1)
WHAT IS THE PRINCIPLE OF UV-VIS SPECTROSCOPY?
In UV-VIS, by passing
the UV light through the analyte solution, Based on changes in electronic
transition (electronic energy levels) of analyte we can determine
conjugation present in the sample.
2) WHAT ARE BEER-LAMBERTSLAW? EXPLAIN?
Beers law: Beer's law states that
for a parallel beam of monochromatic radiation passing through homogeneous
solutions of equal path length,
The absorbance is
proportional to the concentration. (A œ C)
Lamberts law: Lambert's law states that for a parallel beam of monochromatic
radiation passing through homogeneous solutions of equal concentration,
The absorbance is proportional to the path length. (A œ t)
By combining both laws
A œ
Ct => a = ε ct (This is known as Beer lamberts law)
A is Absorbance, C is
concentration, t is thickness and ε is molar
extinction coefficient.
Beer lamberts law states
that for
a parallel beam of monochromatic radiation passing through homogeneous
solutions then the absorbance is proportional to concentration and path
length of the solution.
3) WHAT IS LIMIT OF STARY LIGHT? HOW TO MEASURE LIMIT OF STRAY
LIGHT?
1.
Stray light is defined as detected light of any wavelength that
is outside the bandwidth of the selected
wavelength.
- To
measure stray light, some kind of filter is required that absorbs all
light of the wavelength at which the measurement is to be made and
transmits higher and lower wavelengths.
- In
practice such filters do not exist, so “cut-off” filters which transmit
all light above or below a certain wavelength and block all light in
the wavelength range.
Measure of Stray light:
# Stray light can measure by following material.
Material
Cut off
Concentration
NaNO2-Sodium Nitrite -----------------390
nm-------------------------- 5% aqueous
KI-Potassium Iodide
-------------------- 260 nm ------------------------- 1%
aqueous
NaI-Sodium
Iodide------------------------ 260 nm-------------------------- 1%
aqueous
Li2CO3-Lithium Carbonate -------------227
nm--------------------------- Saturated aqueous
NaCl-Sodium Chloride --------------------205
nm--------------------------- 1% aqueous
KCl-Potassium Chloride ------------------200
nm--------------------------- 1.2% aqueous
☼Stray
light is usually checked with a 1.2% potassium chloride solution, where
the absorbance for 1 cm path length should exceed 2.0 at 200 nm against
a water reference. (Absorbance more than 2 means % of Transmittance less than 1)
☼1.2% potassium chloride solution UV Cut off is 200 nm.It means
that it should absorb all the light below 200 nm and transmit all the light
above 200 nm.
a)
If we get absorbance of 1.2% KCl solution more than 2
(Transmittance less than 1.0%) then there is no stray light.
b)
b) If we get absorbance of 1.2% KCl solution less than 2
(Transmittance more than 1.0%) then that transmitted light is known as Stray
light.
A = log10 100 / %T (or)
A = 2 - log10 %T
4) WHICH PARAMETERS DO WE
CHECK IN UV-VIS CALIBRATION?
1. Control of wavelength by HOLMIUM PERCHLORATE SOLUTON (Holmium oxide in
perchloric acid)
2. Control of absorbance by POTASSIUM DICHROMATE (In 0.005M
sulphuric acid)
3. Limit of stray light by POTASSIUM CHLORIDE SOLUTION (In water)
4. Wavelength accuracy
5. Resolution by TOLUENE IN n-HEXANE.
6. Resolution power by TOLUENE IN METHANOL.
7. Absorption of corvettes (Cuvettes).
Why:
Why do we use holmium per chlorate in control of wavelength test?
Holmium per chlorate solution can give very sharp and accurate
peaks at constant places throughout the UV-VIS region(At
241.15nm,287.15nm,361.5nm,536.3nm) and it is suggested by NIST (National
Institute of Standards and Technology) and Pharmacopeia.
Why do we use potassium dichromate in control of absorbance test?
Potassium dichromate in sulphuric acid can give more maxima in the
UV-VIS region (At 235nm, 257nm, 313nm, 350nm, 430nm) so that we can check the
whole UV-VIS region with a single run and it is suggested by NIST and
Pharmacopeia.
Potassium dichromate itself is stable and available in high
purity. In dilute perchloric acid solution, it has a linear response with
temperature.
Why do we use potassium chloride in limit of stray light test?
The UV cut off for potassium chloride is 200 nm so it is neutral
for UV-VIS region. Means It cannot absorb in UV-VIS region.
Why we use Toluene in hexane in resolution test?
Toluene can give one maxima at 269nm and minima at 266.These two
are very close to each other if any small change in the resolution we can
easily identify the absorbance differences. Hexane cannot absorb uv light and it is
miscible with Toluene.
5) WHAT IS THE PRINCIPLE
OF IR SPECTROSCOPY?
In IR, by passing the IR light through the analyte,
based on changes in vibration & rotational movements of the atoms present
in the sample we can determine functional groups present in the
sample.
6)
WHY WE USE KBR FOR PELLET MAKING IN IR?
1. It does not
absorb IR radiation in whole region (400-4000 cm-1). (RX does not absorb IR
radiation).
2. Due to its high mechanical strength. (With very small amount of KBr we
can make a very transparent pellet).
3. It does not react with the sample due to its neutral properties.
NaCl (mostly for liquids), NaI, KCl can also use for IR pellet making.
7) WHAT IS THE THICKNESS OF POLYSTYRENE FILM USED FOR IR?
Thickness:
Generally we use 38 micron (0.038mm=0.04mm) thickness polystyrene
film for FTIR calibration. There are some other micron thickness films also available
(like 76 micron).
8) WHAT IS THE UNITS OF WAVE
NUMBER IN FTIR?
Units:
The units of wave number in FTIR are cm-1
9)
WHY WE USE POLYSTYRENE FILM FOR CALIBRATION OF FT-IR?
Polystyrene film for
calibration:
☼In polystyrene
film all bands executed which covers whole IR region. And the bands are at
constant places with sharp intensities.
☼polystyrene film is
made by the polymer of styrene. So it’s having highly durability
and stabled at any temperature.
10) WHAT ARE THE ADAVNTAGES OF FTIR OVER IR?
Below are some of major advantages of FT-IR over dispersive IR.
1. Speed: All the measurements are made simultaneously most
measurement are made by FTIR are made in a matter of seconds rather than in minutes
as in dispersive.
2. Sensitivity: Sensitivity is dramatically improved in FTIR for
many reasons. Detectors employed are much more sensitive. Sensitivity directly
prepositional to number of scans, If number of scans increases sensitivity increases.
Noise is low.
3. Mechanical Simplicity: The moving mirror in the interferometer is only
continuously moving part in the instrument. Then there is very little
possibility of mechanical breakdown.
4. Internally Calibrated: This instrument employs a helium-neon
laser light as an internal wavelength calibration standard. FTIR instruments
are self calibrating and never need to be calibration check by user.
These advantages along
with several other make measurements made by FTIR extremely accurate and reproducible.
Thus it is very reliable technique for positive identification of virtually any
sample.The sensitivity benfits enables identification of even the smallest of
contaminants.
11) WHAT IS THE PRINCIPLE OF
POLARIMETER?
By passing plane
polarized light (moves in a single direction) through the sample, Based on
the change in the direction of plane polarized light we can identify the
optical isomers present in the sample. That means whether it is dextro or levo.
If light rotates toward right we confirm it as Dextro and if to left we can
confirm it as Levo.
12) WHAT IS THE DIFFERENCE BETWEEN OPTICAL
ROTATIONS & SPECIFIC ROTATION (or) SPECIFIC OPTICAL ROTATION?
Optical Rotation
(OR): α
It
is the angle of rotation of a plane of polarized light when passed through
an optically active substance.
· It is measured by the
instrument Polari meter and is denoted by α.
Specific optical Rotation
(SOR): [α]
· It is measured from the
optical rotation and is denoted by [α].
[α] =
100α /cl
c = Concentration of sample in Gram/100 ml
l = Length of cell in dm (decimeter) (1dm=10 cm=100 mm)
13) ON WHICH FACTORS OPTICAL
ROTATIONS & SPECIFIC OPTICAL ROTATION DEPENDS?
1.
The optical rotation depends upon:
·
The type of sample (example: sugar solution).
·
Concentration of the
optically active component(s).
·
The length of the sample tube.
·
The wavelength of the light
source.
·
Temperature of the sample.
·
Solvent used for dilution.
2. The specific optical rotation depends on
·
Type of sample
·
Wavelength of light source
and
·
Temperature
But it does depend on,
concentration of sample and cell length. That means
if we analyze a sample with different concentrations or different cell lengths
under same conditions the OR valves change but SOR values are same.
14) WHY WE
USE SUCROSE FOR POLARIMETER CALIBRATION? EXPLAIN MUTAROTATION?
1. It is a non
mutarotating compound. (It has no isomeric forms to get mutarotation)
2. It is easily soluble
in water up to 30% and soluble up to 50%.
Mutarotation:
The change in the
specific rotation between isomeric forms of a compound is known as
Mutarotation.
Explanation:
D (+)-Glucose exists in two isomeric forms which
undergo mutarotation.
a) Crystals of ordinary D(+)-Glucose of melting point 146°C are dissolved in
water the specific rotation gradually drops from an initial +112 to +52.7.
b) Crystals of ordinary D(+)-Glucose of melting point 150°C (obtained by
crystallization at temp 98°C) are dissolved in water the specific rotation
gradually rises from an initial +19 to +52.7.
The form with higher positive rotation is called Alpha D (+)-Glucose. And with lower positive
rotation is called Beta D (+)-Glucose .The change in the specific rotation of each of
these is known as Mutarotation.
15)
WHAT IS THE PRINCIPLE OF KARL FISCHER TITRATION?
In Karl Fischer
titration one mole of iodine reacts with one mole water present in the sample
and forms two moles of HI. Based on this reaction we can determine water
present in the sample.
I2 +
H2O ---> 2HI
ROH
+ SO2 +R’N à[R’NH] SO3R
+ H2O + I2+ 2R’N à 2[R’NH] I + [R’NH] SO4R
Mechanism:
Step
1: 3R’N+ SO2 + I2 + H2O à [R’N]
SO3 + 2[R’NH] I
Step
2: [R’N] SO3 + ROH à [R’NH]
SO4R {R’N = Pyridine}
16)
WHY WE USE DST FOR KF REAGENT STANDARDIZATION & KF APPARATUS
CALIBRATION?
Standardization of KF
reagent:
Standardization
of KF reagent means checking of concentration of KF reagent. As per Karl
Fischer 5mg of water is neutralized by 1 ml of KF reagent (If
the KF reagent is good).That’s why we get the KF factor around 5 mg of
water/ml.
So we need a standard
which contains following properties.
1. It must be
a primary standard
2. It should Contains a
known & constant percent of water in it at room temperature.
3. Should dissolve in methanol easily.
Generally we use
Water - 100% of water (For volumetric)
Disodium
Tartrate Dihydrate - 15.66% of water (For volumetric)
Lactose
Mono hydrate - 5.0% of water (For Coulometric)
KF Apparatus Calibration:
We use KF
Apparatus for determination of water present in the sample. Calibration means
to check the instrument whether it works properly or not. So any sample which
contain constant amount of water in it can use for KF Apparatus calibration.
Generally we use above mentioned samples (Mostly DST)
17) WHY WE ARE USING EXCESS METHANOL IN KF
TITRATION?
The reaction in KF
titration is
3 Im +I2 + SO2 + H2O ------> 2 Im.
HI + Im.SO3
Im.SO3 +
CH3OH --------> Im.(H)SO4CH3.
(Stochiometri of I2 &
H2O is 1:1)
In the 2nd step if methanol is not present the reaction is like below.
3 Im +I2 + SO2 + H2O ------> 2 Im. HI + Im.SO3
(Im= Imidazole)
Im.SO3 + H2O --------> Im.NH
+ SO4H
(Stochiometri of I2 &
H2O is change from1:1 to 2:1)
During the titration, If Excess methanol is not present
Im.SO3 can react with H2O which varies the Stochiometri of H2O
& I2 from 1:1 to 2:1.
Note:
1.The above mentioned is the most suitable reason. And some other benefits
also there for methanol like it can dissolve most of the organic solvents, low
cost & availability.
18) WHY WE ARE USING IPA or PYRIDINE IN PLACE OF
METHANOL IN THE DETERMINATION OF WATER CONTENT PRESENT IN CARBONYL COMPOUNDS BY
KARLFISCHER TITRATION?
If we use
methanol for carbonyl compounds, Methanol (Primary alcohol - Very
reactive) reacts with carbonyl compounds and forms acetals, ketals and
water.
This means side reaction takes place.
☻If we use any secondary alcohol (less
reactive than Primary alcohol) like IPA in place of Methanol we can easily
stop the side reaction (Acetal or ketals with water formation) means
IPA only reacts with water but not with carbonyl compounds.
☻We use this reaction for determination of
water. But in above case water is produced in the reaction which leads to a
wrong assumption.
☻ Pyridine also used in place of methanol.
Note:
1. We have the only choices of Alcohol (Primary,
secondary & tertiary) and Basic buffers (like pyridine &
Imidazole).Because karl fischer reagent is miscible with these two.
2. Tertiary alcohol is very less reactive so
it is not suitable. So the remaining choices are Primary & secondary
alcohols and pyridine & Imidazole.
19) WHY WE ARE USING SILICONE OIL IN MR APPARATUS?
Silicone oil is chosen for its ecofriendlyness by having below
mention properties.
·
It cannot transfer electricity.
·
Non flammable and fire resistant.
·
Thermally stable in both cold and hot extremes·
·
No toxicity
