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Pharmaceutical Analysis
Absorption spectroscopy • Fluorimetry • Flame photometry • Potentiometry • Conductometry • Plarography • Pharmacopoeial assays • Principles of NMR • Principles of ESR • Principles of Mass spectroscopy • X-ray diffraction analysis • Chromatographic methods

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Absorption spectroscopy
Visible and Ultraviolet Spectroscopy

1. Background
An obvious difference between certain compounds is their color. Thus, quinone is yellow; chlorophyll is green; the 2,4-dinitrophenylhydrazone derivatives of aldehydes and ketones range in color from bright yellow to deep red, depending on double bond conjugation; and aspirin is colorless. In this respect the human eye is functioning as a spectrometer analyzing the light reflected from the surface of a solid or passing through a liquid. Although we see sunlight (or white light) as uniform or homogeneous in color, it is actually composed of a broad range of radiation wavelengths in the ultraviolet (UV), visible and infrared (IR) portions of the spectrum. As shown on the right, the component colors of the visible portion can be separated by passing sunlight through a prism, which acts to bend the light in differing degrees according to wavelength. Electromagnetic radiation such as visible light is commonly treated as a wave phenomenon, characterized by a wavelength or frequency. Wavelength is defined on the left below, as the distance between adjacent peaks (or troughs), and may be designated in meters, centimeters or nanometers (10-9 meters). Frequency is the number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second, or hertz (Hz). Visible wavelengths cover a range from approximately 400 to 800 nm. The longest visible wavelength is red and the shortest is violet. Other common colors of the spectrum, in order of decreasing wavelength, may be remembered by the mnemonic: ROY G BIV. The wavelengths of what we perceive as particular colors in the visible portion of the spectrum are displayed and listed below. In horizontal diagrams, such as the one on the bottom left, wavelength will increase on moving from left to right.

• • • • • • •

Violet: Indigo: Blue: Green: Yellow: Orange: Red:

400 - 420 nm 420 - 440 nm 440 - 490 nm 490 - 570 nm 570 - 585 nm 585 - 620 nm 620 - 780 nm

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Prepared B y D hruw ansh When white light passes through or is reflected by a colored substance, a characteristic portion of the mixed wavelengths is absorbed. The remaining light will then assume the complementary color to the wavelength(s) absorbed. This relationship is demonstrated by the color wheel shown on the right. Here, complementary colors are diametrically opposite each other. Thus, absorption of 420-430 nm light renders a substance yellow, and absorption of 500-520 nm light makes it red. Green is unique in that it can be created by absoption close to 400 nm as well as absorption near 800 nm.

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red REd red

O range

vio let indigo

yellow

b lue

green

Early humans valued colored pigments, and used them for decorative purposes. Many of these were inorganic minerals, but several important organic dyes were also known. These included the crimson pigment, kermesic acid, the blue dye, indigo, and the yellow saffron pigment, crocetin. A rare dibromo-indigo derivative, punicin, was used to color the robes of the royal and wealthy. The deep orange hydrocarbon carotene is widely distributed in plants, but is not sufficiently stable to be used as permanent pigment, other than for food coloring. A common feature of all these colored compounds, displayed below, is a system of extensively conjugated pi-electrons.

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2. The Electromagnetic Spectrum
The visible spectrum constitutes but a small part of the total radiation spectrum. Most of the radiation that surrounds us cannot be seen, but can be detected by dedicated sensing instruments. This electromagnetic spectrum ranges from very short wavelengths (including gamma and x-rays) to very long wavelengths (including microwaves and broadcast radio waves). The following chart displays many of the important regions of this spectrum, and demonstrates the inverse relationship between wavelength and frequency The energy associated with a given segment of the spectrum is proportional to its frequency. The bottom equation describes this relationship, which provides the energy carried by a photon of a given wavelength of radiation.

3. UV-Visible Absorption Spectra
To understand why some compounds are colored and others are not, and to determine the relationship of conjugation to color, we must make accurate measurements of light absorption at different wavelengths in and near the visible part of the spectrum. Commercial optical spectrometers enable such experiments to be conducted with ease, and usually survey both the near ultraviolet and visible portions of the spectrum. The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole, and the near ultraviolet region, out to 200 nm, extends this energy range to 143 kcal/mole. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is seldom used as a routine tool for structural analysis. The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole, and the near ultraviolet region, out to 200 nm, extends this energy range to 143 kcal/mole. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is seldom used as a routine tool for structural analysis. The energies noted above are sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, absorption spectroscopy carried out in this region is sometimes called "electronic spectroscopy". A diagram showing the various kinds of electronic excitation that may occur in organic molecules is shown on the left. Of the six transitions outlined, only the two lowest energy ones (left-most, colored blue) are achieved by the energies available in the 200 to 800 nm spectrum. As a rule, energetically favored electron promotion will be from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), and the resulting species is called an excited state. When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. An optical spectrometer records the wavelengths at which absorption occurs, together with the degree of absorption at each wavelength. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength, as in the isoprene spectrum shown below. Since isoprene is colorless, it does not absorb in the visible part of the spectrum and this region is not displayed on the graph. Absorbance usually ranges from 0 (no absorption) to 2 (99% absorption), and is precisely defined in context with spectrometer operation. Because the absorbance of a sample will be proportional to the number of absorbing molecules in the spectrometer light beam (e.g. their molar concentration in the sample tube), it is necessary to correct the absorbance value for this and other operational factors if the spectra of different compounds are to be compared in a meaningful way. The corrected absorption value is called "molar absorptivity", and is particularly useful when comparing the spectra of different compounds and determining the relative strength of light absorbing functions (chromophores). If the isoprene spectrum on the right was obtained from a dilute hexane solution (c = 4 * 10 -5 moles per liter) in a 1 cm sample cuvette, a simple calculation using the above formula indicates a molar absorptivity of 20,000 at the maximum absorption wavelength. Indeed the entire vertical absorbance scale may be changed to a molar absorptivity scale once this information about the sample is in hand. Clicking on the spectrum will display this change in units.

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Chromophore C=C C≡C C=O N=O C-X X=Br X=I

Example Ethene 1-Hexyne Ethanal Nitromethane Methyl bromide Methyl Iodide

Excitation π π n π n π n n
__ __

λmax, nm 171 180 290 180 275 200 205 255

ε 15,000 10,000 15 10,000 17 5,000 200 360

Solvent hexane hexane hexane hexane ethanol ethanol hexane hexane

> π* > π* > π* > π* > π* > π*

__ __ __ __ __ __

> σ* > σ*

From the chart above it should be clear that the only molecular moieties likely to absorb light in the 200 to 800 nm region are pi-electron functions and hetero atoms having non-bonding valence-shell electron pairs. Such light absorbing groups are referred to as chromophores. A list of some simple chromophores and their light absorption characteristics is provided on the left above. The oxygen non-bonding electrons in alcohols and ethers do not give rise to absorption above 160 nm. Consequently, pure alcohol and ether solvents may be used for spectroscopic studies. The presence of chromophores in a molecule is best documented by UV-Visible spectroscopy, but the failure of most instruments to provide absorption data for wavelengths below 200 nm makes the detection of isolated chromophores problematic. Fortunately, conjugation generally moves the absorption maxima to longer wavelengths, as in the case of isoprene, so conjugation becomes the major structural feature identified by this technique. Molar absorptivities may be very large for strongly absorbing chromophores (>10,000) and very small if absorption is weak (10 to 100). The magnitude ofε reflects both the size of the chromophore and the probability that light of a given wavelength will be absorbed when it strikes the chromophore.

4. The Importance of Conjugation
A comparison of the absorption spectrum of 1-pentene, λmax = 178 nm, with that of isoprene (above) clearly demonstrates the importance of chromophore conjugation. Further evidence of this effect is shown below. The spectrum on the left illustrates that conjugation of double and triple bonds also shifts the absorption maximum to longer wavelengths. From the polyene spectra displayed in the center diagram, it is clear that each additional double bond in the conjugated pi-electron system shifts the absorption maximum about 30 nm in the same direction. Also, the molar absorptivity (ε) roughly doubles with each new conjugated double bond. Spectroscopists use the terms defined in the table on the right when describing shifts in absorption. Thus, extending conjugation generally results in bathochromic and hyperchromic shifts in absorption. The appearance of several absorption peaks or shoulders for a given chromophore is common for highly conjugated systems, and is often solvent dependent. This fine structure reflects not only the different conformations such systems may assume, but also electronic transitions between the different vibrational energy levels possible for each Prepared B y

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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

To understand why conjugation should cause bathochromic shifts in the absorption maxima of chromophores, we need to look at the relative energy levels of the pi-orbitals. When two double bonds are conjugated, the four patomic orbitals combine to generate four pi-molecular orbitals (two are bonding and two are antibonding). In a similar manner, the three double bonds of a conjugated triene create six pi-molecular orbitals, half bonding and half antibonding. The energetically most favorable π __> π* excitation occurs from the highest energy bonding pi-orbital (HOMO) to the lowest energy antibonding pi-orbital (LUMO). The following diagram illustrates this excitation for an isolated double bond (only two pi-orbitals). In each case the HOMO is colored blue and the LUMO is colored magenta. Increased conjugation brings the HOMO and LUMO orbitals closer together. The energy (ΔE) required to effect the electron promotion is therefore less, and the wavelength that provides this energy is increased correspondingly .

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Many other kinds of conjugated pi-electron systems act as chromophores and absorb light in the 200 to 800 nm region. These include unsaturated aldehydes and ketones and aromatic ring compounds. A few examples are displayed below. The spectrum of the unsaturated ketone (on the left) illustrates the advantage of a logarithmic display of molar absorptivity. The π __> π* absorption located at 242 nm is very strong, with an ε = 18,000. The weak n __> π* absorption near 300 nm has an ε = 100.

Benzene exhibits very strong light absorption near 180 nm (ε > 65,000) , weaker absorption at 200 nm (ε = 8,000) and a group of much weaker bands at 254 nm (ε = 240). Only the last group of absorptions are completely displayed because of the 200 nm cut-off characteristic of most spectrophotometers. The added conjugation in naphthalene, anthracene and tetracene causes bathochromic shifts of these absorption bands, as displayed in the chart on the left below. All the absorptions do not shift by the same amount, so for anthracene (green shaded box) and tetracene (blue shaded box) the weak absorption is obscured by stronger bands that have experienced a greater red shift. As might be expected from their spectra, naphthalene and anthracene are colorless, but tetracene is orange. The spectrum of the bicyclic diene (above right) shows some vibrational fine structure, but in general is similar in appearance to that of isoprene, Closer inspection discloses that the absorption maximum of the more highly substituted diene has moved to a longer wavelength by about 15 nm. This "substituent effect" is general for dienes and trienes, and is even more pronounced for enone chromophores.

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Fluorimetry
Quantitative Fluorimetry Introduction Light emission from atoms or molecules can be used to quantitate the amount of the emitting substance in a sample. The relationship between fluorescence intensity and analyte concentration is: F = k * QE * Po * (1-10[-epsilon*b*c]) where F is the measured fluorescence intensity, k is a geometric instrumental factor, QE is the quantum efficiency (photons emitted/photons absorbed), Po is the radiant power of the excitation source, epsilon is the wavelengthdependent molar absorptivity coefficient, b is the path length, and c is the analyte concentration (epsilon, b, and c are the same as used in the Beer-Lambert law). Expanding the above equation in a series and dropping higher terms gives: F = k * QE * Po * (2.303 * epsilon * b * c) This relationship is valid at low concentrations (<10-5 M) and shows that fluorescence intensity is linearly proportional to analyte concentration. Determining unknown concentrations from the amount of fluorescence that a sample emits requires calibration of a fluorimeter with a standard (to determine K and QE) or by using a working curve.

Limitations

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Flame photometry
Background Flame photometry is an atomic emission method for the routine detection of metal salts, principally Na, K, Li, Ca, and Ba. Quantitative analysis of these species is performed by measuring the flame emission of solutions containing the metal salts. Solutions are aspirated into the flame. The hot flame evaporates the solvent, atomizes the metal, and excites a valence electron to an upper state. Light is emitted at characteristic wavelengths for each metal as the electron returns to the ground state. Optical filters are used to select the emission wavelength monitored for the analyte species. Comparison of emission intensities of unknowns to either that of standard solutions, or to those of an internal standard, allows quantitative analysis of the analyte metal in the sample solution. Flame photometry is a simple, relatively inexpensive, high sample throughput method used for clinical, biological, and environmental analysis. The low temperature of the natural gas and air flame, compared to other excitation methods such as arcs, sparks, and rare gas plasmas, limit the method to easily ionized metals. Since the temperature isn't high enough to excite transition metals, the method is selective toward detection of alkali and alkali earth metals. On the other hand, the low temperatures renders this method susceptible to certain disadvantages, most of them related to interference and the stability (or lack thereof) of the flame and aspiration conditions. Fuel and oxidant flow rates and purity, aspiration rates, solution viscosity, concomitants in the samples, etc affect these. It is therefore very important to measure the emission of the standard and unknown solutions under conditions that are as nearly identical as possible. This experiment will serve as an introduction to sodium analysis by flame emission photometry and will demonstrate the effects of cleanliness and solution viscosity on the observed emission intensity readings. The instrument is calibrated with a series of standard solutions that cover the range of concentrations expected of the samples. Standard calibrations are commonly used in instrumental analysis. They are useful when sample concentrations may vary by several orders of magnitude and when the value of the analyte must be known with a high degree of accuracy. This experiment does not produce hazardous waste. Procedure Consult your Teaching Assistant for operating instructions for the Buck PFP-7 Flame Photometer. Allow a sufficient warm-up period. Be sure to aspirate deionized-distilled water between samples to clean out the sample tube and aspirator. Sodium is ubiquitous. It is imperative that you use scrupulously cleaned glassware to obtain good results. Prepared B y

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Prepare sodium chloride standard solutions by volumetric dilution of the stock solution. The following approximate concentrations should be made: 5, 10, 25, 50, 75, and 100 mg/mL as Na. Be sure to use clean methods. Use ultra-pure deionized-distilled water to clean your glassware and for dilution of the 1000 mg/mL standard. Prepare these standards in scrupulously clean volumetric glassware and transfer the solutions to plastic bottles. Glass often is made from high sodium glass. Allowing extremely high or low pH solutions to stand in glass could alter the sodium concentrations in solution. Prepare 25 mg/mL Na solutions in other solvents, 10% Ethanol, 50% Ethanol, 50% Glycerin. Standard solutions may be pre-prepared by the laboratory instructor or may be made up as a class or group project. Unknown Preparation Obtain a sodium unknown from your instructor in a scrupulously clean 50 mL volumetric flask. Dilute to the mark with distilled water. Instrument Calibration Set the readout to zero using distilled water as a blank. Set the peak reading according to the instrument instructions using the most concentrated sodium solution (100 mg/mL). Measure the emission intensity of each of the remaining sodium standard solutions, and of the sodium unknown solution. Check for accuracy and repeatability by measuring the standards several times. Be sure to aspirate deionized distilled water between measurements. Cleanliness Check Dip two fingers into a clean beaker containing about 20 mL of distilled water. Measure and record the sodium emission intensity. Measure and record the sodium emission intensity of tap water. Be sure to aspirate deionized distilled water between measurements. Viscosity Variations Measure and record the sodium emission intensity of each of the 25 mg/mL Na solutions in various solvents. Be sure to aspirate deionized distilled water between measurements. Reproducibility Check Remeasure the emission intensity of two or three of the standard solutions. If a significant change has occurred, READJUST the zero and peak readings, and RE-MEASURE the emission intensity of the standards and the unknown. Results Plot a working curve from the data obtained in the Instrument Calibration step. Calculate and report the sodium concentration of the unknown, "fingered" solution, and tap water in units of mg/ml. Plot intensity reading as a function of viscosity for the data of Viscosity Variation step. Turn in the two graphs and the sodium concentrations of the unknown, "fingered" solution, and tap water. Grades will be based 80% on the reported unknown concentration and 20% on graphs. Tap water and "fingered" solution data is for our future reference. Prepared B y

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Sawyer, Heineman, Beebe, Chemistry Experiments for Instrumental Methods, Wiley, New York, 1984. D. C. Harris Quantitative Chemical Analysis 4th Ed., W. H. Freeman and Company, New York 1995 Chapter 21

Potentiometry
Introduction Potentiometry is the field of electroanalytical chemistry in which potential is measured under the conditions of no current flow. The measured potential may then be used to determine the analytical quantity of interest, generally the concentration of some component of the analyte solution. The potential that develops in the electrochemical cell is the result of the free energy change that would occur if the chemical phenomena were to proceed until the equilibrium condition has been satisfied.

This concept is typically introduced in quantitative analysis courses in relation to electrochemical cells that contain an anode and a cathode. For these electrochemical cells, the potential difference between the cathode electrode potential and the anode electrode potential is the potential of the electrochemical cell.

If the reaction is conducted under standard state conditions, this equation allows the calculation of the standard cell potential. When the reaction conditions are not standard state, however, one must utilize the Nernst equation to determine the cell potential. Prepared B y

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Physical phenomena which do not involve explicit redox reactions, but whose initial conditions have a non-zero free energy, also will generate a potential. An example of this would be ion concentration gradients across a semipermeable membrane. This can also be a potentiometric phenomena, and is the basis of measurements that use ionselective electrodes.

Nuclear Magnetic Resonance Spectroscopy
1. Background Over the past fifty years nuclear magnetic resonance spectroscopy, commonly referred to as nmr, has become the preeminent technique for determining the structure of organic compounds. Of all the spectroscopic methods, it is the only one for which a complete analysis and interpretation of the entire spectrum is normally expected. Although larger amounts of sample are needed than for mass spectroscopy, nmr is non-destructive, and with modern instruments good data may be obtained from samples weighing less than a milligram. To be successful in using NMR as an analytical tool, it is necessary to understand the physical principles on which the methods are based. The nuclei of many elemental isotopes have a characteristic spin (I). Some nuclei have integral spins (e.g. I = 1, 2, 3....), some have fractional spins (e.g. I = 1/2, 3/2, 5/2 ....), and a few have no spin, I = 0 (e.g. 12C, 16O, 32S, ....). Isotopes of particular interest and use to organic chemists are 1H, 13C, 19F and 31P, all of which have I = 1/2. Since the analysis of this spin state is fairly straightforeward, our discussion of nmr will be limited to these and other I = 1/2 nuclei. The following features lead to the nmr phenomenon: 1. A spinning charge generates a magnetic field, as shown by the animation on the right. The resulting spin-magnet has a magnetic moment (μ) proportional to the spin.

2. In the presence of an external magnetic field (B0), two spin states exist, +1/2 and -1/2. The magnetic moment of the lower energy +1/2 state is alligned with the external field, but that of the higher energy -1/2 spin state is opposed to the external field. Note that the arrow representing the external field points North.

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3. The difference in energy between the two spin states is dependent on the external magnetic field strength, and is always very small. The following diagram illustrates that the two spin states have the same energy when the external field is zero, but diverge as the field increases. At a field equal to B x a formula for the energy difference is given (remember I = 1/2 and μ is the magnetic moment of the nucleus in the field).

Strong magnetic fields are necessary for nmr spectroscopy. The international unit for magnetic flux is the tesla (T). The earth's magnetic field is not constant, but is approximately 10-4 T at ground level. Modern nmr spectrometers use powerful magnets having fields of 1 to 20 T. Even with these high fields, the energy difference between the two spin states is less than 0.1 cal/mole. To put this in perspective, recall that infrared transitions involve 1 to 10 kcal/mole and electronic transitions are nearly 100 time greater. For nmr purposes, this small energy difference (ΔE) is usually given as a frequency in units of MHz (10 6 Hz), ranging from 20 to 900 Mz, depending on the magnetic field strength and the specific nucleus being studied. Irradiation of a sample with radio frequency (rf) energy corresponding exactly to the spin state separation of a specific set of nuclei will cause excitation of those nuclei in the +1/2 state to the higher -1/2 spin state. Note that this electromagnetic radiation falls in the radio and television broadcast spectrum. Nmr spectroscopy is therefore the energetically mildest probe used to examine the structure of molecules. The nucleus of a hydrogen atom (the proton) has a magnetic moment μ = 2.7927, and has been studied more than any other nucleus. The previous diagram may be changed to display energy differences for the proton spin states (as frequencies) by mouse clicking anywhere within it. 4. For spin 1/2 nuclei the energy difference between the two spin states at a given magnetic field strength will be proportional to their magnetic moments. For the four common nuclei noted above, the magnetic moments are: 1H μ = 2.7927, 19F μ = 2.6273, 31P μ = 1.1305 & 13C μ = 0.7022. These moments are in nuclear magnetons, which are 5.05078•10-27 JT-1. The following diagram gives the approximate frequencies that correspond to the spin state energy separations for each of these nuclei in an external magnetic field of 2.35 T. The formula in the colored box shows the direct correlation of frequency (energy difference) with magnetic moment (h = Planck's constant = 6.626069•10-34 Js).

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This important and well-established application of nuclear magnetic resonance will serve to illustrate some of the novel aspects of this method. To begin with, the nmr spectrometer must be tuned to a specific nucleus, in this case the proton. The actual procedure for obtaining the spectrum varies, but the simplest is referred to as the continuous wave (CW) method. A typical CW-spectrometer is shown in the following diagram. A solution of the sample in a uniform 5 mm glass tube is oriented between the poles of a powerful magnet, and is spun to average any magnetic field variations, as well as tube imperfections. Radio frequency radiation of appropriate energy is broadcast into the sample from an antenna coil (colored red). A receiver coil surrounds the sample tube, and emission of absorbed rf energy is monitored by dedicated electronic devices and a computer. An nmr spectrum is acquired by varying or sweeping the magnetic field over a small range while observing the rf signal from the sample. An equally effective technique is to vary the frequency of the rf radiation while holding the external field constant.

As an example, consider a sample of water in a 2.3487 T external magnetic field, irradiated by 100 MHz radiation. If the magnetic field is smoothly increased to 2.3488 T, the hydrogen nuclei of the water molecules will at some point absorb rf energy and a resonance signal will appear. An animation showing this may be activated by clicking the Show Field Sweep button. The field sweep will be repeated three times, and the resulting resonance trace is colored red. For visibility, the water proton signal displayed in the animation is much broader than it would be in an actual experiment. Since protons all have the same magnetic moment, we might expect all hydrogen atoms to give resonance signals at the same field / frequency values. Fortunately for chemistry applications, this is not true. By clicking the Show Different Protons button under the diagram, a number of representative proton signals will be displayed over the same magnetic field range. It is not possible, of course, to examine isolated protons in the spectrometer described above; but from independent measurement and calculation it has been determined that a naked proton would resonate at a lower field strength than the nuclei of covalently bonded hydrogens. With the exception of water, chloroform and sulfuric acid, which are examined as liquids, all the other compounds are measured as gases. Why should the proton nuclei in different compounds behave differently in the nmr experiment ? The answer to this question lies with the electron(s) surrounding the proton in covalent compounds and ions. Since electrons are charged particles, they move in response to the external magnetic field (Bo) so as to generate a secondary field that opposes the much stronger applied field. This secondary field shields the nucleus from the applied field, so Bo must be increased in order to achieve resonance (absorption of rf energy). As illustrated in the drawing on the right, Bo must be increased to compensate for the induced shielding field. In the upper diagram, Prepared B y

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The magnetic field range displayed in the above diagram is very small compared with the actual field strength (only about 0.0042%). It is customary to refer to small increments such as this in units of parts per million (ppm). The difference between 2.3487 T and 2.3488 T is therefore about 42 ppm. Instead of designating a range of nmr signals in terms of magnetic field differences (as above), it is more common to use a frequency scale, even though the spectrometer may operate by sweeping the magnetic field. Using this terminology, we would find that at 2.34 T the proton signals shown above extend over a 4,200 Hz range (for a 100 MHz rf frequency, 42 ppm is 4,200 Hz). Most organic compounds exhibit proton resonances that fall within a 12 ppm range (the shaded area), and it is therefore necessary to use very sensitive and precise spectrometers to resolve structurally distinct sets of hydrogen atoms within this narrow range. In this respect it might be noted that the detection of a part-per-million difference is equivalent to detecting a 1 millimeter difference in distances of 1 kilometer.

π-Electron Functions An examination of the proton chemical shift chart (above) makes it clear that the inductive effect of substituents cannot account for all the differences in proton signals. In particular the low field resonance of hydrogens bonded to double bond or aromatic ring carbons is puzzling, as is the very low field signal from aldehyde hydrogens. The hydrogen atom of a terminal alkyne, in contrast, appears at a relatively higher field. All these anomalous cases seem to involve hydrogens bonded to pi-electron systems, and an explanation may be found in the way these pi-electrons interact with the applied magnetic field. Pi-electrons are more polarizable than are sigma-bond electrons, as addition reactions of electrophilic reagents to alkenes testify. Therefore, we should not be surprised to find that field induced pi-electron movement produces strong secondary fields that perturb nearby nuclei. The pi-electrons associated with a benzene ring provide a striking example of this phenomenon, as shown below. The electron cloud above and below the plane of the ring circulates in reaction to the external field so as to generate an opposing field at the center of the ring and a supporting field at the edge of the ring. This kind of spatial variation is called anisotropy, and it is common to nonspherical distributions of electrons, as are found in all the functions mentioned above. Regions in which the induced field supports or adds to the external field are said to be deshielded, because a slightly weaker external field will bring about resonance for nuclei in such areas. However, regions in which the induced field opposes the external field are termed shielded because an increase in the applied field is needed for resonance. Shielded regions are designated by a plus sign, and deshielded regions by a negative sign. The anisotropy of some important unsaturated functions will be displayed by clicking on the benzene diagram below. Note that the anisotropy about the triple bond nicely accounts for the relatively high field chemical shift of ethynyl hydrogens. The shielding & deshielding regions about the carbonyl group have been described in two ways, which alternate in the display. Prepared B y

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Spin-Spin Interactions The nmr spectrum of 1,1-dichloroethane (below right) is more complicated than we might have expected from the previous examples. Unlike its 1,2-dichloro-isomer (below left), which displays a single resonance signal from the four structurally equivalent hydrogens, the two signals from the different hydrogens are split into close groupings of two or more resonances. This is a common feature in the spectra of compounds having different sets of hydrogen atoms bonded to adjacent carbon atoms. The signal splitting in proton spectra is usually small, ranging from fractions of a Hz to as much as 18 Hz, and is designated as J (referred to as the coupling constant). In the 1,1dichloroethane example all the coupling constants are 6.0 Hz.

The splitting patterns found in various spectra are easily recognized, provided the chemical shifts of the different sets of hydrogen that generate the signals differ by two or more ppm. The patterns are symmetrically distributed on both sides of the proton chemical shift, and the central lines are always stronger than the outer lines. The most commonly observed patterns have been given descriptive names, such as doublet (two equal intensity signals), triplet (three signals with an intensity ratio of 1:2:1) and quartet (a set of four signals with intensities of 1:3:3:1). Four such patterns are displayed in the following illustration. The line separation is always constant within a given multiplet, and is called the coupling constant (J). The magnitude of J, usually given in units of Hz, is magnetic field independent.

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The splitting patterns shown above display the ideal or "First-Order" arrangement of lines. This is usually observed if the spin-coupled nuclei have very different chemical shifts (i.e. Δν is large compared to J). If the coupled nuclei have similar chemical shifts, the splitting patterns are distorted (second order behavior). In fact, signal splitting disappears if the chemical shifts are the same. Two examples that exhibit minor 2nd order distortion are shown below (both are taken at a frequency of 90 MHz). The ethyl acetate spectrum on the left displays the typical quartet and triplet of a substituted ethyl group. The spectrum of 1,3-dichloropropane on the right demonstrates that equivalent sets of hydrogens may combine their influence on a second, symmetrically located set. Even though the chemical shift difference between the A and B protons in the 1,3-dichloroethane spectrums is fairly large (140 Hz) compared with the coupling constant (6.2 Hz), some distortion of the splitting patterns is evident. The line intensities closest to the chemical shift of the coupled partner are enhanced. Thus the B set triplet lines closest to A are increased, and the A quintet lines nearest B are likewise stronger. A smaller distortion of this kind is visible for the A and C couplings in the ethyl acetate spectrum.

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ESR Spectroscopy
EPR Spectroscopy. Introduction. Objective: You will learn what kind of information ESR can provide both, spectroscopic and kinetic, and investigate some organic and inorganic radicals and ion radicals and gather information anot only bout their hyperfine structure but also what may affect it (e.g. concentration, oxygen etc.) Electron Paramagnetic Resonance (EPR), often called Electron Spin Resonance (ESR), is a branch of spectroscopy in which electromagnetic radiation (usually of microwave frequency) is absorbed by molecules, ions, or atoms possessing electrons with unpaired spins, i.e. electronic spin S > 0. EPR is similar to Nuclear Magnetic Resonance (NMR) which you will get acquainted with in the Lab 8. The NMR technique deals with nonzero nuclear spins, I > 0. In both EPR and NMR, the sample material is immersed in a strong static magnetic field and exposed to an orthogonal low- amplitude high-frequency field. ESR usually requires microwave-frequency radiation (GHz), while NMR is observed at lower radio frequencies (MHz). With ESR, energy is absorbed by the sample when the frequency of the radiation is appropriate to the energy difference between two states of the electrons in the sample, but only if the transition satisfies the appropriate selection rules.

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Prepared B y D hruw ansh Most of materials in a bulk form at normal conditions have net zero electronic spin and, thus, are EPR silent, but below:

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some are can provide an EPR signal. In this lab will analyse some representative species from the three groups

Transition-metal and rare-earth species which contain unpaired nd and/or mf electrons. This experiment will offer you to examine liquid solutions of Mn2+, Fe3+ and Cu2+ ions with some ligands, as well as Cr3+ as a dilute impurity in a solid Al2O3 host (ruby crystal).

A small number of organic molecules are called free radicals because they contain a single unpaired electron, 1,1'-Diphenyl-2-picryl-hydrazyl (DPPH) and 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) are the examples; the latter will be used in a first part of this experiment. All but one of the electrons of these molecules are paired so there is only the orbital and spin motion of one electron present per molecule. Organic ion-radicals, very reactive species which are created along a course of redox reactions. They can be stabilized at certain conditions. You will make and analyze perylene+ cation-radical and benzosemiquinone anion-radical.

Triplet-state organic molecules and biradicals.

Theory Energy Level Structure, g-factor and hyperfine interaction

In EPR, because of the interaction of the unpaired electron spin moment (given by two projections, ms = ± 1/2, for a free electron) with the magnetic field, the so-called Zeeman effect, different projections of the spin gain different energies, as shown below and on the figure to the right: Ems = gmBBoms (1) Here Bo is the field strength of the external magnetic field. The SI units for magnetic field is tesla,T, but, historically in EPR, gauss (1 G = 0.0001 T) is still used. Other terms in Eq.(1): ms - is a spin projection on the field (ms = ± 1/2 for a free electron), mB is the Bohr magneton: mB = |eh/ 4pme|= 9.2740 x 10-24 J/T (2) with e and me beeing electron charge and mass, respectively, and h-Planck's constant. Parameter g for free electron, ge, has the value close to two: ge = 2.0023193. If the electron has nonzero orbiatl angular moment, L, the g-value (sometimes called factor Landé) becomes: g=1+ S(S + 1) - L(L + 1) + J(J + 1) (3) Prepared B y

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Prepared B y D hruw ansh 2J(J + 1) The overall magnetic momentun, meff , can be expessed via overall angular momentum, J, and the g-value: meff = gmB[J(J + 1)]1/2 (4) For most of organic radicals and radical ions, unpaired electrons have L close to zero and the total electron angular momentum quantum number J is pretty much the spin quantum number, S. As result, their g-values are close to 2. Situation becomes much more complicated with transition metals. Not only they have large L's and S's, but these values depend on the surrounding electric fields of ligands, making everythings messier but also more interesting. In this case, Eq.(1) should be written as E mJ = gJmBBomJ, stating that the Zeeman splitting appears in accordance with the total angular momentum projection, mJ. If L = 0 then J = S, and Eq.(1) will define the energies of all the possible projection of ms from -S to S - 1, S (2S + 1 of such). If the molecule contains nuclei with magnetic moments, such as protons, their interaction with external field and the electronic magnetic moment will change stationary energies of Eq.(1). The nuclear angular momentum quantum number I determines the nuclear magnetic moment the same way as for the electron: m = gNmN[I(I + 1)]1/2 (5) with N now being the nuclear Bohr magneton: N = eh/ 4  p = 5.051 x 10-27 J/T m (6)

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much smaller value because of the ~2000 times more heavier proton m p. The nuclear g factor, gN, is obtained from a knowledge of the structure of the nucleus. Interaction with external field splits the nuclear sublevels due to Zeeman interaction the same way as for electron spin: mI = gNNBomI (7)

where mI is one of the 2I + 1 projections of the nuclear spin. Electron-nuclear interaction will depend on the projections of both, electron and nuclear spins: electron-nuclear = A mI ms (8)

where coefficient A, a so-called hyperfine coupling constant/interaction (hfi), depends not only on the g-values for the electron and the nucleus but also on the distance between them and their orientation with respect to the external field (dipole-dipole interaction). In solutions, the anisotropic part of this interaction averages out because of the fast molecular rotation. The remaining isotropic part is given by the Fermi contact interaction in the form: A = (8/3)gNNgeB(0) (9)

where (0) = |(0)|2 is the unpaired electron density at the nucleus. For pure 1s electron on hydrogen atom, A equals h x 1420 MHz. As a matter of convinience, hyperfine constants are usually given in Gauss, a = A/geB. For

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E = geBms(Bo +  aimIi) - gNNBomI You should note two important things:

(10)

the signs in front of ge and gNare different because of the opposite charges of the electron and the proton, causing  spin of the electron to be higher in energy than  but N spin of the proton to be higher in , energy than N the magnetic field on the electron differs from the Bo by a integer times ai and the sign of this cange is opposite for  and  electron spins

Hydrogen atom example is given on the right. In EPR, allowed transitions correspond to the change of only ms:  s = ± 1, mI = 0 m (11) i.e. electronic spin "flips" but the nuclear one remains unchanged. Thus, in the figure on the right for hydrogen atom, only two transitions can be observed for a single resonant frequency h  (This is a typical mode of EPR . signal detection: microwave frequency is kept constant and magnetic field is varied). These transitions are separated by the hyperfine constant a (508 G for hydrogen atom). If there are more than one hydrogens, each of them will contribute to the change in magnetic field on the electron.

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Prepared B y D hruw ansh All combinations of the nuclear spin projections should be included but (EPR allowed) transitions only between sublevels with the same configurstions of nuclear spins will be realized. For the case of four equivalent protons, a simple ladder propagation scheme (Figure on the left) illustrates how to construct the EPR spectrum. Each new step corresponds to one more proton added. In the first step, two lines appear corresponding to the two different orientations of mI = -1/2 and mI = + 1/2. In the second step, each line splits into two, corresponding to the two different orientations of mI = -1/2 and mI = + 1/2, of the second proton.Since the two protons are equivalent, the splitting is the same on each step, resulting in three lines of 1:2:1 intesity ratio. For the four protons, there will be 5 lines with the 1:4:6:4:1 intensity ratio. Lines with the lengths of these ratio separated by appropriate constants are called to represent a stick diagram for the radical. If the spin of the nucleus is greater than 1/2, as in the case of 14N ( I = 1), it splits each of the electronic levels,  and , into the 2I + 1 sublevels resulting in 2I + 1 obeserved lines of equal intensity. In most cases, one observes a combination of different nuclei with different constants which sometimes is not as easy to interpret.

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Example of a radical with two different groups of hfi constants and different nuclei is shown on the right for a radical with one nitrogen and two equivalent hydrogens (e.g. NH2). The magnitude of the hyperfine constants in a radical or radical ion reflects the extent of s character in its orbital (only s-orbital has nonzero density at the nucleus). Sometimes it is quite tricky how s -orbital gets populated. For example, we think of aromatic ion radicals as being purely -systems, as you will see for yourselves, there are substantial hfi constants on there hydrogens. McConnell6 showed that a aprt of the unpaired -electron density is transferred through the C-H sigma bonding electrons to the H nucleus via exchange interactions. He provided a simple formular which links the aH on a hydrogen with the unpaired electron spin density on the nearest carbon atom : aH (G) = - 22.5  (12) The signal intensity in EPR depends on how much absorption of the microwave power takes place. This value will be the larger the greater is the difference between populations of the and electron spin states. Following   Boltzmann statistics, the latter can be estimated : E/kT (n- n)/n = e  - 1 ~ gBBo/kBT (13) a small value for regular X-band EPR spectrometer (9.5 GHz), which can be substanially increased by lowering the temperatute. The Lineshape and Kinetics

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Figure on the right shows how EPR signal shape of a stable radical, TEMPO (see its structure on the left), varies with concentration (intensities were scaled to the same hight). It illustrates that the linewidths of the resonant transitions are not infinitely small and can be altered by the environment. At low concentrations, each line has a width because of the heterogeneous broadening, i.e. unresolved structure of the small hyperfine constants and the homogeneous broadening, Go: Go = 1/geT2 (14) which is caused by a limited lifetime T2 (also called a spin-spin relaxation time) of a radical at a particular spin state (uncertainty principle related). The constant ge is called the magnetogyric (or gyromagnetic) ratio and relates to Bohr's magneton, mB, ge = 4pmB/h = e/mec = 1.77 x 107 s-1 G-1 (15) 1/2 The full width half maximum, G, is 3 /2 = 0.866 times greater than the measured distance between maximum and minimum of the experimentaly detective derivative of absorption, i.e. G = 0.866 D. This lifetime can be affected by the dipole-dipole (and exchange) interaction with other radicals. If another unpared electron is at a distance r, the magnitude of its field at the spin can take any value between ± 2mB/r3, depending on orientation. Due to the fast rotation of both radicals, the interaction averages this field to zero except for a very short period of time during collision after which the radical can end up at a random configuration of its nuclear spins. Result of such randomization is equivalent to the jumping from one spectral position to another. For nitroxyl radical, such as TEMPO, with three equivalent spectral lines (S = 1), only 2/3 of such collisions result in a shift. Beacuse of that, the width will increase only at 2/3 of the rate of collision: Slow Exchange: G = Go + 2k[R]/3ge (16) where k is the collision rate constant and [R] is the concentration of radicals. With further increase of the radical concentration, the continuing broadening will be accompanied by a measureable decrease in separation between the peaks, DB: Intermediate Exchange: (aN2 - DB2 )1/2 = 2k[R]/3ge? (17) where aN is the hyperfine constant on nitrogen (separation between lines). After they coalesce, the overall width of now a single-line spectrum, starts decreasing: Rapid Exchange: G = ge(aN2)/k[R] (18) The radicals can exchange spins not on themselves but on any other radical as well. Oxygen has nonzero spin and can also cause the line broadening by the same mechanism. So if your solvent is nonviscous and you want to detect linewidths below 3 G, you may want to get rid of O2. Prepared B y

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The ESP-300 ESR spectrometer (Bruker) consists of an electromagnet with power supplies to generate and modulate a uniform magnetic field of several thousand Gauss, as well as the components that generate and detect microwave power. The operation manual is awailable on the web, courtesy of the University of Illinois at UrbanaChampaign. I am putting our version as well. A static magnetic field is provided by an electromagnet with a current- regulated power supply. A homogeneous field is required for best results. A Hall probe, driven from a stable constant-current power system, with a digital multimeter (DMM) reading the Hall voltage, is used to measure the value of the magnetic field between the poles of the magnet. The microwave system consists of a microwave power supply (on a table above the magnet) which uses a clystron. The ouput of the microwave power (mw) supply is connected via rectangular waveguide (10 GHz) and through a circulator to a high-Q resonant cavity. The samples to be investigated are mounted in the middle of the cavity, where magnetic component of the microwave power has a maximum and is oriented perpendicular to the static field. A microwave diode, which detects the mw resides inside the same box as the power supply. The higher the quality of the resonant cavity the greater the microwave field can be obtained on the sample. Very polar solvents like water absorb efficiently on microwave frequencies themselves (remember microwave ovens?). Because of that, water samples have to be made in narrow cappilaries and carefully located in the center of the cavity. Most of our measurements will be done in the 0.1 - 1.0 mW range.

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There are variety of schemes for detecting resonant EPR transitions. Historically, most popular became a detection of absorption of the microwave power. To minimize the noise from the mw diode in steady state measurements, a magnetic field modulation scheme with phase sensitive detection is usually employed. As a result, the detected signal appears as a first derivative (see figure on the right). In the Bruker spectrometer there is choice between 12.5 kHz and 100 kHz modulation frequency. The amplitude of field modulation (MA) can be varied up to 40 G (4mT). Yo have to be careful in choosing appropriate MA. If MA is to big, the width of detected signal derivative would be wider than the real width of absorption, but if it is too small, the signal intensity would be less. For most of your case it is good first approximation to set MA between 0.5 and 1.0 G

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Mass Spectrometry
1. The Mass Spectrometer In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer, and the associated components, are: 1. A small sample of compound is ionized, usually to cations by loss of an electron. The Ion Source 2. The ions are sorted and separated according to their mass and charge. The Mass Analyzer 3. The separated ions are then detected and tallied, and the results are displayed on a chart. The Detector Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum. Atmospheric pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to 10-8 torr (less than a billionth of an atmosphere). Each of the three tasks listed above may be accomplished in different ways. In one common procedure, ionization is effected by a high energy beam of electrons, and ion separation is achieved by accelerating and focusing the ions in a beam, which is then bent by an external magnetic field. The ions are then detected electronically and the resulting information is stored and analyzed in a computer. A mass spectrometer operating in this fashion is outlined in the following diagram. The heart of the spectrometer is the ion source. Here molecules of the sample (black dots) are bombarded by electrons (light blue lines) issuing from a heated filament. This is called an EI (electron-impact) source. Gases and volatile liquid samples are allowed to leak into the ion source from a reservoir (as shown), but non-volatile solids and liquids may be introduced directly. Cations formed by the electron bombardment (red dots) are pushed away by a charged repellor plate (anions are attracted to it), and accelerated toward other electrodes, having slits through which the ions pass as a beam. Some of these ions fragment into smaller cations and neutral fragments. When the ion beam experiences a strong magnetic field perpendicular to its direction of motion, the ions are deflected in an arc whose radius is inversely proportional to the mass of the ion. Lighter ions are deflected more than heavier ions. By varying the strength of the magnetic field, ions of different mass can be focused progressively on a detector fixed at the end of a curved tube (also under a high vacuum). When a high energy electron collides with a molecule it often ionizes it by knocking away one of the molecular electrons (either bonding or non-bonding). This leaves behind a molecular ion (colored red in the following diagram). Residual energy from the collision may cause the molecular ion to fragment into neutral pieces (colored green) and smaller fragment ions (colored pink and orange). The molecular ion is a radical cation, but the fragment ions may either be radical cations (pink) or carbocations (orange), depending on the nature of the neutral fragment. An animated display of this ionization process will appear if you click on the ion source of the mass spectrometer diagram.

2. The Nature of Mass Spectra A mass spectrum will usually be presented as a vertical bar graph, in which each bar represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar indicates the relative abundance of the ion. The most intense ion is assigned an abundance of 100, and it is referred to as the base peak. Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself. Modern mass spectrometers Prepared B y

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Prepared B y D hruw ansh easily distinguish (resolve) ions differing by only a single atomic mass unit (amu), and thus provide completely accurate values for the molecular mass of a compound. The highest-mass ion in a spectrum is normally considered to be the molecular ion, and lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound. The following diagram displays the mass spectra of three simple gaseous compounds, carbon dioxide, propane and cyclopropane. The molecules of these compounds are similar in size, CO2 and C3H8 both have a nominal mass of 44 amu, and C3H6 has a mass of 42 amu. The molecular ion is the strongest ion in the spectra of CO2 and C3H6, and it is moderately strong in propane. The unit mass resolution is readily apparent in these spectra (note the separation of ions having m/z=39, 40, 41 and 42 in the cyclopropane spectrum). Even though these compounds are very similar in size, it is a simple matter to identify them from their individual mass spectra. By clicking on each spectrum in turn, a partial fragmentation analysis and peak assignment will be displayed. Even with simple compounds like these, it should be noted that it is rarely possible to explain the origin of all the fragment ions in a spectrum. Also, the structure of most fragment ions is seldom known with certainty. Since a molecule of carbon dioxide is composed of only three atoms, its mass spectrum is very simple. The molecular ion is also the base peak, and the only fragment ions are CO (m/z=28) and O (m/z=16). The molecular ion of propane also has m/z=44, but it is not the most abundant ion in the spectrum. Cleavage of a carbon-carbon bond gives methyl and ethyl fragments, one of which is a carbocation and the other a radical. Both distributions are observed, but the larger ethyl cation (m/z=29) is the most abundant, possibly because its size affords greater charge dispersal. A similar bond cleavage in cyclopropane does not give two fragments, so the molecular ion is stronger than in propane, and is in fact responsible for the the base peak. Loss of a hydrogen atom, either before or after ring opening, produces the stable allyl cation (m/z=41). The third strongest ion in the spectrum has m/z=39 (C3H3). Its structure is uncertain, but two possibilities are shown in the diagram. The small m/z=39 ion in propane and the absence of a m/z=29 ion in cyclopropane are particularly significant in distinguishing these hydrocarbons.

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Most stable organic compounds have an even number of total electrons, reflecting the fact that electrons occupy atomic and molecular orbitals in pairs. When a single electron is removed from a molecule to give an ion, the total electron count becomes an odd number, and we refer to such ions as radical cations. The molecular ion in a mass spectrum is always a radical cation, but the fragment ions may either be even-electron cations or odd-electron radical cations, depending on the neutral fragment lost. The simplest and most common fragmentations are bond cleavages producing a neutral radical (odd number of electrons) and a cation having an even number of electrons. A less common fragmentation, in which an even-electron neutral fragment is lost, produces an odd-electron radical cation fragment ion. Fragment ions themselves may fragment further. As a rule, odd-electron ions may fragment either to odd or even-electron ions, but even-electron ions fragment only to other even-electron ions. The masses of molecular and fragment ions also reflect the electron count, depending on the number of nitrogen atoms in the species. Ions with no nitrogen odd-electron ions even-electron ions or an even # N atoms even-number mass odd-number mass Ions having odd # N atoms an odd-electron ions even-electron ions odd-number mass even-number mass

This distinction is illustrated nicely by the follwing two examples. The unsaturated ketone, 4-methyl-3-pentene-2one, on the left has no nitrogen so the mass of the molecular ion (m/z = 98) is an even number. Most of the fragment ions have odd-numbered masses, and therefore are even-electron cations. Diethylmethylamine, on the other hand, has one nitrogen and its molecular mass (m/z = 87) is an odd number. A majority of the fragment ions have even-numbered masses (ions at m/z = 30, 42, 56 & 58 are not labeled), and are even-electron nitrogen cations. The weak even -electron ions at m/z=15 and 29 are due to methyl and ethyl cations (no nitrogen atoms). The Prepared B y

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4-methyl-3-pentene-2-one

N,N-diethylmethylamine

When non-bonded electron pairs are present in a molecule (e.g. on N or O), fragmentation pathways may sometimes be explained by assuming the missing electron is partially localized on that atom. A few such mechanisms are shown above. Bond cleavage generates a radical and a cation, and both fragments often share these roles, albeit unequally. 3. Isotopes Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. This is manifested most dramatically for compounds containing bromine and chlorine, as illustrated by the following examples. Since molecules of bromine have only two atoms, the spectrum on the left will come as a surprise if a single atomic mass of 80 amu is assumed for Br. The five peaks in this spectrum demonstrate clearly that natural bromine consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu respectively. Thus, the bromine molecule may be composed of two 79Br atoms (mass 158 amu), two 81 Br atoms (mass 162 amu) or the more probable combination of 79Br-81Br (mass 160 amu). Fragmentation of Br2 to a bromine cation then gives rise to equal sized ion peaks at 79 and 81 amu.

Fig(2) Vinyl Chlorid Fig(1) Bromine

Fig(3) Methylene Chloride

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Prepared B y D hruw ansh The center and right hand spectra show that chlorine is also composed of two isotopes, the more abundant having a mass of 35 amu, and the minor isotope a mass 37 amu. The precise isotopic composition of chlorine and bromine is: Chlorine: 75.77% 35Cl and 24.23% 37Cl Bromine: 50.50% 79Br and 49.50% 81Br The presence of chlorine or bromine in a molecule or ion is easily detected by noticing the intensity ratios of ions differing by 2 amu. In the case of methylene chloride, the molecular ion consists of three peaks at m/z=84, 86 & 88 amu, and their diminishing intensities may be calculated from the natural abundances given above. Loss of a chlorine atom gives two isotopic fragment ions at m/z=49 & 51amu, clearly incorporating a single chlorine atom. Fluorine and iodine, by contrast, are monoisotopic, having masses of 19 amu and 127 amu respectively. It should be noted that the presence of halogen atoms in a molecule or fragment ion does not change the odd-even mass rules given above. 4. Fragmentation Patterns

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The fragmentation of molecular ions into an assortment of fragment ions is a mixed blessing. The nature of the fragments often provides a clue to the molecular structure, but if the molecular ion has a lifetime of less than a few microseconds it will not survive long enough to be observed. Without a molecular ion peak as a reference, the difficulty of interpreting a mass spectrum increases markedly. Fortunately, most organic compounds give mass spectra that include a molecular ion, and those that do not often respond successfully to the use of milder ionization conditions. Among simple organic compounds, the most stable molecular ions are those from aromatic rings, other conjugated pi-electron systems and cycloalkanes. Alcohols, ethers and highly branched alkanes generally show the greatest tendency toward fragmentation.

The mass spectrum of dodecane on the right illustrates the behavior of an unbranched alkane. Since there are no heteroatoms in this molecule, there are no non-bonding valence shell electrons. Consequently, the radical cation character of the molecular ion (m/z = 170) is delocalized over all the covalent bonds. Fragmentation of C-C bonds occurs because they are usually weaker than C-H bonds, and this produces a mixture of alkyl radicals and alkyl carbocations. The positive charge commonly resides on the smaller fragment, so we see a homologous series of hexyl (m/z = 85), pentyl (m/z = 71), butyl (m/z = 57), propyl (m/z = 43), ethyl (m/z = 29) and methyl (m/z = 15) cations. These are accompanied by a set of corresponding alkenyl carbocations (e.g. m/z = 55, 41 &27) formed by loss of 2 H. All of the significant fragment ions in this spectrum are even-electron ions. In most alkane spectra the propyl and butyl ions are the most abundant. The presence of a functional group, particularly one having a heteroatom Y with non-bonding valence electrons (Y = N, O, S, X etc.), can dramatically alter the fragmentation pattern of a compound. This influence is thought to occur because of a "localization" of the radical cation component of the molecular ion on the heteroatom. After all, it is easier to remove (ionize) a non-bonding electron than one that is part of a covalent bond. By localizing the reactive moiety, certain fragmentation processes will be favored. These are summarized in the following diagram, where the green shaded box at the top displays examples of such "localized" molecular ions. The first two fragmentation paths lead to even-electron ions, and the elimination (path #3) gives an odd-electron ion. Note the use of different curved arrows to show single electron shifts compared with electron pair shifts. Prepared B y

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The charge distributions shown above are common, but for each cleavage process the charge may sometimes be carried by the other (neutral) species, and both fragment ions are observed. Of the three cleavage reactions described here, the alpha-cleavage is generally favored for nitrogen, oxygen and sulfur compounds. Indeed, in the previously displayed spectra of 4-methyl-3-pentene-2-one and N,N-diethylmethylamine the major fragment ions come from alpha-cleavages. Further examples of functional group influence on fragmentation are provided by a selection of compounds that may be examined by clicking the left button below. Useful tables of common fragment ions and neutral species may be viewed by clicking the right button. The complexity of fragmentation patterns has led to mass spectra being used as "fingerprints" for identifying compounds. Environmental pollutants, pesticide residues on food, and controlled substance identification are but a few examples of this application. Extremely small samples of an unknown substance (a microgram or less) are sufficient for such analysis. The following mass spectrum of cocaine demonstrates how a forensic laboratory might determine the nature of an unknown street drug. Even though extensive fragmentation has occurred, many of the more abundant ions (identified by magenta numbers) can be rationalized by the three mechanisms shown above. Plausible assignments may be seen by clicking on the spectrum, and it should be noted that all are even-electron ions. The m/z = 42 ion might be any or all of the following: C3H6, C2H2O or C2H4N. A precise assignment could be made from a high-resolution m/z value (next section).

Odd-electron fragment ions are often formed by characteristic rearrangements in which stable neutral fragments are lost. Mechanisms for some of these rearrangements have been identified by following the course of isotopically

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Prepared B y D hruw ansh labeled molecular ions. A few examples of these rearrangement mechanisms may be seen by clicking the following button. 5. High Resolution Mass Spectrometry

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Formula Mass

C6H12 84.0939

C5H8O 84.0575

C4H8N2 84.0688

In assigning mass values to atoms and molecules, we have assumed integral values for isotopic masses. However, accurate measurements show that this is not strictly true. Because the strong nuclear forces that bind the components of an atomic nucleus together vary, the actual mass of a given isotope deviates from its nominal integer by a small but characteristic amount (remember E = mc2). Thus, relative to 12C at 12.0000, the isotopic mass of 16O is 15.9949 amu (not 16) and 14N is 14.0031 amu (not 14). By designing mass spectrometers that can determine m/z values accurately to four decimal places, it is possible to distinguish different formulas having the same nominal mass. The table on the right illustrates this important feature, and a double-focusing high-resolution mass spectrometer easily distinguishes ions having these compositions. Mass spectrometry therefore not only provides a specific molecular mass value, but it may also establish the molecular formula of an unknown compound. Tables of precise mass values for any molecule or ion are available in libraries; however, the mass calculator provided below serves the same purpose. Since a given nominal mass may correspond to several molecular formulas, lists of such possibilities are especially useful when evaluating the spectrum of an unknown compound. Composition tables are available for this purpose, and a particularly useful program for calculating all possible combinations of H, C, N & O that give a specific nominal mass has been written by Jef Rozenski.

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X-Ray Diffraction Analysis
General Description. X-Ray powder Diffraction analysis is a powerful method by which X-Rays of a known wavelength are passed through a sample to be identified in order to identify the crystal structure. The wave nature of the X-Rays means that they are diffracted by the lattice of the crystal to give a unique pattern of peaks of 'reflections' at differing angles and of different intensity, just as light can be diffracted by a grating of suitably spaced lines. The diffracted beams from atoms in successive planes cancel unless they are in phase, and the condition for this is given by the BRAGG relationship. nl = 2 d Sin q l is the wavelength of the X-Rays d is the distance between different plane of atoms in the crystal lattice. q is the angle of diffraction. The X-Ray detector moves around the sample and measures the intensity of these peaks and the position of these peaks [diffraction angle 2q ]. The highest peak is defined as the 100% * peak and the intensity of all the other peaks are measured as a percentage of the 100% peak. 1. J.C.P.D.S. [Joint Committee on Powder Diffraction Standards] This organisation produced standard diffraction patterns for many of the minerals and inorganic structures suitable for analysis by X-Ray Diffraction Spectroscopy, and published these data as standards. The reference standard for Calcium Hydroxylapatite is 9-432. The procedure was carried out by Hodge.cs. The reference standards for other phosphates and for other possible impurities is given in Table 2. There is no specified method for sample preparation quoted in the J.C.P.D.S. handbook, consequently we have outlined below the sample preparation method to be used. 2. Sample preparation. The method preferred for the analysis of Hydroxylapatite powders is the method by which all possible crystal orientations are presented simultaneously to the beam of X-Rays. Some crystallites will be correctly orientated to fulfil the Bragg equation for each value of d, and the detector will measure the intensity of the beams of diffracted X-rays at all locations given by the theoretical values of q. Any impurities will give additional lines which can be identified by looking at the pattern expected from likely impurities such as a Tri-Calcium Phosphate or b TriCalcium Phosphate etc. The material should be removed from the test plate by bending the mild steel plate in a vice, the flaked material then should be ground by hand in a pestle & mortar sufficiently fine so that it will just pass through a 40 micron sieve. The quantity of powder required to fill the sample holder used is 0.35 grams approx and this should be packed into the holder using a standard back fill method sufficiently firmly such that it will not fall out during the 90ø tilt test. The standard backfill method is as follows:Place the sample holder on a glass microscope slide and hold in place with sticky tape. Fill the recess with the ground and sieved sample, tap the holder lightly to ensure the corners are filled, draw another glass microscope slide over the recess to remove excess sample. place the second slide on the sample surface and hold in place with sticky tape. Turn the whole unit over such that the bottom is now the top and remove the first glass slide. The sample is now ready to be inserted into the X-Ray machine. Prepared B y

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Prepared B y 3. Operation method of the X-Ray diffractometer.

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The Xrays used are of the Copper k a wavelength 1.54056 x 10-10m, the scan is taken between 2 theta of 10° and 2 theta of 45° at increments of 0.04° with a count time of 4 seconds for each step. These angles have been selected as this is where the important reflections lie for Hydroxylapatite and other relavent impurities. The count time is selected as 4 seconds to give a good signal to noise ratio and yet to enable the analysis to take place over a reasonable period. The data are collected by computer and stored on floppy discs for later evaluation and hard copy production. See graph 2 enclosed. The intensity of the Xrays are measured on the Y axis, and increasing values of 2 theta are shown on the X axis. The sample is run on the machine and then the Hydroxylapatite standard is run for comparison. 4. Evaluation of data 4a. Relative Crystallinity. Determination of the relative crystallinity for the Hydroxylapatite sample under test utilises an XRD software package and involves integrating the area under the curve on the graph printout between 2 theta of 30°and 2 theta of 35° , the result is then divided by a value obtained similarly from a control sample. (standard P120 hydroxylapatite powder supplied by Plasma Biotal and X-rayed on the same day (under the same scan parameters). The ratio expressed in percentage terms gives the relative crystallinity of the sample tested. The Standard P120 hydroxylapatite powder is compared with the international standard for a Alumina [Reference Standard material 674A available from US National Institute of Standards and Commerce. Gaithersburg, MD. 20899.USA.] N.B. The height of the peaks (intensity) depends upon the number of crystallites diffracting the X-Rays, thus a sample more finely ground will give higher but narrower peaks than the same sample coarsely ground. The area under the graph (as described above) measuring crystallinity will yield the same result in each case whether the sample is finely or coarsely ground. 4b. Quantification of beta Ortho Tricalcium phosphate (Whitlockite). When equal proportions 50% Hydroxylapatite and 50% beta Whitkockite by weight are mixed then upon analysis the 100% peaks for Hydroxylapatite and for beta Whitlockite are of equal intensity. Graphs have been produced for 10%, 5%, 2%, 1%. mixtures of beta Whitlockite with Hydroxylapatite by weight. these are used to check that the estimate produced for whitlockite by the method below is in the correct range. The assessment of % by weight of beta Whitlockite is found by integrating the area under the 100% whitlockite peak (2 theta of 31.027) and the 100% Hydroxylapatite peak (2 theta of 31.774). this fraction when multiplied by the overall relative crystallinity give the percentage (weight by weight) of whitlockite present in the sample (to the nearest significant percentage.) 4c. Unit Cell Dimensions. The Unit cell dimensions are not measured on a regular basis however from many analyses performed (1990-1993) it has become clear that the unit cell dimensions are consistently different in Hydroxylapatite analysed from coating samples, from those obtained by analysing the starting material, this is indicative of minor microstructural changes. These are likely to have occurred during the heating and cooling stages of the process. 4d. Preferred Orientation. * [When analysing Hydroxylapatite it may be possible under certain circumstances for the `100%' peak, normally at Interplanar spacing 2.814 angstroms units to be reduced height and the `40%' peak at Interplanar spacing 3.440 Prepared B y

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Prepared B y D hruw ansh angstroms units to be of increased height such that the 40% peak is higher than the 100% peak. This is thought to arise due to 'preferred orientation' which is described as follows:The crystallites of Hydroxylapatite can be regular 'needle shaped' particles which tend to all lie in a similar direction in the sample holder, this is especially true of the Hydroxylapatite which has been thermally processed by the coating procedure. The X-Ray powder diffraction method requires that the crystallites be randomly packed to give the correct intensity of reflections, thus on occasions the sample may need to be reground and the holder repacked

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Table 1 J.C.P.D.S. Standard for Hydroxylapatite 9-432 Interplanar spacings and peak intensity data Identity IPS-1 IPS-2 IPS-3 IPS-4 IPS-5 IPS-6 IPS-7 IPS-8 IPS-9 IPS-10 IPS-11 IPS-12 IPS-13 IPS-14 IPS-15 IPS-16 IPS-17 IPS-18 IPS-19 IPS-20 IPS-21 IPS-22 IPS-23 IPS-24 IPS-25 IPS-26 d (Andstoms) 8.170 5.260 4.720 4.070 3.880 3.510 3.440 3.170 3.080 2.814 2.778 2.720 2.631 2.528 2.296 2.262 2.228 2.148 2.134 2.065 2.040 2.000 1.943 1.890 1.871 1.841 Intensity/Max Intensity 100 12 6 4 10 10 2 40 12 18 100 60 60 25 6 8 20 2 10 4 8 2 30 16 6 40 h 1 1 1 2 1 2 0 1 2 2 1 1 2 3 2 3 2 3 3 1 4 2 2 3 2 2 k 0 0 1 0 1 0 0 0 1 1 1 1 0 0 1 1 2 1 0 1 0 0 2 1 2 1 l 0 1 0 0 1 1 2 2 0 1 2 2 2 1 2 0 1 1 2 3 0 3 2 2 0 3

Prepared B y

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Prepared B y IPS-27 IPS-28 IPS-29 IPS-30 IPS-31 IPS-32 IPS-33 IPS-34 IPS-35 IPS-36 IPS-37 IPS-38 IPS-39 IPS-40 IPS-41 IPS-42 IPS-43 IPS-44 IPS-45 IPS-46 IPS-47 1.806 1.780 1.754 1.722 1.684 1.644 1.611 1.587 1.542 1.530 1.503 1.474 1.465 1.452 1.433 1.407 1.348 1.316 1.306 1.208 1.265 20 12 16 20 4 10 8 4 6 6 10 12 4 13 9 4 3 5 4 7 3 9 1 11 9 3 4 4 0 1 3 3 5 4 3 2 5 5 3 3 5 4 4 5 4 4 5 2 4 3 6 2 4 5 5

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2 1 0 0 0 2 1 0 2 3 1 0 1 0 2 1 2 1 1 3 0 2 0 2 2 0 1 3 1 2 1 0 2 4 4 2 3 1 0 1 4 2 0 4 3 1 2 3 2 1 4 0 5 3 4 2 5 2 3 2

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IPS-48 1.257 IPS-49 1.249 IPS-50 1.235 IPS-51 1.221 plus other minor lines of less than 1% . System: Hexagonal Table 2 Material Alpha Calcium Orthophosphate Beta Calcium Orthophosphate Calcium Hydrogen Orthophosphate

Formulae Ca3(PO4)2 Ca3(PO4)2 Ca(H2PO4)2.H2O

J.C.P.D.S.* reference 9-169 9-390

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Prepared B y Calcium Phosphate Alpha Calcium Di-Phosphate Beta Calcium Di-Phosphate Beta Calcium Metaphosphate Calcium Hydrogen Orthophosphate Calcium Oxide Calcium Carbonate Hydrate Silicon Dioxide CaP4O11 Ca2P2O7 Ca2P2O7 (CaP2O6)x CaHPO4 CaO CaCO3.H20 SiO2

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21-839 9-345 9-346 11-39 9-77 4-777 22-147 5-0490

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Chromatography
Introduction Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the the components in a mixture. A column (or other support for TLC, see below) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column they can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on-line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC-MS and LC-MS), Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array UV-VIS absorption spectroscopy (HPLC-UVVIS). Specific chromatographic methods: Gas chromatography (GC) Applied to volatile organic compounds. The mobile phase is a gas and the stationary phase is usually a liquid on a solid support or sometimes a solid adsorbent. High-performance liquid chromatography (HPLC) A variation of liquid chromatography that utilizes high-pressure pumps to increase the efficiency of the separation. Liquid chromatography (LC) Used to separate analytes in solution including metal ions and organic compounds. The mobile phase is a solvent and the stationary phase is a liquid on a solid support, a solid, or an ion-exchange resin. Prepared B y

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Prepared B y Size-exclusion chromatography (SEC)

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Also called gel-permeation chromatography (GPC), the mobile phase is a solvent and the stationary phase is a packing of porous particles. Thin-layer chromatography (TLC) A simple and rapid method to monitor the extent of a reaction or to check the purity of organic compounds. The mobile phase is a solvent and the stationary phase is a solid adsorbent on a flat support.

Prepared B y

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