Spectroscopy Measures light (radiation) absorbed by species in solution. Some radiation is absorbed by ground state electrons in atoms or molecules. Radiation NOT ABSORBED by the component being analysed reaches a detector. This detector measures intensity of radiation that is not absorbed by the sample. Absorbance readings are related to concentration. Samples are generally diluted.

Types of chemical analysis GC – smaller, volatile hydrocarbons HPLC – larger macromolecules (proteins, drugs) Flame test – presence of metal (emission) AAS – quantitative concentration of metal UV – coloured compounds IR – Bend & stretching of bonds in molecules (presence of different functional groups) NMR – nuclear spin states (exact order of atoms)

IR – Infrared Spectroscopy Expensive, but simple are available at lower cost. Similar to UV spectrophotometer but operates in IR region – lower energy than UV light IR radiation does not have enough energy to promote electrons to higher level, but causes changes in covalent bond s in molecules. The technique is based on the idea that different bonds are of different lengths and therefore absorb IR radiation differently. Difference in radiation between a reference cell and the sample cell is recorded.

Cells are made from NaCl or KBr as glass and plastic absorb IR, Fast once sample is prepared Gases, liquids & solids can be used Absorption spectroscopy Qualitative, rarely quantitative Plot of transmittance against wave number (spectra look like upside down UV spectra).

IR Transmittance: measures how much light has been transmitted. 100% transmittance means no energy has been absorbed. Absorbance is the intensity of light remaining after some has been absorbed. Used to identify organic compounds, in particular functional groups, design of new drugs, protein analysis, checking quality of wine products, tea leaves that have a smell, forensic identification of oils, fibres, paint flakes

All IR Spectra has what's known as an unique fingerprint region. This is generally only useful for comparing an unknown spectra with that of a known substance IR spectra. Note cm -1 is called the fingerprint region. It is generally hard to ID stretches within this region. It is the peaks that appear after the fingerprint region that we are interested in. The high absorbance readings at the larger wave numbers correspond and can inform us pretty clearly of the function groups that are present in the sample. It must be noted that symmetric molecules do not absorb, cannot be ID. Molecules with stronger bonds absorb high energy. Problems with IR Spectra: Some bands may overlap others.

IR

Amines show a similar broad peak closer to 3500 cm -1 (due to N-H stretch). C=O group has an absorption peak between 1670 and 1750 cm -1. This peak would appear for carboxylic acids and esters. The O-H group shows a broad peak ~3000cm -1, this may be seen in carboxylic acids and alcohols.

Can you find the functional group that is showing the maximum absorbance in the following spectra? C=O C=O O-H Broad!

Make sure you are using your data book! C-H O-H or N-H *Cannot be O- H (acid) as no presence of C=O!

NMR – Nuclear Magnetic Resonance Spectroscopy Expensive Hazardous because of the strong magnetic field Operates in the radio region Very fast (milliseconds) Qualitative Absorption spectrum Spectra can be low resolution or high resolution

How does NMR work? Protons, neutrons & electrons can spin if provided with radiation of the correct wavelength. If there is a even number of protons and neutrons in the nucleus (called nucleons) – no overall spin is exhibited. If there is a odd number of nucleons – there will be an overall spin. Atoms of this kind will align either with or against a magnetic pole when placed in a strong magnetic field. The atoms will act like a magnet when the correct amount of radiation is absorbed. The atom will then flip to a higher or lower energy state. This variation in energy is called the chemical shift and is measurable in ppm.

NMR Continued A flip or chemical shift can only occur when some of the odd nucleon atoms (e.g. 1H and 13C) absorb radiation in the radio region. When nuclei return to their lower energy level, the difference in radiation is released at a specific radio frequency. The chemical shift is measured relative to an organic compound called TMS< whose absorbance is plotted as zero on a NMR spectra.

LOW RESOLUTION PROTON NMR Tells us the type of bond (using chemical shift value) Tells us the type of bond (using chemical shift value) The number of peaks tell us how many different H environments there are. The number of peaks tell us how many different H environments there are. Height of peak tells us how many H atoms in each environment (a higher peak means there is a greater number of H nuclei in that particular environment). Height of peak tells us how many H atoms in each environment (a higher peak means there is a greater number of H nuclei in that particular environment). The area under the peak is called the integration value. The area under the peak is called the integration value.

HIGH RESOLUTION PROTON NMR Same info can be gained from such spectra. Each peak is split into smaller peaks (a number of peaks) The number of peak splits tells us the number of H atoms the carbon adjacent to the group we are looking at has. No splits = no neighbouring H atoms or may be a O-H or N-H group. 3 peak splits = 2 H on next group. 4 peak splits = 3 H atoms on next group.

Each peak in a 13 C NMR Spectra represents a difference Carbon environment. The chemical shift away from the TMS reference is used to determine the nature of the carbon environment The chemical shift values are greater than those for proton NMR. Carbon NMR spectra show no splitting.

How many Carbon environments? How many Hydrogen environments? Answer: 2 Carbon Environments 2 Hydrogen Environments Answer: 5 Carbon Environments 4 Hydrogen Environments

Answer: 3 Carbon Environments 3 Hydrogen Environments Answer: 3 Carbon Environments ONLY 1 Hydrogen Environment *BOTH CH 3 groups are said to be equivalent Answer: 3 Carbon Environments 3 Hydrogen Environments Equivalent C and H atoms…

Mass Spectrometry (MS) Expensive, but portable models are cheaper Very sensitive – uses µg samples Qualitative and Quantitative Operates at low pressures Sample is vaporised and high energy electrons knock off electrons of molecules & cause fragmentation. The different species are separated on passing though an electric field and a magnetic field.

Used to determine relative atomic mass on periodic table! Also used to identify organic molecules, qualitative and quantitative analysis of proteins, determine protein structure and abundance and mass of isotopes.

We can utilise MS to confirm molecular mass and organic structures using the produced fragmentation patterns. Samples are bombarded with high speed electrons. Ions are formed & accelerated to high speeds into a magnetic field. Lighter ions are deflected more than heavy ions. +2 charge are deflected more than +1 charged ions. A mass charge ration (m/z or m/e) is produced and ALWAYS plotted on the horizontal axis.

More than one positive ion can be formed (this is what we see as fragmentation patterns). In the first ionisation, where one electron is knocked off, a molecular or parent ion is formed. Once molecules form the parent (molecular ion) they can continue to break down into smaller pieces or molecules of lower molecular weight. It is important to remember that only charged species will produce a line on a mass spectrograph. The highest peak (also called base peak) is found for the most stable fragment that is 100% absorbed. Common ions formed: CH mass/charge = 15 Also CH 2 Ch 3 +, OH - and COOH +

From the mass spectra above: Q1. Which ion is most likely to be the molecular (or parent) ion? Q2. Which ion is most abundant (has the highest base peak)? MS

Combining Techniques