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Unit 29

Spectroscopy & Structure

Techniques & Instrumentation

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Overview

One primary consideration in selecting an analytical technique for identification purposes is the need for either a qualitative or a quantitative determination. The former relates just to the identity of the material, while the latter requires the precise determination of amounts, quantities, and percent compositions by weight.

Now that the basic components of matter have been defined, the proper selection of analytical techniques that will allow the analytical chemist to identify or compare matter can best be understood by classifying all substances into one of two very broad chemical groups: organics and inorganics. Organic substances are based the element carbon, and comprise all forms of matter and energy which live and breathe. Inorganic substances encompass all other known chemical substances. Each of these two broad categories has both chemical and physical properties that are quite distinctive and characteristic. 

Organic compounds consist of carbon atoms, hydrogen atoms, and functional groups. The valence of carbon is 4, and hydrogen is 1, functional groups are generally 1. From the number of carbon atoms and hydrogen atoms in a molecule the degree of unsaturation can be obtained. Many, but not all structures can be envisioned by the simple valence rule that there will be one bond for each valence number. The knowledge of the chemical formula for an organic compound is not sufficient information because many isomers (different structural forms with the same chemical formula) can exist.

Techniques of elemental analysis are generally destructive in their use for the determination of elemental composition of individual compounds and molecules. Spectroscopy is the method most often used, it all its various forms. The instruments most directly concerned with our primary interest, molecular structure, are the spectrometers - measurers of spectra. Of the various spectra, we shall work chiefly with only two kinds: infrared (IR) and nuclear magnetic resonance (proton NMR and CMR), since they are the workhorses of the modern organic chemistry laboratory. Of these, we shall devote an entire chapter to NMR. We shall look briefly at three other kinds of spectra: mass, ultraviolet (UV) and electron spin resonance (ESR).

Organic compounds often exist as mixtures. Because many organic compounds have relatively low boiling points and/or dissolve easily in organic solvents there exist many methods for separating mixtures into pure constituents that are specific to organic chemistry such as distillation, crystallization and chromatography techniques. Thus, there exist a number of different methods for deducing the structure and/or chemical composition an organic compound. These include:

IR Spectroscopy, Atomic Spectroscopy, Mass Spectrometry, Nuclear Magnetic Resonance (NMR), X-Ray Diffraction, UV/Vis Spectroscopy, and Chromatography, all of which will be briefly described in this unit. 

Spectroscopy

Spectroscopy is the study of matter and its properties by investigating light, sound, or particles that are emitted, absorbed or scattered by the matter under investigation. Spectroscopy may also be defined as the study of the interaction between light and matter. Historically, spectroscopy referred to a branch of science in which visible light was used for theoretical studies on the structure of matter and for qualitative and quantitative analyses. The definition has broadened, however, as new techniques have been developed that utilize not only visible light, but many other forms of electromagnetic and non-electromagnetic radiation, including microwaves, radiowaves, x-rays, electrons, phonons (sound waves) and others.

Impedance spectroscopy is a study of frequency response in alternating current. Spectroscopy is often used for the identification of substances through the frequency spectrum emitted or absorbed by them. A device for recording a spectrum is a spectrometer. Spectroscopy can be classified according to the physical quantity which is measured or calculated or the measurement process.

I. IR Spectroscopy

Of all the properties of an organic compound, the one that gives the most information about the compound's structure is its infrared spectrum. Thus, infrared spectroscopy is used to determine the presence of certain functional groups. Infrared frequencies of light are used to study fundamental vibrations and associated rotational-vibrational structure via vibrational resonance and selective absorption.

Infrared spectroscopy works because chemical bonds have specific frequencies at which they vibrate corresponding to energy levels. In order for a vibrational mode in a molecule to be IR active, it must be associated with changes in the permanent dipole. The resonant frequencies are determined by the normal modes of vibration. (Normal modes are also known as natural frequencies or resonant frequencies. There is a set of these frequencies that are unique to each molecular structure). These natural frequencies can be related to the strength of the bond, and the mass of the atoms (or harmonic oscillators) at either end of it. Thus, the frequency of the vibrations can be associated with a particular bond type.

Changes in the vibrational frequencies of a molecule are caused by the absorption of infrared light. A particular portion of the infrared spectrum is referred to either by its wavelength (in microns or Angstroms) of frequency (not Hz, but rather in wavenumbers, or reciprocal centimeters. The wavenumber is simply the number of wave periods per centimeter, and is equal to the reciprocal of the wavelength in centimeters. 

A particular group of atoms gives rise to characteristic absorption bands. For example, the OH group absorbs strongly at 3200-3600 per cm; the C=O group of ketones absorbs strongly at 1710 per cm; the C=N group absorbs strongly at 2250 per cm; and the CH3 group absorbs at both at 1450 and 1375 per cm (2 different modes of vibration).  

Interpretation of an IR spectrum is not a simple matter. Bands may be obscured by the overlapping of other bands. Overtones (or harmonics) may appear at exactly twice the frequency of the fundamental band. The absorption band of a particular group may be shifted by various structural features, such as conjugation, electron withdrawal by a neighboring substituent, angle strain or van der Waals strain, or hydrogen bonding.

                               Characteristic IR Absorption Frequencies

 

Simple diatomic molecules have only one bond, which may stretch. More complex molecules may have many bonds, and vibrations can be conjugated, leading to infrared absorptions at characteristic frequencies that may be related to chemical groups. The atoms in a CH2 group, commonly found in organic compounds can vibrate in six different ways, symmetrical and asymmetrical stretching, scissoring, rocking, wagging and twisting.

In order to measure a sample, a beam of infrared light is passed through the sample, and the amount of energy absorbed at each wavelength is recorded. This may be done by scanning through the spectrum with a monochromatic beam, which changes in wavelength over time, or by using a Fourier transform (FTIR) instrument to measure all wavelengths at once. From this, a transmittance or absorbance spectrum may be plotted, which shows at which wavelengths the sample absorbs the IR, and allows an interpretation of which bonds are present.

The infrared portion of the electromagnetic spectrum is divided into three regions; the near-IR, mid-IR and far-IR (relative to the visible spectrum).

1) The far-IR (~ 400-10 cm-1). Adjacent to the microwave region, the longer wavelength far-IR has lower energy and may be used for rotational spectroscopy.

2) The mid-IR (~ 4000-400 cm-1) may be used to study the fundamental vibrations and associated rotational-vibrational structure.

3) The near-IR (14000-4000 cm-1) with its higher energy level and higher frequencies can excite overtone or harmonic vibrations.

This technique works almost exclusively on covalent bonds, which are those found predominantly in organic compounds. Clear spectra are obtained from samples with few IR active bonds and high levels of purity. More complex molecular structures lead to more absorption bands and more complex spectra. The technique has been used for the characterization of very complex mixtures.

 

II. Atomic Spectroscopy

Atomic spectroscopy, such as flame atomic absorption, graphite furnace atomic absorption, and inductively coupled plasma atomic emission, are used to probe the outer electronic structure of atoms. Other spectroscopic methods which are used to probe the inner electronic structure of atoms include X-ray fluorescence, particle induced x-ray emission, x-ray photoelectron spectroscopy, and Auger electron spectroscopy.

Atomic spectroscopy is the determination of elemental composition either by its electromagnetic spectrum or its mass spectrum. Atomic spectroscopy is closely related to other forms of spectroscopy. It can be divided by atomization source or by the type of spectroscopy used. In the latter case, the main division is between optical spectroscopy and mass spectroscopy (or spectrometry).

Mass spectrometry generally gives significantly better analytical performance, but is also significantly more complex. This complexity translates into higher purchase costs, higher operational costs, more operator training, and a greater number of components that can potentially fail. Because optical spectroscopy is generally less expensive and has performance adequate for many tasks, it is far more common. Atomic absorption spectrometers are one of the most commonly sold and used analytical devices.

Optical Spectroscopy

Electrons exist in energy levels within an atom. These levels have well defined energies and electrons moving between these levels must absorb or emit an energy equal to the difference (or "quantum of energy") between them. In optical spectroscopy, the energy absorbed to move an electron to a more energetic level and/or the energy emitted as the electron moves to a less energetic energy level is in the form of a photon (a particle of light). Because this energy is well-defined, an atom's identity (i.e. what element it is) can be identified by the energy of this transition. The wavelength of light can be related to its energy. It is usually easier to measure the wavelength of light than to directly measure its energy.

Optical spectroscopy can be divided into absorption, emission, and fluorescence.

In atomic absorption spectroscopy, light is passed through a collection of atoms. If the wavelength of the light has energy corresponding to the energy difference between two energy levels in the atoms, a portion of the light will be absorbed. The relationship between the concentration of atoms, the distance the light travels through the collection of atoms, and the portion of the light absorbed is given by the Beer-Lambert law.

The energy stored in the atoms can be released in a variety of ways. When it is released as light, this is known as fluorescence. Atomic fluorescence spectroscopy measures this emitted light. Fluorescence is generally measured at a 90° angle from the excitation source to minimize collection of scattered light from the excitation source, often such a rotation is provided by a Pellin-Broca prism on a turntable which will also separate the light into its spectrum for closer analysis. The wavelength once again tells you the identity of the atoms. For low absorbances (and therefore low concentrations) the intensity of the fluoresced light is directly proportional to the concentration of atoms. Atomic fluorescence is generally more sensitive (i.e. it can detect lower concentrations) than atomic absorption.

Strictly speaking, any measurement of the emitted light is emission spectroscopy, but atomic emission spectroscopy usually does not include fluorescence and rather refers to emission after excitation by thermal means. In a manner similar to fluorescence, the wavelength of the emitted photon once again tells you the identity of the atoms. And again, the intensity of the emitted light is directly proportional to the concentration of atoms.

Atomic Mass Spectrometry

Atomic mass spectrometry is similar to other types of mass spectrometry in that it consists of an ion source, a mass analyzer, and a detector. The identities of atoms are determined by their mass-to-charge ratio (via the mass analyzer) and their concentrations are determined by the number of ions detected. Although considerable research has gone into customizing mass spectrometers for atomic ion sources, it is the ion source that differs most from other forms of mass spectrometry. These ion sources must also atomize samples, or an atomization step must take place before ionization. Atomic ion sources are generally modifications of atomic optical spectroscopy atom sources.

 

III. Mass Spectrometry

Mass spectrometry is used to determine the molecular weight of a compound, as well as its molecular structure from the fragmentation pattern. The device measures the mass-to-charge ratio of ions, or m/e value. Each kind of ion has a particular m/e value. This measurement is achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring relative intensities of ion flux. Techniques such as inductively coupled mass spectrometry is used to probes the masses of individual atoms.

Thus, different compounds have different atomic masses, and this fact is used in a mass spectrometer to determine what chemicals are present in a sample. For example, table salt (NaCl) is vaporized and then ionized into electrically charged ions (Na+ and Cl-). The sodium cations (Na+) and chloride anions (Cl-) have specific atomic weights. They also have an electrical charge, which means that their path can be controlled with an electric or magnetic field.

The ions are sent into an acceleration chamber and passed through a slit in a metal sheet. A magnetic field is applied to the chamber. The field pushes each ion perpendicular to the plane defined by the particles direction of travel and the magnetic field lines. They are then deflected onto a detector. The lighter ions are deflected more than the heavier ions because (Newton's second law of motion) the acceleration of a particle is inversely proportional to its mass. Likewise, the magnetic field can push the lighter ions further, thereby giving them a larger deflection, than the heavier ions. The detector measures exactly how far each ion has been deflected. From this data, the ion's 'mass-to-charge ratio' can be worked out. Thus, it is possible to determine with a high level of certainty the chemical composition of the original sample.

The set of ions is analyzed in such a way that a signal is obtained for each value of m/e that is represented by the data. The intensity of each signal reflects the relative abundance of the ion producing the signal. The largest peak is called the base peak. Its intensity is taken as 100, and the intensities of the other peaks are expressed relative to it. Thus, the results are 'normalized' with respect to the largest peak.

A plot of the relative peak intensities vs. m/e value is called a mass spectrum, and is highly characteristic of a particular compound. Mass spectra can be used to:

1) Establish the identity of two compounds

2) Help identify the structure of an new or unknown compound.

Two compounds are shown to be identical by the fact that they have identical physical properties (m.p., b.p., density, refractive index, etc.). The greater the number of physical properties measured, the stronger the evidence. Now, a single mass spectrum amounts to dozens of physical properties, since it shows the relative abundance of dozens of different fragments. Thus, if we measure the mass spectrum of an unknown compound and find it to be identical with the spectrum of a previously reported compound of known structure, then we can conclude that the two compounds are identical.  

The mass spectrum can give a precise molecular weight, as well as indicating the presence of certain structural units.

Results

The mass spectrum of benzene is relatively simple and illustrates some of the information that mass spectrometry provides. The most intense peak in the mass spectrum is called the base peak and is assigned a relative intensity of 100. Ion abundances are proportional to the peak intensities and are reported as intensities relative to the base peak. The base peak in the mass spectrum of benzene corresponds to the molecular ion (M+) at m/z = 78.

Benzene does not undergo extensive fragmentation. None of the fragment ions in its mass spectrum are as abundant as the molecular ion. The small peak at m/z = 79 is due to the presence of the isotope C-13 in place of one of the C-12 atoms in the ring. Indeed, because all hydrocarbon compounds contain carbon and most contain hydrogen, similar isotopic clusters will appear in the mass spectra of all organic compounds.  

Unlike the case of benzene, in which ionization involves the loss of a pi electron from the ring, electron-impact-induced ionization of chlorobenzene involves the loss of an electron from an unshared pair of electrons. The molecular ion then fragments by C-Cl bond cleavage.

The peak at m/z = 77 in the mass spectrum of chlorobenzene is attributed to this fragmentation and the resulting phenyl cation. The peak at m/z = 77 + 35 = 112 would then correspond to the molecular ion of chlorobenzene.   

Some classes of compounds  so prone to fragmentation that the molecular ion peak is very weak. The base peak in most unbranched alkanes, for example, is located at m/z = 43 (corresponding to the propyl cation). This relatively weak base peak is followed by peaks of decreasing intensity at m/z values of 57, 71, 85, etc. corresponding to cleavage of each possible C-C bond in the molecule. The pattern is evident in the mass spectrum of decane (below), and the points of cleavage are indicated in the diagram below.

 

Ion /Atom Sources

Sources can be adapted in many ways, but the lists below gives the general uses of a number of sources. Of these, flames are the most common due to their low cost and their simplicity. Although significantly less common, inductively-coupled plasmas, especially when used with mass spectrometers, are recognized for their outstanding analytical high performance and their versatility.

For all atomic spectroscopy, a sample must be vaporized and atomized. Alternatively, for atomic mass spectrometry, a sample must also be ionized. Vaporization, atomization, and ionization are often, but not always, accomplished with a single source. Alternatively, one source may be used to vaporize a sample while another is used to atomize (and possibly ionize). An example of this would be laser ablation inductively-coupled plasma atomic emission spectrometry. A laser is used to vaporize a solid sample and an inductively-coupled plasma is used to atomize the vapor.

With the exception of flames and graphite furnaces (commonly used for atomic absorption spectroscopy) most sources are used primarily for atomic emission spectroscopy.

Liquid-sampling sources include:

Solid-sampling sources include

Gas-sampling sources include

 

IV. Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) spectroscopy is one of the principal non-destructive techniques used to obtain physical, chemical, electronic and structural information. This method identifies different nuclei (& elements) from their magnetic properties, or their magnetic moment. This is the most important and commonly used spectroscopic technique for organic chemists, often permitting complete assignment of atom connectivity and even stereochemistry given the proper set of spectroscopy experiments (e.g. correlation spectroscopy).

Nuclear magnetic resonance (NMR) is a physical phenomenon based upon the quantum mechanical magnetic properties of an atom's nucleus. All nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. The most commonly measured nuclei are hydrogen-1 (the most receptive isotope at natural abundance) and carbon-13 (CMR), although nuclei from isotopes of many other elements can also be observed. NMR studies magnetic nuclei by aligning them with a very powerful external magnetic field and perturbing this alignment using an electromagnetic field. The resulting response to the external perturbing electromagnetic magnetic is the phenomena that is exploited in nuclear magnetic resonance spectroscopy and magnetic resonance imaging.

NMR spectroscopy is one of the principal techniques used to obtain physical, chemical, electronic and structural information about molecules. It is a powerful technique that can provide detailed information on the topology, dynamics and three-dimensional structure of molecules in solution and the solid state. Also, nuclear magnetic resonance is one of the techniques that has been used to build elementary quantum computers.

 

V. Crystalline Structure: Diffraction

This is the most precise method for determining molecular geometry. However, it is very difficult to grow crystals of sufficient size and high quality to get a clear picture. Thus it remains a secondary form of analysis. Crystallography has seen especially extensive use in biochemistry (for protein structure determination) and in the characterization of organometallic catalysts, which often possess significant symmetry. Primary methods include X-ray diffraction, neutron diffraction and electron diffraction.

95% of all solid matter which exists on the surface of the earth has a distinctive and identifiable crystalline structure. This means there is a certain degree of long-range order which exists between the individual molecules or molecular building blocks. Thus the molecules in a single crystal can be thought of as being composed of a series of parallel planes.

                             www.ill.fr/dif/3D-crystals/index.html

X-ray diffraction is a technique in crystallography in which the pattern produced by the diffraction of X-rays through the closely spaced lattice of atoms in a crystal is recorded and then analyzed to reveal the ordered nature of the crystal lattice. This generally leads to an understanding of the material and molecular structure of a substance. The spacing in the crystal lattice can be determined using Bragg's law. The electrons that surround the atoms, rather than the atomic nuclei themselves, are the entities that physically interact with the incoming X-ray photons.

X-ray diffraction thus relies on the principle of superposition of waves. When waves interact with one another, they can do so either destructively or constructively. In the former case, their incoherent structural features (peaks and troughs) are not “in sync” with each other, and the result is “noise” (destructive interference) as opposed to “music”. When the coherent waves are ‘in sync”, their peaks overlap in space to produce a single amplified wave of identical wavelength. This process is known as constructive interference.

Multiple X-rays entering a highly ordered crystalline environment find themselves reflected from parallel planes whose spacing (or distance of planar separation) is similar to the wavelength of the incident radiation. This ideal condition results in constructive interference of the X-rays. The result is the characteristic periodic arrangement of light and dark bands of local intensity maxima and minima known as the diffraction pattern of the X-rays as they are reflected form the planes inside of the crystal. You might think of these concentric bands as being analogous to the ripples (peaks and troughs) in a pool as you drop in a stone. All waves behave in such a similar fashion.  

                                 X-ray diffraction pattern


Thus, when a material (sample) is irradiated with a parallel beam of monochromatic X-rays, the atomic lattice of the sample acts as a three dimensional diffraction grating causing the X-ray beam to be diffracted to specific angles. The diffraction pattern includes both the position (angles) and intensities of the diffracted beam.

Angles are used to calculate the distance d of planar separation or atomic spacing (d-spacing). The atomic spacing (or lattice parameter) d is related to the angle of incidence  by the following relationship, which his known as Bragg’s equation:

                                                       

                                          n = some whole number integer

                                   =  wavelength of incident radiation

Because every crystalline material will give a characteristic diffraction pattern and can act as a unique fingerprint, the position (d) and intensity (I) information are used to identify the type of material by comparing them with patterns for over 80,000 experimental data entries. These entries can be accessed in the International Powder Diffraction File (PDF) database, complied by the Joint Committee for Powder Diffraction Standards (JCPDS).

By this method, identification of any crystalline compounds, even in a complex sample, can be made. The position (d) of diffracted peaks also provides information about how the atoms are arranged within the crystalline compound (unit cell size or lattice parameter). The intensity information is used to assess the type and nature of atoms. Determination of lattice parameter can also be helpful in understanding the extent of solid solution: complete or partial substitution of one element for another, as in some metal alloys.

Thus, X-ray diffraction is a versatile, non-destructive technique that reveals detailed information about the chemical composition and crystallographic structure of natural and manufactured materials. Areas of application are quite wide and include metals, organic and inorganic compounds. Also included are airborne dusts and particulate matter, hazardous inorganic chemicals, asbestos, metals, bio-materials (bone or dental implants), pharmaceuticals, catalysts, polymers, ceramics and composites, rocks and minerals, clays & soils, semiconductors, and corrosion products.

Unfortunately, all glassy (or vitreous) solids are non-crystalline in nature. Thus their molecular arrangements do not reflect anything but some limited degree of short-range order, and do not create the type of constructive interference necessary to produce a recognizable X-ray diffraction pattern. Thus the use of X-ray diffraction for chemical identification is limited to crystalline solids only.

Even though X-rays are the most common choice. For some purposes electrons or neutrons are used, which is possible due to the wave properties of the particles. These methods are referred to as:  neutron diffraction and electron diffraction.

Since neutrons are electrically neutral (not charged), they do not interact with the electron cloud surrounding the atom (unlike x-ray and electron diffraction). The neutrons will only interact with the nucleus of the atom. Thus, neutron diffraction reveals the atomic structure only, and not charge distribution around the atom (although the two are usually very similar).

Neutron diffraction reveals structural details of the target material, which are measured by recording the way in which neutrons are deflected. Neutrons can also change their speed during the scattering experiment. This is used to study the types of vibrations that can occur in the solid. One important difference between neutron and X-ray diffraction is that neutrons are sensitive to magnetic forces in the material.

Electron diffraction experiments are usually performed in a transmission electron microscope (TEM), or a scanning electron microscope (SEM) as electron backscatter diffraction. In these instruments, the electrons are accelerated by an electrostatic potential in order to gain the desired energy and wavelength before they interact with the sample to be studied.

 

VI. UV / Vis Spectrophotometry

Spectrophotometry is more specific than the general term spectroscopy, in that spectrophotometry deals with visible (white) light and ultraviolet (UV) radiation as opposed to the entire electromagnetic spectrum. In between the IR and UV portions of the electromagnetic spectrum, light of wavelength between about 400 nm and 750 nm is visible to the naked eye. UV spectrometers commonly measure absorption of light in the visible and near UV region (200 -750 nm). This light is of higher frequency (and greater energy) than IR light and, when it is absorbed by a molecule, the changes it produces require greater energy. These are typically changes in electronic states. The absorption of light is typically due electronic transitions in molecules (or "color centers"). 

Conjugation / Color Centers

The UV spectrum is not used primarily ot show ht presence of individual functional groups. It is rather used to show relationships which exist between functional groups. UV/VIS spectroscopy is often used to determine the degree of conjugation in the system:

1) Conjugation between two or more C=C double (or triple) bonds.

2) Conjugation between C=C and C=O double bonds.

3) Conjugation between double bonds and an aromatic ring.

4) The presence of an aromatic ring.

It can, in addition, reveal the number and location (but not identity) of substituent groups attached to the carbon atoms of the conjugated system.

In a transition to a higher electronic level, a molecule can go from any of a number of sublevels (vibrational and rotational states) to any of a number of other sublevels. Thus UV absorption bands are more broad than those seen in a typical IR spectrum. As opposed to the many sharper peaks of an IR spectrum, a typical UV spectrum shows only a few broad humps. Parameters typically used to describe such a spectrum are the position of the top of the hump (λmax) and the intensity of that absorption (ε, the extinction coefficient).

A sigma electron is held tightly, and a good deal of energy is required to excite it (corresponding to short wavelength UV radiation). It is often excitations of the comparatively loosely held n and pi electrons that appear in the longer wavelength (near UV) spectrum. And, of these, only jumps to the lower (more stable) excited states.

Conjugation of double bonds lowers the energy required for an electron to shift from a stable (bonding) orbital to a more unstable (non-bonding) orbital. Thus, absorption moves to longer wavelengths, where it can be more conveniently measured. If there are enough double bonds in conjugation, absorption will move into the visible region, and the compound will be colored. Beta-carotene is a yellow pigment found in carrots and green leaved plants and vegetables (such as spinach) and is a precursor of vitamin A. It contains 11 C=C double bonds in conjugation, and owes its color to absorption at the violet end of the visible spectrum  λmax  = 451 nm.

Molar Concentration

An essential piece of information about any molecular species is how much of it is present. Quantitative measures of concentration are essential in all scientific fields. Of all the methods that have been devised for measuring concentration, by far the most widely applied is absorption spectrophotometry (especially in liquid samples). 

A spectrophotometer (or photometer) is a device for measuring light intensity. The instrument typically measures the  intensity as a function of the color, frequency or wavelength of light. The amount of light that a sample absorbs at a particular wavelength is measured and used to determine the concentration of the sample by comparison with appropriate standards or reference data.

The most useful measure of light absorption is the absorbance (A), also commonly called the optical density (OD). The absorbance is defined as:

                                               

                            =   Intensity of light incident on the sample

                             =   Intensity of light transmitted by the sample



                                 1

 The absorbance of a sample at any given wavelength can be related to the concentration of the absorbing species through Beer's law:                                           

                                                A  =  c ε

                c is concentration (usually measured in moles per liter)

             ε is a proportionality constant (the molar extinction coefficient) 

The value of ε is a function of both the particular compound being measured and the wavelength. Chlorophylls typically have an ε value of about 100,000 liters / mole. When more than one component of a complex mixture absorbs at a given wavelength, the absorbances due to the individual components are generally additive. For a review of the mechanisms responsible for the absorption of light:        

           www.shu.ac.uk/schools/sci/chem/tutorials/molspec/uvvisab1.htm
 

The essential parts of a spectrophotometer include a light source, a wavelength selection device such as a monochromator or filter, a sample chamber, a light detector, and a readout device (often include a computer for data storage and spectral analysis). The most useful machines scan the wavelength of the light that is incident on the sample and produce, as output, spectra of absorbance versus wavelength.  

                 1         

The choice of a source will vary depending on what type of radiation is desired. For visible radiation, an ordinary tungsten bulb provides a convenient source. In the UV region, a hydrogen or deuterium discharge lamp is normally used. For IR radiation, a heated molded rod containing a mixture of rare earth oxides provides a good source.

The function of the monochromator is to select a single wavelength or frequency from the light source. Colored glass filters can provide a convenient solution to this problem. Higher precision may require a prism or diffraction grating. 

While glass and quartz are often used for as cell materials, special sample cells composed of basic salts, such sodium chloride or potassium bromide, are commonly used because they will not absorb light over a wide range of the IR portion of the electromagnetic spectrum.

The detector measures the quantity of radiation that passes thru the sample by converting it to an electrical signal. UV and visible spectrophotometers employ photoelectric tube detectors. A signal is generated when the photons strike the tube surface to produce a current that is directly proportional to the intensity of the light that is transmitted through the sample. When this signal is compared to an experimental “blank” (the intensity of light transmitted to he detector in the absence of an absorbing material), the absorbance of a substance can be determined at selected frequencies or wavelengths.

The signal from the detection system is then fed into a recorder, which plots absorbance as a function of wavelength or frequency. Modern spectrophotometers are designed to trace an entire absorption spectrum automatically. Many are equipped with Fourier transform capabilities, which are used to decode the measured signals of all wavelengths simultaneously and record the generated data.

An ultraviolet-visible spectrum is essentially a graph of light absorbance vs. wavelength in a range of ultraviolet or visible regions. Such a spectrum can often be produced by a more sophisticated spectrophotometer. Wavelength is often represented by the symbol λ. Similarly, for a given substance, a standard graph of extinction coefficient ε vs. wavelength λ may be made or used if one is already available. Such a standard graph would be effectively "concentration-corrected" and thus independent of concentration. For the given substance, the wavelength at which maximum absorption in the spectrum occurs is called λmax (Lambda-max).

Woodward-Fieser rules are a set of empirical observations which can be used to predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as dienes and ketones.

 

VII. Chromatography

Although spectrophotometry is exceedingly useful in the chemical identification of many organic materials, its optimum utilization requires that a material be in a relatively high state of chemical purity. Because the impurity levels of any given sample is often beyond the control of the analytical chemist, this criterion is often not met. For this reason, the analytical technique of chromatography is widely applied for the analysis of physical evidence. Chromatography is a means of separating and tentatively identifying the components of a chemical mixture. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be chemically isolated.

For example, illicit drugs and other controlled substances which are sold on the street are not manufactured to meet government labeling standards. Instead the may be “diluted” with practically any material that is readily available to the dealer in order to increase the quantity and associated street value of the product. Hence, the task of identifying an illicit drug preparation would be an arduous one without the aids of chromatographic methods to first separate the mixture into its components.  

The theory of chromatography has as its basis the observation that chemical substances have a tendency to partially escape into the surrounding environment when dissolved in a liquid or when absorbed on a sold surface. In short, the distribution or “partitioning” of a gas between the liquid and gas phases is determined by the solubility of the gas in the liquid. The higher its solubility, the greater the tendency of the gas molecules to remain in the liquid phase.

More importantly, if two different gases (analytes A & B) are dissolved in the same liquid, each will reach a state of chemical equilibrium independently of one another. Their solubilities will be determined solely by their respective solubilities in the liquid. 

The essence of chromatography is that some (but not all) substances will actually have a tendency to move in a specific direction over a stationary (or fixed) liquid phase in response to an applied flow. This motion is enhanced by forcing the motion of the air over the liquid.  

Interestingly, those components containing the highest number of molecules in the mobile gas phase (or present at higher number densities) will move more rapidly in response to the air flow. Eventually, when the entire gas phase (air + analytes) has advanced a reasonable distance, the component with the higher mobility will become entirely separated from the others.

The three different types of chromatography commonly used in the laboratory include: Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), and Thin Layer Chromatography (TLC). We briefly outline those here.

Gas Chromatography (GC)

Gas chromatography (or gas-liquid chromatography) separates mixtures on the basis of their distribution between a stationary liquid phase and a mobile gas phase. A chemically inert carrier gas (e.g. nitrogen or helium) flows through a column made of stainless steel or glass. The liquid sample under investigation is injected into a heated injection port by syringe, where it is immediately vaporized and swept into the column by the carrier gas. As the carrier gas flows through the column, it carries along with it the components of a mixture that have been injected into the column. Components will be separated according to their relative mobilities (and corresponding solubilities) in the mobile gas phase.

As each component emerges from the column, it enters a detector. One type of detector uses a flame to ionize the emerging chemical substance, thus generating an electrical signal. The signal is recorded onto a strip-chart recorder as a function of time. This record is referred to as a chromatogram. 

A typical gas chromatogram will show a series of peaks, each one corresponding to a specific component of the chemical mixture. The time required for a component to emerge form the column from the time of its injection is known as the retention time. This experimentally determined parameter serves as a useful identifying characteristic of a material (tho it may vary for different instrument configurations).

Gas chromatography has an added advantage in that it is extremely sensitive and can yield quantitative results. The amount of substance passing thru the GC detector is directly proportional to the area underneath the recorded peak. The amount of sample can often be determined to within the nearest billionth of a gram.

One important extension of this application in forensic science is the technique of Pyrolysis GC. Many solid materials commonly encountered as physical evidence (paint chips, fibers, plastics, etc.) cannot be readily dissolved in a solvent for liquid injection. However, materials such as these can be heated or pyrolyzed to high temperatures (500 – 1000 degrees C) so that they will decompose into numerous gaseous products. Pyrolyzers have been designed in order to permit these gaseous products to enter the carrier gas stream, flowing into and thru the GC column.    

Liquid Chromatography (HPLC)

In the case of high–performance liquid phase chromatography (HPLC), the stationary phase is a finely ground particulate solid. The mobile (carrier) phase is a liquid that is pumped thru a column filled with fine solid particles whose surfaces have been chemically treated. The major advantage of HPLC is that the entire process takes place at room temperature. Examples of materials where HPLC may be the preferred method of choice may include organic explosives, which are generally heat sensitive. Also included are heat sensitive psycho-active drugs, such as LSD.

Thin-Layer Chromatography

The technique of thin-layer chromatography (TLC) incorporates a solid stationary phase and a mobile liquid phase to effect the separation of the constituents of a mixture. A thin-layer plate is prepared by coating a glass plate with a thin film of granular material, which is bonded to the plate with plaster of Paris.

Solid samples must first be dissolved and a few microliters of the solution spotted with a capillary tube or syringe onto the granular surface near the lower edge of the plate. A liquid sample may be applied directly to the plate in a similar manner. The plate is then placed upright into a closed chamber that contains a selected liquid, with care that the liquid does not touch the sample spot.

The liquid will slowly begin to rise up along the surface of the plate by capillary action. The rising liquid serves as the mobile carrier phase. As it moves past the sample spot, the components of the sample become distributed between the stationary solid phase and the mobile liquid phase. Those components with a greater solubility in the mobile liquid phase will travel up the surface of the plate at a faster speed. When the liquid front has moved a sufficient distance (usually 10 cm) the development is complete, and the plate is removed from the chamber and dried.

Because most components are colorless (exceptions: organic pigments such as chlorophyll and beta-carotene) no separation will be noticeable after development unless the materials are visualized. To accomplish this, the plates are placed under UV light, revealing those materials that fluoresce as bright spots on a dark background. For identification purposes, a high confidence level necessitates the use of experimental standards which can be compared to the results for confirmation of the suspect material’s chemical composition.  

Migration distances must therefore be matched with similar experiments on similar compounds using similar experimental configurations and apparatuses. If the distances are identical, then a tentative identification can be made. However, it must be cautioned that such identification cannot be considered definitive for the possibility always exists that numerous other substances can migrate the same distance up the plate when chromatographed under similar conditions. Thus, thin-layer chromatography cannot by itself provide an absolute and undisputed identification. Thus, TLC must be used in conjunction with other testing procedures in order to provide absolute proof of chemical identity.    

GC / MS

Many of the uncertainties associated with vapor phase chromatography (GC) have been overcome by linking the gas chromatograph to a mass spectrometer to yield an extremely powerful combination known as gas chromatography / mass spectrometry (GC / MS).

The separation of a mixture’s components is first accomplished on the gas chromatograph. A direct connection between the GC column and the mass spectrometer then allows each component to flow into the spectrometer as it emerges form the gas chromatograph.

In the mass spectrometer, the material enters a high-vacuum chamber where a beam of high-energy electrons is aimed at the sample molecules. The electrons collide with the molecules, causing them to lose electrons and to acquire a positive charge. These positively charged (+) cations are highly unstable and rapidly decompose into smaller fragments. These fragments then pass through an electric or magnetic filed, where they are separated according to their masses. The advantage of this technique is that no two substances produce the same fragmentation pattern.   

Hence, this fragmentation pattern serves as a “fingerprint” of a specific chemical substance. It is also sensitive to minute concentrations. With the data obtained from a GC/ MS determination, a forensic analyst can, with one instrument, separate the components of a complex drug mixture and then unequivocally identify each substance present in the mixture.