Part 5-Instrumentation: Introduction to Spectroscopy for Chemical

AnalysisKR

LSU

The Spectrophotometer- Instruments

IMPORTANT: Absorption spectrophotometer

(a)

(b)

FFYI: Double-beam spectrophotometer (better than single beam see previous page): Light passes alternately through the reference and sample cuvettes. A chopper is a mirror that rotates in and out of the light path diverting the light between the reference and sample cuvettes. Routine procedure is to first record a baseline spectrum with two reference cuvettes. The absorbance of the reference is then subtracted from the absorbance of the sample to obtain the "true" absorbance at each wavelength.

FYI: ….Or diode array spectrophotometer

Light Sources

FYI: Sources of radiation objects

Any object that is heated emits radiation. Emission from real objects such as a tungsten filament light bulb emulate blackbody radiation (the emission is a continuous spectral distribution).

 Visible and infrared lamps as light sources approach blackbody radiators. The radiation

from an object's surface expressed as power per unit area is the excitance (emittance), M.

M = T4

 Where is the Stefan-Boltzmann constant ~ 5.7 X 10-8 W/(m2K4), T = Temperature (K)

Spectral distribution of blackbody radiation

IMPORTANT: Lamps for absorption spectrometersTypically they are inexpensive and stable.

 

i) Visible and Near Infrared: Tungsten Lamp, Xenon lampii) Ultraviolet and Visible: Quartz Halogen Lamp, Xenon lampiii) Ultraviolet: Deuterium Arc Lampiv) Infrared: nichrome wire , silicon carbide (globar)

v) VIS and UV Atomic absorption Hollow Cathode Lamps !!! (see later)

Examples:

FYI: Lasers provide ~ single Very bright sources for spectroscopyProperties of Lasers:

·  Monochromatic (only one wavelength)·  Collimated (emit in one direction)·  Polarized (only one electric field vector) ·  Coherent (electric/magnetic fields in phase)·  Expensive hHigh maintenance, but some He-Ne, and many solid state lasers may be less

expensive

Laser operate on the principle of trapping a large number of physical objects to a new state in a cavity, and simultaneous release of these objects to a new or old state with emission

Monochromators and other devices for separation of radiation objects

Slits +MonochromatorsSlits are constructed by machining a sharp edge onto two metal pieces. These lie in a plane and the spacing between them, the slit width, can be adjusted. The smaller the slit width, the better the spectral resolution. (example)

 Filters:

Filters are used to pass on only desired wavelengths of light. A filter could be colored glass. Most likely they are also based on constructive or destructive interference of light waves. (example)

Prisms Separation of wavelengths on some commercial instruments.    

Prisms were used in older instruments, Quartz or salt crystals.

II)       Monochromators separate wavelengths of light; they consist of both entrance & exit slits, mirrors, and diffraction grating or refraction lens/prisms, and filters.

FYI: selection of colors

Grating–monochromators Polychromatic light is collimated (focused) into a beam of parallel rays by a concave mirror

(monochromatic-one wavelength; polychromatic-many wavelengths).Rays strike the reflection grating (see next figure) and different wavelengths are diffracted (separated) at different angles.Diffracted light is focussed by a second concave mirror so that only one wavelength passes through the exit slit at a time.

 

 

Grating Equation: n = d(sin – sin) 

  = angle of incidence

n = diffraction order (1…n) ; d = groove spacing; = angle of reflection;

Components of a Grating Spectrophotometer i 

diffraction grating is ruled with a series of closely spaced parallel grooves separated by distance d.

 These are often constructed from aluminum metal and coated with a non oxidative coating applied.

 When light is reflected from the grating, each groove behaves as a source of light. When adjacent rays are in phase, they reinforce each other. When adjacent rays are out of phase, the partially or completely cancel each other. Thus can be aligned to allow only certain wavelengths to pass through.

 

Wavelength selector (monochromator) passes a narrow bandwidth of radiation (if more narrow , higher resolution!!)

EXPERIMENTAL PARAMETERS : how to get the most from measurements with absorption spectroscopy

C Choice of the Wavelength and

Bandwidth – 

Effects of monochromator’s slit: 0.25nm,1.0nm, 2.0nm, 4.0nm

INTERFEROMETERS: from time and length observables to frequency/energy observables

1) allows for signal averaging2) allows all wavelengths to be monitored simultaneously3) mathematical process that converts data obtained in the time domain to be converted into the frequency domain.

·        Allows all wavelengths to simultaneously reach the detector ·        Radiation from source reaches beam splitter, where half of the radiation hits the

moving mirror and half hits the fixed mirror.·        The beams reflect and re-combine, the emerging radiation for a wavelength exhibits

constructive or destructive interference. ·        With constant mirror velocity, the wavelength modulates in a regular sinusoidal

manner. ·        Both the sampling rate of radiation reaching the detector and the mirror velocity is

modulated by a helium-neon laser. ·        The resulting detector signal typically is stored as a time domain spectrum

(interferogram). Converted to a spectrum in the frequency domain using the mathematical process of

Fourier Transform.

Infra-Red spectroscopy, NMR spectroscopy: FT is a standard technique

Detectors

-detectors

•The phototube is used frequently as a detector in UV-Vis spectrometers. •The cathode consists of a photo-emissive surface. •Electrons are ejected from the cathode proportional to the radiant power (photons) striking its surface. •The emitted electrons are attracted to the anode. •The accompanying voltage is fed to an amplifier and converted to a signal.

•The Photo Multiplier Tube, (PMT) is similar to the photo tube, but is a vast improvement. •In addition to the cathode and anode, the PMT has dynodes, which produce a cascade effect on the electron emission production. •Each photon causes a ~ 107 additional electrons to be produced. •The PMT possesses high sensitivity, good S/N ratio, and excellent dynamic range.•PMTs are highly sensitive to visible and UV excitations at extremely low power conditions, (very low concentrations of analyte). •Intense light sources (such as daylight or stray light) can destroy and damage PMTs.

FYI: PHOTO DIODE ARRAY: diode

•PDAs are a series of silicon photo diodes, with each having a storage capacitor, and a switch that are combined in a integrated circuit on a silicon chip.•The number of sensors (silicon photodiodes) in a PDA range from 64 to 4096. •The slit width of the instrument allows the radiation to be dispersed over the entire array, allowing the spectral information to be accumulated simultaneously. •PDAs are not as sensitive nor have the same S/N ratio as the PMT, but one gains the advantage of gathering multi-channel information (all of spectrum collected simultaneously). •Advantage of the PDA is recording the entire spectrum in a fraction of the time required for a conventional scanning spectrometer to scan one wavelength at a time. An example of PDA use is in atomic emission spectroscopy (AES), UV-vis spectrophotometry, fluorescence spectrometry, Raman spectrometry.

CCD detectors: read textbook

FTIR detectors : read textbook

Noise

Eliminating noise: signal averaging

Stray Light, Electrical Noise, Cell Positioning-  Stray light can cause problems:  

Stray light arises from two major sources:1) Misdirected rays coming from the monochromator. 2) Light coming from outside the instrument such as the sample compartment lid not closed properly. Other concerns;

 

•the correct choice of the sample cell (does it require glass or quartz?)• the alignment of the sample cell (and/or the sample cell holder) •dust & fingerprints on the cell 

Part 5- From VUV to IR-Introduction to Spectroscopy for

Chemical AnalysisKR

LSU

MOLECULAR SPECTROSCOPY: IR

absorption, UV/VIS electronic absorption and emission

Atomic vs. Molecular

• What is same what is different

IMPORTANT: Most atomic spectra are discrete:

IMPORTANT - MOLECULES: Molecular spectra are broader because of the close electronic and vibrational energies

One must take into account all molecular

energies from different “degrees of freedom” : translational (motion),

electronic and here vibrational and

rotational motion of nuclei

Here are some of the degrees of freedom of

H20

FYI: MOLECULES: also electronic molecular orbitals and…..

Harmonic oscillator models

"What Happens When a Molecule Absorbs Light?" What happens when the absorption process takes place for molecules and compounds?  Molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe

the distribution of electrons in an atom. As example; :CO: localized (Lewis) vs delocalized (MO)

In an electronic transition, an electron from one molecular orbital moves to another orbital, with a concomitant increase or decrease in the energy of a molecule.

Molecular Spectroscopy 

The VIS-visible absorption methods rely on complexes or compounds forming a color and it must be easily distinguishable from other species present.

  The UV methods may be less specific in that typically most compounds

absorb UV radiation; thus the results maybe limited to only quantitative detection (information). That is, how much is there.

We have a solution containing different proteins, which absorb certain wavelengths of light (ie 254 nm). But if we require each protein's identification a more specific technique must be chosen.

  On the other hand the IR methods depend upon vibrational and rotational

absorptions. They can give both quantitative,(how much is present) and qualitative, (compound identification) information.

IMPORTANT - MOLECULES: Absorption of photon: electrons gain energy (ground to excited state)

IMPORTANT

Emission of photon: electrons lost energy (excited to ground state)

EXAMPLES : ABSORPTION IR and UV., atoms and molecules

Absorption/excitation

Emission/Luminescence (fluorescence and phosphorescence)

W What is the fate of the absorbed electronic energy associated with UV-vis spectrometry? Sometimes it results in the emission of another photon of light (Luminescence)

  Excess energy is dissipated by the excited molecule through;        collisions with other molecules (solvent)        vibrations        rotations         heat        produce a photon and relax back to the ground state. This emission process is termed

luminescence.What kinds of molecules typically exhibit luminescence?

•Highly degree of conjugation (multiple double bonds)•Aromatic molecules• Molecules with atoms, which have unpaired nonbonding valence electrons•Molecules with molecular rigidity (polycyclic)• Metal chelates

The emission spectrum typically resembles the mirror image of the absorption spectrum, but is shifted to longer wavelengths.

SSinglet state (S) – when the electron in the excited state is still paired with the ground-state electron. The spins of the two electrons remains opposed.

 Triplet state (T) – when the electron in the excited state becomes unpaired with the ground-state electron. The spins of the two electrons are now parallel.

 Electronic absorption bands are broad due to the large number of vibrational and rotational states present at each electronic state.

 

We have discussed the instrumental procedure, components and design or UV-vis spectroscopy.

  The visible adsorption methods rely on complexes or compounds forming a color

and it must be easily distinguishable from other species present.   The UV methods may be less specific in that typically most compounds absorb UV

radiation; thus the results maybe limited to only quantitative detection (information). That is, how much is there.

For example: We have a solution containing different proteins, which absorb certain wavelengths of light (ie 254 nm). But if we require each protein's identification a more specific technique must be chosen.

  On the other hand the IR methods depend upon vibrational and rotational absorptions. They

can give both quantitative,(how much is present) and qualitative, (compound identification) information.

(a)

What can happen when a molecule absorbs light and an electron is promoted from the ground state, So, to a vibrationally and rotationally excited level of the excited electronic state S1?

vibrational relaxation is a radiationless (does not produce photon) transfer of energy to other molecules (typically the solvent) by collisions – manifested as heat

 internal conversion is a radiationless transition between states with the

same spin quantum numbers (e.g., S1 S0) 

intersystem crossing is a radiationless transition between states with different spin quantum numbers (e.g., T1 S0)

 fluorescence is a radiational transition between states with the same

spin quantum numbers (e.g., S1 S0) 

phosphorescence is a radiational transition between states with different spin quantum numbers (e.g., T1 S0)

phosphorescence is a radiational transition between states with different spin quantum numbers (e.g., T1 S0)

In general, fluorescence and phosphorescence are observed at a lower energy than that of the absorbed radiation (the excitation energy).

 

em > ab

Luminescence Rrefers to emission of light by any mechanism from any type of molecule. LLuminescence measurements are inherently more sensitive than absorption measurements.

It is much easier to detect and measure a small signal rather than the difference in changes of signals associated with absorption.

 Luminescence (light emission detection methods) instrumentation has been developed around fluorescence methods rather than phosphorescence methods since fluorescence is much more sensitive.

AApplications of Fluorescence spectroscopyi) determination of biomolecules: enzymes, steroids, drugs: for example, as little as 1 ng/L (pp trillion) of the drug LSD in 5 mL blood sample.ii) determine trace contaminants; ppb of benzopyrene in air pollution samplesiii) "Electronic dog at airports", check air samples that contain TNT explosive; detection in the 600 ng/mL range.iv) determination of the Fluoride ion, [F-], indirectly by its ability to quench(inhibit) fluorescence in the Al+3 -Alizarin garnet R complex.

Principle Components of a Fluorescence Spectrometer

i)Light sources: mercury arc lamp, xenon arc lamp, lasers

ii) absorption and interference filters

iii) Grating monochromators: excitation and emission (90geometry) (remember, the UV-vis experiment is 180o configuration)

iv) Detectors - PMT, photodiode arrays

v) sample cells - silica glass

vi) amplifier and read out

As we see in the instrumental diagram: two optical units are required. The excitation monochromator selects the wavelength from the source and directs it onto the sample cell. The emitted luminescence is directed through the emission monochromator to the detector (As seen in the diagram, they are at 90 degrees to the lamp source).

In emission spectroscopy, we measure the intensity of emitted radiation, not the fraction of radiant power striking the detector as we do in absorption methods. Since

 Emission Intensity: I = k P0 c

  We can decide the excitation monochromator and the emission

monochromator settings by looking at: 

Emission spectrum: constant ex and variable em

 Excitation spectrum: constant em and variable ex

As shown above right, a standard curve can be constructed, similar to a Beer's law plot for an absorption measurement. The points represent readings at different concentrations. At higher concentrations the curve becomes nonlinear.

At low concentrations, the absorbance effects are small and the emission intensity (I) is directly proportional to the sample concentration (c) and to the incident radiant power (P0); where the constant k depends on the quantum efficiency of the fluorescence process, cell path length, etc.

 Emission Intensity: I = k P0 c

  Factors that inhibit fluorescence:

i) temperature: increased temperature increases collisions ii) solvent: halogen compounds, pH, dissolved oxygeniii) concentration dependent (too much)

  Some analytes are naturally fluorescent and can be analyzed directly. For example: Vitamin B2 , riboflavin Most compounds are not naturally luminescent enough to be analyzed

directly. However, coupling a fluorescent moiety provides an easy route to this sensitive analysis.

A = bc = (10000 M-1cm-1)(1 cm)(0.050 mM) = 0.50  

A = –log10 T ‹ T = 10–A = 10-0.50 = 0.32 or %T = 32% 

ExampleAt a given wavelength, a solution of a colored compound has a molar absorptivity of 104 M-1cm-

1. Calculate for a 1 cm cell containing a 0.050 mM solution of this compound, at that wavelength: (a) the absorbance, and (b) the transmittance.

ExampleAt a given wavelength, a cuvet filled with a sample solution has a transmittance of 63.1%. A reference cuvet filled with the solvent has a transmittance of 94.7% at that same wavelength. What is the corresponding absorbance of the sample?

 A = –log10 T so Asample = –log(0.631) = 0.20

Aref = –log(0.947) = 0.02 

Acorrected = Asample – Aref = 0.20 – 0.02 = 0.18

ExampleA 0.267 g quantity of a compound with a molecular weight of 337.69 g/mol was dissolved

in 100 mL of ethanol. Then 2.000 mL was withdrawn and diluted to 100 mL. The spectrum of this solution exhibited a maximum absorbance of 0.728 at 438 nm in a 2.000-cm cell. What is ?

Constructing a Calibration Curve - This method is used in other chemical analyses not just spectrophotometric ones.

 A calibration curve is a graph showing how the experimentally measured property depends on the known concentrations of the standards.

 We prefer calibration procedures with a linear response, in which the corrected analyte signal is proportional to the quantity of analyte.

 

Procedure for Constructing a Calibration Curve 

Step #1. Prepare known samples of analyte, covering a convenient range of concentration, and measure the response of the analytical procedure to these standards.

 Step #2. Subtract the average response of the three blank samples from each measured responses to obtain the corrected response. The blank measures the response of the procedure when no analyte is present.

 Step #3. Make of graph of corrected response vs. quantity of analyte analyzed.

 

See examples under linear calibration –previous material and Excel

IR spectroscopy

-methods, instruments (including ATR)-scales, physics

-applications

Infrared Spectroscopy (looking at vibrational transitions):Most organic as well as inorganic compounds, which are covalently bonded and exhibit a dipole moment absorb infrared radiation. The absorption process is quantized and as opposed to UV-vis, results in the excitation of a vibrational process in the molecule, not an electronic one. An absorbed energy matches the energy for a vibrational mode of a molecular bond. Several vibrational modes are possible: stretching, bending, twisting, etc.  A) IR active compounds have a dipole moment and are not symmetric. A dipole is merely an unequal distribution of charge (e-) due to the nature of the atoms in the molecule (electronegativity). (+) (-)

HCl e- density "pulled" towards ClMolecules like N2, O2, or Cl2 do not have a net dipole change when they vibrate or rotate. They are IR inactive. CO2 does have a dipole change though.Background on vibrations in molecular species(Normally vibrating in the environment) (After absorption) Y<---- M ----> X and Y <---- M ----> X Y <---- M ----> X

E1 E2E2 - E1 = ∆E = hor

∆E = hc/ = hc is usually in µm   (wave numbers) = 1(in cm) is in cm-1 cm-1 = [1/µm] * 104 or µm = [1/(cm-1)] * 104 

We can treat the two portions of the molecule like two masses connected by a spring with a force constant ƒ. 

m1 m2 m1 m2

r1 r2

Hook's Law states: F = -ƒx  F is the restoring force, x is ∆r, the distance from eq. position If we integrate these, we get the Potential Energy: E = 1/2 ƒx2 The frequency at which a molecule vibrates is

= (1/2π) (ƒ/µ)1/2 where µ = (m1m2)/(m1 + m2) (Reduced Mass)   = (1/2πc) * (ƒ/µ)1/2 = 5.3 x 10-12 * (ƒ/µ)1/2

So we can calculate an approximate wavelength for an absorption in IR!

 mC = 12 g/mole = 1.1 x 10-23 g/atom

6.02 x 1023 atom/mole 

mO = 2.7 x 10-23 g/atom

µ = (2.0 x 10-23) (2.7 x 10-23) = 1.1 x 10-23 4.7 x 10-23 A double bond has a force constant ƒ ≈ 1 x 106 dynes/cm

  = (5.3 x 10-12) [(1 x 106)/(1.1 x 10-23)]1/2

= 1.6 x 103 cm-1

Experimentally found in the 1500 ----> 1900 cm-1 region

 Single bonds, ƒ ≈ 5 x 105 dynes/cm Thus, these are very characteristic of the environment of the chemical bonds!

We can use these very characteristic stretching frequencies to deduce structures of molecules. 

1 Newton = 10^5 dyne

NOTE: NO dipole, NO IR transitions !!!

NOTE: small mass higher energy, vibrational frequency

B) Since different bonds possess different vibration modes, different compounds should not have identical IR spectra. Thus, each IR active compound has its own unique IR fingerprint spectrum. 

Group (cm-1) Intensity

C-H2850-3000 (s)

C=O 1650-1750 (s)

N-H ~3500 (m)

C-N ~2250 (m)

O-H~3200 (vs)

C) IR spectra are often displayed as % Transmission verses frequency units

(cm-1) wavenumber rather than (nm) wavelength units.

D) Two types of IR spectrometers: the dispersive instrument and the nondispersive (Fourier Transform) instrument

i) Common light sources for both types of instruments are :1) Nernst Glower - rod consisting of fused mixture of Zr, Y, & Th2) Globar - rod consisting of silicon-carbide3) nichrome wire coilii) Common detectors: 1) Thermocouple - junction potential between two conductors changes with temperature2) Thermistor, (bolometer) - consists of metal oxide flakes whose resistance changes with temperature3) Golay cell - thermal expansion of gas changes a flexible mirror (this changes radiant power reference

reaching a photocell4) Pyroelectric crystal - solid state circuit - crystal polarization change 5)Photoconductive - semiconductor, IR radiation changes conductance

4) deuterated triglycine sulfate (DTGS)iii) FTIR optics(on dispersive Instruments): use filters and diffraction grating, BUT ARE NOT USED MUCH IN AC iv) Sample cells for typical applications are: salt plates(NaCl, KBr, etc. or CaF2) for liquids, ATR or salt pellets for solids, and special gas cells for gases.

F) Fourier Transform (nondispersive) instrument, see the notes, is different from the conventional dispersive instrument in that it has an interferometer. The radiation exiting the interferometer is a complex mixture of modulation frequencies, which after passing through the sample, are focused onto the detector. The resulting signal, an interferogram, is either stored or it is transformed using the Fourier algorithm to produce the spectrum. The Pyroelectric crystal and the Photoconductive detectors are used since the response time of the other thermal detectors is slow.

ATR -Attenuated total reflection (ATR)

ATR based on total internal reflection that can form an evanescent waveIR light is passed through the ATR crystal in such a way that it reflects at least once off the internal surface which is in contact with the sample. This reflection forms the evanescent wave which extends into the sample. The penetration depth into the sample is typically between 0.5 and 2 (angle, wavelength dependent and n-s).

Incident IR Reflected IR

I mportant Uses for I R Spectroscopy i) Structural information and compound identification are two important uses for I R spectroscopy. ii) I R spectrometers have been combined with separation techniques such as GC (gas liquid chromatography), HPLC (High Performance chromatography) and SFC (SuperCritical Fluid Chromatography). I n these instances, the I R acts as the detector to identif y the separated compounds. The advantages of analyte separation and their peak identification will be obvious when we discuss these separation methods. Remember, IR cannot analyze mixtures, only pure compounds. iii) As Government atmospheric regulations prolif erate, I R seems to be one method summoned to meet the goals for the rapid monitoring of atmospheric contaminants. iv) An I R technique with a diff erent approach for extracting information about diff erent types of solids (such as polymer fi lms, pastes, powders, papers, adhesives, threads, etc.) is I nternal- Reflection IR spectroscopy. Useful information to a manufacturing process can be accomplished using this technique. I t deals with the angle of reflection, the angle of the interface, the penetration of the radiation beam and the index of ref raction of the material.

v) FTIR has been combined with optical microscopy to analyze samples with small size. Some applications: impurities found in polymer films, paper, & semiconductors; forensic examination of paint chips, drugs, explosives; and studying biological samples (plant leaves, etc).

ATR

Some mixtures can be analyzed, more complex process.

Both qualitative and quantitative analysis with FTIR.

CO measurements, see textbook.

Additional: Scattering

-non-resonant scattering-active transitions (virtual vs. IR)

-microscopies