Instrumental Analysis Fundamentals of Spectroscopy 1 Absorption Spectrophotometry

Instrumental Analysis Fundamentals of Spectroscopy 1

(Absorption) Spectrophotometry n General Stuff: • Qualitative: Spectrum (a plot of A vs. ) is characteristic of a specific species • Quantitative: Absorbance at a particular can be related to the amount of absorbing species Definitions and units . monochromatic wavelength (cm) Po. incident radiant power (erg cm -2 s -1 ) P. transmitted radiant power (erg cm -2 s -1 ) b. absorption pathlength(cm) 2

Qualitative analysis: The spectrum 3

Molecular and Atomic Spectrometry is the study of electromagnetic radiation (EMR) and its applications To begin to understand theory and instrumental application of spectrometry requires an understanding of the interaction of EMR (i. e. light) with matter 4

Questions What is nature of light? Are their different types of light? – How are they the same? – How are they different? How does light propagate? 5

What is Light? Light is a form of energy Light travels through space at extremely high velocities – The speed of light (c) ~ 3 x 1010 cm/sec or 186, 000 miles per second Light is classified as electromagnetic radiation (EMR) 6

Characteristics of Light behaves like a wave. – That is, it can be modeled or characterized with wave like properties. Light also behaves like a particle. – The photon and photoelectric effect. Today, we envision light as a selfcontained packet of energy, a photon, which has both wave and particle like properties. 7

The Electromagnetic Spectrum 8

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The Electromagnetic Spectrum 10

The EMR Spectrum Different portions of the EMR spectrum and different types of spectroscopy involve different parts (quantum states) of the atom 11

EMR Wave Characteristics Wavelength ( ) is the distance from one wave crest to the next. Amplitude is the vertical distance from the midline of a wave to the peak or trough. Frequency (v) is the number of waves that pass through a particular point in 1 second (Hz = 1 cycle/s) 12

EMR Wave Characteristics The frequency of a wave is dictated (or fixed) by its source, it doesn’t change as the wave passes through different mediums. The speed of a wave (u), however, can change as the medium through which it travels changes umedium = v = c/n Where n = refractive index nvacuum = 1 nair = 1. 0003 (vair = 0. 9997 c) nglass ~1. 5 (vgas ~ 0. 67 c) Since v is fixed, as decreases, u must also decrease 13

Wave Properties of Electromagnetic Radiation EMR has both electric (E) and magnetic (H) components that propagate at right angles to each other. 14

Particle Properties of EMR The energy of a photon depends on its frequency (v) Ephoton = hv h = Planck’s constant h = 6. 63 x 10 -27 erg sec or 6. 63 x 10 -34 Js 15

Relationship between Wave and Particle Properties of EMR Ephoton = hv ; umedium = v = c/n With these two relationships, if you know one of the following, you can calculate the other two – – – Energy of photon Wavelength of light Frequency of light Ephoton = 16

Relationship between Wave and Particle Properties of EMR Example: What is the energy of a 500 nm photon? n = c/ = (3 x 108 m s-1)/(5. 0 x 10 -7 m) n = 6 x 1014 s-1 E = hn =(6. 626 x 10 -34 J • s)(6 x 1014 s-1) = 4 x 10 -19 J 17

How Light Interacts with Matter. Atoms are the basic blocks of matter. They consist of heavy particles (called protons and neutrons) in the nucleus, surrounded by lighter particles called electrons 18

How Light Interacts with Matter. An electron will interact with a photon. An electron that absorbs a photon will gain energy. An electron that loses energy must emit a photon. The total energy (electron plus photon) remains constant during this process. 19

Characteristics of Absorption is defined as the process by which EMR is transferred, in the form of energy, to the medium (s, l, or g) through which it is traveling Involves discrete energy transfers Frequency and wavelength selective – Ephoton = hv = c/ 20

Characteristics of Absorption Involves transitions from ground state energy levels to “excited” states – The reverse process is called emission For absorption to occur, the energy of the photon must exactly match an energy level in the atom (or molecule) it contacts – Ephoton = Eelectronic transition We distinguish two types of absorption – Atomic – Molecular 21

How Light Interacts with Matter. Electrons bound to atoms have discrete energies (i. e. not all energies are allowed). Thus, only photons of certain energy can interact with the electrons in a given atom. 22

How Light Interacts with Matter. Consider hydrogen, the simplest atom. Hydrogen has a specific line spectrum. Each atom has its own specific line spectrum (atomic fingerprint). 23

Energy Transitions and Photons The energy of photon that can interact with a transition jump depends on the energy difference between the electronic levels. 24

Unique Atomic Signatures Each atom has a specific set of energy levels, and thus a unique set of photon wavelengths with which it can interact. 25

Energy Level Diagram Absorption and emission for the sodium atom in the gas phase Illustrates discrete energy transfer ΔEtransition = E 1 - E 0 = hv = hc/ 26

Molecular Absorption More complex than atomic absorption because many more potential transitions exist – Electronic energy levels – Vibrational energy levels – Rotational energy levels Emolecule = Eelectronic + Evibrational + Erotational – Eelectronic > Evibrational > Erotational Result - complex spectra 27

Energy Level Diagram for Molecular Absorption 28

Molecular Absorption Spectra of Benzene in the Gas Phase Electronic Transition Vibrational Transition Superimposed on the Electronic Transition Absorption Band – A series of closely shaped peaks 29

Molecular Absorption Spectra in the Solution Phase In solvents the rotational and vibrational transitions are highly restricted resulting in broad band absorption spectra 30

Beer’s Law or the Beer-Lambert Law Pierre Bouguer discovered that light transmission decreases with the thickness of a transparent sample in 1729. This law was later rediscovered by Lambert, a mathematician, and then by Beer, who published in 1852 what is now known as the Beer-Lambert-Bouguer law. Beer's 1852 paper is the one that is often cited in older textbooks. Bouguer's contribution is rarely mentioned and the law is known as either "Beer's law" or "the Beer-Lambert law".

Consider a beam of light with an (initial) radiant intensity Po The light passes through a solution of concentration (c) The thickness of the solution is “b” cm. The intensity of the light after passage through the solution (where absorption occurs) is P P 0 hv Concentration (c) Spectroscopy Terms Describing Absorption (Beer’s Law) b P

We Define Transmittance (T) = P/P 0 (units = %) Absorbance (A) (units = none) A = log (P 0/P) A = -log (T) = log (1/T) A = abc (or εbc) <--- Beer’s Law a = absorptivity (L/g cm) b = path length (cm) c = concentration (g/L) ε = molar absorptivity (L/mol cm) – Used when concentration is in molar units

Transmittance T => transmittance P T = ----Po b Po P

Example P 0 = 10, 000 P = 5, 000 -b- A = -log T = -log (0. 5) = 0. 3010

Beer’s Law A = abc = ebc A c

Beer’s Law A = e bc Path Length Dependence, b Readout Absorbance 0. 82 Source Detector

Beer’s Law A = e bc Path Length Dependence, b Readout Absorbance 0. 62 Source b Detector Sample

Beer’s Law A = e bc Path Length Dependence, b Readout Absorbance 0. 42 Source Detector Samples

Beer’s Law A = e bc Path Length Dependence, b Readout Absorbance 0. 22 Source Detector Samples

Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0. 80 Source b Detector

Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0. 82 Source Detector

Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0. 30 Source b Detector

Beer’s Law A = ebc Wavelength Dependence, a Readout Absorbance 0. 80 Source b Detector

Non-Absorption Losses "Reflection and scattering losses. " AKA The Guinness Effect

Limitations to Beer’s Law Real – At high concentrations charge distribution effects occur causing electrostatic interactions between absorbing species Chemical – Analyte dissociates/associates or reacts with solvent Instrumental – ε = f(λ); most light sources are polychromatic not monochromatic (small effect) – Stray light – comes from reflected radiation in the monochromator reaching the exit slit.

Chemical Limitations A reaction is occurring as you record Absorbance measurements Cr 2 O 72 - + H 2 O 2 H+ + Cr. O 42 Cr 2 O 72 A 550 300 400 wavelength 500 A 446 concentration

Instrumental Limitations - ε = f(λ) How/Why does ε vary with λ? Consider a wavelength scan for a molecular compound at two different wavelength bands Larger the Bandwidth – larger deviation In reality, a monochromator can not isolate a single wavelength, but rather a small wavelength band

Instrumental Limitations – Stray Light How does stray light effect Absorbance and Beer’s Law? A = -log P/Po = log Po/P When stray light (Ps) is present, the absorbance observed (Aapparent) is the sum of the real (Areal) and stray absorbance (Astray)

Instrumental Limitations – Stray Light Aapp = Areal + Astray = As the analyte concentration increases ([analyte]↑), the intensity of light exiting the absorbance cell decreases (P↓) Eventually, P < Ps

Instrumental Limitations – Stray Light Result – non-linear absorption (Analyte vs. Conc. ) as a function of analyte concentration – Similar to polychromatic light limitations

Emission of EMR is released when excited atoms or molecules return to ground state – Reverse of the absorption process – We call this process “emission” Initial excitation can occur through a number of pathways – Absorption of EMR – Electrical discharge – High temperatures (flame or arc) – Electron bombardment 52

Emission of EMR We distinguish several types of emission 1. 2. 3. Atomic X-Ray Fluorescence Involves molecules Resonance and non-resonance modes 4. Phosphorescence Non-radiative relaxation Similar to fluorescence only relaxation times are slower than fluorescence Involves metastable intermediates 53

Energy Level Diagrams and Emission 54

MO diagram of dinitrogen (N 2)

Luminescence is the emission of light from any substance and occurs from electronically excited states. It is formally divided into two categories: Molecular fluorescence. Molecular phosphorescence. Its attractive feature is the inherent high sensitivity, 3 order of magnitude lower than absorption measurements (ppb).

Fluorescence is emission of light from excited singlet states (the electron in the excited state orbital is spin paired (has the opposite spin) to the electron in the ground state orbital) – therefore, return to the ground state is spin-allowed, and the excited state lifetime is short (1 -10 ns). Phosphorescence is emission of light from excited triplet states (the electron in the excited orbital has the same spin orientation as the ground state electron) –therefore, the transition to the ground state is spin-forbidden, and the excited state lifetime is long (ms to seconds or even minutes!) Molecular chemiluminescence: emission from an excited species that formed in the course of chemical reaction.

Jablonski Diagram

Deactivation Processes Intersystem Crossing: transition with spin change (e. g. S to T). As with internal conversion, the lowest singlet vibrational state overlaps one of upper triplet vibrational levels and a change in spin state is thus more probable. Intersystem crossing is most common in molecules that contain heavy atoms, such as iodine or bromine (the heavy-atom effect). Fluorescence: emission not involving spin change (e. g. singlet→singlet), efficient, short-lived <10 -5 s. Phosphorescence: emission involving spin change. Long-lived> 10 -4 s. A triplet →singlet transition is much less probable than singlet →singlet transition. This transition may persist for some time after irradiation has been. discontinued since the average lifetime of the excited triplet state with respect to emission ranges from 10 -4 to 10 s or more. . Dissociation: excitation to vibrational state with enough energy to break bond. Predissociation: relaxation to state with enough energy to break bond

Fluorescence Quenching is ANY process that decreases the amount of fluorescence for a given number of input photons: Collisional quenching –the excited state is de-activated via diffusional contact with a quencher (dynamic quenching) Fluorophores can form nonfluorescent complexes with quenchers. This process is referred to as static quenching since it occurs in the ground state and does not rely on diffusion or molecular collisions. attenuation of the emitted radiation by the fluorophore

Collisional Quenching methyl viologen

How likely is fluorescence? From the equation, it is clear that 0< φ< 1, and that a high value for kr and a small value for knr lead to the best quantum yield (i. e. , fluorescence is faster than all other competing processes). It should be noted that a change in quantum yield can occur owing to many factors (temperature, p. H, solvent, presence of quenchers, dimerization, etc) and thus fluorescence intensity may not be directly proportional to concentration.

Molecular Luminescence Spectroscopy S 0 -common, diamagnetic (not affected by B fields). D 0 -unpaired electron, many radicals, two equal energy states. T 1 -rare, paramagnetic (affected by B fields). Energy (S 1) > Energy (T 1) (difference is energy required to flip electron spin). Ground state Excited state Singlet, So Doublet, Do Triplet, T 1 S 1 emission So S 1 absorption Excited state Singlet, S 1 So

What about Lifetimes? Absorption S 1 S 0 very fast 10 -15 -10 -13 s Relaxation Resonant emission S 1 S 0 fast 10 -9 -10 -5 s (fluorescence) common in atoms strong absorber - shorter lifetime Non-resonant emission S 1 S 0 fast 10 -9 -10 -5 s (fluorescence) common in molecules, have extremely fast vibrational relaxation red shifted emission (Stokes shift)

Stokes Shifting- The energy of the emission is typically less than that of absorption. Fluorescence typically occurs at lower energies or longer wavelengths. this is called Stokes Shifting.

Non-resonant emission T 1 S 0 slow 10 -5 -10 s (phosphorescence) Transitions between states of different multiplicities are improbable (forbidden) (e. g. T S or T S)

Fluorescence Quantum Yield - ratio of number of molecules fluorescing to number excited.

What Affects the fluorescence quantum yield? (1) Excitation Short l's break bonds increase kpre-dis and kdis rarely observed s* ®s p* ® n most common p* ® p emission is usually from lowest lying excited state (2) Lifetime of state Transition probability measured by e Large e implies short lifetime Largest fluorescence from short lifetime/high e state p*®p > p*® n (10 -9 -10 -7 s > 10 -7 -10 -5 s)

(3) Structure Few conjugated aliphatics fluoresce but Many aromatics fluoresce Desire short lifetime S 1, no/slowly accessible T 1 Fluorescence increased by # fused rings and substitution on/in ring Emission Intensity –the factors that control emission intensity include the presence of heteroatoms, presence of aromatic rings, overall structural rigidity, and resonance stabilization. Presence of heteroatoms: often this can lead to unwanted π*→n transitions that are likely to convert to the triplet state, and give no fluorescence. All of the species shown below are non-fluorescent

(4) Rigidity Rigid structures fluoresce Increase in fluorescence with chelation

Ethidium bromide