level separation also depends on the particular nucleus involved. Each
nucleus (hydrogen, chlorine, and so on) has a different ratio of magnetic moment to angular mo-
mentum, since each has different charge and mass. This ratio, called the magnetogyric ratio, 7, is a
constant for cach nucleus and determines the energy dependence on the magnetic field:

SE =f (yp) =hv Equation 3.4

ve the angular momentum of the nucleus is quantized in units of h/27, the final equation takes
e form

AF (= h ) Be hy Equation 35

Solving for the frequency of the absorbed crierEy,

v= (2) Bo Equation 3.6

THE MECHANISM OF ABSORPTION (RESONANCE)

‘To understand the nature of a nuclear spin transition, the analogy of a child’s spinning top is useful.
Protons absorb energy because they begin tu precess in an applied magnetic ficld. The phenomenon

of precession is similar to that of a spinning top. Owing to the influence of the earth’s gravitational

ficld, the top begins to “wobble,” or precess, about its axis (Fig. 3.7a). A spinning nucleus behaves

in a similar fashion under the influence of an applied magnetic field (Fig. 3.7b).
When the magnetic field is applied, the nucleus begins to precess about its own axis of spin

with angular frequency w, which is sometimes called its Larmor frequency. The frequency at

th

!
“TS

re 1 ao”

(a) ey ae

FIGURE 3.7 (a)A top precessing in the earth’s gravitational field; (b) the precession of a spimung nucleus resulting
from the influence of an applied magnetic field.

which a proton precesses is directly proportional to the strength of the applied magnetic field;
the stronger the applied field, the higher the rate (angular frequency, @) of precession. For a pro-
ton, if the applied field is 1.41 Tesla (14,100 Gauss), the frequency of precession is approxi-
mately 60 MHz,

Since the nucleus has a charge, the precession generaes an oscillating electric field of the same
frequency. If radiofrequency waves of this frequency are supplied to the precessing proton, the en-
ergy can be absorbed. That is, when the frequency of the oscillating electric field component of the
incoming radiation just matches the frequency of the electric field generated by the precessing nu-
cleus, the two ficlds can couple, and energy can be transferred from the incoming radiation to the
nucleus, thus causing a spin change. This condition is called resonance, and the nucleus is said to
have resonance with the incoming electromagnetic wave, Figure 3.8 schematically illustrates the
resonance process.

oo ~ 60 MHz a

» = 60 MHz |) | Absorption occurs

om a Wis
>

hy = fiw . ak
| ?

|| 4 | |
| | __j

6, 14.100 gauss

FIGURE 3.8 The nuclear magnetic resonance process; absorption occurs when v= @

17

INSTRUMENTATION:

A line diagram of the instrument of NMR spectrophotometer along with its components is
shown in Fig. 25.5.

|
Recorder

Pig. 25.5

Magnet : The strong magnet provides stable and homogencous field. The magnet size is 15
inches in diameter and is capable of producing strong fields (upto 23.500 yauss for 100 MHz
work).

If the magnetic field is not homogeneous, the nuclei in the different parts of the sample
precess with different frequencies, thereby producing broad signals.

Radio Frequency Oscillator (Transmitter) and Sweep Generat:o Trhe RF oscillator cvil is
installed perpendicular to the magnetic field and transmits radiowaves of some mixed
frequency such as 60, 100, 220 or 300 MHz.

Since the large magnet as well as the Radio Frequency oscillator both produce fixed fields,
a sweep generator is installed to supply a variable de current to a secondary smaller magnet
This allows us to vary (or sweep? the total applied magnetic field over a small ranye.
RF receiver (detector) and Recorder

The coil of the RF receiver or detector is installed perpendicular to both the magnetic field
and the oscillator coil and is tuned to the same frequency as the transmitter. When the
precession frequency is matched with the radio frequency the nuclei induces electromagnetic
field (emf) in the detector coil by virtue of the change in magnetic flux following nuclear
flipover. This signal is amplified and sent to a recorder.

The recorder gives a spectrum as a plot of the strength of the resonance signal on the Y axis
Vs strength of the magnetic field on X axis. The strength of the resonance signal is directly
proportional to the number of nuclei resonating at that particular ficld strength The area of the
peak is therefore a direct measure of number of resonating nucle: and hence most of the
instruments are equipped with automatic integrator which can record poak arcas in the form of
a superimposed integration trace on the chart

The measurement of exact strength of continuously sweeping magnetic field is difficult
task, hence it is difficult to assign a peak position on absolute scale. Thus, the method used is to
record the peak position in relation to the position of an arbitrary standard lines (internal
standard).

The tetramethyl silane (TMS — (CH,), Si) is used as internal standard for most of protons
and is ndded to the sample before recording the spectrum.

Sample and Sample Holder:

A 1-30 mg sample is generally used in the form of dilute solution (2 – 10%). The solvent
should not contain hydrogen of tts own.

Sample holder is a glass tube about 5 mm in diameter and is 15 – 20 cm in length.

FACTORS AFFECTING CHEMICAL SHIFT

1. Inductive Effect:

A proton is said to be deshielded if it is attached with an electro negative group. Greater the
vlectronegativity of the atom, greater the deshiclding effect and more will be the 6 value.

CH, – CH, 0.95
CH, – Cl 3.05 &
CH; -F 4.26

Thus electronegative groups deshield the proton, As the distance from the electronegative
atom increases, the deshielding effect diminishes.

mn

2. Van der Waal’s Deshielding:

In overcrowded molecules it is possible that some proton may be occupying sterically
hindered positions. Clearly the electronegative cloud of ‘bulky group will tend to repell the
electron cloud surrounding the proton, Thus, such a proton will be deshielded and will
resonate at alightly higher values of 5 than expected. This is considered as Van der Waal’s
deshielding.

3. Anisotropic effect (Space Effect):
Magnetic field developed by x bond is stronger in one direction than other e.g. in alkene it

is oriented in such a way that the plane of the double bond remains right angled to applied field
The induced magnetic field around carbon is dimagnetic and paramagnetic in the direction
of alkene proton. Thus proton will feel greater field strength and hence effect occurs at lower
field.

4. Hydrogen Bonding:

Hydrogen atom exhibiting the property of hydrogen bonding in a compound absorbs at a
low field in comparison to the one which does not show hydrogen bonding. The hydrogen
bonded proton being attached to highly electronegative atom will have smaller electron density
around it (deshielded) hence resonate at downfield.

5. Concentration, Solvent and Temperature Effect :

In CCl, and CdCl, chemical shift of proton attached to carbon is independent of
concentration and temperature while protons of ~ OH, — NH, — SH groups exhibit a
substantial concentration and temperature effect due to hydrogen bonding. Intermolecular
hydrogen bonding is less affected than intermolecular bonding by concentration change. Both
types of hydrogen bonding are affected by temperature variations.

SPIN-SPIN COUPLING:

The spins of neighbouring groups of nuclei in molecule are said to be coupled if their spin states
mutually interact. The interactions, which involve electrons in the intervening bonds, result in
small variations in the effective magnetic fields experienced by one group of nuclei due to different
Orientations of the spin angular momenta and magnetic moments of those in the neighbouring
group or groups, and vice versa, These lead to the splitting of the resonance signal into two or
more components that are shifted slightly upfield and downfield respectively from the position in
the absence of coupling, the probabilities of each being roughly the same because the pernitted
nuclear spin energy levels are almost equally populated. Thus, the resonance signals for two single
adjacent nuclei with substantially different chemical shifts are each split into two component peaks
of equal intensity.

(Spin-Spin Splitting)

On observing the NMR spectra of compounds it in seen that the signals are split into
number of lines. e.g. in CH,CH, OH the signals given are :

Singlet for OH
Quartet for CH,
Triplet for CH,

We say that each signal will split into doublet, triplet, quartet depending on the number of
protons present on adjacent carbons. The multiplicity of lines is related to the number of
protons on neighbouring groups. A simple rule (n + 1 rule) is used to find the multiplicity.
Count the number of neighbouring protona and add 1.

71

Splitting of the spectral lines arise because of coupling interaction between neighbour
protons and is related to the number of possible spin orientations that these neighbours can
udopt. The phenomenon is called either spin-spin splitting/coupling.
Theory of spin—spin splitting H,

Consider Cinnamic acid Ox = t_ COOH

Hs
representing 2 vicinal protons, Hy, and Hy. These protons, having different magnetic
environments, come to resonance at different positions in unit spectrum. They do not give rise
to single peaks (singlets) but doublets. The separation between the lines of each doublet is
equal, this spacing is called coupling constant ‘J’.

The resonance position for A depends on its total magnetic environment, part of its
magnetic environment is the nearby proton B, which is itself magnetic, and the proton B can
either have its nuclear magnet aligned or opposed with proton A. The two spin orientations of B
create 2 different magnetic fields around A. Therefore the proton A comes to resonance, not
once, but twice and proton A gives rise to a doublet.

Similarly with proton B the mutual magnetic influence between protons A and B is not
transmitted through space, but via the electrons in the intervening bonds. The nuclear spin of
A couples with electron spin of C-H, bonding electrons these in turn couple with C-C bonding
electrons and then with C-H, bonding electrons, The coupling is eventually transmitted to the
spin of Hg nucleus.

THE COUPLING CONSTANT

Section 3.15 discussed the splitting pattern of the ethyl group and the intensity ratios of the multi-

plet components but did not address the quantitative amount by which the peaks were split. The dis-

tance between the peaks in a simple multiplet is called the coupling constant, J. The coupling

constant is a measure of how strongly a nucleus is affected by the spin states of its neighbor. The

spacing between the multiplet peaks is measured on the same scale as the chemical shift, and the

coupling constant is always expressed in Hertz (Hz). In ethyl iodide, for instance, the coupling con-

stant J is 7.5 Hz. To see how this value was determined, consult Figures 3.26 and 3.34. . |

The spectrum in Figure 3.26 was determined at 60 MHz; thus, each ppm of chemical shift (d unit)

represents 60 Hz. Inasmuch as there are 12 grid lines per ppm, each grid line represents (60 Hz)/12 =

Singlet |

Doublet I 1

Triplet i 2 4)

Quartet 1 3 3 41

Quintet 1 4 6 4 1

Sextet 1 5 10 10 5 1

FIGURE 3.33 Pascal’s triangle. Septet 1 6 15 20 15 6 1

??

a

~~ +
4s rer >

el Tere

oa

roe rere ro the
ee .: -~+

~~?

$+ > ++? ++
‘ ~rre he
* $+?+ ¢ _e? 7 Toth:

*– owe 4 . Tt het
a

peter “re

–
–

4+e4 + b~ +

f 53445 ed ; ++
—¢ terete

* teem eew bee
+ trie $
. oe ” ever tei+ > ll

ren

erere per este rept ee ttt +t tects tot – – -— ~~ “? vv

7g ao a5 49 30 20 ‘6 ore

FIGURE 3.26 The ‘II NMR spectrum of ethyl iodide (60 MHz),

J J J/
Coupling constant
measured in Hz

Chemical shift is

canotf agrrou p
Chemica! shill difference

FIGURE 3.34 The definition of the coupling constants in the ethy! splitting pattern.

5 Hz. Notice the top of the spectrum. It is ¢ alibrated in cycles per second (cps) . Which are the same as
Hertz, and since there are 20 chart d ivisions per 100 cps, one division equals (100 eps)/20 = 5
5 Hz Now ex amine the multiplets. The spac between the component peaks is approximately
1.5 chart divisions, so

S Hz
J=1.5 x = 7.5 Hz

I div

sede Core boeesdbent

eliee« “)

oT

444e4h
+= -~—-

ea ;

othe

settee

Ht
eeeee

bbe
>.

barae .

+444
ss

p+ ee
° °
tt
oe oe

bo

. “+
– ++ +

. os

That ts, the coupling constant between the methyl and methylene protons is 7.5 Hz. When the pro-
tons interact, the magnitude (in ethyl iodide) is always of this same value, 7.5 Hz. The amount of
coupling is constant, and hence J can be called a coupling constant.

The constant nature of the coupling constant can be observed when the NMR spectrum of ethy!
iodide is determined at both 60 MHz and 100 MHz. A comparison of the two spectra indicates that
the 100-MH7z spectrum is greatly expanded over the 60-MHz spectrum. The chemical shift in Hertz
for the CH and CH; protons is much larger in the 100-MHz spectrum, although the chemical shifts

in 6 units (ppm) for these protons remain identical to those in the 60-MHz spectrum. Despite the
expansion of the spectrum detenmined at the higher spectrometer frequency, careful examination of
the spectra indicates that the coupling constant between the CH, and CH; protons is 7.5 Hz in both
spectra! The spacings of the lines of the triplet and the lines of the quartet do not expand when the
spectrum of ethyl iodide is determined at 100 MHz. The extent of coupling between these two sets
of protons remains constant irrespective of the spectrometer frequency at which the spectrum wes
determined (Fig. 3.35).

For the interaction of most aliphatic protons in acyclic systems, the magnitudes of coupling con-
stants are always near 7.5 Hz. Compare, for example, 1,1,2-trichlorocthane (Fig. 3.25), for which J
= 6 Hz, and 2-nitropropane (Fig. 3.27), for which J = 7 Hz, These coupling constants are typical for
the interaction of two hydrogens on adjacent sp’-hybridized carbon atoms. Different types of pro-
tons have different magnitudes of J. For instance, the cis and trans protons substituted on a double
bond commonly have values of approximately Jj, = 10 Hz and Jireng = 17 Hz. In ordinary com-
pounds, coupling constants may range anywhere from 0 to 18 Hz.

The magnitude of J often provides structural clues, For instance, one can usually distinguish be-
tween a cis olefin and a trans olefin on the basis of the observed coupling constants for the viny]
protons, Table 3.9 gives the approximate values of some representative coupling constants. A more
extensive list of coupling constants appears in Appendix 5.

Before closing this section, we should take note of an axiom: the coupling constants of the
groups of protons that split one another must be identical. This axiom is extremely useful in inter-
preting a spectrum that may have several multiplets, each with a different coupling constant.

J = 75 Hz

” i

305 Hz

183 Hz

FIGURE 3.38 An illustration of the relationship between the chemical shift und the coupling constant

74

NUCLEAR MAGNETIC DOUBLE RESONANCE / SPIN SPIN DECOUPLING:

It is a powerful tool for simplifying a spectra. In a complex molecule if several of the
coupling constants have nearly the same values; or if the long range coupling is present or if
complex absorption gives multiplets then it becomes very difficult to determine structure.

A proton spin couples with neighbouring proton because it has sufficient life time in a
given spin state. If life time of a spin is reduced i.e. if the exchange between spin states of
nuclei is speeded up then little information about the neighbouring nuclei will be obtained,

i:
} |

In case of such compounds, where H, and H), are in different environments – C – C –
ori

Hy Hy
therefore 2 doublets at different field strengths are observed. If Hy is irradiated with strong
correct radio frequency so that the rate of its transition between the two energy states becomes

larger then the life time of this nucleus in any one spin state will be too short to resolve coupling
with Hp. In such a case Hj proton will have one time average view of Hy and hence Hg will
come to resonance only once and Hyp will appear as a singlet and not doublet.

Time d; is needed to resolve the two lines of a doublet which is related to J. Thus, formation
of a doublet is possible if each spin state of Hy has a life time greater than d;. Due to double
irradiation life time becomes still less and thus coupling is not possible. So it results in a
singlet by spin-spin decoupling

FT-NMR OR PULSED NMR:

In NMR, the radiofrequency energy can be introduced cither by continuous wave (CW) scanning
of the frequency range or by pulsing the entire range of frequencies with a single burst of
radiofrequency energy. The two methods result in two distinct classes of NMR spectrometers viz.
CW NMR spectrometers and FT or pulsed NMR spectrometers.

In Fourier transform (FT) or pulse NMR studies, an instrument with a 2.1-Tesla magnetic field
uses a short (1 to 10 1 sec) bursts of 9OMHz energy to accomplish. The source is turned on and
off very quickly, generating a pulse similar to that shown below.

On —r Pf 1=—_ Off

Intensity

_—P
Time

FIGURE 3.14 Ashort pulse.

According to a variation of the Heisenberg Uncertainity Principle, even though the frequency of
the oscillator generating this pulse is set to 9OMHz, if the duration of the pulse is very short, the
frequency content of the pulse is uncertain because the oscillator was not on long cnough to
establish a solid fundamental frequency. Therefore, the pulse actually contains a range of

as

frequencies centred around the fundamental frequency. This range of frequencies is great enough
to excite all of the distinct types of hydrogens in the molecule at once with this single burst of
energy.

When the pulse is discontinued, the excited nuclei begin to lose their excitation energy and retum
to their original spin state or relax, As each excited nucleus relaxes, it emits electromagnetic
radiation. Since the molecule contains many different nuclei, many different frequencies of
electromagnetic radiation are emitted simultaneously. This emission is called a free induction
decay (FID) signal. The intensity of the FID decays with time as all of the nuclei eventually lose
their excitation. The FID is a superimposed combination of all the frequencies emitted and can be
quite complex. The individual frequencies due to different nuclei are extracted by using a computer
and a mathematical method called Fourier transform (FT) analysis.

Therefore, the FID is the superimposition of many different frequencies, each of which could have
a different decay rate. The FT analysis will separate cach of the individual components of this
signal and convert them to frequencies, The FT analysis breaks the FID into its separate sine or
cosine wave components. This procedure is too complex to be carried out by eye or by hand and
it requires a computer, Pulsed FT NMR spectrometers have computers built into them that not only
can work up the data by this method but can control all of the settings of the instrument.

– ih —
WWW

Fig. The appearance of the FID when the decay is removed.

The pulsed FT method described here has several advantages over the CW method. It is more
sensitive, and it cun measure weaker signals. Five to 10 minutes are required to scan and record a
CW spectrum; a pulsed experiment is much faster, and a measurement of an FID can be performed

in a few seconds, With a computer and fast measurement, it is possible to repeat and average the
measurement of the FID signal. This is a real advantage when the sample is small, in which case the
FID is weak in intensity and has a great amount of noise associated with it. Noise is random elec-
tronic signals that are usually visible as fluctuations of the baseline in the signal (Fig. 3.17), Since
noise is random, it normally cancels out of the spectrum after many iterations of the spectrum are

are added together,

ah

Pulsed FT-NMR is therefore especially suitable for the examination of nuclei that are not very
abundant in nature, nuclei that are not strongly magnetic, or very dilute samples.

The most modem NMR spectrometers use supercooled magnets, which can have field strengths
as high as 14 Tesla and operate at 600 MHz. A superconducting magnet is made of special alloys

and must be cooled to liquid helium temperatures. The magnet is usually surrounded by a Dewar
flask (an insulated chamber) containing liquid helium; in turn, this chamber is surrounded by an-
other one containing liquid nitrogen. Instruments operating at frequencies above 100 MHz have su-
perconducting magnets. NMR spectrometers with frequencies of 90 MHz, 200 MHz, 300 MHz, and
400 MHz are now common in chemistry; instruments with frequencies up to 800 MHz are used for
special research projects.

NC NMR:

Carbon-12, the most abundant isotope of carbon, is NMR inactive since it has a spin of zero (see
Section 3.1), Carbon-13, or °C, however, has odd mass and does have nuclear spin, with / = !. Un-
fortunately, the resonances of ‘*C nuclei are more difficult to observe than those of protons (‘H).
They are about 6000 times weaker than proton resonances, for two major reasons.

First, the natural abundance of carbon-13 is very low; only 1.08% of all carbon atoms in nature are
“°C atoms. If the total number of carbons in a molecule is low, it is very likely that a majority of the
molecules in a sample will have no °C nuclei at all. In molecules containing a ”C isotope, it is un-
likely that a second atom in the same molecule will be a °C atom. Therefore, when we observe a °C
spectrum, We are observing a spectrum built up from a collection of molecules, where cach molecule
supplies no more than a single ‘°C resonance. No single molecule supplies a complete spectrum.

Second, since the magnetogyric ratio of a ‘°C nucleus is smaller than that of hydrogen (Table
3.2), °C nuclei always have resonance at a frequency lower than protons. Recall that at lower fre-
quencies, the population of excess nuclei is reduced (Table 3,3); this, in turn, reduces the sensitivity
of NMR detection procedures.

Through the use of modem Fouricr transform instrumentation (Section 3.7B), it is possible to
obtain ‘*C NMR spectra of organic compounds even though detection of carbon signals is difficult
compared to detection of proton spectra. To compensate for the low natural abundance of carbon, u
greater number of individual scans of the spectrum must be accumulated than is common for a pro-
ton spectrum.

Carbon spectra can be used to determine the number of nonequivalent carbons and to identify the
types of carbon atoms (methyl, methylene, aromatic, carbonyl, and so on) that may be present in a
compound. Thus, carbon NMR provides direct information about the carbon skeleton of a mo-
lecule. Some of the principles of proton NMR apply to the study of carbon NMR; however, struc-
tural determination is generally easier with carbon-13 NMR spectra than with proton NMR,
Typically, both techniques are used together to determine the structure of an unknown compound.

The theoretical background for NMR has already been
presented in Chapter 3. Some of the principal aspects
of “C NMR to consider that differ from ‘H NMR are
as follows:

e In the commonly used CPD or broadband proton-
decoupled °C spectrum (see Section 4.2.1), the peaks

are singlets unless the molecule contains other mag-
netically active nuclei such as 7H, “’P. or ‘9F.

¢ The “C peaks are distributed over a larger chemical-
shift range in comparison with the proton range.

e C peak intensities du not correlate with the number
of carbon atoms in a given peak in routine spectra,
duc to longer 7, valucs and NOE.

© The ”C nuclei are much Iess abundant and much Icss
sensitive than protons. Larger samples and longer
times are needed,

e For a given deuterated solvent, the “C and ‘H
solvent peaks differ in multiplicities

Al first glance, some of the above summary would
seem to discourage the use of ”C spectra. However, the
ingenious remedies for these difficultics have made “C
NMR spectrometry a powerful tool, as this chapter will
confirm. In fact, side-by-side interpretation of °C and
‘H spectra provide complementary information.

Applicatoifo nNMsR

1. It is used in identification of structural isomers such as

CH, — CH, – CH, OH CH, – CH — CHy
4 signals

n-propanol OH

3 signals isopropanol

It is employed in determination of Hydrogen bonding. Intermolecular hydrogen
bonding shifts the absorption to downfield. The extent of H-bonding varies with
solvent, concentration and temperature. Intramolecular H-bonding also shifts the
absorption downfield. It is not concentration dependant,

3. This method is used to detect aromaticity of a compound. Proton attached to the benzyl,
polynuclear and heterocyclic compounds whose x-electrons follow Huckel rule (4n + 2x
elec.) are extremely deshielded due to the circulation of sextet of x electron. So signals
appear at very downfield. From this aromatic character of a compound can be
predicted,

4. The NMR technique is used to distinguish between cis and trans isomers. Protons in
cis and trans isomers have different chemical shift values and different coupling
constanta.

Some “So,

Hm

j=u7-12 Ja 3-18

Similarly axial and equatorial protons or groups carrying a proton can be
distinguished because protons/groups are deshieclded in axial.

Detection of electronegative atoms or group is easily done by NMR method. Presence of
electronegative atom in the neighbourhood of proton cause deshielding, so, signal get
shifted to downfield. Greater the electronegative group greater is 5 value.

Determination of some double bond character due to resonance : In some compounds
molecules acquire a little double bond character due to resonance. Due to this, different
Signals are expected. These signals are due to the restricted rotation of the formed
double bond which changes the geometry of molecule e.g. N, N dimethyl formamide.

MASS SPECTROMETRY

he principles that underlie mass spectrometry predate all of the other instrumental techniques
described in this book. The fundamental principles date to 1898. In 1911, J. J. Thomson used
a mass spectrum to demonstrate the existence of neon-22 in a sample of neon-20, thereby es-

tablishing that elements could have isotopes. The earliest mass spectrometer, as we know it today,
was built in 1918. However, the method of mass spectrometry did not come into common use until
quite recently, when reasonably inexpensive and reliable instruments became available. With the
advent of commercial instruments that can be maintained fairly easily, are priced within reason for
many industrial and academic laboratories, and provide high resolution, the technique has become
quite important in structure elucidation studies.

THE MASS SPECTROMETER

In its simplest form, the mass spectrometer performs three essential functions. First, it subjects
molecules to bombardment by a stream of high-energy electrons, converting some of the mole-
cules to ions, which are accelerated in an electric field. Second, the accelerated ions are sepa-
rated according to their mass-to-charge ratios in a magnetic or electric field. Finally, the ions
that have a particular mass-to-charge ratio are detected by a device which can count the number
of ions striking it. The detector’s output is amplified and fed to a recorder. The trace from the
recorder is a mass spectrum—a graph of the number of particles detected as a function of mass-
lo-churge matio,

When we examine each function in detail, we sec that the mass spectrometer is actually some-
what more complex than just described. Before the ions can be formed, a stream of molecules must
be introduced into the ionization chamber where the ionization takes place. A sample inlet system
provides this stream of molecules.

A sample studied by mass spectrometry may be a gas, a liquid, or a solid. Enough of the sample
must be converted to the vapor state to obtain the stream of molecules that must flow into the ion-
ization chamber. With gases, of course, the substance is already vaporized, so a simple inlet system
can be used, This inlet system is only partially evacuated so that the ionization chamber itself is at a
lower pressure than the sample inlet system. The sample is introduced into a larger reservoir, from
which the molecules of vapor can be drawn into the ionization chamber, which is at low pressure.

To ensure that a steady stream of molecules is passing into the tonization chamber, the vapor travels
through a small pinhole, called a molecular leak, before entering the chamber. The same system
can be used for volatile liquids or solids, For less volatile materials, the system can be designed to
fit within an oven, which can heat the sample to provide a greater vapor pressure. Care must be
taken not to heat any sample to a temperature at which it might decompose.

With rather nonvolatile solids, a direct-probe method of introducing the sample may be used.
The sample ts placed on the tip of the probe, which is then inserted through a vacuum lock into the
ionization chamber. The sample is placed very close to the ionizing beam of electrons. The probe
can be heated, thus causing vapor from the sample to be evolved in proximity to the beam of elec-
trons. A system such as this can be used to study samples of molecules with vapor pressures lower
than 107? mm Hg at room temperature.

79

Principloef Mass Spectrometry

In mass spectrometry organic molecules in gaseous state under the pressure between 10″
10°° mm of Hg are bombarded with a beam of energetic electrons (70 eV) using tungsten
rhenium filament. Molecules are broken up into cations and many other fragments.

Meer — M 42e

fast slow

M- M+ M
2

M+ MoM.
j 2

R-H+e — RH +2e

These cations (molecular or parent ions) are formed due the loss of an electron usually
from n or x orbita) from a molecule, which can further break up into smaller ions (fragment
ions or daughter ions). All these ions are accelerated by an electric field, sorted out according
to their mass to charge ratio by deflection in variable magnetic field, and recorded. The output
is known as mass spectrum. The mass spectrum is a plot of relative abundance versus mass to
charge ratio, Each line upon the mass spectrum indicates the presence of atoms or molecules of
a particular mass. The most intense peak in the spectrum is taken aa the base peak. It’s
intensity is taken as 100 and other peaks are compared with it.

The mass spectrum of the compound can be obtained by using very small sample (0.1 to
1 mg). The main disadvantage of this technique is that the sample gets destroyed. Another
disadvantage is the difficulty in introducing very small samples without disturbing the
vacuum system. The instrumentation is complicated, expensive and requires skilled
technicians for operation and maintenance.

INSTRUMENTATION:

A block diagram of a mass spectrometer is shown in Figure 2. It is operated under a vacuum of
10“ to 10°” Nm*® as the presence of air would swamp the mass spectra of samples, and damage
the ion source and detector.

Sample Pressure, 10°’ – 10° Nm~*

Inlet lon Mass
system source analyzer Detector

|

Vacuum Signal

system processor

|
Read-out

Fig. 2. Block diagram of a mass spectrometer.

The principal components are

® a sample inlet, which facilitates the controlled introduction of gaseous or
vaporized liquid samples via a molecular leak (pinhole aperture) and solids
via a heated probe inserted through a vacuum lock;
an ion source to generate ions from the sample vapor;
a mass analyzer which separates ions in space or time according to their
mass-to-charge ratio. lons generated in the source are accelerated into the
analyzer chamber by applying increasingly negative potentials to a series of

metal slits through which they pass.

VW

There are several types of mass analyzer.

(i) A single focusing magnetic mass analyzer (Fig. 3) generates a field at right
angles to the rapidly moving ions, causing them to travel in curved traject-
ories with radii of curvature, r, determined by their mass-to-charge ratio,
m/z, the magnetic field strength, B, and the accelerating voltage, V, as given
by the relation

s 5
m Br*

z: WV

The majority of ions carry a charge of +1, hence m is directly proportional to
r’. For ions of a particular mass there is a specific combination of values of B
and V that allows them to pass along the center of a curved analyzer tube
to a detector positioned at the end. Progressive variation of the field or the
accelerating voltage allows ions of different mass to pass down the center of
the analyzer tube, be detected and a mass spectrum recorded.

To ___10** toe –
purnp Inlet

hot Anode
— — “} filament –

| electron — a Slit A

Sample – souresa Sit B oe
feservor *“—-

\ Path of
WA lighter ions |

YY?
Path of 10″ torr =

heavier sons

Metal NEB
analyzer ta |

tube a |

ton collector

Fig.3 . OQiagram of a single focusing magnetic analyzer mass spectrometer. From Principles of ‘
eet ererial Arcohewe Dred ate ber TVA ChaneF MAL Utert M 1000 Deserted woth weorrriccinanf

(ii) A double focusing mass analyzer employs an electrostatic separator in
addition to a magnetic analyzer to improve the mass resolution. Ions of the
same mass inherently acquire a range of kinetic energies when accelerated
and this leads to overlapping signals from those with similar masses.
Application of an electrostatic field to the moving ions allows the selection
of those with the same kinetic energy so eliminating this problem.

?

(iii) A quadrupole mass analyzer consists of a set of four parallel metal rods

positioned very closely together, but leaving a small space through the
center (Fig. 4). lons are accelerated into the space between the rods at one
end and a DC potential and a high frequency RF signal is applied across

opposite pairs of rods. This results in ions of one particular m/z value
passing straight through the space to a detector at the other end while all
others spiral in unstable trajectories towards the rods. By altering the DC
and RF signals applied to the rods, ions with different m/z ratios can be
allowed to reach the detector in turn. The ion trap is a modified version
with a circular polarizable rod and end caps enclosing a central cavity
which is able to hold ions in stable circular trajectories before allowing
them to pass to the detector in order of increasing m/z value. A particular
feature of quadrupole and ion trap analyzers is their ability to scan through
a wide range of masses very rapidly, making them ideal for monitoring
chromatographic peaks (Section F).

Nonresonant
ion – -°*

a

lon
. = collector

omy “ss HPF sax
, ee ——-

a ‘I | Resonant
oo gee ion

‘ te:

= +U, + Vcos wt

ty,
lonizing In dc and

electron beam (LV) rf voltages
-U.,,. — V cos wt K

Fig. 4. Dsagram of a quecrupole mass analyzer.

se

(iv) Tandem mass analyzers incorporate several mass analyzers in series. This
enables ions selected from the first analyzer to undergo collision induced
dissociation (CID) with inert gas molecules contained in a collision cell
producing new ions which can then be separated by the next analyzer. The
technique, known as tandem mass spectrometry, MS-MS or (MS)”, is used
in the study of decomposition pathways, especially for molecular ions
produced by soft ionization techniques (vide infra). Collision-induced reac-
tions with reactive gases and various scan modes are also employed in
these investigations.

(v) Time of flight:

fime of Flight Analysers :

In this type of analyser the sorting of the ions is done in absence of magnetic field. It
iperates on the principle that, if the ions produced are supplied with equal energy and allowed
o travel predetermined distance then they will acquire different velocities depending on their
nasses. In Bendix time of flight mass spectrometer (Fig. 26.5), a pulse of ions is produced in
he ion source. An accelerating potential of the order of 2000 V is applied to a grid, in the form of
voltage pulse lasting » sec or less and is repeated intermittently. This positive pulse

ceelerates the ions into a long field-free drift tube of about 1 meter, through which the ions
sove at their own velocities. Depending on the momentum, the ions reach the detector in the
rder of increasing mass.

In this instrument specially designed electron multiplier detector is used which consista of
wo glass plates coated with high resistance metallic film which act as dynodes. The sorted

ions get collected at the anode in order of their increasing mass, Time of flight spectrometers
have resolution power of 500 to 600, Fe ion uM ie el

lon source Focusing plate Accelerating plate multiplier (detector)

From ate
sample al ——

_ | — He

Dynodes Anode
“oe Vacuum

pump
Fig. 26.5 : Bendix time of flight mass spectrometer

IONIZATION TECHNIQUES or DIFFERENT TYPES OF IONIZATION:
There are numerous means of ionizing molecules or elements in a sample, the
most appropriate depending on the nature of the material and the analytical
requirements. In addition, mass spectrometry can be directly interfaced with
other analytical techniques, such as gas or liquid chromatography (Topics D4 to
D7) and emission spectrometry (Topics E3 to E5). These hyphenated systems
are described in Section F. The more important ionization techniques are
summarized below.

7.3.1.1 Electron Impact flonization. Electron
impact (EI) is the most widely used method for gener-
ating ions for mass spectrometry. Vapor phase sample
molecules are bombarded with high-energy electrons
(generally 7OeV), which eject an electron from a
sample molecule to produce a radical cation, known as
the molecular ion. Because the ionization potential of
typical organic compounds is generally less than 15 eV,
the bombarding electrons impart 50 eV (or more) of
excess cnergy to the newly created molecular ion,
which is dissipated in part by the breaking of covalent
bonds, which have bond strengths between 3 and 10 eV.

Bond breaking is usually extensive and critically,
highly reproducible, and characteristic of the
compound. Furthermore, this fragmentation process
is also “predictable” and is the source of the powerful
structure clucidation potential of mass spectrometry.
Often, the excess energy imparted to the molecular
ion is too greal, which leads to a mass spectrum with
no discernible molecular ton. Reduction of the ion-
ization voltage is a commonly used strategy to obtain
a molecular ion; the strategy is often successful
because there is greatly reduced fragmentation. The
disadvantage of this strategy is that the spectrum
changes and cannot be compared to “standard” liter-
ature spectra.

To many, mass spectrometry is synonymous with
EI] mass spectrometry. This view is understandable for
two reasons. First, historically, El was universally avail-
able before other ionization methods were developed.
Much of the early work was EI mass spectrometry.
Second, the major libraries and databases of mass spec-
tral data, which are rclied upon so heavily and cited so
often, are of El mass spectra. Some of the readily
accesible databases contain El mass spectra of over
390,000 compounds and they are easily searched by

efficient computer algorithms. The uniqueness of the
El mass spectrum for a given organic compound, even
for stereoisomers, is an almost certainty. This unique-
ness, coupled with the great sensitivity of the method, is

what makes GC-MS such a powerlul and popular
analytical tool.

ww

7.3.1.2 Chemical lonization. Electron impact
ionization often leads to such extensive fragmentation
that no molecular ion is observed. One way to avoid this
problem is to use “soft ionization” techniques, of which
chemical ionization (C1) is the most important. In Cl,
sample molecules (in the vapor phase) are not subjected
to bombardment by high energy electrons. Reagent gas
(usually methane, isobutane, ammonia, but others are
used) is introduced into the source, and ionized. Sample
molecules collide with ionized reagent gas molecules
(CH,*, CyH,*, etc) in the relatively high-pressure Cl
source, and undergo secondary ionization by proton
transfer producing an [M+ 1]” ion, by electrophilic
addition producing [M + 15]°,|M + 24]’,[M + 43]*, or
IM + 18]° (with NH,*) ions, or by charge exchange
(rare) producing a |M]* ion. Chemical ionization spectra
sometimes have prominent [M — 1]* ions because of
hydride abstraction. The tons thus produced are even
electron species. The excess energy transfered to the
sample molecules during the ionization phase is small,

generally less than 5eV, so much less fragmentation
takes place. There are several important consequences,
the most valuable of which are an abundance of molecu-
lar ions and greater sensitity because the total ion
current is concentrated into a few ions There its
however, less information on structure. The quasimolec-
ular ions are usually quite stable and they are readily
detected. Oftentimes there are only one or two fragment
ions produced and sometimes there are none.

Three stages are involved. For methane, for
example:

(i) reagent gas ionized by EI: CH, +e — CH,’* + 2e
(ii) secondary ion formation: CH,** + CH, — CH.’ + CH,°
(iii) formation of molecular species: CH. +M —> MH* +CH,

(pseudomolecular ion)

WM

1.3.2.1 Field Desorption lonization. |n the
field desorption (FD) method, the sample is applied to a
metal emitter on the surface of which ts found carbon
microncedles, The microncedles activate the surface,
which is maintained at the accelerating voltage and func-
tions as the anode, Very high voltage gradients at the tips
of the needles remove an electron from the sample, and
the resulting cation ts repelled away from the emitter.
The ions generated have little excess energy so there is
minimal fragmentation, i.c., the molecular ion is usually
the only significant ion seen. For example with
cholesten-5-ene-3,16,22,26-tetrol the El and Cl do not
see a molecular ion for this steroid. However, the FD
mass spectrum (Figure 1.4) shows predominately the
molecular ion with virtually no fragmentation.

Field desorption was eclipsed by the advent of
FAB (next section). Despite the fact that the method is
often more useful than FAB for nonpolar compounds
and does not suffer from the high level of background
ions that are found in matrix-assisted desorption meth-
ods, it has not become as popular as FAB probably
because the commercial manufacturers have strongly
supported FAB.

7.3.2.2 Fast Atom Bombardment lonization.
Fast atom bombardment (FAB) uses high-enerpy The bombarding beam consists of enon (or argon) at-
xenon or argon atoms (6-10 keV) to bombard samples coms ed hig’ transiational energy (Xe). Thes beam is pro
dissolved in a liquid of low vapor pressure (c.g., glyc- doced by fet innumieg tenon atoms wet electroes fo
erol). The matrix protects the sample from excessive pve lesen tadical Cahoes:

radiation damage. A related method, liquid secondary
Xe “—» Xe”+ 2 e

ionization mass spectrometry, LSIMS, is similar except the glycosidic and peptide bonds, respectively, thereby
that it uses somewhat more energetic cesium ions affording a method of sequencing these classes of
(10-30 keV). compounds

In both methods, positive ions (by cation altach- The upper mass limit for FAB (and LSIMS) joniza-
ment ((M + 1]* or [M + 23, Na]*) and negative ions tion is between 10 and 20 kDa, and FAB is really most
(by deprotonation [M — 1]’) are formed; both types of useful up to about 6 kDa. FAB is seen most often with

ions are usually singly charged and, depending on the double focusing magnetic sector instruments where it
instrument, FAB can be used in high-resolution mode. has a resolution of about 0.3 m/z over the entire mass
FAB is used primarily with large nonvolatile mole- range; FAB can, however, be used with most types of
cules, particularly to determine molecular weight. For mass analyzers. The biggest drawback to using FAB is
most classes of compounds, the rest of the spectrum is that the spectrum always shows a high level of matrix
less useful, partially because the lower mass ranges generated ions, which limit sensitivity and which may
may be composed of ions produced by the matrix obscure important [ragment ions.
itself. However, for certain classes of compounds that
are composed of “building blocks,” such as polysaccha-
rides and peptides, some structural information may
be obtained because fragmentation usually occurs at

v7

MALDI:

Matrix-assisted laser desorption/ionization (MALDIJ) is an tonization technique that uses a laser
energy absorbing matrix to create ions from large molecules with minimal fragmentation. It is
similar in character to electrospray ionization (ESI) in that both techniques are relatively soft (low
fragmentation) ways of obtaining ions of large molecules in the gas phase, though MALDI
typically produces far fewer multi-charged ions.

MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix
material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering
ablation and desorption of the sample and matrix maternal, Finally, the analyte molecules are
ionized by being protonated or deprotonated in the hot plume of ablated gases, and then they can
be accelerated into whichever mass spectrometer is used to analyse them,

The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-
dimethox y-4-hydroxycinnamic acid (sinapinic acid), a-cyano-4-hydroxycinnamic acid (a-CHCA,
alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB).[15] A solution of one of
these molecules is made, often in a mixture of highly purified water and an organic solvent such
as acctonitrile (ACN) or ethanol, A counter ion source such as Trifluoroacetic acid (TFA) ts usually
added to generate the [M+H] tons.

The identification of suitable matrix compounds is determined to some extent by trial and error,
but they are based on some specific molecular design considerations. They are of a fairly low
molecular weight (to allow casy vaporization), but are large enough (with a low enough vapor
pressure) not to evaporate during sample preparation or while standing in the mass spectrometer.
They are often acidic, therefore act as a proton source to encourage ionization of the analyte.

The matrix solution is mixed with the analyte (e.g. protein-sample). A mixture of water and organic
solvent allows both hydrophobic and water-soluble (hydrophilic) molecules to dissolve into the
solution. This solution is spotted onto a MALDI plate (usually a metal plate designed for this
purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte
molecules embedded into MALDI crystals. The matrix and the analyte are said to be co-
crystallized. Co-crystallization is a key issue in selecting a proper matrix to obtain a good quality
mass spectrum of the analyte of interest.

MALDI techniques typically employ the use of UV lasers such as nitrogen lasers. The most
common mass analyzer paired with MALDI is the time of flight (TOF) mass spectrometer. MALDI

produces ions in short bursts due to the use of a pulsed laser, and produces a wide variety of ion
masses that require a detector with a broad mass range.

APCI:

Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass
spectrometry which utilizes gas-phase ion-molecule reactions at atmospheric pressure (105 Pa),
commonly coupled with high-performance liquid chromatography (HPLC). APCI is a soft
ionization method similar to chemical ionization where primary ions are produced on a solvent
spray. The main usage of APCI is for polar and relatively less polar thermally stable compounds
with molecular weight less than 1500 Da. The application of APCI with HPLC has gained a large

popularity in trace analysis detection such as steroids, pesticides and also in pharmacology for
drug metabolites.
A typical APCI usually consists of three main parts: a nebulizer probe which can be heated to 350-
500°C, an ionization region with a corona discharge needle, and an ton-transfer region under
intermediate pressure. The analyte in solution is introduced from a direct inlet probe or a liquid
chromatography (LC) eluate into a pneumatic nebulizer with a flow rate 0.2-2.0mL/min. In the
heated nebulizer, the analyte coaxially flows with nebulizer N2 gas to produce a mist of fine
droplets. By the combination effects of heat and gas flow, the emerged mist is converted into a gas
stream. Once the gas stream arrives in the ionization region under atmospheric pressure, molecules
are ionized at corona discharge which is 2 to 3 kV potential different to the exit counter-electrode.
Sample ions then pass through a small orifice skimmer into the ton-transfer region. lons may be
transported through additional skimmer or ion-focusing lenses into a mass analyzer for subsequent
mass analysis.

ESI:

Electrospray ionization (ESI) ts a technique used in mass spectrometry to produce tons using an
electrospray in which a high voltage is applied to a liquid to create an acrosol. It is especially useful
in producing ions from macromolecules because it overcomes the propensity of these molecules
to fragment when ionized. ESI is different from other atmospheric pressure ionization processes
(e.g. matrix-assisted laser desorption/ionization (MALDI)) since it may produce multiple-charged
ions, effectively extending the mass range of the analyser to accommodate the K1Da-MDa orders
of magnitude observed in proteins and their associated polypeptide fragments.

Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS) or,
less commonly, electrospray mass spectrometry (ES-MS), ESI is a so-called ‘soft ionization’
technique, since there is very little fragmentation. This can be advantageous in the sense that the
molecular ion (or more accurately a pseudo molecular ion) is always observed, however very little
structural information can be gained from the simple mass spectrum obtained. This disadvantage
can be overcome by coupling ESI with tandem mass spectrometry (ESI-MS/MS). Another
important advantage of ESI is that solution-phase information can be retained into the gas-phase.

APPI (Atmospheric Pressure Photoionization):

All mass spectrometers require the molecules to be in the gas phase and charged (ionized either
positive or negative). In this technique, UV light photons are used to ionize sample molecules. The
technique works well with nonpolar or low polarity compounds not efficiently ionized by other
ionization sources,

«9

First the sample (analyte) is mixed with a solvent. Depending on the type used, the solvent could
increase the number of ions that are formed.

The liquid solution is then vaporized with the help of a nebulizing gas such as nitrogen, then enters
an ionization chamber at atmospheric pressure. There, the mixture of solvent and sample molecules
is exposed to ultraviolet light from a krypton lamp. The photons emitted from this lamp have a
specific energy level (10 electron volts, or eV) that is just right for this process; high enough to
ionize the target molecules, but not high enough to tonize air and other unwanted molecules. So
only the analyte molecules proceed to the mass spectrometer to be measured.

First, we’ll look at what happens when just the solvent and analyte molecules are exposed to the
UV light. Then we will look at the slightly more complicated, and much more typical, scenario in
which a dopant (a kind of additive) is introduced into the mixture.

Once they are exposed to the UV light, the analyte molecules are tonized in two ways:

Direct APPI-

A minority of them will be ionized directly by the UV light (Photoionization)

M+hv—M”+e

Dopant Assisted APPI-

The dopant ion can donate a proton to the analyte molecule. The result ts an ionized
sample molecule, Toluene is commonly used as a dopant.

D’ +M — [M+H]’ + (D-H]’

MASS FRAGMENTATION AND ITS RULES:

Fragmentation is the dissociation of energetically unstable molecular tons formed from passing
the molecules in the ionization chamber of a mass spectrometer. The fragments of a molecule
cause a pattern in the mass spectrum used to detennine structural information of the molecule.

The production of a molecular ion is often followed by its dissociation, or frag-
mentation, into ions and neutral species of lower mass, which in tum may
dissociate further. Fragmentation patterns are characteristic of particular molec-
ular structures and can indicate the presence of specific functional groups, thus
providing useful information on the structure and identity of the original mole-
cule. The points of cleavage in a molecule are determined by individual bond
strengths throughout the structure and, additionally, molecular rearrangements
and recombinations can occur.

A number of general rules for predicting promi-
nent peaks in El spectra can be written and rational-
ized by using standard concepts of physical organic
chemistry.

1. The relative height of the molecular ion peak is
greatest for the straight-chain compound and
decreases as the degree of branching increases (sce
rule 3).

2. The relative height of the molecular ion peak usu-
ally decreases with increasing molecular weight in
a homologous series. Fatty esters appear to be an
exception.

3. Cleavage is favored at alkyl-substituted carbon
atoms: the more substituted, the more likely
is cleavage. This is a consequence of the increased
stability of a tertiary carbocation over a secondary,
which in turn is more stable than a primary.

Cation stability order:
CH,’ < R,CH,;* < R,CH* < RC”

Generally, the largest substituent at a branch is
eliminated most readily as a radical, presumably
because a long-chain radical can achieve some stability
by delocalization of the lone electron.

4. Double bonds, cyclic structures, and especially
aromatic (or heteroaromatic) rings stabilize the
molecular ion and thus increase the probability of
its appearance.

§. Double bonds favor allylic cleavage and give the
resonance-stabilized allylic carbocation. This rule
does not hold for simple alkenes because of the
ready migration of the double bond, but it does
hold for cycloalkenes

6. Saturated rings tend to lose alkyl side chains at the
a bond. This is merely a special case of branching
(rule 3). The positive charge tends to stay with the
ring fragment. Sce Scheme 1.4.

R ‘*

Cy –

(Sch 1.4)

Unsaturated rings can undergo a retro-Diels-Alder
reaction Scheme 1.5:

(Sch 1.5)

7. In alkyl-substituted aromatic compounds, cleavage
is very probable at the bond B to the ring, giving
the resonance-stabilized benzyl ion or, more likely,

the tropylium ion (sce Scheme 1.6).

4?

(Seh 1.6)

8 The C—C bonds next to a heteroatom are
frequently cleaved, leaving the charge on the frag-
ment containing the heteroatom whose non-
bonding electrons provide resonance §stabiliza-
tion.

9. Cleavage is often associated with climination
of small, stable, neutral molecules, such as carbon
monoxide, olefins, water, ammonia, hydrogen
sulfide, hydrogen cyanide, mercaptans, ketene, oF
alcohols, often with rcarrangement (Scction
1.5.5),

It should be kept in mind that the fragmentation
rules above apply to El mass spectrometry. Since
other ionizing (Cl. etc.) techniques often produce
molecular ions with much lower energy or quasimo-
lecular ions with very different fragmentation patterns,
different rules govern the fragmentation of these
molecular ions.

METASTABLE IONS:
Stable ions form the conventional mass spectrum. Some of the ions, however do not break down
before reaching the ion collector of a mass spectrometer. These are called as metastable ions. They
appear as broad peaks called metastable ion peaks.
Fragment of a parent ion will give rise to a new jon (daughter) plus either a neutral molecule or a
radical,

M,° — M2’ + non charged particle

An intermediate situation is possible; Mi” may decompose to My’ while being accelerated. The
resultant daughter ion M2° will not be recorded at cither M; or M2 but at apposition M’ as a rather
broad poorly focused peak. Such an ion is called as metastable ion.
Metastable ions have lower kinetic energy than normal ions and metastable peaks are smaller than
the M; and M2 peaks and also broader.

4%

Significance of metastable ions-
¥ Metastable ions are useful in helping to establish fragments routes.
~ Metastable ion peak can also be used to distinguish between fragmentation process,

which occur in few microseconds,

ISOTOPIC PEAKS:

Most elements occur naturally as a mixture of isotopes, all of which contribute
to peaks in a mass spectrum. Examples are given in Table 1 along with their

Table 1. Natural isotopic abundances of some common elements as a percentage of the
most abundant isotope

Element Most abundant Other isotopes Abundance of
isotope heavier isotope’

Hydrogen ‘H “H 0.016
Carbon “Cc a 1.11
Nitrogen “IN **N 0.38
Oxygen “oO i @) 0.04

*O 0.20
Sulfur “S$ =S 0.78

“S 4.40
Fluorine “FE 100
Chiorine *C| “Cl 32.5
Bromine “Br “Br 98.0
lodine 27 100
Phosphorus ‘p 100

percentage relative abundances. Thus, natural carbon consists of a mixture of
98.90% “°C and 1.10% “°C, and natural hydrogen is a mixture of 99.985% ‘H and
0.015% *H (deuterium).

The very small peaks above the molecular ion peak, M’, at m/z 32 in the spec-
trum of methanol (Fig. 1) are due to the heavier isotopes of carbon, hydrogen
and oxygen. These isotope peaks, designated (M+1)*, (M+2)° etc., are of impor-
tance in the interpretation of mass spectra and can be used for two purposes.

(i) To establish an empirical formula for molecules containing C, H, O and N
by comparison of the relative intensities of the M, M+1 and M+2 peaks ina
recorded spectrum with tabulated values. Table 2 is an extract of extensive

tables for all possible combinations of these elements up to an RMM of
several hundred. Theoretical values for any molecular formula are readily
calculated by multiplying the number of atoms of each element by the
corresponding natural isotopic abundances and summing them to obtain
the intensities of the M+1 and M+2 peaks as percentages of the M peak, e.g.
for C,H ,O0-

Cc H © Total

M* 100,00

(M+1)° 24 = 1.108 22 x 0.016 7 x0.04 27.22

(NM+2)° = – 7 x 0.20 1.40

(ii) To determine the numbers of chlorine and bromine atoms in a compound
from the relative intensities of the M, M+2, M44, M+6, etec., peaks in a
recorded spectrum by comparisons with tabulated values or graphical
plots. The presence of one or more atoms of either halogen will give isotope
peaks two mass units apart (Table 3).

APPLICATIONS: ae

Applications of Mass spectrometry ; Ss:
‘einM an, e has : :

several applications in analytical field. Some applications are

1. Mass spectra of a pure compound provides important information for identification by
the help of molecular weight, molecular formula and by fragmentation pattern.
Mass spectrometry is one of the best tool for the determination of molecular weight of a
substance, When a substance is bombarded with moving electrons and it’s mass
spectra is recorded, the mass of the peak at the highest m/e reveals the molecular maxs
accurately. This helps in molecular weight determination. Similarly one can find
molecular formula of a compound.

2. Mass spectrometry is very useful for the preparation of pure isoto j
and natural products can be analysed by Babi pte ys oe an ee

3. Mass spectrometry can also be used to distinguish between cis and trans isomers; since
the stability of ions produced may differ for cis and trans ions significantly,

4. Mass spectrometry is also useful in study of free radicals, determination of bond
strength, evaluation of heat of sublimation etc.

5. Mass spectrometry is extremely useful in analysis of closely related compounds like
hydrocarbons, petroleum products, lubricating oils ete.

6. In inorganic trace analysis, mass spectrometry can be used for trace analysis of
elements in alloys and minerals and in super conductors.

aS

Chapter-4

AFFINITY CHROMATOGRAPHY:
According to the International Union of Pure and Applied Chemistry (IUPAC), affinity
chromatography ts defined as a liquid chromatographic technique that makes use of a “biological
interaction” for the separation and analysis of specific analytes within a sample. For example, a
protein that binds metals such as nickel can be purified in the presence of other non-specific
proteins using a resin containing immobilized nickel, A protein can bind nickel because of the
presence of amino acids such as histidine positioned in a specific manner, which contains
imidazole functionality that can co-ordinate nickel.

The biomolecule of interest interacts reversibly with a specific ligand bound to a matrix allowing
for a specific binding on the matrix in the presence of other contaminants and later clution of the
bound biomolecule. Using this method the biomolecule can be purified in a single step, with
efficient recovery and high purity.

PRINCIPLE:

There are certain specific requirements for an affinity chromatography that must be met. These
requirements are as follows:

e A biospecific ligand that can be covalently attached to a chromatography matrix.
e The bound ligand must be able to bind the target biomolecule specifically.
e The binding between the ligand and target molecule must be reversible to allow the target

molecules to be removed in an active form.

The biological interactions involve mostly non covalent interactions between the reactive groups
of molecule targeted for punfication and ligand with a dissociation constant Ky,

: lA] [B]
~ [AB]

Where, A is assumed as molecule targeted and B as ligand and AB ts the complex formed
between them. Ky varies between 10° to 10°’ M for affinity binding.

The principle of affinity chromatography is that the stationary phase consists of a support medium
(e.g. cellulose beads) on which the substrate (or sometimes a coenzyme) has been bound

covalently, in such a way that the reactive groups that are essential for enzyme binding are exposed.
As the crude mixture of proteins is passed through the chromatography column, proteins with
binding site for the immobilized substrate will bind to the stationary phase, while all other proteins
will be eluted in the void volume of the column,

Some of the examples of types of interactions utilized in the affinity chromatographic
purification include:

e Antigen: antibody
e Enzyme: substrate analogue

df

Binding protein: Ligand
Receptor : ligand
Lectin : polysaccharide, glycoprotein
Nucleic acid : complementary base sequence
Hormone, vitamin : receptor, carrier protein.
Glutathione : glutathione-S-transferase or GST fusion proteins.
Metal ions : Poly (His) fusion proteins, native proteins with histidine or cysteine on their
surfaces.

PROCEDURE:
The steps involved in a typical affinity chromatographic separation are as follows:

i. The ligand is first covalently coupled to a matrix, such as agarose beads.
ii, The matrix is poured into a column.

iii. An impure mixture containing biomolecule of interest is loaded on the affinity column.
iV, Biomolecules sieve through matrix of affinity beads and interact with affinity ligand,

Molecules that do not bind to ligand elute from the column.
Vv. Wash off contaminant molecules that bind to ligand loosely.

Vi. Elute proteins that bind tightly to ligand and collect purified protein of interest using
either a biospeciife or nonspecific clution methods:

a, Biospecific — An inhibitor is added to the mobile phase (free ligand). Free ligand
will compete for the solute.

b. Nonspecific — A reagent is added that denatures the solute. Once denatured, the
solute is released from the ligand.

Matrix
impurities/contaminants

APPLICATIONS:
The technique was developed for purification of enzymes but now affinity chromatography
is used for various other purposes like purification of nucleotides, nucleic acid,
immunoglobulin, membrane receptors ete.

Immunoglobulin purification (antibody immobilization)-Antibodics can also be
immobilized by adsorbing them onto secondary ligands. Alternatively, antibodies can be
directly adsorbed onto a protein A or protein G support due to the specific interaction of
antibodies with protein A and G, Immobilized antibodies on the protein A or G support can
easily be replaced by using a strong eluent, regenerating the protein A/G, and re-applying
fresh antibodies, Generally, this method is used when a high capacity/high activity support
is needed.
Recombinant tagged proteins- Purification of proteins can be easier and simpler if the
protein of interest is tagged with a known sequence commonly referred to as a tag. This
lag can range from a short sequence of amino acids to entire domains or even whole

47

proteins. Tags can act both as a marker for protein expression and to help facilitate protein
purification,
GST tagged purification- Glutathione S-transferase (GST) is a 26 kDa protein (211 amino
acids) located in cytosole or mitochondria and present both in cukaryotes and prokaryotes.
Separation and purifcation of GST-tagged proteins is possible since the GST tag is capable
of binding its substrate, glutathione. The free glutathione replaces the immobilized
glutathione and releases the GST-tagged protein from the matrix allowing its elution from
the column.

Chapter- 5

ELECTROPHORESIS

PAPER ELECTROPHORESIS:
(refer Ravi Sankar page 28)

GEELE CTROPHORESIS:
This technique involves the separation of molecules based on their size, in addition to the electrical
charge. Gel electrophoresis is the core technique for genetic analysis and purification of nucleic
acids for further studies, Nucleic acids are separated and displayed using various modifications of
gel electrophoresis and detection methods.

It is used in:

e Clinical chemistry to separate proteins by charge and/or size.
e Biochemistry and Molecular biology to separate DNA and RNA fragments by length, or

lo separate proteins by charge.

Gel electrophoretic methods provide the highest resolution of all protein separation techniques.

PRINCIPLE-

Electrophoresis is the migration of charged particles or molecules in an electric field. This occurs
when the substances are in aqueous solution. The speed of migration is dependent on the applied
electric field strength and the charges of the molecules. Thus, differently charged molecules will
form individual zones while they migrate. In order to keep diffusion of the zones to a minimum,
electrophoresis is carried out in an anticonvective medium such as a viscous fluid or a gel matrix.
Therefore, the speed of migration is also dependent on the size of the molecules. In this way
fractionation ofa mixture of substances is achieved with high resolution.

GEL TYPES-

There are two types of gel most typically used:

i. Agarose gel

i. = Polyacrylamide gel (PAGE).

Each type of gel is well-suited to different types and sizes of analyte,

1. Agarose gel-
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations
of 0.5 to 2%, The higher the agarose concentration the “stiffer” the gel. Higher percentages
requiring longer run times. Agarose gels have greater range of separation, and are therefore
used for DNA fragments of usually 50-20,000 bp in size.

49

Instrumentation of agarose gel-

e The equipment and supplies necessary tor conducting agarose gel electrophoresis are
relatively simple and include:

°e Anelectrophoresis chamber and power supply
Gel casting trays, which are available in a variety of sizes and composed of UV-
transparent plastic. The open emds of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis

e Sample combs, around which molten medium is poured to form sample wells in the
gel.
Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE),
Loading buffer, which contains something dense (e.g. glycerol) to allow the saniple to
“fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and

Allow visual monitoring or how far the electrophoresis has proceeded.
¢ Staining: DNA molecules are easily visualized under an ultraviolet lamp when

electrphoresed in the presence of the extrinsic fluor ethidium bromide.
Alternatively, nucleic acids can be stained after electrophoretic separation by
soaking the gel in a solution of ethidium bromide. When intercalated into double-
stranded DNA, fluorescence of this molecule increases greatly. It is also possible to
detect DNA with the extrinsic fluor L-anilino S-naphthalene sulphonate. NOTE:
Ethers bromide ts a Lieun mutagen and should te lraandled as a hazardous chennucal –
wear gloves winte landing.

¢ Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in
gels. NOTE: always wear protective eyewear when observing DNA on a
Transilluminator to prevent danuge to the eves from UV light.

Preparation and running of agarose gel-

To prepare gel, agarose powder is mixed with electrophoresis butter to the desired
concentration, and heated in a microwave oven to melt it. Ethidium bromide is added to the
gel (tinal concentration 0.5 ug/ml) to facilitate visualization of DNA after electrophoresis.
After cooling the solution to about 60°C, it is poured into a casting tray containing a sample
comb and allowed to solidify at room temperature.

After the gel has soliditied, the comb is removed, taking care not to np the bottom of the
wells. The gel, still in plastic tray, is inserted horizontally into the electrophoresis chamber
and is covered with buffer. Samples containing DNA mixed with loading buffer are then
pipetted into the sample wells, the lid and power leads are placed on the apparatus (Fig. 2),
and a current is applied. The current flow can be comfinned by observing bubbles coming
off the electrodes. DNA will migrate towards the positive electrode, which is usually
colored red, in view of its negative charge.

The distance DNA has migrated in the gel can be judged by visually monitoring migration
of the tracking dyes like bromophenol blue and xylene cvanol dyes.

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Staining of the bands-

The bands are visualized with fluorescent dyes that are visible in UV light — ethidium bromide or
SYBR Green, SYBR Green is less mutagenic and more sensitive than ethidium bromide. The best
results and highest resolutions are obtained when the gels are stained after the run.

Recovery of DNA fragments from gels-

Several different procedures are used for the isolation of nucleic acids from agarose gels :

Electroelution
absorption to DEAE paper
absorption to glass powder or resins
digestion of agarose with enzymes.

For preparative electrophoresis, it is very important to use highly purified agarose that is free from
polymerase and other enzyme inhibitors. Since the advent of polymerase chain reaction (PCR)

technology, tiny amounts of DNA fragments can easily be amplified for further experiments.

ii. Polyacrylamide gel (PAGE)-
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains
is dictated by the concentnation of acrylamide used, which is typically between 3.5 and
20%. It is used for separating proteins ranging in size from 5 to 2,000 kDa due to the
uniform pore size provided by the polyacrylamide gel. In contrast to agarose,
polyacrylamide gels are used extensively for separating and characterizing mixtures of
proteins.

1

Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be
handled with gloves due to the possible presence of free acrylamide. Acrylamide is a potent
neurotoxin and should be handled with care.
Preparation and running of polyacrylamide gel-

Polyacrylamide gels are prepared by chemical copolymerization of acrylamide
monomers with a crosslinking reagent, usually N,N’-methylenebisacrylamide.
A clear transparent gel is obtained, which is chemically inert, mechanically stable
and without electroendosmosis.
Polymerization of the acrylamide monomers and the cross-linker molecules occurs
in the presence of free radicals. These are provided by ammonium persulfate as
catalyst; tertiary amino groups, usually N, N, N’, N’-tetramethylethylenediamine
(TEMED), are required as accelerators.
Because oxygen is a scavenger of free radicals, polymerization is performed in
closed cassettes.
Sample application wells for vertical gels are formed at the upper edge of the gel
during polymerization with the help of an inserted comb (see Figure).
Sample wells for flatbed gels are made by using self-adhesive tape glued onto one
of the glass plates.

(a)

(b)

Pigure § Schemeastes drawirg of chambers for ped yectylamiutc

Figure 4 Schematic drawing of a casette with stampk well pel chectrogtiomesis (a) Matted charmer with cooleng plate, the
ccenh and a caster for potyacry lamide eel clectrmdc rowcrvoirs being contamead m depoeahle pol yactylamidke

estroge, ord (th) wertical charter ware biguad bealler

Ss?

The samples are denatured just prior to loading the gel. Sample DNA may re-anneal if
denatured for an extended time before loading and may produce indeterminate
fragments.
For electrophoresis in vertical systems, the complete gel cassettes are placed into the
buffer tanks; the gels are in direct contact with the electrode buffers.
Gels for flatbed systems are polymerized on a film support and removed from the
cassette before use.

Detection of bands-

Silver Staining:

Ethidium bromide and SYBR Green staining are rarely used for polyacrylamide gels, because the
signals are weaker than in agarose gels.

The most sensitive staining for protein ts silver staining. This involves soaking the gel in Ag NO3
which results in precipitation of metallic silver (AgO) at the location of protein or DNA forming a
black deposit in a process similar to that used in black and white photography,

FACTORS AFFECTING GEL ELECTROPHORESIS:

The higher the voltage/current, the faster the DNA migrates.
High voltage causes a tremendously increase in buffer temperature and current in very
short time. The high amount of the heat and current built up in the process leads to the
melting of the gel. Therefore, it is highly recommended not exceed 5-8 V/cm and 75 mA
for standard size gels or 100 mA for minigels.
Electrophoresis is performed in buffer solutions (Electrophoresis buffers TBE) to reduce
pH changes due to the electric field, which is important because the charge of DNA and
RNA depends on pH.
Running for too long can exhaust the buffering capacity of the solution so it should be
changed from time to time.

APPLICATIONS OF GEL ELECTROPHORESIS:

Agarose gel electrophoresis technique is extensively used for investigating the DNA
cleavage efficiency of small molecules and as a useful method to investigate various

binding modes of small molecules to supercoiled DNA,
It is also a useful method to investigate various binding modes of small molecules to
supercoiled DNA.
Development of new metallonucleases as small molecular models for DNA cleavage
at physiological conditions. Since DNA cleavage is a biological necessity, these small
molecular models have provided much of our most accurate information about nucleic
acid binding specificity.
Examples of metallonucleases:

e = [Cu(II)(hist)(tyr)]’

si

e = [Cu(II)(phen)(his-leu)]”

Capillary electrophoresis (CE) is a family of related techniques that employ narrow-bore (20-
200 jim i.d.) capillaries to perform high efficiency separations of both large and small molecules.
These separations are facilitated by the use of high voltages, which may generate electro-osmotic
and electro-phoretic flow of buffer solutions and ionic species, respectively, within the capillary.
The properties of the separation and the ensuing electropherogram have characteristics resembling
a cross between traditional polyacrylamide gel electrophoresis (PAGE) and modern high
performance liquid chromatography (HPLC).

PRINCIPLE-

One of the fundamental processes that drive CE is electroosmosis. This phenomenon is a
consequence of the surface charge on the wall of the capillary. The fused silica capillaries that are
typically used for separations have ionizable silanol groups in contact with the buffer contained
within the capillary. Therefore, the inside walls of the capillary negatively charged because the
inside wall has silanol groups (SO,°). This means that the inner wall has a net negative charge.

= Ss So oS S

25 to 100 gern =—
Wy

S = = = 5‘
The buffer solution in cach reservoir has equal amounts of cations and anions, and the capillary
ends are each placed in a buffer reservoir. Each reservoir also has an electrode connected to the
power supply.
When the voltage is applied to the circuit, one electrode become net positive and the other net
negative. The (wall’s) immobile silanol anions pair with mobile buffer cations, forming a double
layer along the wall (wall-—>buffer cations–>buffer anions–>bulk buffer solution). The remaining
buffer cations are attracted to the negative electrode, dragging the bulk buffer solution with them.
This is electroosmotic flow, For an uncoated capillary, the electroosmotic force (EOF) is toward
the negative electrode.

If the analyst wants the EOF (to flow) in the opposite direction then the capillary can be purchased
coated with a cationic surfactant, or one is added to the buffer, and the capillary walls will be
negatively charged and the electroosmotic flow will be reversed, that is, toward the positively
charge electrode. This might be chose based on a specific analyte separation, In the case below the
wall is uncoated, the wall is net negatively charged and the EOF is toward the negative electrode.

So everything injected into the buffer ows with the EOF. But, like the flow of analytes in a gas
chromatographic carrier gas, separation wouldn’t occur unless the analytes flow towards the
detector at different speeds. In GC this occurs because of interaction with the GC columns
stationary phase. In CE this occurs because analytes have different electrophoretic mobilities. In
the simplest approximation, electrophoretic mobility can be because of analyte charge and size.
Large, singly charged analytes will travel slower than small, singly charge analytes, and small,
doubly charged ions will travel faster than larger, doubly charged analytes, etc. In other forms of
CE separation is more complicated, The electrical potential also effects this process,

INSTRUMENTATION-

e The basic instrumental configuration for CE is relatively simple.
e The requirements are:

i. fused-silica capillary with an optical viewing window
il. A controllable high voltage power supply
iii. |§$ Two electrode assemblies
iv. Two buffer reservoirs
v, An ultraviolet (UV) detector.

e The ends of the capillary are placed in the buffer reservoirs and the optical viewing
window is aligned with the detector.

e After filling the capillary with buffer, the sample can be introduced by dipping the end of
the capillary into the sample solution and elevating the immersed capillary a foot or so
above the detector-side buffer reservoir.

e Virtually all of the pre-1988 work in CE was carried out on homemade devices following
this basic configuration. While relatively easy to use for experimentation, these carly
systems were inconvenient for routine analysis and too imprecise for quantitative analysis.

e <A diagram of a modem instrument, the P/ACE™ 2000 Series, is illustrated in the figure
below, Compared to the early developmental instruments, this fully automated instrument
offers computer control of all operations, pressure and electrokinetic injection, an
autosampler and fraction collector.

e Automated methods development, precise temperature control, and an advanced heat
dissipation system. Automation is critical to CE since repeatable operation is required for
precise quantitative analysis.

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A fundamental term in chromatography is retention time. In electrophoresis, under ideal
conditions, nothing is retained, so the analogous term becomes migration time. The
migration time ™ is the time it takes a solute to move from the beginning of the capillary
to the detector window.

FACTORS AFFECTING CE:

The Capillary Surface-
The inner surface of a capillary is an extremely important factor in CE. The inner wall
is in contact with the separation chemistry and the samples. As noted carlicr, the
capillary wall is the site of the mechanism by which EOF is created,
Surface modifications-
Capillaries perform best when they are “dedicated” to a specific type of buffer species,
This dedication of a capillary to one type of system is a relatively inexpensive way to
improve results,
Separation buffers-
The significance of the capillary wall in controlling the process of separation in CE
cannot be overstated, The separation, however, takes place in the separation buffer. It
is here that the conditions are such that the differences in mobility can exist. Even the
best instrument system will not perform properly with a poorly prepared buffer.

iv. Significance of pH-
In CE it is extremely important to properly control pH since it affects analyte charge,
electroosmotic flow, and, by affecting current, heat production. Thus small changes in
pH tend to have greater impact in CE,

Y. Additives-
Other reagents are frequently added to the buffer systems used in CE. The most
common are detergents, such as sodium dodecyl sulfate (SDS), viscosity modifiers,
such as linear polyacrylamide, organic solvents, such as acetonitrile, denaturants, such
as urea, or combinations of these additives.
The addition of detergent to a buffer used in CE can change dramatically the separation
properties of the system. Detergents can aid in solubilizing analytes and in reducing
analyte-wall interactions. They may also bind to the capillary wall, affecting the EOF.

vi. Temperature-

Temperature control is crucial to reproducible separations in CE. However,
temperature regulation is complicated by several factors.
First is that the passage of electrical current through the buffer-filled capillary results
in the production of heat. This self-heating effect is inherent in electrophoretic
separations and is called Joule heating. Thus temperature control in CE is as much a
task of removing heat as it is maintaining a constant temperature environment.
Second is that the temperature of the contents inside the capillary is difficult to measure.

APPLICATIONS-

i. It is very useful for the separation of proteins and peptides since complete resolution
can often be obtained for analytes differing by only one amino acid substituent.

i, It is particularly important in tryptic mapping where mutations and post-translational
modifications must be detected.

ill. Other applications where CE may be useful include separation of inorganic anions and
cations such as those typically separated by ion chromatography.

Iv. Small molecules such as pharmaceuticals can often be separated provided they are
charged.

Capillary electrophoresis comprises a family of techniques that have dramatically different
Operative and separative characteristics. The techniques are:

Capillary zone electrophoresis
[Isoelectric focusing
Capillary gel electrophoresis
Isotachophoresis
Micellar electrokinetic capillary chromatogmphy

Therefore Zone Electrophoresis (ZE) or Capillary Zone Electrophoresis (CZE) is one of the
types of capillary electrophoresis.

Capillary zone electrophoresis (CZE), also known as free solution capillary electrophoresis,
is the simplest form of Capillary electrophoresis (CE). The separation mechanism is based on
differences in the charge-to-mass ratio. Fundamental to CZE are homogeneity of the buffer
solution and constant field strength throughout the length of the capillary.

PRINCIPLE-

The separation mechanism is based on differences in the charge-to-mass ratio. Following injection
and application of voltage, the components of a sample mixture separate into discrete zones as
shown in the figure.

Figure S Capillary Zowe Electrogrooretis

57

The fundamental parameter, electrophoretic mobility, pics, can be approximated from Debcye-
Huckel-Henry theory.

Ne 6rnR
Where,

q is the net charge
R is the Stokes radius, and

1) is the viscosity.
The net charge is usually pH dependent. For example, within the pH range of 4-10, the net charge
on sodium is constant as is its mobility. Other species such as acetate or glutamate are negatively
charged within that pH range and thus have negative mobilities (they migrate towards the positive
electrode). At alkaline pH, their net migration will still be towards the negative electrode because
of the EOF, Zwitterions such as amino acids, proteins, and peptides exhibit charge reversal at their
pl’s (Isoelectric point) and, likewise, shifts in the direction of electrophoretic mobility.
Separations of both large and small molecules can be accomplished by CZE. Even small
molecules, where the charge-to-mass ratio differences may not be great, may still be separable.

INSTRUMENTATION-
(same as previous)

APPLICATIONS-
(same as previous)

Capillary electrophoresis comprises a family of techniques that have dramatically different
operative and separative characteristics. The techniques are:

Capillary zone electrophoresis
Isoelectric focusing
Capillary gel electrophoresis
lsotachophoresis
Micellar electrokinetic capillary chromatography

Therefore Isoelectric focusing (IEF) is one of the types of capillary electrophoresis.

The fundamental theory of isoelectric focusing (IEF) is that a molecule will migrate so long as it
is charged. Should it become ncutral, it will stop migrating in the electric field. IEF is run in a pH
gradient where the pH ts low at the anode and high at the cathode (Figure 7). The pH gradient is
generated with a series of zwitterionic chemicals known as carricr ampholytes.

Low pH High pH

Figure 7. lsovlectric Focusing

PRINCIPLE-

Molecules that carry both positively and negatively charged groups exhibit, at a specific pH, an
equal number of positive and negative charges. At this pH, known as the isoelectric pH or pl, the
molecule, although charged, behaves as if it is neutral because its positive and negative charges
cancel each other. The molecule, therefore, has no tendency to migrate in an electrical field. In
isoelectric focusing, special reagents called ampholytes are used to create a pH gradient within the
capillary. These ampholytes are mixtures of buffers with a range of pKa values. In an electrical
field, ampholytes will arrange themselves in order of pKa; this gradient is trapped between a strong
acid and a strong base. Analytes introduced into this gradient will migrate to the point where the
pH of the gradient equals their pl. At this point the analyte, having no net charge, ceases to migrate.
It will remain at that position so long as the pH gradient is stable, typically as long as the voltage
is applied.
The pH of the anodic buffer must be lower than the pl of the most acidic ampholyte to prevent
migration into the analyte. Likewise, the catholyte must have a higher pH than the most basic
ampholyte.

INSTRUMENTATION AND WORKING-
(instrumentation similar to previous)

The three basic steps of IEF are:
L. Loading
il. Focusing
i. Mobilization.

i. Loading- The sample is mixed with the appropriate ampholytes to a final concentration
of 1-2% ampholytes. The mixture is loaded into the capillary cither by pressure or
vacuum aspiration.

ii. Focusing- The buffer reservoirs are filled with sodium hydroxide (cathode) and

phosphoric acid (anode). Field strengths on the order of 500-700 V/cm are employed.
As the focusing proceeds, the current drops to less than | mA. Overfocusing can result
in precipitation due to protein aggregation at high localized concentrations. Dispersants
such as nonionic surfactants or organic modifiers such as glycerol or ethylene glycol
may minimize aggregation. These agents are mild and usually do not denature the
protein. Urea could also be used, but the protein will become denatured. Because of
precipitation problems, very hydrophobic proteins are not usually separated by IEF.
Gel-filled capillaries are sometimes useful for separating troublesome proteins.

iii. |©§ Mobilization- Mobilization can be accomplished in either the cathodic or anodic
direction, For cathodic mobilization, the cathode reservoir is filled with sodium

hydroxide/sodium chloride solution. In anodic mobilization, the sodium chloride is
added to the anode reservoir. The addition of salt alters the pH in the capillary when
the voltage is applied since the anions/cations compete with hydroxyl/hydronium ion
migration. As the pH is changed, both ampholytes and proteins are mobilized in the
direction of the reservoir with added salt. As mobilization proceeds, the current rises
as the saline tons migrate into the capillary. Detection is performed at 280 nm for
proteins since the ampholytes absorb strongly in the low UV range.

APPLICATIONS-
i. In addition to performing high resolution separations, IEF is useful for determining the

pl of a protein.
ii. IEF is particularly useful for separating:

¢ Immunoglobulins
e Hemoglobin variants and
e Post-translational modifications of recombinant proteins.

ae? Vas” SO UIV EF ial SO£8 O6F2. O i.
Moving boundary electrophoresis (MBE) was developed by Ame Tiselius in 1930. Tiselius was
awarded the 1948 Nobel Prize in chemistry for his work on the separation of colloids through
electrophoresis.

MBE its a technique for separation of chemical compounds by electrophoresis in a free solution.

PRINCIPLE-

The moving boundary electrophoresis apparatus includes a U-shaped cell filled with buffer
solution and electrodes immersed at its ends, The sample applied could be any mixture of charged
components such as a protein mixture. On applying voltage, the compounds will migrate to the
anode or cathode depending on their charges, The change in the refractive index at the boundary
of the separated compounds is detected using Schliren optics at both ends of the solution in the
cell,

INSTRUMENTATION:
Consists of a U shaped glass cell of rectangular cross section, with electrodes placed on
the one each of the limbs of the cell.

Sample solution is introduced at the bottom or through the side arm, and the apparatus is
placed in a constant temp. bath at 40°C.

Detection is done by measuring refractive index throughout the solution.(Schlieren optical
system).

Fig. MBE

2x Focal Length

Ee
Camera er —

————
——___

Point Light a

Source

Parabolic Mirror

Fig. Schlieren photography

APPLICATIONS-

e To study the homogenicity of a macromolecular system.

e Analysis of complex biological mixtures.

Al

X-ray crystallography is a tool used for identifying the atomic and molecular structure ofa crystal,
in Which the crystalline atoms cause a beam of incident X-rays to diffract into many specific
directions. By measuring the angles and intensities of these diffracted beams, a crystallographer
can produce a three-dimensional picture of the density of electrons within the crystal. From this
electron density, the mean positions of the atoms in the crystal can be determined, as well as their
chemical bonds, their disorder and various other information.

PRINCIPLE:

X-rays are electromagnetic radiation with wavelengths between about 0.02 A and 100 A. Because
X-rays have wavelengths similar to the size of atoms, they are useful to explore within crystals.
Since X-rays have a smaller wavelength than visible light, they have higher energy. With their
higher energy, X-rays can penetrate matter more easily than can visible light. Their ability to
penetrate matter depends on the density of the matter, and thus X-rays provide a powerful tool in
medicine for mapping internal structures of the human body (bones have higher density than tissue,
and thus are harder for X-rays to penetrate, fractures in bones have a different density than the
bone, thus fractures can be seen in X-my pictures).

Brage’s law- When X-rays are scattered from a crystal lattice, peaks of scattered intensity are
observed which correspond to the following conditions:

e The angle of incidence = Angle of scattering
e The pathlength difference is equal to an integer number of wavelength.

A?

en Seed
a .

nec ,ni= = 2AB | ey : E a a a

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Lae .-

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Ba 5 | nt ae ‘ r : . .

; 4 FF ML 4 *: ;7

PRODUCTION OF X-RAYS:

X-rays are produced when high speed electrons collide with a metal target. High speed clectrons
are generated from hot tungsten filament (cathode) and after applying a high accelerating voltage
(15-60kV) and are made to fall on metal target (anode) like copper/aluminium, molybdenum or
magnesium. Water is circulated as coolant o copper block containing desired target metal.

Az

DIFFERENT X-RAY METHODS:

1. Laue Photographic Method-
A single small crystal is placed in the path of a narrow beam of X-rays from a tungsten
anticathode and the resulting diffracted beam is allowed to fall on a photographic plate.
When the photographic plate is developed, a characteristic pattem, known as Laue pattem
of spots is seen. From the positions of the spots of the distance of the photographic plate
from the crystal, 9 is calculated and the relative spacing between the planes is estimated,
Laue pattern can be used to orient crystals for solid-state experiments and to determine the
symmetry of single crystal. However, the significance of reflection intensities is uncertain
duc to non-homogencous nature of the incident X-rays.

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Lue photographic method.

2. Rotating Crystal Method-
The method developed by Schiebold (1919) and M. Polanyl (1921), is perhaps the most
widely used method in the study of crystal structure. In this method a beam of
homogencous X-ray is allowed to penctrate a small crystal at right angles. The crystal being
rotated around an axis parallel to one of the crystal axes, During the rotation of the crystal
various planes come successively into suitable positions for diffraction to occur and the
corresponding spots are observed on a photographic plate.
In fig. 14, A and B show points on two successive lattice planes. For diffraction maxima
to occur the difference (BR) in the path of two diffracted mys must be equal to whole
number of wavelengths i.e., equal to nA. Since the value of n depends on the angle 0 (BAR),
scries of directions of diffraction corresponding to increasing value of n is obtained on the
photographic plat. Horizontal lines are seen for all lattice planes having the same spacing
(AB) in the direction parallel to the axis of rotation. Such lines are referred to as layer lines.
If the X of incident X-rays is known and the distance from the crystal to the photographic
plate and vertical distance between the layer lines is determined, it is possible to calculate
9 and hence the spacing of the planes AB. A set of spots in a transverse direction called
row lines is also observed on the photograph which is used to deduce lattice spacings and
the size of the unit cell,

nN . = a

me ‘

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”

fe

ftertcating crystsl macthod

3. Oscillating Crystal Method-
In oscillation method the crystal ts oscillated through an angle of 15° to 20°. But the number
of reflecting positions exposed to the incident X-rays is limited. The oscillations of the
crystal are synchronized with the movement of the cylindrical photographic film. The

position ofa spot on the plate indicates the orientation of the crystal at which the spot was
formed.

4. Powder Crystal Method-
Powder method was devised independently by P. Debye and P. Scherrer (1916) and A. W.
Hill (1917). This method employs powdered samples in which the crystals are oriented in
all directions so that some of the crystals will be properly oriented for a observable
reflections. A narrow beam of monochromatic X-rays is allowed on the finely powdered
specimen, The diffracted rays are then passed on to a strip of film which almost complete
surrounds the specimen. The random orientation of crystals produces diffraction rings or

AS

caves rather than spots, The method is commonly employed for identification purposes by
comparing the observed spacing of the axes produced on the film. Extensive files of
spacings from powder photographs are available for comparison. For a cubic crystal the
identification of lines in the powder photograph is relatively simple. Also the indexing of
lines in hexagonal, rhombohedral, tetrahedral etc. is not very complicated. However, in
crystals of lower symmetry a large number of lines are observed which cannot be accurately
identified.

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TYPES OF CRYSTALS:

Crystal structure
* Seven different possible geometries for the unit cell,
e There are 14 Bravais lattices, with each point representing the same atom or

collection of atoms. a

* Pure metals are Lt
usually FCC, BCC or Pe
H C 4 ° ae one

« Except for
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1/8 at corners

1/2 at face centers “tetragonal
All of body centered én ‘a

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F ey
Simple BaseL-cenfter ed Trictinic

APPLICATIONS OF X-RAY DIFFRACTION:
i. Study of polymorphism in drugs.

il, Measurement of the average space ‘d’ between layers of atoms.
iil. Determination of the orientation of a single crystal and crystal structure of unknown

material.
iV. Measurement of size, shape and internal stress of small crystalline regions.
Vv. Measurement of thickness of thin films and multi layers.

Vi, Determination of each type in mixed crystals.
Vil. Size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences

among various materials, especially minerals and alloys.
Vili. Used to study many materials which form crystals like salts, metals, minerals,

semiconductors, as well as various inorganic, organic and biological molecules.

A7

6. IMMUNOLOGICAL ASSAY

antigen and the same quantities of antibody and
RADIOIMMUNOASSAY labelled antigen.

Radioimmunoassay (RIA) was developed in The labelled antigen-antibody (Ag*-Ab)
1959 by Solomon, Benson and Rosalyn Yalow complex is separated by precipitation. The
for the estimation of insulin in human serum. radioactivity of ‘3’! present is Ag*-Ab is
This technique has revolutionized the estimation determined.
of several compounds in biological fluids that
are found in exceedingly low concentrations Applications

(nanogram of picogram). RIA is a highly sensitive RIA is no more limited to estimating of
and specific analytical tool, hormones and proteins that exhibit antigenic

properties. By the use of Aapfens (small
Principle

molecules such as dinitrophenol, which, by
Radioimmunoassay combines the principles themselves, are not antigenic), several substances

of radioactivity of isotopes and immunological can be made antigenic to elic® specific antibody
reactions of antigen and antibody, hence the responses. in this way, a wide variety of
hare. compounds have been brought under the net of

RIA estimation, These include peptides, stero
The principle of RIA is primarily based on the id

hormones, vitamins, drugs, ant
competition between the labelled and unlabelled ibiotics, nucteic

acids, structural proteins and hormone r
amigens to bind with antibody to form antigen- eceptor

proteins.
antibody complexes (either labelled of
unlabelled). The unlabelled antigen is the Radioimmunoassay has tremendous
substance (say insulin) to be determined. The application in the diagnosis of hormonal
antibody to it is produced by injecting the disorders, cancers and therapeutic monitoring of
antigen to a goat or a rabbit. The specific drugs, besides being useful in biomedical
amtibody (Ab) is then subjected to react with research.
unlabelled antigen in the presence of excess

amounts of isotopically labelled (2’l antigen
(Ag*) with known radioactivity. There occurs a

ENZYME-LINKED
competition between the antigens (Ag* and Ag)
to bind the antibody. Certainly, the labelled Ag* INIMUNOSORBANT ASSAY

will have an upper hand due to its excess
Enzyme-linked immunosorbant assay (ELISA)

presence.
i$ 2 Non-isotopic immunoassay. An enzyme is
used as a label in ELISA in plofa racdioaecti ve

Ag’ «Ab-———?> Ag Ab
isotope employed in RIA. ELISA is as sensitive

Ag as of even more sensitive than RIA. In addition.
there is no risk of radiati

-,* on hazards (as is the
case with RIA) in ELISA.

vr

Ag-Ab
Principle

As the concentration of unlabelled antigen
(Ag) increases the amount of labelled antigen- ELISA is based on the immunochemical
antibody complex (Ag*-Ab) decreases. Thus, the principles of antigen-anretacitbioon.d Tyhe
concentration of Ag’-Ab is inversely related to sages of ELISA, depicted in Fig.417.11, are
the concentration of unlabelled Ag Le. the summarized.
substance to be determined. This relation is 1. The antibody against the protein to be
almost linear. A standard curve can be drawn by determined is fixed on an inert solid such as
using different concentrations of unlabelled polystyrene.

2. The biolsaomplge iconctaianinlg t he

protein to be estimated is applied on the
antibody coated surface.

3. The protein antibody complex is
then reacted with a second protein specific
antibody to which an enzyme is covalently
linked. These enzymes must be easily
assayable and produce preferably coloured
products. Peroxidase, amylase and alkaline

phosphatase are commonly used.

4. After washing the unbound antibody
linked enzyme, the enzyme bound to the
second antibody complex is assayed.

5. The enzyme activity is determined
by its action on a substrate to form a

product (usually coloured), This is related
to the concentration of the protein being
estimated.

The principle tor the use of the enzyme
peroxidase in ELISA ts illustrated next.

Poroccase

H,O, 7H) +O

(substrate) (nascent oxyper) Fig. 41.11 : Daagrammate represeonf tenazytmei otinke d
imemunosaogsrayb (aELrIStA)

Dammobenrkine Oxxized haye the desired progerties but are found
(colourless) Giaminobeazidine

(brown) mans ea ther antvbodied which undoulyal® are
not requil , A simple, fonvenienpEnd desirable

Applications method for ( Nc la rge scUle prog& tion of specific

antibodies remai a for immunologists
ELISA is widely used for the determinatioonf

for a long period,
‘

stall quantities
of proteins (hormones, antigens,

Ce ein (N
antibodies} ¢ 19 84) made this

and other biological substances. The
dream a reality crea y b r id cells that

most commonly used pregnancy test for the
will make ur ited quan#ties of Notibodies with

detection of human chorionic gonadotropin
defined Fecificities, wiych are as

(hCG) in urine is based on ELISA, By this test,

J / onal antibodies ( b). This d@ w g ye ry,
pregnancy can be detected within few days after

ni te” referred to as hybri technology,
conception. ELISA is also been used for the

Molutionized methods for antibody production
diagnosis of AIDS.

Bioluminescence is the production and emission of light by a living organism, It is a form of
chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as
well as in some fungi, microorganisms including some bioluminescent bacteria and terrestrial
invertebrates such as fireflies.

Bioluminescence assays involve the use of the property of bioluminescence for measuring cell
proliferation, apoptosis, drug metabolism, kinase activity, etc.

PRINCIPLE:

Bioluminescence is a form of chemiluminescence where light energy is released by a chemical
reaction. This reaction involves a light-emitting pigment, the luciferin, and a luciferase, the
enzyme component. Because of the diversity of luciferin/luciferase combinations, there are very
few commonalities in the chemical mechanism.

For example, the firefly luciferin/luciferase reaction requires magnesium and ATP and produces
carbon dioxide (CO), adenosine monophosphate (AMP) and pyrophosphate (PP) as waste
products. Other cofactors may be required for the reaction, such as calcium (Ca*’) for the
photoprotein aequorin, or magnesium (Mg*’) ions and ATP for the firefly luciferase, Generically,
this reaction could be described as:

Luciferase
Lucificferrii n o O, Othe csoaifaacciooes Ox‘y lucifieferri n + Liiggh t e. nergy

HARNESSING BIOLUMINESCENCE:

1. Food testing using ATP (Bioluminescence) Technology-
The test kit for this test contain firefly luciferase and luciferin which are used to detect the
presence of ATP, a compound that is found in all living cells; this includes any live microbes
like Salmonella or E. coli that might be present in food and food products.
The intenisty of the light produced from the reaction is detected by a luminometer.

Luciferin +ATP +Luciferase ——> Visible Light (Intensity detected by luminometer)

The more the ATP, the brighter the light, so the intensity of luminescence reveals how many

bacteria are present. This test is able to detect even tiny amounts of microbial contamination,
using very senstitive instruments to measure light production. It requires mere minutes instead
of the days needed to detect contaminated food by growing bacterial cultures.

Bioluminescent imaging-
Fireflies have helped scientists develop real-time, noninvasive imaging to see what’s happening
inside living organisms.
When LUC (luciferase) genes are used to label particular cell or tissue types, very sensitive
cameras can be used to detect their light inside the live animal.

~

Mice bearing tumors are tagged with LUC gene which express luciferase and are further
injected with luciferin. Researchers then use a sensitive camera system to view, without killing
the mice, the tumor and any effects of the different cancer agents.
Tumor cells can be grown in culture medium and then treated with different drugs. Using
luminescence-based tests to measure cell viability, those drugs most effective at killing tumor
cells can be quickly identified, This way, potential new chemotherapies for treating cancers
can be tested.

Luciferase (LUC) gene as reporter for the activity of other genes-
Here, the researchers splice the LUC gene together with a specific gene they want to study,
and then insert this spliced DNA into living cells.
Whenevr the spliced DNA gets transcribed, the cells will manufacture luciferase. When
luciferin is added, these cells will respond by lighting up. This technque has been used, for
instance, to find out exactlty when and where specific plant genes get tumed on,
To learn about particular genes regulating plant growth, biologists have spliced the LUC gene
into different bits of plant DNA. When plants are sprayed or fed with luciferin-containing
water, the leaves will glow whenever LUC gene gets turned on. This allows researchers to
identify specific genes regulating plant growth at different times and locations.
Such reporter genes have also provided tools for studying diseases, for developing new
antibiotic drugs and for gaining new insights into many human metabolic disorders.

Development of new treatment for antibiotic-resistant tuberculosis-
To help discover new treatments for antibiotic-resistant tuberculosis, scientists have infected
mice with luciferase-labeled tuberculosis bacteria. They then treat the mice with various anti-
tuberculosis drugs and use bioluminescence imaging to monitor the bacteria inside.

Measurement of calcium changes inside cells- The photoprotein aequorin requires Ca2+, it
is often used to measure calcium changed indside cells.

6. Discovery of green protein (GFP)-
One of the most well-known developments to come out of bioluminescence research ts the
discovery of the green fluorescent protein (GFP). While GFP is not a bioluminescent protein,
it Serves as an accessory emitter by receiving energy from a luciferin-luciferase reaction and
re-emitting it as green light.

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7

REFERENCES:

Hollas, J. M. Modern Spectroscopy. John Wiley & Sons, Inc, England. 2004 (4).

Silverstein, R. M, Webster, F. X. Spectrometric Identification of Organic Compounds. John

Wiley & Sons, Inc, England, Sixth Edition.

3. Douglas, A.S, West, D. M. Principles of Instrumental Analysis. Saunders College, USA. 1980.

4. Pavia, D. L, Lampman, G. M, Kriz, G. S. /ntroduction to Spectroscopy. Thomson Leaming,

Inc. USA. 2001 (3).

Kasture, A. V, Mahadik, K. R, Wadodkar, S. G, More, H. N. A Texthook of Pharmaceutical

Analysis Instrumental Methods. Nirali Prakashan, Pune, 2008 (77).

Sankar, S. R. Textbook of Pharmaceutical Analysis. Rx Publications, Tirunelveli. 2016 (4).

Satyanarayana, U, Chakrapani, U. Biochemistry. Books and Allied Pvt. Ltd Kolkata. 2007 (3).

Kealey, D, Haines, P. J. /nstant Notes Analytical Chemistry. BIOS Scientific Publishers Ltd.

UK. 2002.

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