MOLECULAR ABSORPTION SPECTROSCOPY : THEORY, INSTRUMENTATION & APPLICATION CHAPTER 2

MOLECULAR ABSORPTION SPECTROSCOPY : THEORY, INSTRUMENTATION & APPLICATION CHAPTER 2

COMPONENTS OF INSTRUMENTS FOR OPTICAL SPECTROSCOPY

General Design of Optical Instruments Absorption Emission

Five Basic Optical Instrument Components 1) Source A stable source of radiant energy at the desired wavelength (or range). 2) Sample Container A transparent container used to hold the sample (cells, cuvettes, etc). 3) Wavelength Selector A device that isolates a restricted region of the EM spectrum used for measurement (monochromators, prisms & filters). 4) Detector/Photoelectric Transducer Converts the radiant energy into a useable signal (usually electrical). 5) Signal Processor & Readout Amplifies or attenuates the transduced signal and sends it to a readout device as a meter, digital readout, chart recorder, computer, etc.

I. Sources of Radiation Generate a beam of radiation that is stable and has sufficient power. A. Continuum Sources emit radiation over a broad wavelength range and the intensity of the radiation changes slowly as a function of wavelength. This type of source is commonly used in UV, visible and IR instruments. - Deuterium lamp is the most common UV source. - Tungsten lamp is the most common visible source. - Glowing inert solids are common sources for IR instruments.

B. Line Sources Emit a limited number lines or bands of radiation at specific wavelengths. - Used in atomic absorption spectroscopy - Usually provide radiation in the UV and visible region of the EM spectrum. - Types of line source: 1) Hollow cathode lamps 2) Electrodeless discharge lamps 3) Lasers-Light amplification by stimulated emission of radiation

II. Wavelength Selectors Wavelength selectors output a limited, narrow, continuous group of wavelengths called a band. Two types of wavelength selectors: 1) Filters 2) Monochromators

A. Filters - Two types of filters: 1) Interference Filters 2) Absorption Filters B. Monochromators - Wavelength selector that can continuously scan a broad range of wavelengths - Used in most scanning spectrometers including UV, visible, and IR instruments.

III. Radiation Transducer (Detectors) Early detectors in spectroscopic instruments were the human eye, photographic plates or films. Modern instruments contain devices that convert the radiation to an electrical signal. Two general types of radiation transducers: a. Photon detectors b. Thermal detectors

A. Photon Detectors - Commonly useful in ultraviolet, visible, and near infrared instruments. - Several types of photon detectors are available: 1. Vacuum phototubes 2. Photomultiplier tubes 3. Photovoltaic cells 4. Silicon photodiodes 5. Diode array transducers 6. Photoconductivity transducers

B. Thermal Detectors - Used for infrared spectroscopy because photons in the IR region lack energy to cause photoemission of electrons. - Three types of thermal detectors: 1. Thermocouples 2. Bolometers 3. Pyroelectric transducers

IV. Sample Container Sample containers, usually called cells or cuvettes must have windows that are transparent in the spectral region of interest. There are few types of cuvettes: - quartz or fused silica - silicate glass - crystalline sodium chloride Quartz or fused silica - required for UV and may be used in visible region Silicate glass - Cheaper compared to quartz. Used in UV. Crystalline sodium chloride - Used in IR.

Spectrometer - is an instrument that provides information about the intensity of radiation as a function of wavelength or frequency. Spectrophotometer - is a spectrometer equipped with one or more exit slits and photoelectric transducers that pemits the determination of the ratio of the radiant power of two beams as a function of wavelength as in absorption spectroscopy.

SUMMARY Types of source, sample holder and detector for various EM region REGION SOURCE SAMPLE HOLDER DETECTOR Ultraviolet Deuterium lamp Quartz/fused silica Phototube, PM tube, diode array Visible Tungsten lamp Glass/quartz Phototube, PM tube, diode array Infrared Nernst glower (rare earth oxides or silicon carbide glowers) Salt crystal e.g. crystal sodium chloride Thermocouples, bolometers

ULTRAVIOLET-VISIBLE SPECTROSCOPY

In this lecture, you will learn: Absorption process in UV/VIS region in terms of its electronic transitions Molecular species that absorb UV/VIS radiation Important terminologies in UV/VIS spectroscopy

INSTRUMENTATION Important components in a UV-Vis spectrophotometer 1 2 3 4 Source lamp Sample holder selector Detector 5 Signal processor & readout UV region: -Deuterium lamp; H 2 discharge tube Quartz/fused silica Prism/monochromator Phototube, PM tube, diode array Visible region: - Tungsten lamp Glass/quartz Prism/monochromator Phototube, PM tube, diode array

Instrumentation UV-Visible instrument 1. Single beam 2. Double beam

Single beam instrument

Single beam instrument - One radiation source - Filter/monochromator ( selector) - Cells - Detector - Readout device

Changing of wavelength is accompanied by a change in light intensity. Thus spectral scanning is not possible. Single beam instrument Disadvantages: Two separate readings has to be made on the light. This result in some error because the fluctuations in the intensity of the light do occur in the line voltage, the power source and in the light bulb btw measurements.

Double beam instrument Double-beam instrument with beams separated in space

Double-beam instrument Advantages: 1. Compensate for all but most short-term fluctuations in the radiant output of the source as well as for drift in the transducer and amplifier. 2. Compensate for wide variations in source intensity with. 3. Continuous recording of transmittance or absorbance spectra.

ORGANIC COMPOUNDS INORGANIC SPECIES MOLECULAR SPECIES THAT ABSORB UV/VISIBLE RADIATION CHARGE TRANSFER

Definitions: Organic compounds Chemical compound whose molecule contain carbon E.g. C 6 H 6, C 3 H 4 Inorganic species Chemical compound that does not contain carbon. E.g. transition metal, lanthanide and actinide elements. Cr, Co, Ni, etc Charge transfer A complex where one species is an electron donor and the other is an electron acceptor. E.g. iron (III) thiocyanate complex

PERIODIC TABLE OF ELEMENTS

ULTRAVIOLET-VISIBLE SPECTROSCOPY In UV/VIS spectroscopy, the transitions which result in the absorption of EM radiation in this region are transitions between electronic energy levels.

Molecular absorption In molecules, not only have electronic level but also consists of vibrational and rotational sub-levels. This result in band spectra.

Types of transitions 3 types of electronic transitions -, and n electrons - d and f electrons - charge transfer electrons

What is σ, and n electrons? single covalent bonds (σ) H + O + H H O H or H O H lone pairs(n) O C O or O C O double bonds ( ) N N or N N triple bond ( )

Sigma ( ) electron Electrons involved in single bonds such as those between carbon and hydrogen in alkanes. These bonds are called sigma ( ) bonds. The amount of energy required to excite electrons in bond is more than UV photons of wavelength. For this reason, alkanes and other saturated compounds (compounds with only single bonds) do not absorb UV radiation and therefore frequently very useful as transparent solvents for the study of other molecules. For example, hexane, C 6 H 14.

Pi ( ) electron Electrons involved in double and triple bonds (unsaturated). These bonds involve a pi ( ) bond. For exampel: alkenes, alkynes,conjugated olefins and aromatic compounds. Electrons in bonds are excited relatively easily; these compounds commonly absorb in the UV or visible region.

Examples of organic molecules containing bonds. CH 2 CH 3 H CH 3 C C H H C C C H propyne H C C C H H H H H C C ethylbenzene benzene C C H H H 1,3-butadiene

n electron Electrons that are not involved in bonding between atoms are called n electrons. Organic compounds containing nitrogen, oxygen, sulfur or halogens frequently contain electrons that re nonbonding. Compounds that contain n electrons absorb UV/VIS radiation.

Examples of organic molecules with nonbonding electrons. : NH 2.. O : C R H 3 C H C C aminobenzene Carbonyl compound :.. Br.. H If R = H aldehyde 2-bromopropene If R = C n H n ketone

ABSORPTION BY ORGANIC COMPOUNDS UV/Vis absorption by organic compounds requires that the energy absorbed corresponds to a jump from occupied orbital to an unoccupied orbital of greater energy. Generally, the most probable transition is from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO).

Energy * * n * n * Electronic energy levels diagram * * Antibonding Antibonding Unoccupied levels n Nonbonding Bonding Occupied levels Bonding

Electronic transitions Increasing energy * * n * n * In alkanes In alkenes, carbonyl compounds, alkynes, azo compounds In oxygen, nitrogen, sulfur and halogen compounds In carbonyl compounds

Electronic transitions * transitions The energy required to induce a * transition is large (see the arrow in energy level diagram). Never observed in the ordinarily accessible ultraviolet region. This type of absorption corresponds to breaking of C-C, C-O, C-H, C-X,.bonds

n * transitions - Saturated compounds containing atoms with unshared electron pairs (non-bonding electrons). - Compounds containing O, S, N and halogens can absorb via this type of transition. - Absorption are typically in the range, 150-250 nm region and are not very intense. - range: 100 3000 cm -1 mol -1 - Absorption maxima tend to shift to shorter in polar solvents. e.g. H 2 O, CH 3 CH 2 OH

Some examples of absorption due to n * transitions Compound max (nm) max H 2 O 167 1480 CH 3 OH 184 150 CH 3 Cl 173 200 CH 3 I 258 365 (CH 3 ) 2 O 184 2520 CH 3 NH 2 215 600

n * transitions - Unsaturated compounds containing atoms with unshared electron pairs (nonbonding electrons) - These result in some of the most intense absorption in range, 200 700 nm - Unsaturated functional group - to provide the orbitals - range: 10 100 Lcm -1 mol -1

* transitions - Compounds with unsaturated functional groups to provide the orbitals. - These result in some of the most intense absorption in range, 200 700 nm - range: 1000 10,000 Lcm -1 mol -1

Examples n * and * H O H C C H H * at 180 nm n * at 290 nm

MOLECULAR SPECIES THAT ABSORB UV/VISIBLE RADIATION (A) Absorption by organic compounds 2 types of electrons are responsible: i. Shared electrons that participate directly in bond formation ( and bonding electrons) ii. Unshared outer electrons (nonbonding or n electrons)

Absorption by organic compounds The shared electrons in single bonds, C-C or C-H ( electrons) are so firmly held. Therefore, not easily excited to higher E levels. Absorption ( *) occurs only in the vacuum UV region ( 180 nm). Electrons in double & triple bonds (electrons) are more loosely held. Therefore, more easily excited by radiation. Absorptions ( *) for species with unsaturated bonds occur in the UV/VIS region ( 180 nm)

Absorption by organic compounds CHROMOPHORES Unsaturated organic functional groups that absorb in the UV/VIS region.

Typical organic functional groups that serve as chromophores Chromophores Chemical structure Type of transition Acetylenic -C C- * Amide -CONH 2 *, n * Carbonyl >C=O *, n * Carboxylic acid -COOH *, n * Ester -COOR *, n * Nitro -NO 2 *, n * Olefin >C=C< *

Absorption by organic compounds AUXOCHROME Groups such as OH, -NH 2 & halogens that attached to the double bonded atoms cause the normal chromophoric absorption to occur at longer (red shift).

Effect of Multichromophores on Absorption More chromophores in the same molecule cause bathochromic effect ( shift to longer ) and hyperchromic effect (increase in intensity). In conjugated chromophores, * electrons are delocalized over larger number of atoms. This cause a decrease in the energy of * transitions and an increase in due to an increase in probability for transition.

Absorption by organic compounds Factors that influenced the : i) Solvent effects (shift to shorter : blue shift) ii) Structural details of the molecules

Absorption spectra for typical organic compounds

Important terminologies Hypsochromic shift (blue shift) - Absorption maximum shifted to shorter Bathochromic shift (red shift) - Absorption maximum shifted to longer

Terminology for Absorption Shifts Nature of Shift To Longer Wavelength To Shorter Wavelength To Greater Absorbance To Lower Absorbance Descriptive Term Bathochromic Hypsochromic Hyperchromic Hypochromic

(B) Absorption by inorganic species Involving d and f electrons absorption 3d & 4d electrons - 1 st and 2 nd transition metal series e.g. Cr, Co, Ni & Cu - Absorb broad bands of VIS radiation - Absorption involved transitions between filled and unfilled d-orbitals with energies that depend on the ligands, such as Cl -, H 2 O, NH 3 or CN - which are bonded to the metal ions.

Absorption spectra of some transition-metal ions and rare earth ions Most transition metal ions are colored (absorb in UV-VIS) due to d d electronic transitions

Absorption by inorganic species 4f & 5f electrons - Ions of lanthanide and actinide elements - Their spectra consists of narrow, welldefined characteristic absorption peaks.

(C) Charge transfer absorption Absorption involved transfer of electron from the donor to an orbital that is largely associated with the acceptor. an electron occupying in a or orbital (electron donor) in the ligand is transferred to an unfilled orbital of the metal (electron acceptor) and vice-versa. e.g. red colour of the iron (III) thiocyanate complex

Absorption spectra of aqueous charge transfer complexes

Quantitative Analysis The fundamental law on which absorption methods are based on Beer s Law (Beer- Lambert Law).

Measuring Absorbance You must always attempt to work at the wavelength of maximum absorbance ( max ). This is the point of maximum response, so better sensitivity and lower detection limits. You will also have reduced error in your measurement.

Quantitative Analysis Calibration curve method Standard addition method

Calibration curve method - A general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard sample of known concentration.

Absorbance Standard Calibration Curve How to measure the concentration of unknown? Practically, you have measure the absorbance of your unknown. Once you know the absorbance value, you can just read the corresponding concentration from the graph.

Absorbance How to produce standard calibration curve Prepare a series of standard solution with known concentration. Measure the absorbance of the standard solutions. Calibration standard Plot the graph Abs vs concentration of std. Find the best straight line. Stock solution 100 ppm

The slope of the line, m: m = y 2 y 1 x 2 x 1 The intercept, b: b = y mx Thus, the equation for the least-square line is: y = mx + b

Concentration, x 5 10 15 20 25 y = mx + b From the least-square line equation, you can calculate the new y values by substituting the x value. Then plot the graph.

Standard addition method - used to overcome matrix effect - involves adding one or more increments of a standard solution to sample aliquots of the same size. - Each solution is diluted to a fixed volume before measuring its absorbance.

Absorbance Standard Addition Plot

How to produce standard addition curve? 1. Add same quantity of unknown sample to a series of flasks. 2. Add varying amounts of standard (made in solvent) to each flasks, e.g. 0, 5, 10, 15 ml). 3. Fill each flask to line, mix and measure.

Standard Addition Methods Single-point standard addition method Multiple standard addition method

Standard addition - if Beer s Law is obeyed, A = bv std C std + bv x C x V t V t = kv std C std + kv x C x k is a constant equal to b V t

Standard Addition - Plot a graph: A vs V std A = mv std + b where the slope m and intercept b are: m = kc std ; b = kv x C x

C x can be obtained from the ratio of these two quantities: m and b b = kv x C x m kc std C x = bc std mv x

Example: 10 ml aliquots of raw-water sample were pipetted into 50.0 ml volumetric flasks. Then, 0.00, 5.00, 10.00, 15.00 and 20.00 ml respectively of a standard solution containing 10 ppm of Fe 3+ were added to the flasks, followed by an excess of aqueous potassium thiocyanate in order to produce the red ironthiocyanate complex. All the resultant solutions were diluted to volume and the absorbance of each solution was measured at the same.

The results obtained: Vol. of std added (ml) Absorbance (A) 0 0.215 5.00 0.424 10.00 0.625 15.00 0.836 20.00 1.040 Calculate the concentration of Fe 3+ (in ppm) in the raw-water sample

Absorbance Absorbance vs Vol. of std added 1.2 1 0.8 b = 0.24 (V std ) 0 = -6.31 ml 0.6 0.4 Slope, m = 0.0382 0.2 0-10 -5 0 5 10 15 20 25 Vol. of std Note: From the graph, extrapolated value represents the volume of reagent corresponding to zero instrument response.

The unknown concentration of the analyte in the solution is then calculated: C sample = -(V std ) 0 C std V sample C x = bc std mv x

SELF-EXERCISE The chromium in an aqueous sample was determined by pipetting 10.0 ml of the unknown into each of 50.0 ml volumetric flasks. Various volumes of a standard containing 12.2 ppm Cr were added to the flasks, following which the solutions were diluted to the mark. Volume of unknown (ml) Volume of standard (ml) Absorbance 10.0 0.0 0.201 10.0 10.0 0.292 10.0 20.0 0.378 10.0 30.0 0.467 10.0 40.0 0.554 i) Plot a suitable graph to determine the concentration of Cr in the aqueous sample.

Visible Spectroscopy The portion of the EM spectrum from 400-800 is observable to humans- we (and some other mammals) have the adaptation of seeing color at the expense of greater detail. 400 500 600 700 800, nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780

Visible Spectroscopy When white (continuum of λ) light passes through, or is reflected by a surface, those λs that are absorbed are removed from the transmitted or reflected light respectively. What is seen is the complimentary colors (those that are not absorbed). This is the origin of the color wheel.

Visible Spectroscopy Organic compounds that are colored are typically those with extensively conjugated systems (typically more than five). Consider b-carotene. b-carotene, max = 455 nm λ max is at 455 nm in the far blue region of the spectrum. This is absorbed. The remaining light has the complementary color of orange.

Visible Spectroscopy lycopene, max = 474 nm O H N λ max for lycopene is at 474 nm in the near blue region of the spectrum this is absorbed, the compliment is now red. λ max for indigo is at 602 nm in the orange region of the spectrum. This is absorbed, the compliment is now indigo! N H O indigo