SPECTROMETRIC METHODS OF ANALYSIS: UV-VIS AND FTIR SPECTROSCOPY

CHEM 28 Prof. Kurt W. E. Sy Piecco

The Rise of Spectroscopy

Before the beginning of the twentieth century most quantitative chemical analyses used gravimetry or titrimetry as the analytical method. With these methods, analysts achieved highly accurate results, but were usually limited to the analysis of major and minor analytes. Other methods developed during this period (1856) extended quantitative analysis to include trace level analytes.

One such method was colorimetry where the samples color is compared against the colors of a range of standards to determine the analytes concentration.

Colorimetry, in which a sample absorbs visible light, is one example of a spectroscopic method of analysis. During the twentieth century, spectroscopy has been extended to include other forms of electromagnetic radiation; X-rays, microwaves, radio waves, and energetic particles (e.g. electrons and ions).

Electromagnetic Radiation (EM)

EM radiation consists of electric and magnetic components, which are perpendicular to each other.

EM radiation is a form of energy.

Energy has both wave-like and particle-like properties.

Wavelike properties: Propagates (moves) through space in a straight line at constant speed (the speed of light). Oscillations in the electric and magnetic fields are perpendicular to each other, and to the direction of the waves propagation. Can be refracted, reflected, or diffracted Can be described by these parameters; frequency, wavelength, amplitude, etc.

Particle-like properties: EM radiation can also be pictured as being made up of energetic particles, called photons. Photons have momentum (p = mv) p is momentum, m is mass, v is velocity The energy of a photon is related to its frequency (E = h) E is energy, h is Planks constant, is frequency h = 6.626 x 1034 J s De Broglies equation Relates the wave and particle properties of EM radiation

The EM Spectrum

Interactions of EM Radiation with Matter

When a sample absorbs electromagnetic radiation it undergoes a change in energy.

When a photon is absorbed by a sample, the photon is destroyed, and the photons energy acquired by the sample. Substances can only absorb discrete amounts of energy. The amount differs from one substance to another.

After absorption of energy, the samples atoms, molecules, ions or radicals become excited. When these excited particles return to their ground states, the absorbed energy is released (as heat or light, or both).

Different parts or motions of the atoms are affected depending on the energy (which is proportional to the frequency) of the incident EM radiation.

Two Broad Classes of Spectroscopy

1. Involves transfer of energy between photons and the sample: absorption and emission of radiation.

In absorption spectroscopy the energy carried by a photon is absorbed by the analyte, promoting the analyte from a lower-energy state to a higherenergy, or excited, state. The source of the energetic state depends on the photons energy. The intensity of photons passing through a sample containing the analyte is attenuated because of absorption. The measurement of this attenuation, which we call absorbance, serves as our signal. Absorption only occurs when the photons energy matches the difference in energy, E, between two energy levels. A plot of absorbance as a function of the photons energy (proportional to its frequency or wavelength) is called an absorbance spectrum

Emission of a photon occurs when an analyte in a higher-energy state returns to a lower-energy state. The higher-energy state is produced after the analyte has absorbed of photon, or after the analyte has undergone a chemiluminescent reaction.

2.

Involves changes in amplitude, polarization, phase angle or direction of propagation of the incident radiation due to refraction, reflection, scattering, diffraction, or dispersion by the sample.

Measuring EM Radiation

Absorbance

Attenuation (lessening) of radiation due to absorption by the analyte.

Absorbance only at the max (quantitative analysis) Full absorption spectrum (qualitative analysis)

Emission

Occurs when a photon is ejected (emitted) as the excited particle relaxes to a lower energy state. Photoluminescence emission occurs after absorption of a photon. Chemiluminescence emission occurs due to a chemical reaction.

The wavelength of the emitted radiation is usually longer than the wavelength of the absorbed radiation.

Basic Components of Spectrometers

1.

Energy source
Source of EM radiation or thermal energy Wavelength/frequency selector

2.

Selects a very narrow band from the continuum radiation spectrum to interact with the sample.

3.

Detector
Senses changes in the intensity, polarization, phase angle or direction of propagation of the incident radiation. Signal processor

4.

Electronic component that calculates and converts the electric signals to numerical or graphical data.

The Energy Source

1.

Electromagnetic radiation (see table on the next slide) Continuum source emits a limited spectrum of EM radiation Line source emits only a few selected, narrow wavelength ranges Thermal energy Flame (2,000-3,400K) Plasma (6,000-10,000K)
Chemical sources of energy Exothermic reactions Chemiluminescent reactions

2.

3.

The choice of lamp to use depends on the properties of the sample and on the type of analysis needed (that is, on the frequency required by the type of analysis).

Each kind of lamp listed in the table in the previous slide has its own emission (continuum or line) spectrum. Observe that the intensity at each wavelength (figures on the right) is different. When the lamp is new, the intensities are high and the spectrometer is accurate even at high sample concentrations.

However, as the lamp wears out, the intensities get weaker. This is one reason why spectrometers have to be calibrated often. Some spectrometer models are equipped with a feature that tests the lamps performance.

Wavelength Selection

To eliminate or minimize interferences from other substances that also absorb radiation, the spectrometer has to be set at a wavelength range where (ideally) only the analyte will absorb. A high nominal wavelength produces a better signal-to-noise ratio. A narrow effective bandwidth provides better resolution. Improving one of these two parameters, however, results in the deterioration of the other. Therefore, the two parameters must be balanced.
A high nominal wavelength is better for quantitative analysis. A narrow effective bandwidth is better for qualitative analysis.

Wavelength Selectors

Filters

Absorption filter absorbs radiation from a narrow band of the EM Spectrum (e.g. colored glass)

Interference filter uses constructive and destructive interference to isolate a narrow band of the EM spectrum (better, but more costly than absorption filters)
Unlike filters in which the selected band is fixed, monochromators can be adjusted to any band needed. Two types: fixed wavelength (for quantitative analyses only) and scanning. Instead of filtering or dispersing the electromagnetic radiation, an interferometer simultaneously allows source radiation of ALL wavelengths to reach the detector.

Monochromators

Interferometers

The Monochromator
Functions of each part

Collimating mirror reflects a parallel beam of radiation Diffraction grating optically reflecting surface with a large number of parallel grooves. Diffraction by the grating disperses the radiation in space.

Wavelength selection is done by rotating the diffraction grating. In some, a prism is used instead.

Polychromatic EM radiation source

Focusing mirror combines radiation of the same wavelength.

Exit slit improves the resolution by narrowing the bandwidth.

Observe the effect of narrow and broad bandwidths (can be controlled by adjusting the width of the opening of the exit slit) on the noise and resolution of a samples spectrum.

Too much noise

OK OK

Bad resolution some spectral features almost gone

The Interferometer

The beam splitter splits the radiation beam in two. Each beam is reflected on either of the two mirrors. Then both beams are recombined on the sample/detector. The recombination of the two beams creates an interference pattern (an interferogram) for ALL WAVELENGTHS SIMULTANEOUSLY. The amount of radiation absorbed by the sample, at every wavelength, is calculated (Fourier Transformations) with the aid of a modern computer. Advantages over monochromators:

Jacquinots advantage: higher throughput of source radiation because of interferometers have fewer parts (no slits, and other components from radiation is scattered or lost). Resulting to a very high nominal wavelength (that is, better signal-to-noise ratio) Fellgetts advantage: much faster because all wavelengths are analyzed simultaneously

Detectors

Modern detectors use sensitive transducers that convert light or heat into electrical signals. Ideally the detectors signal should be a linear function of the electromagnetic radiations power.

Photon transducers Silicon photodiodes can be miniaturized and arranged in an array

Thermal transducers Frequently used for IR spectrometers (because IR radiation has insufficient energy to activate photon transducers)

Photon detectors with photosensitive coating: Photon detectors that are semiconductors:

phototubes and photomultipliers silicon photodiodes, photoconductors and photovoltaic cells

Absorption of EM Radiation

When radiation passes through a sample, most of it is transmitted without loss of intensity.

However, intensity is attenuated at certain frequencies due to absorption of the incident radiation by the sample.

Two requirements for absorption:

There must be a mechanism by which the radiations electric field or magnetic field interacts with the analyte The energy of the electromagnetic radiation must exactly equal the difference in energy, E, between two of the analytes quantized energy states.

Vibrational states

Electronic states

Transmittance

Transmittance is defined as the ratio of the electromagnetic radiations power exiting the sample, PT, to that incident on the sample from the source, P0.

Transmittance is related to Absorbance;

Beers Law

Relates Absorbance to analyte concentration;

A = abC

or

A = bC

where A is absorbance, b is the path length or the diameter of the sample cell, C is the analyte concentration and, a and are the absorptivity and molar absorptivity (respectively) of the sample.

For non-reacting multicomponent samples;

Fundamental limitations of Beers Law:

Beers law is a limiting law that is valid only for low concentrations of analyte.

At high concentrations, intermolecular interactions become significant which changes . Higher concentrations also changes the refractive index of the sample, which changes . Chemical reactions and ionization changes .

Chemical limitations:

Instrumental limitations:

Errors due to narrow bandwidth Beers Law is strictly valid only for monochromatic spectrometry. Polychromatic spectrometry always gives a negative deviation from Beers Law, but is minimized if over the wavelength range selected. Therefore, it is advisable to choose the max from broad peaks. When measurements must be made on the slope, linearity is improved by choosing a narrower effective bandwidth (that is, a narrower exit slit).

Stray radiation Due to imperfections in the wavelength selector that allows some radiation to leak and reach the detector. Stray radiation causes negative deviations from linearity if the analyte concentration is too high. Therefore, sample concentrations must be kept low.

A Quick Look at Emission Spectroscopy

energy

Standardization methods

Calibrating signals determines the mathematical relationship between the absorbance and the concentration of the analyte solution.

Weight correction for buoyancy in air:

Primary reagents high purity solids used to precisely determine the concentration of secondary standard solutions (which is used to quantitatively react directly/indirectly with the analyte)

External standards Solutions containing known quantities of the analyte (usually used for constructing calibration curves)
Method of Standard additions (see next slides) Useful for minimizing matrix errors

The concentration of the analyte can be calculated from the x-intercept

Internal standards Substances added to all samples and standards, whose signal is measured in addition to the signal produced by the analyte.

Calibration curves (using statistical methods)

Slope:
= 2

Y-intercept:

Blank corrections

Total Youden Blank:

UV-Vis Spectroscopy

When a molecule or ion absorbs ultraviolet or visible radiation it undergoes a change in its valence electron configuration.
Instrument Designs Filter Photometer Single-Beam Fixed-Wavelength Spectrophotometer Double-Beam In-Time Scanning Spectrophotometer Diode Array Spectrophotometer Instruments using monochromators for wavelength selection are called spectrometers. In absorbance spectroscopy, where the transmittance is a ratio of two radiant powers, the instrument is called a spectrophotometer.

The Filter Photometer

Either an absorption or interference filter

A single-beam instrument.

Portable, rugged, easy to maintain and inexpensive.

Calibrated to 0%T with the shutter closed, then to 100%T with the blank sample. Must be recalibrated every time the filter is changed.

The Single-Beam Fixed-Wavelength Spectrophotometer

Calibrated and used in the same way as photometers.

More appropriate for a quantitative analysis than for a qualitative analysis. Fixed-wavelength single-beam spectrophotometers are not practical for recording spectra since manually adjusting the wavelength and recalibrating the spectrophotometer is awkward and time-consuming. The accuracy of a single-beam spectrophotometer is limited by the stability of its source and detector over time.

The Double-Beam In-Time Scanning Spectrophotometer

The chopper controls the radiations path, alternating it between the sample, the blank, and a shutter. The signal processor uses the choppers known speed of rotation to resolve the signal reaching the detector into that due to the transmission of the blank (P0) and the sample (PT). By including an opaque surface as a shutter it is possible to continuously adjust the 0%T response of the detector.

The limitations of fixed-wavelength, single-beam spectrophotometers are minimized by using the double-beam intime spectrophotometer.
The effective bandwidth of a double-beam spectrophotometer is controlled by means of adjustable slits at the entrance and exit of the monochromator. A scanning monochromator allows for the automated recording of spectra. Double-beam instruments are more versatile than single-beam instruments, being useful for both quantitative and qualitative analyses; they are, however, more expensive.

The Diode Array Spectrophotometer

A linear photodiode array consists of multiple detectors, or channels, allowing an entire spectrum to be recorded. Source radiation passing through the sample is dispersed by a grating. The linear photodiode array is situated at the gratings focal plane, with each diode recording the radiant power over a narrow range of wavelengths. One advantage of a linear photodiode array is the speed of data acquisition, which makes it possible to collect several spectra for a single sample. Individual spectra are added and averaged to obtain the final spectrum. Signal averaging improves the signal-to-noise ratio.

The Sample Compartment

Sample compartments of modern spectrophotometers prevent loss of radiation due to scattering and reflection. The addition of stray radiation is likewise prevented.
Liquid or dissolved samples are placed in UV-Vis transparent cells made of fused-silica, quartz, glass or plastic. For analyses performed below 300nm wavelengths, quartz or fused-silica cells must be used.

Commonly, cells have 1cm internal diameters (path-lengths). Cells with longer path-lengths are used for very dilute solutions and gaseous samples. High quality cells have rectangular shapes to reduce radiation losses to reflection. These cells are also usually matched (having identical optical properties). Cylindrical cells are of lower quality and are usually used for single-beam instruments. Fiber optic probes are used for real time monitoring or for remote measurements of a samples spectrum.

Sample cells used for UV-Vis spectroscopy

Examples of fiber optic probes

Sample Preparation for UV-Vis Spectrophotometry

The solvent must not absorb in the same region as the analyte. The most common solvents used are listed in the table on the next slide. The way the solvent influences the shifts in the wavelength of absorbed radiation must also be noted. In choosing the right solvent, one must also consider its possible interactions with the analyte (such as, H-bonding).

The figure on the next slide shows the absorption spectra of phenol in two different solvents: ethanol and isooctane. There is a loss of resolution when ethanol is the solvent due to H-bonding with the analyte, phenol. Other interactions that must be considered include complex formation, ionization and possible chemical reactions.

Chromophores

Chromophores are groups of atoms that absorb in the UV-Vis region of the EM spectrum.
Auxochromes are groups that increase the absorption intensity or that shift the absorption wavelength.

Bathochromic (red) shift shift to longer (lower energy) wavelengths Hypsochromic (blue) shift shift to shorter (higher energy) wavelengths

Hypochromic effect decrease in absorption intensity

Hyperchromic effect increase in absorption intensity

The greater the conjugation of -bonds, the greater the shift of spectra to longer (lower energy) wavelengths Lone pairs shift spectra to shorter (higher energy) wavelengths

UV-Vis Spectrum Generalizations

These generalizations only serve as a guide and could be more useful if combined with FTIR and NMR data:
A single band of low to medium intensity at < 220nm usually indicates an n* transition. Possibilities are amines, alcohols, ethers, thiols and cyano (weak n*) groups provided these are not part of conjugated systems. A single band of low intensity within 250 and 360nm with no absorption bands within 200-250nm usually indicates n* transition. A simple (unconjugated) chromophore, generally that contains an O, N or S atom is present. Examples are azides, nitriles, amides, esters, carboxylic acids, nitrates, aldehydes, and ketones. Two bands of medium intensity both with max above 200nm generally indicate the presence of an aromatic system. A third band near 200nm generally means a polynuclear aromatic system is present. In non-polar solvents, fine spectral structure is found in longer wavelengths.

1.

2.

3.

4. High intensity bands above 210nm generally represent an ,-unsaturated ketone, a diene or a polyene. The longer the conjugated system, the longer the observed wavelength. 5. Simple ketones, acids, esters, amides, and other compounds containing both systems and unshared electron pairs show two absorptions: an n* transition at longer wavelengths (>300 nm, low intensity) and a * transition at shorter wavelengths (<250 nm, high intensity).
6. Compounds that are highly colored (have absorption in the visible region) are likely to contain a long-chain conjugated system or a polycyclic aromatic chromophore. However, some simple nitro, azo, nitroso, -diketo, polybromo, and polyiodo compounds may also exhibit color, as may many compounds with quinoid structures.

Infrared (IR) Absorption

Absorption of low energy IR radiation is only enough to promote a molecule or polyatomic ion to a higher vibrational energy state.
Only molecules and polyatomic ions absorb IR radiation because only these have chemical bonds that vibrate. Symmetric bonds in molecules do not absorb IR radiation. Only bonds that have a dipole moment can absorb IR radiation.

Chemical bonds between different pairs of atoms vibrate at different frequencies and, therefore, will absorb at different frequencies.

Infrared Spectroscopy and Chemical Bonding

Note: Infrared spectra of compounds are often complicated by overtones, combination bands and difference bands all on top of the fundamental absorption vibrations.

IR Spectrophotometers

IR spectrophotometers have similar block diagrams as UV-Vis spectrophotometers

Filter photometers portable, dedicated analyzers for gaseous samples. Single-beam

Double-beam preferred over single-beam optics because IR sources and detectors are less stable than those for UV-Vis. Also, error corrections for IR absorption by CO2 and H2O are easier for double-beam optics. Fourier-transform (with interferometer) Single-beam instrument, therefore, a background spectrum has to be taken and subtracted from the sample spectrum to correct for atmospheric CO2 and H2O absorbance. Rapid data acquisition, which allows improvement of the signal-to-noise ratio through signal averaging.

IR instruments are generally classified into two groups:

Dispersive (those that have gratings, prisms or monochromators) and Fourier Transform (those with interferometers)

FTIR Analysis Methods

Transmission

Gaseous samples Placed in a small chamber with NaCl or KBr windows Path-length is usually 10cm (longer, if mirrors are used)

Liquid samples A thin film of a non-aqueous non-volatile sample is prepared by placing a drop between two NaCl plates. Volatile samples and sample solutions (both non-aqueous) must be placed in sealed cells; two NaCl plates separated by a teflon tube. The tube length is equal to the pathlength. Solid samples Transparent samples can be placed directly in the path of the IR beam using the appropriate sample holder. Opaque samples can be dissolved in a suitable solvent (e.g. Nujol or CCl4) and analyzed as described above. Alternatively, the sample can be mixed with KBr and pressed into a transparent pellet.

Attenuated Total Reflectance (ATR)

Aqueous samples must be prepared by applying a thin film on an ATR crystal (e.g. ZnSe, which is an IR transparent crystal with a high refractive index).

Solid samples can also be analyzed with ATR.

ATR spectra are similar (but, not identical) to transmission spectra. Radiation from the source enters the ATR crystal, where it undergoes a series of total internal reflections before exiting the crystal. During each reflection, the radiation penetrates into the sample to a depth of a few microns. The result is a selective attenuation of the radiation at those wavelengths at which the sample absorbs.

Diffuse Reflectance

Powdered samples are mixed with KBr. Diffuse reflection occurs when light penetrates the solid and is scattered by refraction and reflection towards neighboring crystals or towards the detector, where it is analyzed.

The spectra are similar (but, not identical) to transmission spectra.

The disadvantage of using KBr is the possibility of moisture contamination. (Also likely in pelletization).

The IR Background Spectrum

CO2

H2O

The two absorptions at 2350 cm1 are due to the asymmetric stretching modes of carbon dioxide. The groups of peaks centered at 3750 cm1 and 1600 cm1 are due to the stretching and bending modes of atmospheric (gaseous) water molecules (that is, water vapor).
The bell-shaped curve is due to differences in the output of the IR source corrected by a feature called, autobaseline.

Infrared Spectra

Continued on next slide

Main References:

Harvey, D. Modern Analytical Chemistry. McGraw-Hill, 2000.

Pavia, D. et al. Introduction to Spectroscopy, 4th ed. Brooks/Cole, Cengage Learning 2009.
Skoog, D. et al. Fundamentals of Analytical Chemistry, 8th edition. Thomson-Brooks/Cole, 2004. Silberberg. Chemistry: The Molecular Nature of Matter and Change, 4 th ed.