WHAT WE ARE GOING TO LEARN?

PRINCIPLE

 INSTRUMENTATION  APPLICATIONS

Definition

Spectroscopy – Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules or atoms or ions of a sample move from one energy state to another.

PRINCIPLE OF SPECTROSCOPY

Property Excitation

Excited state

molecule

Rel axa tion

SIGNAL (E m TO BE issi on)DETECTED

Electromagnetic Spectrum

Electromagnetic Spectrum
Hz
1021 10-3 1018 1 200 1015 500 1012 106 109 109 106 1012

λ

(nm)

Microwave

Ultraviolet

Cosmic

Infrared

Visible

Radio

X-ray

Absorption vs. Emission
hν En En

Eo

Eo

Absorption

Emission

PRINCIPLE IN COLORIMETRY
 Study

of absorption of visible radiation whose wavelength ranges from 400nm800nm.  Colored substances absorb color of different wavelength and hence we get absorption curve by plotting absorbance vs wavelength.

Beer-Lambert Law
 BEER’S

LAW: related to concentration of absorbing species  LAMBERT’S LAW: related to thickness/pathlength of absorbing species

Beer’s law
 Absorbance

& Beer’s Law

Increasing absorbance

 Io

= intensity of light through blank  IT = intensity of light through sample  Absorption = Io - IT  Transmittance = IT/Io  Absorbance = log(Io/IT)
Io IT

Io

IT

Io

IT

pathlength b

pathlength b

PRINCIPLE : ULTRA VIOLET SPECTROSCOPY
 UV

radiation ranges 200nm-400nm  Any molecule has either n,π or σ or combination of these electrons  These electrons absorb radiation and undergo transition from ground state to excited state  characteristic absorption peaks are formed

Types of Electronic Transitions
1. 2. 3.

Transitions involving π , σ , and n electrons Transitions involving charge-transfer electrons Transitions involving d and f electrons

Absorbing species containing π , σ , and n electrons

Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The spectrum of a molecule containing these chromophores is complex.

σ

− σ

*

Transitions

 An

electron in a bonding σ orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergo σ − σ * transitions) shows an absorbance maximum at 125 nm.

n − σ

*

Transitions

Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n − σ * transitions. These transitions usually need less energy than σ − σ * transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups with n − σ * peaks in the UV region is small.

n − π * and π Transitions
 Most

− π

*

absorption spectroscopy of organic compounds is based on transitions of n or π electrons to the π * excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 700 nm). These transitions need an unsaturated group in the molecule to provide the π electrons.

 Molar

absorbtivities from n − π * transitions are relatively low, and range from 10 to100 L mol-1 cm-1 . π − π * transitions normally give molar absorbtivities between 1000 and 10,000 L mol-1 cm-1 .

Solvent effect
 The

solvent in which the absorbing species is dissolved also has an effect on the spectrum of the species.  Peaks resulting from n − * transitions π are shifted to shorter wavelengths (blue shift) with increasing solvent polarity.

 The

reverse (i.e. red shift) is seen for π − π * transitions. This is caused by attractive polarisation forces between the solvent and the absorber, which lower the energy levels of both the excited and unexcited states.

Choice of Solvent
Solvent Minimum Wavele-ngth (nm) Solvent Minimum Wavelength (nm) Solvent Minimum Wavelength (nm)

Acetonitrile hexane ether

190 195 215

water methanol

191 201

cyclohexane ethanol Chloroform

195 204 237

methylene 220 chloride

carbon 257 tetrachloride

UV spectra and molecular structure
      

The absorbing groups in a molecule are called chromophores A molecule containing a chromophore is called a chromogen An auxochrome does not itself absorb radiation, but can enhance the absorption Bathochromic shift – red shift Hypsochromic shift – blue shift Hyperchromism – an increase in absorption Hypochromism – a decrease in absorption


○ ○ ○ ○ ○ ○ ○ ○

Chromophore λ
Alkanes ~ 150 Alkenes ~ 175 Alkynes ~ 170 Carbonyls ~ 188 alcohols, ethers Amines ~ 195 sulfur compounds Carbonyls ~ 285

mx a

Transition
∗ ∗

σ to σ π to π

~ 185 ~ 195 η to π

η to σ

INTERPRETATION OF UV SPECTRA

α,β UNSATUREATED KETONES
Increment in λ max
+10 mμ +20 mμ +5 mμ -11 mμ +30 mμ

Structural variation
α- alkyl substituent β -alkyl substituent Exocyclic c=c Cyclopentenone system C=C extending congugatation

II. Conjugated dienes:
Structural variation
alkyl substituent Exocyclic c=c Presence of homoannular diene C=C extending conjugatation Increment in λ max
+5 +5 +39

+30

Compounds isolated double bonds
Acetaldehyde
acetone acetonitrile ethylene

λmax
293 mμ
271 mμ 160 mμ 193 mμ

UV-vis Spectrophotometer
 Single-Beam

UV-Vis Spectrophotometer  Single-Beam spectrophotometers are often sufficient for making quantitative absorption measurements in the UV-Vis spectral region.  Single-beam spectrophotometers can utilize a fixed wavelength light source or a continuous source.

Single-Beam UV-Vis Spectrophotometer
The simplest instruments use a singlewavelength light source, such as a lightemitting diode (LED), a sample container, and a photodiode detector. Instruments with a continuous source have a dispersing element and aperture or slit to select a single wavelength before the light passes through the sample cell.

Dual-Beam uv-vis Spectrophotometer

In single-beam Uv-vis absorption spectroscopy, obtaining a spectrum requires manually measuring the transmittance of the sample and solvent at each wavelength. The doublebeam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously.

How Do UV spectrometers work?

Cuvettes (sample holder)
   

Polystyrene
340-800 nm

Methacrylate
280-800 nm

Glass
350-1000 nm

Suprasil Quartz
160-2500 nm

Array-Detector Spectrophotometer
Array-detector spectrophotometers allow rapid recording of absorption spectra.  Dispersing the source light after it passes through a sample allows the use of an array detector to simultaneously record the transmitted light power at multiple wavelengths.  There are a large number of applications where absorbance spectra must be recorded very quickly. Some examples include HPLC detection, process monitoring, and measurement of reaction kinetics.

Instrumentation
These spectrometers use photodiode arrays (PDAs) or charge-coupled devices (CCDs) as the detector. The spectral range of these array detectors is typically 200 to 1000 nm. The light source is a continuum source such as a tungsten lamp.  All wavelengths pass through the sample. The light is dispersed by a diffraction grating after the sample and the separated wavelengths fall on different pixels of the array detector.

The resolution depends on the grating, spectrometer design, and pixel size, and is usually fixed for a given instrument.  Besides allowing rapid spectral recording, these instruments are relatively small . Portable spectrometers have been developed that use optical fibers to deliver light to and from a sample.

Diode Array Detectors
Diode array alternative puts grating, array of photosensitive Semiconductors after the light goes through the sample. Advantage, speed, sensitivity, The Multiplex advantage Disadvantage, resolution is 1 nm, vs 0.1 nm for normal UV

These instruments use only a single light beam, so a reference spectrum is recorded and stored in memory to produce transmittance or absorbance spectra after recording the sample spectrum.

Ideal spectrometer has
Good Scan Speed  Resolution  Software Features  Ease of Operation  Data Storage  Customized Calculations

Practical Applications
 Pharmacy

Practice

Ultraquin (psoriasis med. Needs UV. Act. Pregnancy tests (colorimetric assays) Blood glucose tests, ELISA’s

 Pharmaceutics
pH titrations, purity measurement concentration measurement

pKa Measurement with UV
n

i

Titration of Phenylephrine pKa = pH + log Ai - A A - An

 Medicinal

Chemistry

compound ID (steroids, nucleosides) monitoring isomerization, chirality

 Pharmaceutical

Biotechnology

concentration/purity measurements monitoring conformation of protein drugs

 Pharmacokinetics/Med.

Chem.

HPLC monitoring and purification

Pharmaceutical Apps.
On Line Analysis of Vitamin A and Coloring Dyes for the Pharmaceutical Industry  Determination of Urinary Total Protein Output  Analysis of total barbiturates  Comparison of two physical light blocking agents for sunscreen lotions

 

Determination of acetylsalicylic acid in aspirin using Total Fluorescence Spectroscopy Automated determination of the uniformity of dosage in Quinine Sulfate tablets using a Fibre Optics Autosampler Determining Cytochrome P450 by UV-Vis Spectrophotometry Light Transmittance of Plastic Pharmaceutical Containers