When a molecule
absorbs visible or ultraviolet energy, an electron or electrons will be raised
to a higher energy level if the energy requirement for that transition is equal
to the energy of the incoming photon. The electrons is the inner shells of atom
and those that are shared by two adjacent atoms not affected to the same degree
by incoming radiation as those that can’t be localized within the molecule.
Electrons of the latter type give rise to spectra in the UV and visible regions
of the electromagnetic spectrum.
Saturated double bond
will not absorb ultraviolet energy. Presence of chromophore is essential in a
saturated compound for absorption of radiations at UV and visible regions.
Presence of auxochrome cause a bathochromic shift i.e. absorption maxima shifts
to longer wave length.
In this way
electromagnetic waves are absorbed by a compound which can be determined by the
spectrophotometer and thus qualitative and quantitative of compound can be
done.
Types of transition
states
According to the
molecular orbital theory when a molecule in excited by the absorption of energy
(UV or Visible light), its electrons are promoted from a bonding to an
anti-bonding orbital. The energy required for various transitions obey the
following order: ----------
A transition in which
a bonding ( electron is excited to an anti-bonding ( orbital is referred to as
transition. It is a
high energy processes because of ( bonds are generally very strong. In organic
compounds, all the valence shell electrons are involved in the formation of
sigma bonds resulting do not show absorption in the normal ultra-violet region
(180 – 400nm). For saturated hydrocarbons, like methane (CH4) absorption occurs
near 150nm (high energy). Consider ( ( (* transition in a saturated
hydrocarbon: -
The excitation of
sigma bond electron to (* (anti-bonding) level occurs with net retention of
electronic spin. It is called excited singlet state which may in turn gets
converted to excited triplet state.
n ( (*: This type of
transition takes place in saturated compounds containing one hetero atom with
unshared pair of electrons (n electrons). Such transitions require
comparatively less energy than that required for ( ( (* transitions. In
saturated alkyl halides, the energy required for such a transition decreases
with the increase in the size of the halogen atom (or decrease in the
electronegativity of the atom.)
For n ( (* transition
in methyl chloride and methyl iodide. The absorption maximum for methyl
chloride is 172 – 175nm whereas methyl iodide is 258nm because the
electronegativity of chlorine is greater than iodide. Thus the excitation of
chlorine atom is comparatively difficult than iodide. On the otherhand methyl
iodide has higher molar extinction coefficient than methyl chloride.
n ( (* transitions
are very sensitive to hydrogen bonding. Alcohols as well as amines form
hydrogen bonding with the solvent molecules due to the presence of non bonding
electrons on the hetero atom and thus transition requires greater energy.
( ( (*: This type of
transition occurs in the unsaturated centers of the molecule; i.e. in compounds
containing double or triple bonds and also in aromatics. The excitation of (
electron requires smaller energy and hence transition of this type occurs at
longer wavelength. An ( electron of a double bond is excited to (* orbital.
Consider ( ( (* transition in an alkene: -
This transition
requires still lesser energy as compared to n ( (* transition and therefore,
absorption occurs at longer wavelengths. Absorption usually occurs within the
region of ordinary ultra-violet spectrophotometer. In unconjugated alkenes,
absorption bands appear around 170 – 190nm. In carbonyl compounds, the band due
to ( ( (* transition appears around 180nm and is most intense, i.e. the value
of extinction coefficient is high.
n ( (*: In this type
of transition an electron of unshared electron pair on hetero atom gets excited
to (* antibonding orbital. This type of transition requires least amount of
energy out of all the transition and hence occurs at longer wavelength.
Saturated aldehydes show both the types of transitions, i.e. low energy n ( (*
and high energy ( ( (* occurring around 290nm and 180nm respectively.
Example:
Increasing Energy
n ( (*
In carbonyl compounds
n ( (*
In oxygen, nitrogen,
sulfur and halogen compounds
( ( (*
In alkenes, carbonyl
compounds, alkynes, azo compounds etc.
( ( (*
In carbonyl
compounds.
( ( (*
In alkanes.
Instrumentation
The modern ultra-violet
visible spectrometers consist of: ------
Light source
Monochromator
Detector
Amplifier and
Recording device.
The most suitable
sources of light are:
Tungsten filament
lamp: Tungsten filament lamp is particularly rich in red radiations, i.e.
radiations with wavelength 375nm.
Deuterium discharge
lamp: The intensity of the deuterium discharge source falls above 360nm.
The primary source of
light is divided into two beams of equal intensity with the help of a rotating
prism. The various wavelengths of a light source are separated with a prism and
then selected by slits for recording purposes. The selected beam is
monochromatic which is then divided into two beams of equal intensity. Light
from the first dispersion is passed through a slit and then sent to the second
dispersion. After the second dispersion, light passes through the exit slit
result in increase the band width of the emergent light which is almost
monochromatic.
One of the beams of
selected monochromatic light is passed through the sample solution and the
other beam of equal intensity is passed through the reference solvent. The
solvent as well as solution of the sample may be contained in cells made of a
material which is transparent. Each absorbance measurement on the solution is
accompanied by a simultaneous measurement on the pure solvent.
After the beams pass
through the sample cell as well as the reference cell, the intensities of the
respective transmitted beams are then compared over the whole wavelength range
of the instrument. The spectrometer electronically subtracts the absorption of
the solvent in the reference beam from the absorption of the solution. Hence
the effects due to the absorption of light by the solvent are minimized.
In this way, the
absorbance or the transmittance characteristic of the compound alone can be
measured. The signal for the intensity of absorbance Vs corresponding
wavelength is automatically recorded on the graph. The spectrum is usually
plotted as absorbance A (log10 I0/I) against wavelength ( (abscissa). The plot
is often represented as (mas (Extinction coefficient) against wavelength.
Beers and Lambert’s
Law
When light
(monochromatic or heterogenous) fall upon a homogenous medium, a portion of the
incident light is reflected, a portion is absorbed within the medium and the
remainder is transmitted.
The change of
absorption of light with the thickness and concentration of the medium is
described by Beer’s and Lambert’s Law (a combination of two laws).
Lambert’s Law:
This law relates the
absorptive capacity to the thickness of the absorbing medium. According to this
law: ------
“When a
monochromatic radiation or light passes through a homogenous transparent medium
the rate of decrease of intensity of radiation with the thickness of the
absorbing medium is directly proportional to the intensity of the incident
light.”
Mathematically, the
law is expressed as: ---------
Where,
(- ve) sign
indicates, the intensity of the incident light decreases as the thickness of
the medium increases.
I = intensity of
radiation after passing through a thickness x, of the medium.
dI = Infinitesimally
small decrease in the intensity of radiation on passing through infinitesimally
small thickness dx, of the medium.
dI/dx = The rate of
decrease of intensity of radiation with
the thickness of the medium.
K1 = Proportionality
constant or Absorption coefficient.
By changing the
variable, we get: ----------
Integrating this
equation between the limits “I0 to It” and “0 to x”, we get: ----------
Beer’s Law:
This law relates
absorptive capacity to the concentration of the solute in the solvent.
According to the law: ------
“When a monochromatic
radiation or light passes through a homogenous transparent medium the rate of
decrease of intensity of radiation with the concentration of the solute in that
system is directly proportional to the intensity of the incident light.”
Mathematically, the
law is expressed as: ---------
Where,
(- ve) sing
indicates, the intensity of the incident light decreases as the thickness of
the medium increases.
Intensity of the
radiation dI decreases as it passes through each increase of concentration dc..
K2 = Proportionality
constant or Absorption coefficient.
By changing the
variable, we get: ----------
Integrating this
equation between the limits “I0 to It” and “0 to c”, we get: ----------
Combine the equations
(1) and (2) we get: ---------
Where, K = a new
constant which is equal to absorptivity and denoted by “a”
log I0/It is termed
as absorbance and denoted by “A”. Therefore we can rewrite the equation as
follows: -----
If “x” thickness is
constant this equation represents a equation of straight line (y = mx) which
passes through the origin and slop of which is equal to ac.
Beer’s – Lambert’s
Law applies to a solution containing more than one kind of absorbing
substances, provided there is no interaction among the various species. Thus
for a multiple component system: ------------
Where, the subscripts
refer to absorbing components 1,2, -----------------,n.
Transmittance “T”:
Transmittance is the quotient of the radiant power It transmitted by a sample
divided by the radiant power I0 incident upon the sample. The percent
transmittance (%T) is equal to 100(
[T = It/I0].
Abosrptivity “a”:
Absorptivity is the quotient of the absorbance (A) divided by the concentration
(C) of the solution (gm/liter) and the absorption path length (l) in
centimeter. We know: ------
The absorptivity
values vary with the wavelength of the incident energy. However at a specified
wavelength the absorptivity value for a drug is a constant if Beer’s Law is
obeyed.
Molar absorptivity
“(”: Molar absorptivity is the quotient of the absorbance divided by the
concentration (c) of the solution (moles/liter) and the absorption path length
(l) in cm. it is also the product of the absorptivity and the molecular weight
(M) of the substance.
Significance:
The molar
absorptivity (() is a property of the molecule undergoing an electronic
transition.
“(” signifies the
light absorption capacity of molecules. “(” is directly proportional to the
light absorption capacity of molecules.
“(” is an identifying
character of a molecule.
The following
equation gives the relationship of “(” with absorbance (A) and concentration
(c).
Limitation of the
Beer’s – Lambert’s Law
The limitations are
due to the nature of the solution being examined. Others are due to chemical
changes in the solution or to the type of radiant energy used in the
measurement process.
Actual limitation:
If interaction occurs
at higher concentration (( 0.01M) Beer’s – Lambert’s Law will not be obeyed.
Because at higher concentrations the charge distribution on the molecule.
Since the
absorptivity “a” is dependent upon the refractive index of the solution,
changes of concentration cause significant alteration in the refractive index
“n” of a solution result in Beer’s – Lambert’s Law deviation. A correction for
this effect can be made by the multiplication of absorptivity value (a) by
n/(n+2)2.
The beer’s –
Lambert’s Law is rigorously obeyed when a single species gives rise to the
observed absorption.
Chemical limitations:
The Beer’s – Lambert’s Law may not be obeyed: ------
When different forms
of the absorbing molecule are in equilibrium.
When the solute in
the solution ay associate, dissociate or react with the solvent.
When there is thermal
equilibrium between the ground electronic state and a low lying excited state.
When there one
fluorescent compound or compounds which are changed by irradiation.
Example:
Since the molar
absorptivity values for the dichromate ion and the two chromate species are
different at the wavelength of maximum absorption. Chromate solutions, when
diluted with H2O deviate from
Beer’s – Lambert’s
Law.
Instrumental
deviation: Beer’s law is observed only when the radiation employed is
monochromatic. Polychromatic beam may cause deviation from Beer’s law. All
spectrophotometers isolate theoretically, the wavelength specified on the scale
of the monochromator. However under actual operating condition, solutions are
exposed to several wavelength of radiant energy.
Deviation caused by
the solvent effects: The absorption spectrum of a drug depends on the solvent
used to solubilize the substance. A drug may absorb a maximum of radiant energy
at one wavelength in one solvent but will absorb little at he same wavelength
in another solvent. These changes in spectrum are due to: ------
The nature of the
solvent
The nature of the
absorption
The nature of the
solute
Chromophore:
A chromophore is a
group which when attached to a saturated hydrocarbon, produce a molecule that
absorbs a maximum of visible or ultraviolet energy at some specific wavelength.
Many molecules may
contain two or more chromophores. The interaction of radiant energy with the
molecule then depends upon the relative positions of the two chromophores in
the molecule.
When two chromophores
are separated by more than one carbon atom, total absorption is the sum of the
absorption of each of the two chromophores.
When two chromophores
are adjacent to each other, the absorption maximum shift to longer wavelength
and the intensity of absorption is increased.
When two chromophores
are attached to the same carbon atom, there is a summation of absorption and a
shift toward longer wavelength, but the degree of change is less than that
shown by conjugated coromophores.
Types:
Chromophores are
classified into two groups according to their molar absorptivity value and
formation of bands: -
Simple: The simple
chromophores give rise to R bands and the molar absorptivity value for this
type of band is usually less than 100.
Complex: There are
two kinds of complex chromophores.
The first type is
found in aromatic compounds whose structure contain a benzene ring. These
chromophores give rise to B bands and the molar absorptivity values for these
bands range from 250 – 3000.
The second type has
the following formula: [A – (CH=CH)n – CH=B]
Where A = H, R, OR, SR, NR2, O-,S-
or –NR and B = CH2CHR, CR2, NR, O, S, +NR2, +OR & +SR. theses chromophores
give rise to K bands. The molar absorptivity values for these bands are more
than 10,000.
Chromophores are
further divided into two groups according to their transition states: -------
First type: This type
of chromophores contain ( electrons and undergo ( ( (* transition. Eg.
Ethylene.
Second type: This
type of chromophores contain both ( and n (nonbonding) electron and undergo two
types of transitions i.e. n ( (* and ( ( (*. Eg. Carbonyl, Azo compounds and
Nitriles etc.
Auxochromes:
An auxochrome is a
group which does not itself act as a chromophore but whose present brings about
a shift of the absorption band towards the red end of the spectrum.
An suxochromic group
is called color enhancing group.
Auxochromic group do
not show characteristic absorption above 200nm
The effect of the
auxochrome is due to its ability to extend the conjugation of a chromophore by
the sharing of non-bonding electrons.
Example: - OH, - OR,
- NH2, - NHR, - NR and - SH.
Bathochromic shift:
It is an effect by
virtue of which the absorption maximum is shifted towards longer wavelength due
to the presence of an auxochrome or by the change of solvent. Such an
absorption shift towards longer wavelength is called “Red shift” or
“Bathochromic shift.”
Example: The n ( (*
transition for carbonyl compounds undergoes bathochromic shift when the
polarity of the solvent is decreased.
Hypsochromic shift:
It is an effect by
virtue of which the absorption maximum is shifted towards shorter wavelength.
The absorption shift towards shorter wavelength is called “Blue shift” or
“Hypsochromic shift”.
Causes: It may occur
By the removal of
conjugation
By changing the
polarity of the solvent.
Example: In the case
of aniline, the absorption maximum occurs at 280nm because the pair of
electrons on nitrogen atom is in conjugation with the ( bond system of the
benzene ring. In its acidic solution a blue shift is caused and absorption
occurs at shorter wavelength (203nm). In acidic solution is formed and the electron
pair is no longer present and hence conjugation is removed.
Hyperchromic shift:
It is an effect due
to which the intensity of absorption maximum increases i.e. (max increases. It
is usually caused by the introduction of an auxochrome. Example: The B band for
pyridine at 257nm (max 2750 is shifted to 262nm (max 3560 for 2-methyl
pyridine.
Hypochromic shift:
It is an effect due
to which the intensity of absorption maximum decreases, i.e. extinction
coefficient (max decreases. It is usually caused by the introduction of a group
which distorts the geometry of the molecule. Example: Biphenyl absorbs at
250nm, (max19000 whereas 2-methyl biphenyl absorbs at 237nm, (max 10250. It is
due to the distortion caused by the methyl group in 2-methyl biphenyl.
Types of absorption
band:
K – band: K – bands
originate from a compound containing a conjugated system. Such type of bands
arises in compounds like: ---- dienes, polyeness, enones etc. K – bands also
appear in an aromatic compound which is substituted by a chromophore. The
intensity of K – bands is usually more than 104. The K – bands absorption due
to conjugated “enes” and “enones” are affected differently by changing the
polarity of the solvent.
R – band: R – bands
originate due to n ( (* transition of a single chromophoric group and having at
least one lone pair of electrons on the hetero atom. R – bands are also called
forbidden bands. There are les intense with (max value below 100.
B – band: B – bands
originate due to ( ( (* transition in aromatic or hetero aromatic molecules.
When a chromophoric group is attached to the benzene ring the B – bands are
observed at longer wave lengths than the more intense K – bands. For example, K
– bands appears at 244nm, (max 12000 and B – bands at 282nm, (max 450. The fine spectrum of B – bands may be missing
in case of: -------
Substituted aromatic
compounds
By the use of polar
solvent.
E – band: E – bands
originate due to the electronic transitions in the benzenoid system of three
ethylenic bonds which are in closed cyclic conjugation. These are further
characterized as1 and E2. E1 and E2 bands of benzene appear at 184 and 204nm
respectively.
Some terms and their
brief description:
Radiant energy:
Radiant energy is the energy transmitted as electromagnetic radiation. The sun
is out most important source of radiant energy. Absorption spectrophotometry is
the measurement of the absorption of radiant energy by various substances.
Wavelength: The
wavelength is the linear distance from any point on one wave to the
corresponding point on the adjacent wave. The dimension of wavelength is length
(l). It is mentioned by the Greek letter lambda ((). Wavelength can be
expressed in centimeter (cm) or more commonly in the following units.
1 angstrom (A0) =
10-8cm = 10-10m.
1 nanometer (nm) =
10-9m = 10-7cm = 1m( = 10A0
1 micrometer ((m) =
10-6m = 10-4cm = 1micron (().
Frequency: Frequency,
denoted by ( (nu), of a beam is the number of cycles occurring per second. The
usual unit of it is sec-1 which may also be denoted by cycles per second (CPS)
or hertz (Hz). The relationship between the wavelength (in cm) and frequency is
stated mathematically as: -------
Wave number: Wave
number that is signified by Greek letter sigma ((), is the reciprocal of
wavelength (when wavelength is expressed in cm). It dimension is therefore
reciprocal length (l-1) and its unit is cm-1. The relationship between wave
number and wavelength could be expressed mathematically as: ------
The electromagnetic
spectrum
The range of
spectropic interest
Approx. wavelength
range (cm)
Region of spectrum
Region
Wavelength region
10-12 – 10-11
Cosmic rays
Far ultraviolet
100 – 200nm
10-11 – 10-8
Gamma rays
Ultraviolet
200 – 400nm
10-8 – 10-6
X – rays
Visible
400 – 750nm
10-6 – 10-5
Ultraviolet
Near infrared
0.75 – 4(m
10-5 – 10-4
Visible
Infrared
4 - 25(m
10-4 – 10-2
Infrared
10-2 – 10
Microwave
10 – 108
Radio frequency
Correlation of color
with wavelength of visible light
Wavelength (nm)
Color
Wavelength (nm)
Color
400 – 450
Violet
575 – 590
Yellow
450 – 480
Blue
590 – 625
Orange
480 – 490
Green blue
625 – 750
Red.
500 – 560
Green
Concentration
determination of unknown solution:
If a compound follows
the Beer’s – Lambert’s Law then its concentration in a supplied sample could be
known from the calibration curve drawn for the various strength reference
standard solution of that compound. Calibration curve of the Beer’s – Lambert’s
Law means the curve in which absorbance or percent transmittance is plotted
against concentration. If a compound follows the Beer’s – Lambert’s Law the
calibration curve for this compound shows a straight line going through the
origin.
Procedure:
In this process the
following steps should be operated for getting the concentration of the
supplied sample: -----
To prepare a solution
of concentration ( 0.01M by authentic sample of which concentration is to be
determined by using a suitable optically transparent solvent.
Then (max for the
compound must be determined and to ensure 100% transmittance at (max for the
using solvent.
Various strength of
the reference standard solution of that compound is made by dilution. Eg.
10(g/ml, 20(g/ml, 30(g/ml ---------- 100(g/ml.
Now the absorption or
percent transmittance for these various strength in the (max is measured.
These measured
absorption or % transmittance are plotted against concentration of the
reference standard solution and draw the “A vs c” curve.
If the curve gives a
straight line going through the origin we could understand that the compound
follows the Beer’s – Lambert’s Law.
Then the unknown
sample with proper dilution is supplied in curette to measure the absorption or
% transmittance in the (max. This value will indicate a point on the straight
line drawn for the reference standard solution.
Now a horizontal line
drawn from this point, situated on straight line, on the Y-axis of
concentration axis of the curve will show the concentration of the compound of
the supplied sample.
Finally, the
determined concentration is multiplied by the dilution factor and thus the
actual concentration is measured.
Choice of solvent:
The choice of solvent
to be used in ultraviolet spectroscopy is quite important: -----------
The first criterion
for a good solvent is that it should not absorb ultraviolet radiation in the
same region as the substance whose spectrum is being determined. Usually
solvents which do not contain conjugated system are most suitable for this
purpose.
A second criterion is
the effect of polar and non polar solvent on the fine structure of an
absorption band. A non polar solvent does not hydrogen bond with the solute and
the spectrum of the solute closely approximates what it would be in a gaseous
state. In a polar solvent the hydrogen bonding form a solute solvent complex
and the fine structure may disappear.
The third property of
a solvent which must be considered is the ability of a solvent to influence the
wavelength of ultraviolet light which will be absorbed.
The polar solvents
may not from hydrogen bond as readily with excited states as with ground states
of polar molecules and the energies of electronic transitions in this molecules
will be increased by this polar solvents.
Transition of the n ( (* type are shifted to shorter wavelength by polar
solvent.
On the otherhand, in
some cases the excited states may form stronger hydrogen bonds than the
corresponding ground states. In such cases, a polar solvent would shift
absorption to longer wavelength, since the energy of the electronic transition
would be decreased. Transition of the ( ( (* type are shifted to longer
wavelength by polar solvents. Thus it has been found that increase in polarity
of the solvent generally shifts
n ( (* and n ( (*
bands to shorter wavelength and ( ( (* bands to longer wavelength.
Solvent must not
contain trace impurities. Many impurities (eg. Benzene in absolute alcohol)
absorb radiant energy and complicate the analysis.
PH of the solvent: PH
has a noticeable effect on absorption spectrum. This can be described by the
following example: -----
In 1N HCl solution,
sulfonamides absorb little of the radiant energy above 230nm. The auxochrome
under this condition is – +NH3. But in 1N NaOH solution, a more efficient
auxochrome is formed (- NH2) and the substance now exhibits an absorption
maximum at 251nm. Buffer that used in this condition must transmit ultraviolet
energy if they are to be used for the determination of the spectral
characteristics of drugs.
Formation of
tautomeric forms: Under acidic condition, Phenobarbital does not absorb
ultraviolet energy, to a significant degree of 240nm. The tautomeric forms of
Phenobarbital are shown in the following equation.
Under alkaline
conditions, the chromophoric systems are . At a PH 9, a high
intensity absorption band may be observed at 240nm. However, in 0.1N NaOH this
band appears at 255nm.
Questions:
Theoretically there should be sharp band in UV spectroscopy, but practically,
broad bands are absorbed. Why?
Answer:
Nature is very
specific, if a molecule absorbs UV radiation it usually causes electronic
transition. In this case, the molecule absorbs radiation of a particular
wavelength. So the UV spectrum should contain a sharp band.
But at normal
conditions, UV radiation causes vibrational and/or rotational transitions too.
So molecules usually absorbed radiation at a relatively wide range. That is
why, a broad band is observed.
Questions: What is
the effect of increasing the number of double bonds of compounds on its
UV-Visible spectrum?
Answer:
Compounds having
double or triple bonds contain electrons which are excited relatively easily.
In molecules containing a series of alternating double bonds, the ( electrons
are delocalized and require less energy for excitation. So that the absorption
occurs into higher wavelengths. When there are more conjugation the compound
will absorb less energy radiation.
So the effect of
increasing number of double bonds of a compound on its UV-Visible spectrum is
increasing the absorption characteristics more easily will less energy.
Questions: What is
the effect of chromophore on absorption of spectrum?
Answer:
The chromophore is a
functional group which has a characteristics absorption spectrum in the visible
or UV region. Such groups invariably contain double or triple bonds and include
the C=C linkage. The C(C triples bonds, the nitrogroup, the azo group, the carbonyl
group.
If the chromophore is
conjugated with another of the same or different kind, then the absorption is
enhanced and a new absorption band appears at a higher wavelength.
Difference between
chromophore and auxochrome
Chromophore
Auxochrome
A chromophore is a
group which attached to the saturated hydrocarbon.
Auxochrome are either
co-coordinately saturated or co-coordinately unsaturated.
It shows a
characteristic absorption in UV or Visible region.
It does not show a
characteristic absorption in UV or Visible region.
It is responsible for
color imparting to this compound.
It is responsible for
color enhancing.
There are two group
of chromophore: (1) Simple & (2) Complex
Auxochrome has no
classification.
Chromophore may cause
either bathochromic or hpsochromic shift, hyperchromic or hypochromic shift.
Auxochrome only show
bathochromic and hyperchromic shift
The range of
wavelength of chromophore is 200nm to 800nm
The range of
wavelength of auxochrome is below 200nm
It is conjugated with
another, same or different kind
It is conjugated by
sharing the non bonding electron
Example: carbonyl,
azo and nitro group
Example: - OR, - NHR,
- NH2 etc.
Application of
UV-Visible spectroscopy:
Determination of the
drug content in a sample.
Determination of
functional group with other methods also.
Extent of
conjugation.
Distinction in
conjugated and un-conjugated compounds.
Identification of the
unknown compound.
Elucidation of the
structure of Vit. A and K.
Preference over two
tautomeric forms.
Identification of a
compound in different solvents.
Determination of
configurations of geometrical isomers.
Prepared by Md.
Badrul Alam (Prince)
PAGE
NUMPAGES
It
-------------------------- (1)
I
dI
dx
= K1
I
dI
I
= K1
dx
dI
Or
EMBED ChemDraw.Document.5.0
n ( (*
( ( (*
EMBED ChemDraw.Document.5.0
( ( (*
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Fig: Various
electronic transitions
Fig: Electromagnetic
energy levels
n ( (*
n ( (*
( ( (*
( ( (*
( ( (*
a = A/lc
A = alc
I
(
dx
dI
Transmitted radiation
Monochromatic
radiation
Ir
It
I0
Solution
(
( ( (* ( n ( (* ( ( (
(* ( n ( (*
Energy
(*
(*
n
(
(
(*
(*
n
(
Fig: Ultra-Violet
spectrophotometer.
Recorder
Amplifier
Solution
Solvent
Mirror
Slits
Rotating prism
Light source
B
A
(Wavelength, nm) ((
(
Intensity
EMBED ChemDraw.Document.5.0
EMBED ChemDraw.Document.5.0
Concentration of the
supplied sample (diluted)
Absorption for the
supplied sample (diluted)
Concentration (c)
Absorbance (A)
( = 1/(
Where,
( = Frequency
C = Velocity
( = Wavelength of a
radiation
( = C/(
(
RED
BLUE
Hypsochromic shift
Bathochromic shift
Hypochromic shift
Hyperchromic shift
EMBED ChemDraw.Document.5.0
It
I0
= K1
dx
x
0
In
or,
x
= K1
I0
It
In
or,
x
= K1
It
I0
or,
log
x
=
It
I0
K1
2.303
dI
dc
(
I
Or
dI
dc
= K2
I
-------------------------- (2)
dI
I
= K2
dc
It
I0
dI
I
= K2
dc
c
0
It
I0
= K2
c
In
or,
I0
It
= K2
c
In
or,
I0
It
=
c
log
or,
K2
2.303
= axc
-------------------------------- (3)
log
log
I0
= Kxc
It
I0
Cr2O7-2 + H2O = 2HCrO4- = 2H+ + 2CrO4-
A = (lc
So, “(” can be used
in UV – Visible Spectroscopy for the determination of A and/or c.
Where,
c = concentration of
the solution (moles/liter)
l= length in cm
a = absorptivity
M = molecular weight.
( = a ( M
A
= A1 + A2 + ------------ +An
= a1xc1 + a2xc2 + ----------------- + anxcn
= axc
-------------------------------- (4)
Atotal
( = A/lc
So,
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