Ultraviolet-VisibleAbsorption Spectroscopy

X-ray: core electron excitation

UV:valance electronic excitation

IR: molecular vibrations

Radio waves:Nuclear spin states(in a magnetic field)

Electronic Excitation by UV/Vis Spectroscopy :

Rays Frequency Wavelength

γ rays - 0.01-10nmX rays 1020 -1016 10-50nmFar UV 1016 -1015 50-200nmNear UV 1015 -7.5X1014 200-400nmVisible 7.5X1014 4.0 X1014 400-800nmNear IR 4.0 X1014 -1.2 X1014 0.8-2.5µmMid IR 1.2 X1014 - 6 X1012 2.5-25µmFar IR 6 X1012 -1011 25-400µmMicro waves 1011 -108 400-25cmRadiowaves 108 - 105 25cm-1000m

 Spectroscopic Techniques

 

UV-vis UV-vis region bonding electrons  

Atomic Absorption UV-vis region atomic transitions (val. e-)

FT-IR IR/Microwave vibrations, rotations

Raman IR/UV vibrations  

FT-NMR Radio waves nuclear spin states

X-Ray Spectroscopy X-rays inner electrons, elemental

X-ray Crystallography X-rays 3-D structure  

Ultraviolet and visible spectroscopyIt is used to measure the multiple bonds or atomic conjugation within the molecule.

The UV-Visible region is subdivided as below– Vacuum UV: 100-200 nm– Near UV: 200 to 400 nm– Visible region: 400 to 800 nm

Vacuum UV is so named because molecule of air absorb radiation in these region. The radiation is assessable only in special vacuum equipments.

Spectroscopy:

Synonyms: Spectrometry or spectrophotometry

Spectroscopy is made up of

Spectrum + Skopien → Given by Isaac Newton

He did simple experiment

Sunlight

V I B

G Y

O R

According to Newton experiment, Spectrum is a band of color or pattern of colors or arrangement or array of colors.

But extended definition of spectrum is, Separation of wave or pattern of waves or arrangement /array of wave.

Today, Spectrum is defined as arrangement of array or pattern of anything.

Skopein: Evaluation/examination

Spectroscopy: Evaluation/ Examination of spectrum

Metry: Measurement

Spectrometry: Measurement of spectrum

Photo: EMR (Electro magnetic radiation)

Spectrophotometry: Measurement of EMR spectrum

Fundamental principle of spectroscopy:

Drug + EMR → Drug* + EMR*We measure difference between EMR and EMR*

EMR:

EMR is a radiant energy, that is transmitted through space in normal velocity

Radiant energy has wave nature and being associated with electric as well as magnetic field so called EMR

All EMR propagated through space with same speed (3X 1010 cm/sec) called speed of light

EMR is alternating electric field and associated magnetic field in space

The two components oscillate in planes perpendicular to each other and perpendicular to the direction of propagation of radiation

Direction of propagation of radiation

Trough

Crest

Magnetic Component

Electrical component

EMR is vibration of wave produced by oscillating or vibrating of electron in particular direction.

EMR are produced by periodic motion of charge particles like electrons

Characteristics of EMR:Wavelength FrequencyVelocity Wave number

Wavelength (λ) is defined as the distance between adjacent peaks (or crest/troughs). Designated in meters, centimeters or nanometers (10-9 meters).

Energy α 1/λ max

Frequency (µ) is the number of wave cycles passes a given point in 1 sec. Expressed as cycles per second, or hertz (Hz).

µ α 1/λ, , µ = c/λ

Velocity (c) The distance traveled by wave in 1 sec.

c= λ x µ, c= 3x 1010 cm/sec

Wavenumber (σ) – the number of waves spread in length of 1 centimeter.σ = 1/ λ

Energy α Frequency (µ)E= h µ, E = hC/ λ

• H= plank const= 6.626 x 10 -27 erg/sec

λ max decrease → E increases and frequency also increases

Spectroscopy: it is a branch of science deals with interaction of EMR with matter

Spectroscopy can be divided in,

Study at molecular/atomic level

1) Atomic spec e.g.: AAS, flame photometryChange in E at atomic levelDeals with interaction of EMR with atoms which are in their lowest energy state, i.e., ground state

2) Molecular spec e.g.: UV, IR, fluorimetryChange in E at molecular levelTransition between rotational and vibrational E levels in addition to electronic transition

Study based on absorption/emission

1) Absorption Spe e.g.: UV, IR, X-ray, ESR, NMR

2) Emission Spe e.g.: flame photometry, fluorimetry

Study at electric/ magnetic level

• Electric Spe e.g.: UV, Colorimetry, fluorimetry (without magnetic field)

2) Magnetic Spe e.g.: NMR, ESR

UV Visible Spectroscopy also known as Molecular absorption/ electronic absorption spectroscopy

Importance of UV spectra: Simple in operationHigh sensitivitySpeedy AnalysisQualitative and quantitative application

Limitation: Non Selective

Molecular absorption spectrum/ UV Visible spectrum is band spectrumAtomic spectrum involves line spectrum

Molecular absorption spectrum/ UV Visible spectrum is band spectrum

Why we get ∩ (band) and not ∏ on x-axis even though conc. is same

B’se all the time energy absorbed is not the same…

A molecular energy state is the sum of

an electronic, vibrational, rotational, and translational component

E = E electronic + E vibrational

+E rotational +E translational

Electronic E level: Molecule possess an electronic configuration & the electronic energy depends on the electronic state of the molecule

Vibrational E level: The parts of molecule, i.e., atoms or groups of atoms may move with respect to each other. This motion is called vibration and associated energy is called vibrational E level

Rotational E level: The molecule may rotate about an axis & such rotation is characterized by the rotational E level

Translational E level: The molecule as a whole may move & this is called as transition & the associated E is called translational E level

Eele, Ev, Er are the quantized/internal energy.

In order to absorption to occur, the E difference between two e levels must be equal to conc. of photons absorbs.

Means E2-E1= hv

The e- are mainly involved in absorbing E, so called electronic absorption spectroscopy

Both UV and Visible Spe. Only the valance e- absorbs E, thereby, the molecule undergoes transition fro GS to ES

The intensity of absorption depends on the conc. And path length given by Beer-Lambert's law.

For Absorption in UV – visible intensity of absorption depends on the conc., and hence given by Beer’s law

While in IR absorption Spe., it concerns with jumping of e- from one vibrational E level to other vibrational E level so called Vibrational Abn. Spe./ IR Abn. Spe.

For IR abn. Rotational spectrum is not important analytically.

e- jump from one E level to another ----- Transition

If a molecule passes from one of its allowed E level to a lower one, some of E must be released, which may be lose as radiation.----Emission of radiation

If a molecule passes from one of it’s allowed E level to higher one, some E must be absorbed, which may be absorbed as radiation -------Absorption of radiation

Valance e- : The e- which are required for bond formation present in outer most orbital.

Types of valance e- : 1) σ 2) π 3) n

1) σ e- :

Present in saturated hydrocarbon e.g.: paraffin ( C-C, C-H) Highly stable

Required higher E for excitation and λ req. for excitation is very low

Such e- do not absorbs near UV but absorbs vacuum UV radiation < 200 nm

Hence the comp. ctg σ bond doesn't absorb in UV region ------transparent in UV range -----so used as a solvent. For e.g.: Hexane

2) π e-: Present in unsaturated hydrocarbon e.g.: Double or triple bond…>C=C<,-C≡C- (alkenes, alkynes, aromatic compd., and carbonyl compds such as aldehydes and ketones, cyanides, azo compds etc.

Relatively unstable and highly reactiveRequired less E for excitation

3) n e- : non bonding e-

These e- are not involved in bond formatione.g.: S,O,N & halogen (X), such n e- are excited by

UV radiation

Principle of UV radiation

Any molecule has either n, σ, π or a combination of these e-.

These bonding (σ and π) and non bonding (n) e- absorbs characteristic radiation

undergoes transition from GS to ES

By the characteristics absorption peaks, the nature of e- present, the mol. Stru. can be elucidated

Electronic Transition & Excitation process

A: Bonding Molecular Orbital: if e- are in these region: bond formation takes place, which is highly stable

B: Non Bonding Molecular Orbital: bond formation doesn't take place. Atomic orbital are themselves non bonding molecular orbital prior to bond formation

C: Anti bonding molecular orbital: here, if e- are present in these region, highly unstable---so no bond formation

C

B

A

LUMO

HOMO

σ, π (bonding) and n (non-bonding) electrons

The E required for excitation for different transition are,

n→π* < π→π* < n→σ* < σ→σ*

n→π* ----required lowest E

σ→σ* ----required highest E

Electronic Transition

σ→σ* transition: Required highest E Absorption in ~150 nm

E.g. Hydrocarbons

Methane: λmax = 125 nm (High E compare to ethane, Lower λ)

Ethane: λmax = 135 nm (less E compare to methane, Higher λ)

B’se strength of C-C bond is < C-H bonds..

Propane: maxi abs. At 135 nm…

most of spectrophotometer doesn’t shows abs. < 180-200 nm, σ→σ* not observed

Exception: Cyclopropane: shows abs. at 190 nm

In far UV (Vacuum) region, Oxygen present in the air absorbs strongly, so to study σ→σ* air must be evacuated from the instrument, specially in case of Saturated HC

Since UV operated above 200nm, Saturated hydrocarbon used as a solvent (non polar) as it doesn’t give solvent peak

n→σ* transition:

Saturated compd ctg. atoms with unshared pair of e- (O,N,S/ X/ non bonding e-)

Majority of comps in this class doesn’t shows abs. in near UV regionTransition in region of 150-250 nm, with most abs peak < 200 nmMost commonly used solvent: Alcohols and ethers (abs < 185nm)E.g.: Alkyl Halides

The E req. for n→σ* transition ↓es with ↑es in size of halogen atom/ ↓es in the electro negativity of atom. (F, Cl, Br, I)

E.g.: Methyl chloride (λmax=173) and methyl iodide (λmax=259)B’se of > electro negativity of chlorine than iodine, the n e- on chlorine atom are comparatively difficult to excite, while n e- on iodine atoms are loosely bound

Magnitude of molar extinction coefficient (εmax) for a particular absorption αnal prob. of particular electronic transition.

εmax for CH3I= 400, εmax for CH3Cl= 200

n→σ* transitions are sensitive to H-bonding

For E.g.: alcohols and amines forms H-bonding with solvent molecule (due to n bonding e-)

So, greater E req. for excitation and hence, H-bonding shifts abs. towards shorter λ.

Amines abs at higher λ as compared to alcoholsB’se n bonding e- on N atoms in amine are loosely bound as compared to o atoms in alcohol (B'se higher electro negativity of O than N)

E.g.: Tri methyl amine: in aq. solution doesn't shows abs due to n→σ* B'se protonated amine does not contain any n bonding e-

Absorption Spe. Of org. compd. are based upon, n→π* & π→π*

B’se E req. for these processes brings Abs peaks in to spectral region (200-700nm).

Both transitions req. the presence of an

unsaturated functional group to provide π orbital.

π→π* transition:In simple alkenes, several transitions are available. abs. band between 170-190 nm in un conjugated alkenes

E.g.: Ethylene in vapor phase absorbs at 165 nm, & gives second band at 193 nm due to π→π*,

The intensities of olefinic double bonds is independent of solvents due to non polar nature of double bond Band in π→π* transition also called as K-band.K-band is obtained in the spectra of conjugated π systems.e.g. Butadiene, Mesityl oxideK-band is also known as E-band (Ethylinic) & B band (Benzenoid)

n→π* transition:

required lowest E (longer λ)The peak due to this transition also called as R-band ( longer λ)Peak seen due to: n bonding e- is present in compound ctg = or ≡.e.g. aldehyde, ketone and nitro compd.

n→π*

Blue shift observed with an increase in solvent polarity ( due to salvation and H-bonding to the lone pair, large shift of 30 nm) n→π* transition is characterized by taking spectrum in acid solution.

E.g.: Pyridine: bands due to n→π* in pyridine disappear in acid solution b’se of the formation of bond between acidic proton and n e-

C6H5N: + H+ → C6H5NH+

Peak appear Peak disappear

Characteristic difference between n→π* & π→π* is found with effect of solvent

Blue shift observed with an increase in solvent polarity in n→π*

Red shift observed with an increase in solvent polarity in π →π*

135 nm

165 nm

n183 nm weak

150 nmn188 nmn279 nm weak

A

180 nm

279 nm

C C

C C

C O

C OH

Visible Spectroscopy

Apparent color of the solution is always complementary of the color absorbed

Beer’s Law:

When a monochromatic radiation passed thro’ a transparent medium, a rate of decrease in intensity of radiation with concentration of medium is proportional to the intensity of the incident light.

Intensity of transmission radiation decreases as the conc. of absorbing subs increases arithmetically

The wavelength and amount of light that a compound absorbs depends on its molecular structure and the concentration of the compound used.

where

I = intensity of monochromatic light transmitted through the solution

I0 = intensity of light transmitted through the blank

A = absorbance

b = path length (usually in cm)

a = Absorptivity, a constant for a given solution and a given wavelength

C = concentration in g/100 ml

abcAI

I0log

Deviation from Beer’s Law

Positive deviation: when a small change in conc. produce greater change in absorbance.

Negative deviation: when a large change in conc. produce small change in absorbance.

The law strictly followed for dilute system, as conc. increases causes deviation

Environmental Deviation: Temp, solvent

Instrumental Deviation: Relative Conc. error, Stray radiation, stability of radiation source, wavelength selector, slit control (SSW), electronics and reliability of optical parts

Chemical Deviation: change in chemical equilibrium and change in pH, ionization, presence of complexing agent, competitive metal ion reactions and conc. Dependence (hydrolysis, association, polymerization, ionization, H-bonding).

Refractive index of the sampleNon monochromacity of radiation

Beer’s law is not applicable to suspension, coagulated particle system, impurity that fluorescence / absorbed

Methods to find Conc. By Beer’s law

1) Using Standard equation (using standard A(1%, 1 cm) and ε

A= ε b cThe absorbance of the solution, A, is defined asA= - log T, A= -log [I/Io], T= -log [I/Io] %T, of the solution is expressed as,A = 2.00 – log (% T) = ε b c

ε = A(1%, 1 cm) x Mol weight10

A(1%, 1 cm)/a (Spe. absorptivity) is given than conc. Is in g/100ml

ε is given than conc. Is in mol/lit 2) Calibration Method)

(by serial dilution method) 3) Using Formula method

Au / As = Cu / Cs

Different terminologies used in UV-Visible spectroscopy

• Chromophore: A chromophore is the part of a molecule responsible for its color. The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others.

• Auxochrome: An auxochrome is a group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light. (hydroxyl, amino, nitro group)

• Bathochromic shift: It is a change of spectral band position in the absorption, reflectance, transmittance, or emission spectrum of a molecule to a longer wavelength

• Hypsochromic shift: It is a change of spectral band position in the absorption, reflectance, transmittance, or emission spectrum of a molecule to a shorter wavelength.

• Hyperchromic shift: Increase in absorbance

• Hypochromic shift: Decrease in absorbace

Important Terms in UV Spe.

Chromophore

1) Dependant

2) Independent

Auxochrome

3) Bathochromic gr.

4) Hypsochromic gr.

Bathochromic shift (Red shift)

Hypsochromic shift (Blue shift)

Hyperchromic shift

Hypochromic shift

End absorption

ChromophoreChromo (color) + Phore (producing)

It is covalently unsaturated group responsible for electronic absorption

Minimum requirement for absorption in Uv: Minimum two conjugated double bonds

C=C-C=C

C=C-C=O

C=C-C=N

C=C-C=S

Independant: single chromophore is sufficient to impart color to compd.e.g: -N=N-, -NO-, o & p quinoid gr.

Dependent: More than one chromophore is required to produced color in chromogen>C=O, C=C

e.g.: CH3COCH3 Colorless (acetone)CH3COCOCH3 Yellow (Diacetyl keton)CH3COCOCOCH3 Orange (Tri keto pentane)

Structures having same chromophoric group shows same absorption in UV and can’t analyzed by UV, so UV is non selective method

AuxochromeAuxo: Auxiliary

Auxochrome is a functional group having non bonding electrons which itself has no absorption but when attached to chromophoric group, enhance the absorption properties of chromophoric groups An auxochrome contains unshared pair of electrons

e.g.: NH3, SH, RNH2, OH

Benzene -no chrom. group -colorlessNitro benzene -NO2 chromophore -pale yellowP-nitro aniline -NO2 and -NH2 (auxo.) - dark yellow

Auxochrome: two types

1) Bathochromic groups: deepen the color of chromogen, shift to longer λ, e.g.: 1°, 2°, 3° amine

2) Hypsochromic groups: lighten the color of chromogen , shift to shorter λ, e.g.: acetylation of OH and NH2 i.e. OCOCH3, NHCOCH3

Different Shifts

Hy

po

ch

rom

ic

Hypsochromic

Hy

pe

rch

rom

ic

Bathochromic

TerminologyBathochromic shift: (Red shift) shift of lambda max to longer side or less energy is called bathochromic shift or read shift. This is due to substitution or solvent effect.Hypsochromic shift: (Blue shift) shift of lambda max to shorter side and higher energy is called hypsochromic or blue shift. e.g. solvent effect.Hyperchromic effect: an increase in absorption intensityHypochromic effect: an decrease in absorption intensity

End Absorption

It is special phenomenon of increases in absorption intensity as the λ decreases towards 200nm (End of UV range), which is due to n→σ*

200nm λ 400nm

A

Significance:

End absorption is possible due to only compd having n electrons & having σ bonds. e.g. n electrons present in most of the solvent like water and alcohol.

N electrons have higher E and lower λ.

absorption spectra

1. λmax: Position of Spectra

2. Intensity: Amt of radiation

Factors affecting λmax/ Position of Spectra

Two types

1.Internal

(Structural)

A) Substitution

B) Unsaturation

C) Geometry

D) Resonance

2.External (Non Structural)

A) Solvent

B) pH

C) Effect of metal ion

D) Molecular aggregation/ Charge transfer complex

E) Temperature

A) Resonance

B) Intensity of incident radiation

C) Conc.

D) Thickness

E) Some fundamental factors like SSW & stray light

Factors affecting absorption intensity(εmax/A(1%,1cm)