rmory acer meuivus any separ kare RBA SPECTROSCOPY Lf Spectroscopy, spectrometry or spectrophotometry isthe measurement of eleetromagneti radiation absorbed, scattered, reflected or emitted by atoms, molecules or other chemical species. It isthe interaction of EMR with matter. ELECTROMAGNETIC RADIATION Radiation is a form of energy and we are constantly reminded of its presence via ow sense of sight and ability ee Padua wane, muconassy wine gh, 7-047? tus pig etaclormagrelic wause Yat olgfor 127 tach oto wn wanelsaglh-. Hecrmmagrelé wauss asx pocuscest by we momen Sf “cuclacally charged paclict", Thay tamed +orayh — Consists of electrical and magnetic fields Orange light, for example, has a frequency of about 5 x 10" He (often quoted as 5 x 10° MHz - megahertz). ‘That means that 5 x 10'* wave peaks pass a given point every second, ‘There isa simple relationship between the wavelength, frequency ofa particular colour of light and the speed of light? Page 1 of 11 Scanned with CamSeanner(creo te, v a < ‘c=Av’ ! : revenge ._. and you can rearrange this to work out the wavelength from a given frequency and vice versa: ‘These relationships mean that if you increase the frequency, you must decrease the wavelength. higher trequency means shorerwavelengn iM VV short wavel ahigher frequency wavelength means a Jow frequenc) For example, if you were told that a particular colour of red light had a wavelength of 650 nm, and a green had a wavelength of 540 nm, which has the higher frequency? Quantum Theory FOR raving dual'penonectty? “The quantum theory is based on radiation being thought of as a stream of particles known as photons travelling ina wave-like pattern. In 1900, Max Planck proposed that particles such as atoms and molecules exist in defined energy levels and a change of energy level involves the absorption or emission of diserete amounts or packets of energy called ‘quanta’, This is EMR. The energy absorbed or emitted is represented by: E=by ‘This shows that the energy of a photon is directly proportional to the frequency of radiation, Where h = Planck’s constant = 6.626 x 10 Joules (J), v= the frequency of the photon (s' or Hz) Since c=Av then, E=he/). Bis inversely proportional to wavelength Page 2 of 11. ‘Scanned with CamScanner4 pre shorter the, wavelength or the greater the frequency means the greater the energy of the photon. Blectromagnetic Spectrum ‘The electromagnetic spectrum is the range of all the possible frequencies of EMR. The human eye is only sensitive to a tiny proportion of the total EM spectrum between about 380-780 nm (visible region). A quantum of energy absorbed or emitted by an atom corresponds to EMR of a specific frequency or wavelength on the E, : 107 10% 10+ 105 10% 10% 10? 10 sot 108 10" into wavs ‘RADIO WA) catl Towers NUCLEAR MAGNETIC RESONANCE ne ne dagie “aut nents Sour: an aia long waucengiy . ‘MICROWAVES Reaclacs Mera owns ‘ INFRARED People ‘VISIBLE AND ULTRAVIOLET light bulb” * XRAYS Yor machine GAMMA RAYS Ridlie ouive eluents COSMIC RAYS | most eae show wanelenath Fm raduo MOLECULAR ROTATION AND ELECTRON SPIN RESONANCE MOLECULAR ‘VIBRATIONS ATOMIC AND MOLECULAR ELECTRONIC TRANSITIONS DIFFRACTION AND INTERFERENCE IONIZATION OF GASES Scanned with CamScanner‘Spectroscopy . “The sample being analysed is preserved, it is a non-destructive method of analysing substances. ‘A molecule of any substance has an internal energy: Erorat = Eexecrnontc + Evisrationat + Erotationan. When EMR falls on a substance, processes occur in the molecules of the compound. These interactions provide knowledge about the structure of the molecules. These interactions between EMR and a compound lead to spectroscopy. There are different types of spectroscopy and each type differs in the type of EMR, the compound is subjected to, such as: + Ultra Violet/Visible © Infrared ‘+ Nuclear Magnetic Resonance (NMR) Photocopy pg 348-351 Chem for CAPE Ultra Violet-Visible (UV/VIS) Spectroscopy ‘When atoms or molecules are subjected to intense heat orto an electrical discharge, they may absorb energy and their electrons become excited. On returning to the normal or ground state, such excited species may emit radiation. Ultraviolet and visible spectroscopy is possible because outer electrons of atoms or ions in compounds absorb energy in the ultraviolet or visible part of the EM spectrum, when they are excited. Relationship of absorption to structure The molecular orbital theory states that when 2 atomic orbitals join together, the end result must be 2 molecular orbitals, a bonding molecular orbital and an anti-bonding molecular orbital (which is . “node” where heres 2a chance oes 7 sof findng fhe elecrons i Page 4 of 11, ‘Scanned with CamScannernuaryunar metiuus atu Separeuen recnniques: ey i-bonding orbital is always she \f a star after its symbol. Example, o* Notice that when a bonding orbital forms, it is at a lower energy than the original atoms. Energy is released when the bonding orbital is formed. The miolecule formed is more energetically stable than the original atoms. However, an anti-bonding molecular orbital is less energetically stable than the original atoms. ‘A bonding orbital is stable because of the attractions between the nuclei and the electrons. In an anti-bonding orbital there are no equivalent attractions - instead you get repulsions. There is very little chance of finding the electrons between the two nuclei - and in fact half-way between the nuclei there is zero chance of finding them. ‘There is nothing to stop the two nuclei from repelling each other apart. So in the formation of molecule, both of the electrons go into the bonding molecular orbital, because that produces the greatest stability ~ more stable than having separate atoms, and a lot more stable than having the electrons in the ant-bonding orbitals 4, oN Ground state orbitals i | Antibonding 0 (Sigma) M.0_ SFE SELES | 0° (Sigma sar ant-bondin (pi) MO nat Pifuned shaker, wear | n¥ (pi star) anti-bonding . n ate ‘bonding) ca orbital awaataele Ina double bond there are 2 bonding orbitals (one o and one =) exiled tte ot ger en / or non bonding When excitation occurs, an electron from one ofthe bonding molecular érbitals [o, x, n] is promoted to a,vacant anti-bonding orbital (o*, x*]. (0° fant beneing) | hseseromty empty ‘3° (ant-boning) 1n (rorbonding) | These canta ona pas ————_ rr) ‘These contain rrr bonding pars ofelectons — megy : ; “The energy gaps between these levels determine the frequency (or wavelength) of the light absorbed, and those gaps will be different in different compounds. Note: The o electrons are most tightly bound and will require the most-energy to promote or excite them. Transitions: decreasing order of energy is as follows oot > ont >not >a at >not > nat Saturated groups (only having ¢ bonds) do not exhibit strong absorption in the UV/VIS region due to the large amount of energy required to excite o-electrons. Consequently, the following elec tions can occur by . the absorption of ult ic compounds: Page 5 of 11 ‘Scanned with CamScannerF non n>o* not ‘Transitions of the n —> * and m= —» m*type occur in molecules with unsaturated centres; they require less energy and occur at longer wavelengths than transitions to o* anti-bonding orbitals. As such, in order for a compound to absorb UV/VIS radiation, the molecules must have either: © melectronsgr + Jone pairs of electrons Explaining what happens when light is absorbed (0* (enf-boncing) | esses aiy 7¢* ant-boncing) : 1N hon-toncing) | These contain lone pas 3% (bonding) ‘These mntsin normal CG feoncing) | PDF of eccrs egy The larger the energy jump, the lower the wavelength of the light absorbed (higher the frequency). Chromophores dee fecal Grp wat abenies: WW)ViS radiation’ whet er wlour 19 PT Nunsalweled gp of Stans) A chromophore (colour-bearing) is a structural feature (a particular group) within a molecule responsible for absorption of UV/VIS radiation. Itis that part of the molecule containing electrons involved in electronic transition giving rise to the absorption band in the UV/VIS region of the EM spectrum. ‘The wavelength or frequency of UV/VIS radiation absorbed is characteristic of a chromophore. This can be used to identify chromophores. Groups containing the non-bonding electrons enhance the chromophore’s ability to absorb. Increasing the extent of delocalisation in a system containing double bonds increases the = intensity of the absorption. “+001 [ Chromophore “Typical Peas ‘Transitions Alkene C=C 175 Zon ‘Conjugated alkene C= C- 220 Ton ‘Alkyne C=C 180 (large ema) nom 225 (small Emax) Carbonyl C=O 185 zon 280 n+ nt Carboxyl_COOH 205 nm Amide CONH 215 aoe Page 6 of 11 ‘Scanned with CamScanner‘Azo_N=N 340 no Nitro NOz 280 aoa Nitrate NOs = [270 axe ‘Alcohol OH 180 nae Refer to pg 360 table 25.1 Chem for CAPE Colour _ When white light falls upon a sample, the light may be totally reflected, in which case the substance appears white or the light may be totally absorbed, in which case the substance appears black. If, however, only a portion ofthe light is absorbed and the balance is reflected, the colour of the sample is determined by the reflected light. Example: If violet is absorbed, the sample appears yellow-green. “The colours are described as complementary. However, many substances which appear colourless do have absorption spectra. In this instance, the absorption will take place in the infra-red or ultraviolet and not in the visible region. The relationship between light absorption and colour observed is given in the following table: Colour absorbed Colour observed ‘Absorbed radiation/ nm Violet ‘Yellow-green 400-435 Blue Yellow 435-480 Green-blue Orange 480-490 Blue-green Red 490-500 Green Purple 300-560 ‘Yellow-green Violet 560-580 Yellow Blue 580-595 ‘Orange Green-blue 595-605 Red Blue-green 605-750 ‘A close relationship exists between the colour of a substance and its electronic structure. Insert pg 52 CAPE lecture notes An ultraviolet-visible spectrophotometer or a colorimeter is used to quantitatively measure the amount of light absorbed by a solution. Insert fig, 25.6, sample cuvelte. | ——]detector UV and. lamp reference es: cnvelte detector: ‘igure 6 :Schematic of the.components of a typical double-beam UV-Vis spectrophotometer Page 7 of 11 ‘Scanned with CamScanner“analysing samples by UV/VIS ‘The light source is a deuterium or tungsten lamp which supplies a constant amount of light to the sample. The monochromator selects the required wavelength to:be absorbed by the sample. The cell or cuvette is a transparent plastic or glass container for the sample or reference. The beam splitter divides the Jight into 2 paths, one passes through the sample cell and the other passes through the refe¥enice eth eee at the detector which compares the 2 beams and records it. To find the concentration of an unknown sample, the instrument is 1* set at zero absorbance using a blank sample. Then a series of standard solutions of known, concentrations are prepared, The absorbance of each solution and the unknown sample are then measured at a known maximum wavelength. A graph of absorbance versus tlie known concentration of the series of standards is plotted to generate a calibration curve. The curve is then used to determine the concentration of the unknown. Sample Preparation — to determine the concentration of a sample The solvent chosen must be the highest grade of purity and the sample must be soluble in the solvent chosen Prepare a ‘blank’ which contains only the solvent (as reference) For organic compounds which are not water soluble, must be dried thoroughly under vacuum in a desiccator ‘An accurate mass of the sample is weighed and dissolved in the appropriate solvent, made up to the mark in a volumetric flask. Several solutions are made up of the compound, each of accurately known concentration, Those concentrations should bracket the concentration you are trying to find - some less i concentrated; some more concentrated. If the samples have any particulate matter, it must be filtered to ensure a clear, transparent, homogeneous solution The spectrophotometer is blanked or zeroed with the ‘blank’ solution and the sample solution absorbance is quickly checked (at a particular wavelength - acceptable absorbance values are between 0.2 and 0.9) Absorbance is taken at a particular wavelength Once this is achieved, the spectrophotometer is blanked ONCE and the measurements of the samples are made. ‘A graph is plotted of absorbance against concentration — calibration curve Beer-Lambert's Law The Beer-Lambert Law states that the concentration of a substance in solution is directly proportional to the ‘absorbance’ of the solution. — ¢ Sa E seosmann Fen to Fes —— Page 8 of 11 ‘Scanned with CamScannernuoryenat wicurus one sepa p ey 7 eident radiation of power, Po intensity, Je) passes through a solution ofthe absorbing species at concentration ¢ and path length |, the emergent beam has a power of Py (intensity, I), where Po > Pi. When the incident beam hits the wall of the cuvette it experiences a loss of power and intensity. Upon entering ion is absorbed by the molecules of solute and solvent, some scattered and some the cuvette, some of the radi reflected. Therefore, the emergent beam has less power and intensity than the incident beam. = A=escl e- pti : : A=absorbance (unitless) _ ¢ = molar absorptivity (L mol” cm) adoretoury fr 6 Pe = absor = , een oma parnctio wt pur ol seat c=concentration (mol L) 1 = path length (em) When monochromatic radiation passes through a homogeneous solution in a cuvette, the intensity (1), of the emitted radiation depend upon the thickness (1) and the concentration (¢) of the solution. — the law is only true for monochromatic light (light ofa single wavelength or narrow band of wavelengths), provided that the physical or chemical state of the substance does not change with concentration — the absorption process occurs in a volume of uniform cross section = the absorbing species behave independently of solvent or of each other in a multi-component mixture Uses of UV/VIS Spectroscopy + To identify chromophores in a compound To determine the concentration of a substance such as glucose and urea in blood, iron in iron tablets, cyanide in water Organic functional groups can be identified by their typical Imax values. Calibration Curves ‘Accalibration curve is defined as the instrument response (in this case, absorbance), as a fiunction of concentration of a known analyte. This response is linear and follows the general equation: yem +o ‘The curve us obtained by making a series of accurate solutions of increasing concentration of the known analyte. The absorbance of each of these solutions is recorded and 2 points which must be on the graph are concentrations giving absorbance values of 0.00 [blank] and 0.9-1.00 respectively. For any calibration/standard curve to be Vali, it must have a minimum of 5 concentrations plus blink. Page 9 of 11 ‘Scanned with CamScannerif 5 V. best straight line graph is then accurately ploted and labelled. ‘The concentration of an unknown solution containing the same analyte can then be determined from the absorbance value obtained and using the calibration curve via interpolation. Page 10 of 11 ‘Scanned with CamScanner