Slide 1: ARPANKUMAR V. PARMAR DEPARTMENT OF CEMISTRY
SURAT-395007 Date :14-7-2008 Supervisor : Prof. P BAHADUR Slide 2: INFRARED SPECTROSCOPY BASICS
INSTRUMENTION A Talk
on Slide 3: HISTORY OF IR Sir William Herschel Slide 4: Spectroscopy is the branch of science dealing with the study of interaction of electromagnetic radiation with matter The most important consequence of such interaction is that energy is absorbed or emitted by the matter in discrete amount called quanta Slide 5: Electromagnetic Radiation
• Electromagnetic radiation: light and other
forms of radiant energy
• Wavelength (λ): the distance between
consecutive peaks on a wave.
• Frequency (ν): the number of full cycles of
a wave that passing through a fixed point in
• Wave number: The number of waves per
centimeter. Slide 6: low high Frequency (n) Energy X-RAY ULTRAVIOLET INFRARED MICRO-
WAVE RADIO FREQUENCY Ultraviolet Visible Vibrational
resonance 200 nm 400 nm 800 nm 2.5 mm 15 mm 1 m 5 m short long Wavelength (l) high low THE ELECTROMAGNETIC SPECTRUM BLUE RED Slide 7: Infra means beyond and infrared i.e. beyond red region Slide 8: IR-REGION: 12,800 - 10 cm-1 Slide 9: BASIC PRINCIPLE
IR involves absorption phenomenon the absorption of radiation depends on increasing energy of vibration or rotation associated with covalent bond in molecule provide that such an increasing in energy causes a change in the dipole moment of molecule.
Hence in order to absorb IR radiation a molecule must go a net change in dipole moment due to its vibration or rotation motion
This means that nearly all molecules containing covalent bond will show some degree of selective IR absorption
Infra red spectra are usually plotted as % transmittance (%T) rather than as absorbance as the ordinate. Slide 10: This makes absorption band appear as dips in the curve than as maxima as in the case of UV-Vis.
Each dip In the spectrum is called a band or peak and represented absorption of IR radiation at that frequency by the sample.
The transmittance is 0% if all the radiation is absorbed and the transmittance is 100% for no absorption. Slide 11: Francis A. Carey, Organic Chemistry, Fourth Edition. Copyright © 2000 The McGraw-Hill Companies, Inc. All rights reserved. Wave number, cm-1 Infrared Spectrum of Hexane CH3CH2CH2CH2CH2CH3 C—H stretching bending bending bending %T 100 Slide 12: Use of Wave number than wavelength offer several advantages Wave number are directly proportional to frequency and are expressed in much more convenient numbers (in this region of the spectrum), 5000-500 cm-1
Because the wave number is directly proportional to frequency and energy, the use of wave numbers allows spectra to be displayed linear in energy, which is a distinct aid in sorting out related vibrational transitions INFRARED THEORY : INFRARED THEORY - Molecular vibration can occur by 2
- Firstly, quanta of IR radiation can excite
atoms to vibrate directly. The absorption of
IR radiation give rise to the IR spectrum
- Secondly quanta of visible light achieve the
same result indirectly (RAMAN EFFECT) Slide 14: M1 Force constant, k M2 Ball and spring representation of 2 atom of molecule vibrating in the direction of bond Most the organic compound are fairly large & their resultant vibration spectra are complex.
-To introduce the basic concept governing vibrational spectra a simple diatomic covalent bond will be consider as a spring with atomic masses at either end
-The stiffness of the spring is described by a force constant, K Slide 15: If such a simple system is put in to motion ( by stretching and releasing) the induced vibration of the masses are adequately described by Hooke’s law of simple harmonic motion
Frequency of motion Hooke’s law : = frequency
K : Force constant (dynes /cm)
mr = reduced mass (mass of largest atom) Slide 16: However quantum theory governs molecular motion and restricts the energy stored in the vibration Eν ,such that only certain energy transition are allowed ,a determine by quantum number
Ev = (ν+ 1/2)hν
v-vibrational quantum no.
v-0, 1, 2, 3, . . . For, instance if molecule were to undergo a transition from the lowest level (v=0) to the first level (v =1) by absorption of IR radiation , frequency of that exciting radiation would be given by the Bohr principle
hv = E1-E0
At room temp. the majority of molecule are in the ground state
E0 = (1/2)hν
Now, promotion to the first excited state
E1 = (3/2)hν
By substitution , ΔE= E1- E0= hv
The frequency of radiation v that will bring about this change is identical to the classical vibration frequency of the bond vm Slide 17: i.e. ΔEradiation=hv=hvm : Frequency in cm-1 c : Velocity of light => 3 * 1010 cm/s K : Force constant => dynes /cm m : masses of atoms in grams If we wish to express the radiation in wave number or v=vm Slide 18: From above eq. IR measurements permits
the evaluation of force constant for various types of chemical bond
But generally above eq. can be used to estimate the wave number of the fundamental absorption peak, or the absorption peak due to the transition from the ground state to first excited state for a variety of bond types
Now we calculate the approximate wave number and WL of the fundamental absorption peak due to the stretching vibration Slide 19: larger K,
higher frequency larger atom masses,
lower frequency constants 2150 1650 1200 C-H > C-C > C-O > C-Cl > C-Br 3000 1200 1100 750 650 increasing K increasing m Slide 20: Calculating stretching frequencies Hooke’s law : : Frequency in cm-1 c : Velocity of light => 3 * 1010 cm/s K : Force constant => dynes /cm m : masses of atoms in grams C—C K = 5* 105 dynes/cm C=C K = 10* 105 dynes/cm CC K = 15* 105 dynes/cm Slide 21: Calculating stretching frequencies C=C K = 10* 105 dynes/cm C—H K = 5* 105 dynes/cm C—D K = 5* 105 dynes/cm SELECTION RULE : SELECTION RULE 1) ABSORPTION OF CORRECT WAVELENGTH OF RADIATION (MATCHING OF FREQUENCY
A molecule will absorb suitable radiation when its natural frequency
of vibration matches with the frequency of incident radiation (i.e. a net transfer of energy takes place and it results in a change in the amplitude
of molecular vibration)
Natural frequency of HCl molecule is 8.7 X 1013Hz(vib/sec)or(2890 cm-1)
When IR radiation will allow to pass on HCl sample and transmitted radiation is analyzed .It is observed that part of radiation which has same frequency 8.7X1013 HZ is absorb.
Thus remaining has been transmitted so gives characteristic value of HCl 2) DIPOLE MOMENT : 2) DIPOLE MOMENT “A molecule will absorb IR radiation if the change in the vibrational state is associated with the change in the dipole moment of the molecule” μ = q x r A dipole moment arises from a separation of charges in a molecule : μ =dipole moment (Coulomb ·meters)
q =magnitude of charges
r =vector going from –ve charge to +ve charge Slide 24: H Cl δ+ δ- r A hetronuclear diatomic molecule is composed of 2 diff. atoms. if this atoms exhibit diff. electron withdrawing powers, the e - density will be shifted towards more electronegative atom such molecule are said to be polar and possesses an electric dipole moment
“We can say, dipole moment arise as a consequence of asymmetrical partial charge distribution” Slide 25: μ = q x r q = magnitude of charge
r = Distance between charges
e = 1.602 X 10-19 C Here charge is measured in coulomb & distance in meters
So SI unit of μ = Cm (i.e. coulomb meter) but for convenience μ is often given in unit “DEBYE”
1D = 3.336X10-30Cm but atomic charge is q x e μ = q x e x r q + q - δ+ δ- r Slide 26: If HCl has dipole moment 1.83D bond length
And r is 92 pm from eq.
q = 0.41 Then this value indicates that charge in HCl molecule is distributed (asymmetrically) such that Cl atom has effectively gained 0.41 of e- and H atom has lost 0.41of an electron.
In polyatomic molecule, the to total dipole moment is the vector sum of the dipole moment of the individual bond.
In symmetrical molecule such as CCl4 there is no overall dipole moment,althoug C Cl bond is polar from μ = q x e x r 3)SELECTION RULE OF VIBRATIONAL QUANTUM NUMBER : 3)SELECTION RULE OF VIBRATIONAL QUANTUM NUMBER Based on the harmonic oscillator model (Δv = ±1)
Here + absorption & - emission
Vibrational quantum number change by unity Slide 28: Electronic transitions Vibrational transitions Rotational
transitions Slide 29: Harmonic oscillator Anharmonic oscillator Overtones and fundamental band Slide 30: So far as harmonic oscillator model is consider the lower vibration state can be explain (v - v´=1) but for higher vibrational state the selection rule may fail because vibrational energy levels are less separated as the vibration state increases
(But for harmonic oscillator model it is not true ) Overtones and fundamental band can not be explain by Harmonic oscillator model. So vibrational diatomic molecule can be explain well using anharmonic oscillator model
Anharmonic oscillator model can explain the selection rule for all transition . For real molecule they don’t obey simple harmonic motion. Because the bonds are also real
Therefore anharmonic oscillator model consider
Therefore energy difference goes on decreasing
as quantum no. increasing Δv = ±1, ±2, ±3……
(overtone lines) is possible but they are having
extremely low intensity.
This are known as overtones (they are weaker) TYPES OF VIBRATION : TYPES OF VIBRATION In polyatomic molecules, the atoms or covalent bonds are not rigidly linked together and are able to vibrate from their position of rest.
(In addition there are bond angles enclosed by the various individual diatomic bonds which results in a powerful qualitative method of describing the vibration of polyatomic molecules)
Since each type of chemical bond in a molecule involves different value of force const. and reduced masses, absorption of radiation will occur over a range of frequencies
Thus IR radiation of successive frequencies is passed through a substance, a series of absorption band is recorded the active fundamental modes of vibration. Slide 32: Types of Vibration Stretching Bending Symmetric Asymmetric Scissoring Rocking Wagging Twisting In plane Out of plane TYPES OF MOLECULAR VIBRATION : TYPES OF MOLECULAR VIBRATION Stretching vibration Symmetric
Stretching Symbol nu Symbol nu Isolated vibration Without change bond axis Without change bond angle Slide 34: Bending Vibration In plane Scissoring Rocking Symbol s Symbol rho ρ Slide 35: Out of plane Twisting Wagging Symbol ω Symbol tau,τ Bending Vibration Number of possible vibrational modes : Number of possible vibrational modes CAN WE KNOW THE POSSIBLE VIBRATION? YES, but how? 3N-5 for linear molecules 3N-6 nonlinear molecules N: number of atoms in a molecules BUT WHAT IS 3N, 5 & 6. HOW’S IT COME? Slide 37: Atoms are never fixed in the space but move about continuously
Each atom may be said to posses three degrees of freedom of movement, and thus in N-atom (polyatomic molecule) Molecule there will be 3N degrees of freedom (That means 3 coordinates are needed to locate a point in space. And each coordinate corresponds to one degree of freedom for one of the atom ,so a molecule containing N-atom is said to have 3N degree of freedom ) Slide 38: From this 3 types of motion 1) TRANSLATION MOTION Corresponds to the movement of the entire molecule through space while the position of the atoms relative to each other remain fixed. In this sence, the molecule may be considered as a single particle with the mass of the molecule located at its center of gravity and possesses three degree of translation freedom
Defination of translation motion require 3 coordinates and thus this common motion requires 3 of the 3N degree of freedom 2)ROTATIONAL MOTION : Another 3 degree of freedom are needed to describe the rotational motion of the molecule. (i.e. poly atomic molecule has generally 3 degree of rotation freedom)
But in the special case of linear molecule all the atom lie on a straight line and only 2 rotation can be define (because rotation around the bond axis is not possible) 2)ROTATIONAL MOTION 3)VIBRATIONAL MOTION : Now the rest i.e. substrate transition & rotation motion from 3N degree of freedom
i.e. 3N-6 ( for non linear molecule)
3 of translation motion + 3 of rotational motion=6
& 3N-5(for linear molecule)
3 of translation motion + 2 of rotational motion=5 3)VIBRATIONAL MOTION Slide 41: Now out of this normal vibration no. of stretching and bending vibration can be calculated
For stretching vibration =N -1
For bending vibration =2N -5(non linear)
i.e. [(3N - 6)-(N -1)]=2N -5
i.e. [(3N - 5)-(N -1)] =2N – 4
O2, N2, Cl2: 3N-5 =3x2-5 = 1,
only one vibration mode
CO2 3N-5 =3x3-5 = 4(linear molecule)
H2O 3N-6 =3x3-6 = 3 FACTOR THAT PRODUCED LESS NO. OF PEAKS THAN EXPECTED : FACTOR THAT PRODUCED LESS NO. OF PEAKS THAN EXPECTED The symmetry of a molecular vibration result in no change in the dipole moment (i.e. in co2 only 2 peak out of 4)
The energy of two or more vibrations are identical (same or degenerate)
The absorption intensity is so low as to be undetectable by means (detector)
The vibration energy is in the wL region beyond the range of the instrument HYDROGEN BONDING : HYDROGEN BONDING INTERMOLECULAR H - BONDING INTRAMOLECULAR H - BONDING o - Nitro phenol Formic acid dimer How intermolecular and intramolecular h-bonding can be differentiate with the use of IR spectra? : How intermolecular and intramolecular h-bonding can be differentiate with the use of IR spectra? The inter and intramolecular h-bonding can be distinguished by dilution. The intramolecular H-bonding is independent of the conc. Because it is an internal effects and hence intramolecular h-bonds remains unaffected on dilution and so absorption band also remains unaffected giving bonded O-H absorption
But intermolecular h-bonding is dependent on dilution the H-bonds are broken on dilution (or at low conc.) and hence there is decrease in the bonded O-H absorption
on successive dilution the intensities of the bands due to intermolecular h-bonding gradually decrease and finally disappear INSTRUMENTATION : INSTRUMENTATION Slide 46: SOURCE SAMPLE REFRENCE TRANSDUCER MC INFRARED SOURCE : INFRARED SOURCE IR source consist of an inert solid that is heated electrically to a temperature between 1500 and 2200 K The material is chosen so that its emission approximates
As closely as possible to that of black body radiator. (in candescent solid-the glow due to the great heat) We get continuum radiation approximating that of black body results Slide 48: What is this black body radiation? It is truly continuum radiation is produced when solid are heated incandescence Thermal radiation of this kind, which is called black body radiation, is characteristic of temp. of emitting surface rather than the material of which the surface is composed. Slide 49: Black body radiation is produced by the innumerable atomic and molecular oscillation excited in the condensed solid by thermal energy. It is clear that a very high temp. is needed for thermal excitation
The maximum radiant intensity at this temp. occur between 5000-5900 cm-1 Problems with Infrared from sun 99% of infrared rays are absorbed by water in our atmosphere Slide 50: At higher wave length the intensity fall is smoothly until it is about 1% of max. intensity
But on shorter wavelength side the decrease is much more rapid 10000 5000 670 cm-1 1 2 15 μm 1)THE NERNST GLOWER : 1)THE NERNST GLOWER Nernst Glower is Fabricated from rare earth oxides (e.g. ZrO2+Y2O3) Diameter 1-2mm length 20mm Pt. leads are sealed to the end of cylinder to permit electric connection.
When electric current pass it glows ,at temp 1200-2200 k 2)THE GLOBAR SOURCE : 2)THE GLOBAR SOURCE Silicon carbide rod, 50 mm length ,5 mm D
Electrically heated at 1300-1500 k Advantage of +ve temp. Coefficient of resistance
Water cooling required to prevent arcing
Spectral energy is comparable except in the region 5 μm where provide greater output. 3) INCANDENSCENT WIRE SOURCE(NICHROM WIRE) : 3) INCANDENSCENT WIRE SOURCE(NICHROM WIRE) Tightly wound spiral of NICHROM WIRE heated to about 1100 k by an electric current Lower intensity but larger life than previous 2
A rhodium wire heater sealed in ceramic cylinder has similar property as source
4) MERCURY ARC
Used for far IR region (above 50 μm)
High pressure Hg arc is used (1 atm pressure)
Passage of electricity through the vapour, forms an internal plasma source
that provides comtinuum radiation in far IR 5) THE CO2 LASER : 5) THE CO2 LASER A tunable co2 laser is used as an IR source for monitoring the concentration of certain atmospheric pollutants and for determine absorbing species in aq. Solution Range 900-1100 cm-1 which consist of about 100 of closely spaced discreet line
Imp. For quantitative determination of no. of species like ammonia, butadiene, benzene, ethanol, nitrogen dioxide, trichloroethylene
An important property of the laser source is the radiant power available in each line which is several order of magnitude greater than that of black body source SAMPLE HANDLING TECHNIQUE(SAMPLING) : SAMPLE HANDLING TECHNIQUE(SAMPLING) INTRODUCTION In 1965 Miller has developed appropriate method to handle sample in the gas ,liq and solid phase IR spectroscopy can be used to examine gas ,liq and solid. however a no. of problem exist with regard to adequate sample handling Slide 56: The main problem is that almost all substance absorb IR radiation. This seriously restricted the choice of the material that may be used for construction of the sample cell. the window of the cell must transmit the range of IR. The alkyl halide is most commonly used however this material is hygroscopic and must be stored in decicator and polish periodically. AgCl is the most widely used material for aq solution because it is in soluble in water .
Though it may be darken on exposer to light and easily deformed.
For frequency below 600 cm-1 polyethylene cell and other plastics are appropriate 1) GAS : 1) GAS Gas cell for IR often constructed with a cylindrical glass body and are usually about 10 cm length
To the end of the cell are attached disk of appropriate window material with wax, epoxy cement or pressure plates For trace analysis (at ppm level) such as in air pollution studies ad breath analysis very long path length can be (up to 30m) obtain using multiple reflection cells with gold surface mirror Slide 58: Here beam is directed to 90o angle from its normal direction and thus a longer path length is provide The use of longer cell, even with effective path length as long as 1 km, has been reported.
For some special application for example with corrosive gases or vapors metals such as stainless still, nickel or monel are used for cell bodies. 2) LIQUID : 2) LIQUID Liquid can be sampled in no. of different way depending on the sample characteristics such as volatile, composition and corrosively (towards the cell material) and the overall absorptive.
one of the simplest and most popular method i.e. to sandwich the liquid between 2 IR window typically made from NaCl or KBr thus producing a capillary film this is certainly and rapid procedure. Slide 60: A verity of cell are available for liq. Samples.
The optical path length required for a liq. Is usually less than 1 mm and spacer of different thick ness used with demountable cell to establish a suitable path length.
Liq, cells are thus available as variable or fixed To study the liq, solids or gases (dissolved gas) as a solution it is imp. To select a appropriate solvents that transmit over a wide range of wavelength Slide 62: When the amount of liq sample is small or when a suitable solvent is un available it is common practice to obtain spectra on the pure (neat) liq. Commonly a drop of the neat liq. Is put between 2 rock salt plates to give a layer that has thickness of 0.01mm or less than examined by putting in beam path 3) SOLIDS : 3) SOLIDS There are 2 common techniques for powder solids
1) KBr PELLETE TECHNIQE One of the most popular technique for handling solid samples has been KBr pelleting halide salts be come transparent when sufficient pressure is applied to the finally powder material. Slide 64: About 1 mg or less sample is ground with approximately 300 mg of “infrared quality KBr” product
Mixing can be carried out mechanically in BALL MILL apparatus the mixed then pressed in a special die(13 mm diameter) at pressure 10000-15000 pounds per sqar inch (psi) to yield a transparent disk. The disk is then held in the instrument beam for spectroscopy examination
With many compd. KBr pelleting producing excellent spectra. Slide 65: (in IR, sample is grinding well and being particle size less than IR WL so minimize scattering (less than 2 μm) 2) MULLS IR spectra of solid that are not soluble in IR transparent solvent or are not conveniently pelleted in KBr are often obtain by dispersing the analyte with mulling agent By taking 2-10 mg of finely ground sample is taken with a1-2 droop of mulling agent . Slide 66: Mulling agents are substance that transmit a wide range of IR frequency and help to minimize scattering by surrounding the analyte with a medium whose refractive index more closely matches to that of the sample than does air. Nujol, refined mineral oil is commonly used mulling agents.
Other mulling agent is FLOUROLUBE i.e. halogenated polymer, fluorinated HC mull,perflouro kerosene or hexa flourobutane. Slide 67: Now, with NUJOL (REFIND MINERAL HC OIL)
The problem is arise that it is not suitable for aliphatic
C—C and C—H vibration. Because NUJOL, which is HC oil absorbed IR radiation in the region below 1340 cm-1 and giving a peak of HC Which is interfere in the analysis that’s why it is not used in C—C ,C—H vibration. Slide 68: So, the solution is flourolube (i.e. halogenated polymer)
Fortunately flourolube does not absorb IR radiation in the region (range) below1340 cm-1 where NUJOL absorb.
And where flourolube absorb in the region 1340-4000 cm-1 NUJOL does not absorb So, we can used nujol if analyte also absorb where flourolube absorb (1340-4000 cm-1 )
Or we can used flourolube if analyte absorb where NUJOL absorbs below 1300 cm-1) Thus both are complimentary of each other Thus both are complimentary of each other Slide 69: Drawback
The problem associates with solvent chose are eliminated with mulls. But the spectra generally can not be used for quantitative analysis.
So, a split mull is used split mull consist of mineral oil (NUJOL) and flourolube so whole IR range is covered DETECTORS or TRANSDUCERS : DETECTORS or TRANSDUCERS There are 3 types of detector (transducer)
1) THERMAL DETECTOR
2) PYROELECRIC DETECTOR
3) PHOTON (QUANTUM) DETECTOR THERMAL DETECTOR : THERMAL DETECTOR Thermal detector whose response depend on heating effect.
Which in terns alters the physical properties of transducers such as resistance It is a transducer that changes thermal energy in to an electric signal. The electric signal is amplified and routed to the read out device Slide 72: But the problem of measuring IR radiation by thermal means is compounded by thermal noise from the surrounding for this reason thermal transducers are housing in a vacuum and are carefully shielded from thermal radiation emitted by other near by objects.
Therefore temp.of room is mainted 1)THERMOCOUPLE : 1)THERMOCOUPLE Most widely used IR detector Consist of 2 pieces of metal such as “Bi” which are joined with dissimilar metal such as “sb” (antimony) and form a pair of junction (2 jn) Jn. Is usually blackened (to improve its heat absorb capacity) With black metallic oxides One jn between 2 dissimilar metal is heated with IR radiation other jn. Kept at const. temp.. Slide 74: PRINCIPLE “A CHANGE IN THE TEMP. AT THE BOTH DIFF.JN. BETWEEN 2 UNLIKE METAL CAUSES AN ELECTRIC POTENTIAL TO DEVELOPE BETWEEN SPECIES”
“DUE TO DIFRENCE IN WORK FUNCTION OF METALS WITH TEMP. A SMALL VOLTAGE DEVELOP ACROSS THE THERMOCOUPLE” Slide 75: This potential diff. is depend (proportional) to the amt of IR radiation falling on hot jn. Whole assembly is evacuated in still housing with IR transparent KBr window to minimize conductivity heat loss.
Capable to responding temp. diff. of 10-6 k corresponding to potential diff. of 6-8 μV/ μW
Advantage that independent of response with change in wave length “THERMOPILE” ? Slide 76: 2) THERMISTOR or BOLOMETER When irradiation by IR beam produced an increase in resistance of the metal strip which measured with a whetstone bridge A potential diff. between the 2 elements produced a proportional voltage difference 3)PNEUMATIC DETECTOR or GOLAY DETECTOR : 3)PNEUMATIC DETECTOR or GOLAY DETECTOR Pneumatic detectors respond to change in vol. of non absorbing gas or liq with temp. change In pneumatic device if gas is used as medium called golay detector
Here the absorbing radiation heats an inert gas (usually xenon) in a pneumatic chamber behind the plate and cause the gas to expand As the gas expands the flexible diaphragm at the opposite end of the chamber from the metallic plate is pushed outward PYROELECTRIC DETECTOR : PYROELECTRIC DETECTOR The most recently developed IR detector is pyroelectric detector It is constructed from single crystalline wafers of pyroelectric material which are insulator (dielectric material ) with very thermal and electric properties Slide 79: certain crystal such as
TGS (tri glycine sulphate ((NH2,CH2,COOH)3.H2SO4)
DTGS (deuterated triglycine sulfate
Lithium niobate (LINbO3)
Is the imp. Pyroelectric material used in ir detector This crystal possesses internal electric polarizations. This internal property is taken in account.
Crystal is placed between 2 electrodes one of which is IR transparent Slide 80: When IR radiation fall on crystal its temp. is change, which alters the charge distribution across the crystal, which can be detected as the current in the external electric circuit connecting the 2 side of the capacitor. The magnitude of this current is proportional to the surface area of crystal and the rate of change of polarization with temp.
This is known as pyroelectric effect. Slide 81: Pyroelectric crystals lose their residual polarization when they are heated to a temp. Called the Curie point
For TGC curie point 47 0C DOUBLE BEAM SPECTROPHOTOMETER : DOUBLE BEAM SPECTROPHOTOMETER THANK YOU : THANK YOU