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

molecule

Property

Excitation

Relaxation (Emission)

SIGNALTO BE

DETECTED

Excited state

Electromagnetic Spectrum

Electromagnetic Spectrum

Co

smic

X-r

ay

Ult

ravi

ole

t

Vis

ible

Infr

ared

Mic

row

ave

Rad

io

(nm)

Hz 1021 1018 1015 1012 109 106

10-3 1 200 500 106 109 1012

Absorption vs. Emission

Eo

h

Absorption

En

Eo

h

Emission

En

h

PRINCIPLE IN COLORIMETRY

Study of absorption of visible radiation whose wavelength ranges from 400nm-800nm.

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

pathlength b pathlength b

Io IT Io IT

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 Transitions1. Transitions involving , , and n

electrons

2. Transitions involving charge-transfer electrons

3. Transitions involving d and f electrons

Absorbing species containing , , 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.

* Transitions

An electron in a bonding 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 * transitions) shows an absorbance maximum at 125 nm.

n* Transitions

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

n* and * Transitions Most absorption spectroscopy of organic

compounds is based on transitions of n or electrons to the * 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 electrons.

Molar absorbtivities from n* transitions are relatively low, and range from 10 to100 L mol-1 cm-1 . * 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 are shifted to shorter wavelengths (blue shift) with increasing solvent polarity.

The reverse (i.e. red shift) is seen for * 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 190 water 191 cyclohexane 195

hexane 195 methanol 201 ethanol 204

ether 215 methylene chloride

220 Chloroform 237

carbon tetrachloride

257

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 max Transition○ Alkanes ~ 150 to

○ Alkenes ~ 175 to

○ Alkynes ~ 170○ Carbonyls ~ 188○ alcohols, ethers ~ 185 to

○ Amines ~ 195○ sulfur compounds ~ 195○ Carbonyls ~ 285 to

INTERPRETATION OF UV SPECTRA α,β UNSATUREATED KETONES

Structural variation Increment in λ max

α- alkyl substituent +10 mμ

β -alkyl substituent +20 mμ

Exocyclic c=c +5 mμ

Cyclopentenone system -11 mμ

C=C extending congugatation

+30 mμ

II. Conjugated dienes:

Structural variation Increment in λ max

alkyl substituent +5

Exocyclic c=c +5

Presence of homoannular diene

+39

C=C extending conjugatation

+30

Compounds isolated double bonds

λmax

Acetaldehyde 293 mμ

acetone 271 mμ

acetonitrile 160 mμ

ethylene 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 SpectrophotometerThe simplest instruments use a single-

wavelength light source, such as a light-emitting 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 double-beam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously.

How Do UV spectrometers work?

Cuvettes (sample holder)

Polystyrene340-800 nm

Methacrylate280-800 nm

Glass350-1000 nm

Suprasil Quartz160-2500 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 DetectorsDiode 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 PracticeUltraquin (psoriasis med. Needs UV. Act.Pregnancy tests (colorimetric assays)Blood glucose tests, ELISA’s

PharmaceuticspH titrations, purity measurementconcentration measurement

pKa Measurement with UV

Titration of Phenylephrine

pKa = pH + log

i

n

Ai - A

A - An

Medicinal Chemistrycompound ID (steroids, nucleosides)monitoring isomerization, chirality

Pharmaceutical Biotechnologyconcentration/purity measurementsmonitoring 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