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Spectroscopic methodsLecture 11

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The Electromagnetic Spectrum

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• Wavelength is defined as the distance between adjacent peaks (or

troughs), and may be designated in meters, centimeters or nanometers

(10-9 meters).

• Frequency is the number of wave cycles that travel past a fixed point

per unit of time, and is usually given in cycles per second, or hertz (Hz).

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The energy associated with a given segment of the spectrum is proportional to its

frequency.

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Quiz

1. The shortest length has:

a) gamma rays; b) X-rays; c) UV; d) VIS; e) IR; f) microwave; f) radio wave

2. The smallest energy exhibits….

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Frequency, wavelength, energy

frequency

Velocity of light, 3 . 108 m s-1

Wavelength

energy

Planck’s constant, 6.6 .10-34 J s

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QuizCalculate the energy of UV rays with λ = 340nm.

ΔE = 5.82 . 10-19 J

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Quiz

What is the wavelength of rays with energy 3.2 .10-16 J.

A) 6.19 .10-20m; b) 6.19 .10-10m; c) 61.9 .nm; d) 619 nm

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Physical Chemistry; Understanding our Chemical World. Paul Monk. Manchester Metropolitan University, UK; John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England 9

UV-Vis spectroscopy

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Theoretical background

• An obvious difference between certain compounds is their color.

Thus, carotene is orange; chlorophyll is green; haemoglobin is red

whereas aspirin is colorless.

• In this respect the human eye works as a spectrometer analyzing

the light reflected from the surface of solid solutes or passing

through liquid solutes.

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Violet: 400 - 420 nm

Indigo: 420 - 440 nm

Blue: 440 - 490 nm

Green: 490 - 570 nm

Yellow: 570 - 585 nm

Orange: 585 - 620 nm

Red: 620 - 780 nm

http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/spectrum.htm#uv2

ROY G BIV.

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Violet: 400 - 420 nm Indigo: 420 - 440 nm Blue: 440 - 490 nm

Green: 490 - 570 nm Yellow: 570 - 585 nm

Orange: 585 - 620 nm Red: 620 - 780 nm

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Chlorophyll AChlorophyll Bcarotenoids

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Indigo

Dye of jeans Alizarin, Crimson pigment Tyrian Purple Kermesic acid,

cochineal

Saffron pigment, crocetin

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• The deep orange hydrocarbon carotene is widely distributed in plants, but is not sufficiently stable to be used as permanent pigment, other than for food colouring.

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A common feature of all these coloured compounds, displayed below, is

a system of extensively conjugated pi-electrons (chromophores).18

Chromophores

The functional groups of organic compounds (ketones, amines, nitrogen derivatives, etc.), responsible for absorption in UV/Vis range of the spectrum are called chromophores.

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Chromophore Example Excitation λmax, nm ε Solvent

C=C Ethene π __> π* 171 15,000 hexane

C≡C 1-Hexyne π __> π* 180 10,000 hexane

C=O Ethanaln __> π*

π __> π*

290

180

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10,000

Hexane

hexane

N=O Nitromethanen __> π*

π __> π*

275

200

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5,000

Ethanol

ethanol

C-X X=Br

X=I

Methyl bromide

Methyl

Iodide

n __> σ*

n __> σ*

205

255

200

360

Hexane

hexane

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Isolated chromophores - do not interact with each other

because they are separated by at least two single bonds in the

skeleton, then the overlapping of the effects of each individual

chromophore is observed.

Conjugated chromophore systems - interact with each other

which affects that the absorption spectrum is displaced towards

longer wavelengths (bathochromic effect) with an increase in

the absorption intensity (hyperchromic effect).

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Nature of ShiftDescriptive Term

To Longer Wavelength Bathochromic

To Shorter Wavelength Hypsochromic

To Greater Absorbance Hyperchromic

To Lower Absorbance Hypochromic

Terminology for Absorption Shifts

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The energies of the UV - VIS range of spectrum are sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, absorption spectroscopy carried outin this region is sometimes called "electronic spectroscopy".

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LUMO

HOMO

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Comparison of the transitions met most frequently with simple organic compounds.The four types of transition are united on a single energy diagram in order to situate them with respect to each other and to correlate them with the corresponding spectral ranges concerned.

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The graph of absorbance (A) versus wavelength the isoprene spectrum .Since isoprene is colorless, it does not absorb in the visible part of the

spectrum and this region is not displayed on the graph.

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Food colouring- Red3; Because the λmax of 524 nm falls within the green region of the spectrum, the compound appears red to our eyes

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Bathochromic and hyperchromic effectsThe added conjugation in naphthalene, anthracene and tetracene causes bathochromic shifts of these absorption bands.

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Factors affecting the absorption

•pH

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The pH of the solvent in which the solute is dissolved can have an important effect on its spectrum. Amongst the compounds that present this effect in a spectacular fashion are chemical indicator strips, whose change in color is used during acidimetric measurements.

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Absorption spectra of bromocresol green at different stages of protonation

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Hydrangea in acidic soilHydrangea in alkaline soil.

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1 2 3 4 5 6

1- high pH value (strong base), 2 – base; 3- neutral pH; 4- weak acid; 5 – medium acidic; 6- strong acid (low pH)

Red cabbage juice as the pH indicator

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Solvent type

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• Different compounds may have very different absorption maxima

and absorbances. Intensely absorbing compounds must be examined

in dilute solution, so that significant light energy is received by the

detector, and this requires the use of completely transparent (non-

absorbing) solvents. The most commonly used solvents are water,

ethanol, hexane and cyclohexane. Solvents having double or triple

bonds, or heavy atoms (e.g. S, Br & I) are generally avoided.

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Vis spectrum of 1,2,4,5 tetrazine. Spectrum of solute in a) gaseous stateb) in hexane (nonpolar solvent)c) in water (polar solvent)

From S.F. Mason, J. Chem Soc., 1959, 1265

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In spectroscopy, the transmittance T is a measure of the attenuation of a beam of monochromatic light based upon the comparison between the intensities of the transmitted light (I) and the incident light I0 according to whether the sample is placed, or not, in the optical pathway between the source and the detector.T is expressed as a fraction or a percentage:

T = I/I0

or%T = I/I0 ×100

The absorbance (old name optical density) is defined by:

A=2−log %T ; A = log T

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• If the sample compound does not absorb light of a given wavelength, I = I0.

However, if the sample compound absorbs light then I is less than I0.

• Absorption may be presented as transmittance or absorbance. If no

absorption has occurred, T = 1.0 and A= 0.

• Most spectrometers display absorbance on the vertical axis, and the

commonly observed range is from 0 (100% transmittance) to 2 (1%

transmittance).

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Quiz

Calculate the absorbance of the compound when its transmittance is equal 50%.

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UV-VIS spectroscopy equipment • A diagram of the components of a typical spectrometer are shown in the next

slide.

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http://devarchive.cnx.org/contents/02e7b3d6-cf47-4c92-a380-d011ce5658b1@1/basics-of-uv-visible-spectroscopy

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• All spectrometers require a light source. More than one type of source can be

used in the same instrument which automatically swaps lamps when scanning

between the UV and visible regions:

• for the visible region of the spectrum, an incandescent lamp fitted with a

tungsten filament housed in a silica glass;

• for the UV region a deuterium arc lamp (<350nm);

• a xenon arc lamp can be used for routine apparatuses.

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As the monochromator gratings the following tools may be used:(a) single concave spherical mirror(b) two spherical concave mirrors system (c) the concave grating

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Photodetectors

• Photodiodes

• Phototransistors

• Semiconductors

• photomultiplier

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Quantitative analysis

• The UV/Vis domain has been widely exploited in quantitative

analysis for a particularly long time. The measurements are

based upon the Lambert–Beer law which, under certain

conditions, links the absorption of the light to the

concentration of a compound in solution.

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A= ε l C

• A is an absorbance, an optical parameter without dimension accessible with a

spectrophotometer,

• l is the thickness (in cm) of the solution through which the incident light is passed,

• C the molar concentration

• ε the molar absorption coefficient L mol−1 cm−1 at wavelength , at which the

measurement is made. This parameter, also called the molar absorptivity, is

characteristic of the compound being analyzed and depends among other things upon

the temperature and the nature of the solvent. 55

This law, which concerns only that fraction of the light absorbed can be verified by application of the following conditions:

• the light used must be monochromatic

• the concentrations must be low

• the solution must be neither fluorescent or heterogeneous

• the solute must not undergo to photochemical transformations

• the solute must not undertake variable associations with the solvent.

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Absorption of the light by a homogeneous material and representation of percentage transmittance as a function of the material’s thickness. The light reaching the sample can be reflected, diffused, transmitted or absorbed. Here only this last fraction is taken into account.

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Illustration of the Lambert–Beer law. Spectra of aqueous solutions of increasing concentration in potassium permanganate. Graph of the corresponding absorbances measured at 525nm showing the linear growth of this parameter.

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Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein)

UV-vis spectra of different concentrations of Rose Bengal

Calibration curve of Rose Bengal. Equation of line: y = 0.0977x –0.1492 (R2 = 0.996)

http://devarchive.cnx.org/contents/02e7b3d6-cf47-4c92-a380-d011ce5658b1@1/basics-of-uv-visible-spectroscopy 59

Quiz

Calculate the absorbance of an organic dye C =7×10−4 mol L−1, knowing that the molar absorptivity ε= 650 L mol −1 cm−1 and that the length of the optical path of the cell used is 1cm.

What would happen to the absorbance if the cell used was of triple its present thickness?

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Additive nature of absorbances. For all wavelengths, the absorbance of a mixture is equal to the sum of the absorbances of each component within the mixture (assuming the same molar concentrations in the two experiments).

Experiment: A1 = ε1C1 and A2=ε2C2

A=A1+A2

Additivity of absorbances

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Both NAD+ and NADH absorb at 260 nm. However NADH, unlike NAD+, has a second absorbance band with λmax = 340 nm and ε = 6290 L*mol-1*cm-1. The figure below shows the spectra of both compounds superimposed, with the NADH spectrum offset slightly on the y-axis:

By monitoring the absorbance of a reaction mixture at 340 nm, we can 'watch' NADH being formed as the reaction proceeds, and calculate the rate of the reaction.

http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_04%3A_Structure_Determination_I/Section_4.3%3A_Ultraviolet_and_visible_spectroscopy 63

Quiz

• What is the total absorbance at 450nm of the mixture of twocomponents if the concentration of the first component is 1x10-4M (ε = 4000), the concentration of second compound is 1x10-6M (ε = 45000).

• Both components obey Lamber-Beer law.

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Infrared spectroscopy (IR)

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• Analytical infrared studies are based on the absorption (or reflection) of

the electromagnetic radiation that lies between 1 and 1000 m-1 (780 nm

- 1mm).

• This spectral range is sub-divided into three smaller areas, the near

infrared (near-IR, 1–25μm), the mid infrared (mid-IR, 25–50μm) and the

far infrared (beyond 50μm).

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Absorption of lower energy radiation (from 1 to 15 kcal/mole) causes vibrational and rotational excitation of groups of atoms within the molecule. Because of their characteristic absorptions identification of functional groups is easily accomplished.

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• Some General Trends:

i) Stretching frequencies are higher than corresponding bending

frequencies. (It is easier to bend a bond than to stretch or compress it).

ii) Bonds to hydrogen have higher stretching frequencies than those to

heavier atoms.

iii) Triple bonds have higher stretching frequencies than corresponding

double bonds, which in turn have higher frequencies than single bonds.

(Except for bonds to hydrogen).

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The complexity of infrared spectra in the 1450 to 600 cm-1 region makes it

difficult to assign all the absorption bands, and because of the unique

patterns found there, it is often called the fingerprint region.

Absorption bands in the 4000 to 1450 cm-1 region are usually due to

stretching vibrations of diatomic units, and this is sometimes called the group

frequency region.

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http://www.spectra-analysis.com/infraredspectra/controlled.htm

Gas Phase Infrared Spectrum of Formaldehyde, H2C=O

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Mid-IR spectrum of a polystyrene film

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Infrared spectrum of aspirin, i.e. 2-acetoxybenzoic acid. (aspirin)

Physical Chemistry;Understanding our Chemical WorldPaul Monk.Manchester Metropolitan University, UK; John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester,West Sussex PO19 8SQ, England

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• Infrared spectra may be obtained from samples in all phases (liquid, solid and

gaseous). Liquids are usually examined as a thin film sandwiched between two

polished salt plates (note that glass absorbs infrared radiation, whereas NaCl is

transparent).

• If solvents are used to dissolve solids, care must be taken to avoid obscuring

important spectral regions by solvent absorption. Perchlorinated solvents such as

carbon tetrachloride, chloroform and tetrachloroethene are commonly used.

• Alternatively, solids may either be incorporated in a thin KBr disk, prepared under

high pressure, or mixed with a little non-volatile liquid and ground to a paste (or mull)

that is smeared between salt plates.

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IR instruments can be divided into two categories:

• the Fourier transform spectrometers, which undertake a

simultaneous analysis of the whole spectral region from

interferometric measurements,

• specialized analysers

• Dispersive-type spectrometers are also used for the near-IR.

Equipment

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• Light sources:

• They are either a lamp filament or a hollow rod, made of fused mixtures

of zirconium oxide or rare earth oxides (Nernst source) heated by Joule

effect by the means of an internal resistor (for example Globar™).

IR Detectors

The principle relies upon the thermal effect of IR radiation. Sensors that

measure radiation by means of the change of temperature of an

absorbing material are classified as thermal detectors- thermistors,

thermocouples, thermopiles and other sensors.

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Applications of IR

• In industry as well as in scientific research; quality control and

dynamic measurement.

• Forensics analysis for civil and criminal analysis.

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• Some of the major applications of IR spectroscopy are as follows:

identification of functional group and structure elucidation entire IR region

(range of group frequency is 4000-1500 cm-1 while that for finger print

region is 1500-400 cm-1).

identification of substances

studying the progress of the reaction

detection of impurities

quantitative analysis. The quantity of the substance can be determined

either in pure form or as a mixture of two or more compounds.

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• Many compounds, when excited by a light source in the visible or near ultraviolet regions, absorb energy which is then re-emitted in the form of radiation.

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Bioluminescence, fluorescence and chemiluminescence

Luminescence in general, is the term of emission of light from specific substances/objects.

Chemoluminescence is the emission of light as the result of chemical reactions.

Bioluminescence is the emission light from living/biological organisms.

Phosphorescence occurs then the light is absorbed and emitted. But the later one lasts over a long period of time.

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Luminescence

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The fluorescence of the chlorophyll in UV

Chlorophyll in VIS

An example of fluorescence. Tonic water is clear under normal light, but vividly fluorescent under ultraviolet light, due to the presence of the quinine

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Fluorescence

Bioluminescence

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Glow- worm, fire- fly

Planktonic jellyfish with bright green-fluorescent tentacles. The red fluorescent in the middle comes from chlorophill.

Chemo luminescence

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Phosphorescence

The-glow-in-the dark toysstickersPaintclock dials

Phosphorescent materials: zinc sulfide; strontium aluminate

The Jablonsky diagram illustrating the process involved in the creation of an excited singlet state by optical absorption and subsequent emission of the fluorescence.

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Representation on the same graph of the absorbance and fluorescence spectra of an ethlyenic compound.. Example extracted from Jacobs H.et al., Tetrahedron 1993, 6045.

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According to Stokes’ law, the maximum of the spectral emission

band is located at a longer wavelength than that of the

original excitation light. After excitation, the light intensity

decays extremely rapidly according to an exponential law.

Expression below links the intensity of fluorescence It and the

time passed t since the excitation:

It =I0 · exp [−kt]

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• The extremely rapid extinction of the light intensity when excitation

ceases is the object of analysis. By contrast, phosphorescence is

characterized by a more gradual diminution during time.

Fluorescence is equally employed as the basis of detectors used in

liquid chromatography.

• Although of different origin, chemiluminescence which comprises the

emission of light during certain chemical reactions, has also received

several applications in analytical chemistry.

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Relative position of absorption, fluorescence and phosphorescence bands of chrysene.

Relative position of absorption andfluorescence bands of anthracene. Fluorescing aromatic compounds. The names are followed by their fluorescence

quantum yield Φ, for which the values are obtained by comparison withcompounds of known fluorescence. The measurements are made at 77 K. 8-Hydroxyquinoline is representative of various molecules forming fluorescent chelates with certain metal ions.

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• The intensity of the fluorescence is proportional to the concentration of the analyte and the measurements are made with the aid of fluorometers or spectrofluorometers

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Intensity of fluorescence and concentration. A maximum of fluorescence is observed beyond which, with a continuing increase in concentration, it diminishes. After the maximum the more concentrated the solution then the weaker is the fluorescence – a kind of roll-over or self-quenching. The illustrations correspond to three recordings, at the same scale of biacetyl tetrachloromethane. The curve records the light collected from a small volume situated at the centre of the solution 104

A block diagram of a spectrofluorometer with a xenon arc lamp. The fluorescence is measured, in contrast to studies on fluorescence dynamics, under ‘steady state’ conditions by maintaining the primary excitation source. Below right, a schematic of a xenon arc lamp. The pressure of xenon in the lamp is around 1 MPa. These arc lamps made from a quartz envelope and without a filament, are sources of ‘white light’. The cathode is the finer of the two electrodes.

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Comparison, following a chromatographic separation, of UV and fluorescence detection. Aflatoxins, which are carcinogenic the subject of analysis by HPLC.(reproduced courtesy of a document from Agilent Technologies).

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DyLight 680/800 labeled antibodies used in two-color Western blot.Proteins were separated in 4-20% Precise Protein Gels and transferred to Low-Fluorescence PVDF Transfer Membrane. The membrane was blocked overnight in SEA BLOCK and target proteins were detected following the recommended protocol. Membranes were imaged with the LI-COR Odyssey Infrared Imaging System. Tubulin was detected from the indicated quantity of HeLa cell lysate. Purified TNFα was detected at the indicated quantity.

http://www.piercenet.com/browse.cfm?fldID=5A409573-5056-8A76-4E35-8EE0AA401E7B 107

Application of fluorescence

• Analysis of biological samples:

• Oligonucleotides

• Gens

• Antibodies

• Liquid chromatography detectors

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Quiz • Which of the following statements best defines Luminescence?

A. The emission of light by a substance after absorption of excitation energy

B. Emission of light due to non-thermal process, a chemical reaction, or the absorption of ionizing radiation

C. This light is absorbed by the ground state atoms

D. Emission of light requiring a light source

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Emission methods, atomic methods

• Atomic absorption spectroscopy (AAS) and flame emission spectroscopy

(FES), also called flame photometry, are two analytical measurement

methods relying on the spectroscopic processes of excitation and

emission. Methods of quantitative analysis only, they are used to

measure of around seventy elements (metal or nonmetal).

There exists a broad range of applications, as concentrations to the ppb

level can be accessed for certain elements.110

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The schematics for the optical set-up (collimator,objective), have been simplified for reasons of clarity. 111

The diverse components of a single beam atomic absorption apparatus. Model IL 157(Thermo Jarrell

Ash) constructed during the 1980s. 1, source (spectral lamp); 2, flame burner which provides the

atomic aerosol; 3, monochromator grating; and 4, detector (photomultiplier).

The source illuminates a slit situated at the entrance the dispersive system. The exit slit, is close to the

detector window. It determines a narrow bandwidth of the spectrum, ( of 0.2 to 1 nm), which must not

be confused with either the width of the exit slit or with the image of the entrance slit. 112

Dr. Thomas G. Chasteen; Department of Chemistry, Sam Houston, State University, Huntsville, Texas 77341. Copyright 2000. http://www.shsu.edu/~chm_tgc/primers/pdf/spect.pdf

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• To measure an element by AAS or FES methods, it must be in the form

of free atoms. To this end, the sample is heated to a temperature of at

least 2000oC, in order to dissociate all chemical combinations in which

the element under study is engaged, including the rest of the sample.

This pyrolysis leads to the total concentration of the element without

distinguishing the different chemical structures in which the element

was possibly bound in the original sample (this is therefore opposite to

a speciation analysis).

• The pyrolysis process can be performed with Bunsen burner, with

thermoelectric mode, graphite furnace or with device for hydride

formation.

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Typical model of a hollow cathode lamp. The cathode is a hollow cylinder whose

central axis corresponds to that of the optical axis of the lamp.

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• An alternative to HCL are electrodeless discharge lamps (EDL) whose light

intensity is about 10–100 times greater but are not as stable as HCL. They

are made of a sealed quartz tube that contains a salt of the element of

interest along with an inert gas. These lamps are in general reserved for

elements such as As, Hg, Sb, Bi and P.

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Elements measured by AAS and FES.

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• In AAS, as in FES, the measurement of light intensity is carried out

at a wavelength specific to each element being analysed.

• In AAS, the concentration can be deduced from the measurement

of light absorption by the atoms remaining in the ground state

when they are irradiated by an appropriate source of excitation.

• In FES, conversely, the concentration can be deduced from the

intensity of the radiation emitted by the fraction of atoms that

have passed into excited states.

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• The absorbance (or emision) of the element in the flame depends upon the number of ground state atoms.

• Measurements are made by comparing the unknown to the standard solutions.

• A=k ·C

• where A is absorbance, C is concentration of element and k is a specific coefficient to each element at the given wavelength.

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Examples of calibration graphs in AAS. Left, a straight calibration line at sub-ppb concentrations

obtained with an instrument equipped with a Zeeman effect device for the quantification of sodium. Right, a quadratic curve for the measurement for zinc at concentrations in the ppm range with a burner type instrument. This second graph reveals that when concentrations increase, the absorbance is no longer linear. The quantitative analysis software for AAS provides several types of calibration curves. 121

Application• Atomic Absorption Spectroscopy can be used to measure the

concentration of metals in :

• mining operations and in the production of alloys as a test for purity

• contaminated water, especially heavy metal contamination in industrial waste water

• organisms, such as mercury in fish

• air, e.g. lead

• food

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• Most applications of FES:• determination of trace metals, especially in liquid samples (including

the alkali and alkaline earths, as well as several transition metals such as Fe, Mn, Cu, and Zn; nonmetals: H, B, C, N, P, As, O, S, Se, Te, halogens, and noble gases). FES detectors for P and S are commercially available for use in gas chromatography.

• in agricultural and environmental analysis,

• industrial analyses of ferrous metals and alloys as well as glasses and ceramic materials,

• clinical analyses of body fluids.

• FES can be easily automated to handle a large number of samples. Array detectors interfaced to a microcomputer system permit simultaneous analyses of several elements in a single sample.

http://www.tau.ac.il/~chemlaba/Files/Flame%20supplement.pdf

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Sources1. Physical Chemistry;Understanding our Chemical WorldPaul Monk. Manchester Metropolitan University, UK; John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester,West Sussex PO19 8SQ, England2. http://ull.chemistry.uakron.edu/analytical/3. http://search.conduit.com/Results.aspx?q=spectrophotometry&start=10&hl=pl&SearchSource=13&SelfSearch=1&SearchType=SearchWeb&ctid=CT2475029&octid=CT2475029&FollowOn=True4. http://cfcc.edu/faculty/jjenkins/courses/msc180/lectures/Spe5. http://www.shsu.edu/~chm_tgc/primers/pdf/spect.pdf

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