Marcela A. Oliveira-Antunovich
A new trend has developed where television shows are being geared toward forensic science, (i.e. CSI: New York, CSI: Miami, Numbers, Bones, etc.). As a teacher, I ask my students where their prospects for the future lie and many have indicated an interest in going for forensic science. Who am I to dismiss such an outstanding culmination of scientific inquiry? For this very reason, I have decided to focus the curriculum unit on the investigation of a crime that has taken place New Haven, CT. Through processes of inquiry and laboratory research, the students will be able to identify whether the crime was intentional or a mere freak accident. As a clarification, the unit is not based on any real events but on an "invented incident" that will provide a scenario to facilitate in the explanation and relation of topics.
In order to accomplish the task at hand, students will perform the following tasks:
Collect evidence and run the following tests either through internal procedures or requests for external laboratory assaying:
-
- pH determination--All acidic or alkaline chemicals carry a pH identity. By the measure of the pH, the student may determine how corrosive the chemical, if any, used would have been.
-
- Chromatography--This is the process of separating minute substances from solution through the use of their differences of migration rates through a slightly soluble solvent. In this curriculum unit the focus will be HPLC for its technology applications, but teachers may choose to perform paper chromatography as an introduction to the concept of migration.
-
- Percent Alcohol--through titrations and purification, the student will be able to determine the "blood alcohol level" and see if it the victim was a under the influence of alcohol.
-
- Skid mark calculations--the skid to stop formula may be utilized to determine the speed at which the person was going right as the determinant event occurred.
-
- DNA Analysis--DNA is the fingerprint of every human being. If left behind, DNA is a wonderful source of information that leads to the culprit. DNA analysis to be performed will be based on reports obtained from external requests and will solely be used as comparative analysis rather than complete utilization of the electrophoresis apparatus.
-
- Analysis of spectra--spectra can be obtained from such instruments as an UV/VIS, HNMR, Raman, as well as mass spec. These are useful in identifying the chemical in question.
-
- Introduction to entomology--study of bugs, which can help in aging a body based on the life cycle of the larvae.
-
- Determination of unknown--Unknowns will be used in different scenarios to have the students perform various techniques to identify the evidence.
Description of Methods:
pH Determination:
In terms of classifying chemicals there are many different categories that they can be broken down to. The chemical properties of chemicals are unique and this allows for identification. Even though chemicals can be differentiated between organics and inorganics, natural or synthetic, acids or bases; pH testing is a way to differentiate between acids and bases.
In its simplest form, acids are those chemicals that contain a hydrogen ion (H+) and when placed into water will dissociate into hydrogen ions and its complimentary anion (negative charged ion). A base, on the other hand, is a chemical that contains hydroxide ions (OH-) that when placed into water will dissociate into hydroxide ions and its complimentary cation (positively charged ion). The reaction of an acid coming in contact with a base is called a neutralization reaction where there is the formation of a salt and water as per the following equation:
HX + YOH -> XY + H2O
When segregating the chemicals into the acid/base category, the pH of a solution is utilized. If a chemical has a pH lower than 7, then it is considered an acid; if the pH is greater than 7, then it is considered a base. A pH of seven is neutral and is that of water, the universal solvent.
The pH of a solution may be determined through the use of litmus paper, a pH indicator, or even a pH meter. Depending on the precision and accuracy of the result, different instruments may be used.
Chromatography:
HPLC:
The principle of HPLC involves a stationary and mobile phase under high pressure that acts to separate components of a sample mixture depending on their respective solubility for the mobile phase and affinity for the stationary phase. The most common type of HPLC is reverse phase chromatography, where the mobile phase is a polar mixture like water/methanol, and the stationary phase (analytical column) is a non-polar substrate typically octadecylsilane an 18 carbon molecule, bonded to the inside of a metal cylinder.
The mobile phase sweeps the sample mixture through the column where separations of the individual components occur according to polar/non-polar associations or chemical interactions between the chemicals in the mixture and the mobile/stationary phase. For instance, compounds of a sample mixture that are less polar will have a greater affinity for the stationary phase (remember like dissolves like) and will elute, or leave the column later in time. Compounds that are more polar relatively speaking will spend more time in the mobile phase and will be swept out of the column much faster.
The componentry of an HPLC system begins with the solvent reservoir; this is where the water and methanol are drawn into the system via a pump. Some systems may be of the 4º type and can utilize up to four different solvents to increase separation efficiency. The system can be programmed for isocratic elution or gradient elution depending on the requirements specified during method development regarding the types of components to be separated and their varying solubilities. Isocratic elution involves utilization of a single unchanging mobile phase over time, i.e., 90%H2O/10% MeOH. Gradient elution involves altering the composition of the mobile phase over time, i.e., 70%H2O/30% MeOH to 90%H2O/10%MeOH. As mentioned, utilizing several different solvent concentrations to vary polarity over the course of a run could help separate individual components of a particular nature.
The solvent passes through an inline solvent filter to remove any impurities present in the sample loop where the sample is injected. The sample is carried by the mobile phase through a pre-column filter and then to the guard column that acts to remove any impurities to prevent contaminating the analytical column. The analytical column can be of many dimensions, i.e., 3cm x 3cm (length x inside diameter) and, as mentioned previously, contains the stationary phase where the separations of components occur. Separated compounds elute one by one and pass by a UV light source consisting of one wavelength that irradiates the sample. Compounds will interact and absorb the UV energy. The transmitted energy will pass through the sample and be collected on a photodiode array detector, which in combination with integrators and computer software produce an absorption peak on a chromatogram. The chromatogram has retention time on the x-axis, and absorption intensity on the y-axis.
Usual practice involves the chemist preparing a set of standards, or samples with varying concentrations of the compound of interest to serve as a calibration model, by which concentrations of unknown solutions may be determined. This is possible as the Beer Lambert law governs the absorption of UV radiation:
Absorbance = Concentration x molar absorptivity x path length.
The path length corresponds to the distance through the sample between the UV source and detector. This value is a constant for both unknown and standards, and therefore may be dropped from the equation to give:
Absorbance = Concentration x molar absorptivity
The molar absorptivity term is a constant for a specific molecule at a specific wavelength. The analysis wavelength for HPLC analysis typically remains constant throughout a run, and the compound of interest is assumed to be the same as the compound used to make the standard set. Therefore, the equation can be simplified conceptually to observe that absorbance is proportional to, or in other words, equal to the concentration. This relationship is linear, and therefore a linear regression may be fitted to the data to provide an equation of the form y= mx +b, whose y-value is Absorbance, x-value is concentration, and slope is the combined term molar absorptivity x path length.
So, the chemist may prepare and obtain absorbencies for standards according to a particular HPLC method. An HPLC method sets the parameters of mobile phase concentration, column type, run time, analysis wavelength, etc. The chemist will then proceed to analyze a solution of unknown concentration by the same method. The resulting standard linear model can be used to predict or extrapolate the concentration of the solution.
Percent Alcohol:
When probable cause exists in car accident investigation, police officers utilize breath-test instrumentations to correlate blood alcohol level to percent alcohol in the breath. According to Henry's Law, when a volatile substance is dissolved in a liquid and is brought to the equilibrium with air, there is a fixed ratio between the concentration of the volatile compound in air and is concentration in the liquid, and this ratio is constant for a given temperature. As applied experimentally, when alveolar air leaves the mouth at 34oC, the ratio of alcohol in the blood to alcohol in alveolar air is approximately 2,100 to 1.
(iii)
In cases involving drunk driving, the prosecution has to prove that the defendant's blood alcohol concentration (BAC) at the time of the offense is at or above a statutory concentration. In the majority of jurisdictions it is 0.10% [i.e., 0.1 gram of alcohol per 100 milliliters of blood]. In some jurisdictions, the BAC level may differ pending the jurisdiction's regulations.
(iv)
As it pertains to the realm of this curriculum unit, a breathalyzer test is an inappropriate method of analyzing the blood alcohol concentration as the analysis will be made postmortem. Therefore, a sample of blood is required in order to determine the person's blood alcohol level at the time of the incident. The most widely used method for toxicologists involves Gas chromatography. Another method used is via the oxidation of alcohol to acetaldehyde. The reaction is carried out by utilizing the enzyme alcohol dehydrogenase and a coenzyme nicotin-amide-adenine dinucleotide (NAD). As the reaction proceeds, NAD is converted to NADH, the extent of conversion as measure using a spectrophotometer indicates the original concentration of alcohol. The latter method is used in hospital situations rather than in forensic science.
(v)
Skid Mark Calculations:
In Forensic Science, many accidents produce skid marks. A skid mark is a mark left behind on the traveling surface by the tires of the car that have been locked in position, not rotating. In determining the speed of the car, there are many components that have an influence on the skid to stop formula: skid distance, drag factor for the road surface, and breaking efficiency of the vehicle.
(vi)
The skid mark is created when the driver applies the brakes and locks the tires, once locked; the tires are not able to continue rotating, proceeding in motion. Steering is not possible once the tires are locked. When observing the skid mark, the beginning of the skid mark is called the skid speed. While analyzing the skid mark, it is possible to differentiate between rear tire skids versus front tire skids, where the rear tire skid mark is composed of a darkened center whereas the front skid marks are basically two thin outlines.
The average skid distance is obtained by analyzing all the skid marks present at the scene. When there are four skid marks, it is necessary to add up all the lengths and divide by four; the same is said for three. Furthermore, it is necessary to determine the drag factors by utilizing a vehicle that contains an accelerometer and chalk bumper guns. Professionals who have been formally trained are able to indicate the drag factor as per the performed test. Some of the examples of drag may be found in the appendix (See table 1).
The final component in analyzing the skid marks comes from the braking efficiency. When four skid marks are present, then the skid efficiency is 100%, or having a correlation of 1.00. When the rear tires are not working to their full potential then it is said that there is a lack of 40% in breaking efficiency for a 60% breaking efficiency, or a correlation of 0.60. Some cars are rear-wheel drive, and therefore, it can assume that the break efficiency is 30% for each front tire and 20% for each rear tire.
With all the components at hand, the following formula can be used to determine the velocity of the car:
S = (30*D*f*n)*1/2
(vii)
Where,
S = speed in miles per hour
30 = a constant used in the skid to stop equation
D = skid distance, in decimal feet and inches
f = drag factor for the road surface
n = Breaking efficiency as a percent
DNA Analysis:
Every human is unique and it has to do with macromolecules present in the nucleus of each cell called deoxyribonucleic acid. Deoxyribonucleic acid, DNA for short, encodes for genetic information responsible for all body functions, metabolism, and development. DNA contains building blocks called nucleotides that may contain one of four bases: adenine, cytosine, guanine, and thymine. In addition to one of the four bases, there exists a pentose (five-carbon) sugar and a phosphate
Animal (eukaryotic) cells contain double-stranded DNA that is linked using hydrogen bonding. The molecular conformation of DNA is in the form of double-helix. Each strand is differentiated as the leading or the lagging strand and is has to do with the process of replication as it occurs for each strand. The leading strand has a continuous replication process, whereas the lagging strand has a discontinuous replication through the development of Okasaki fragments.
In forensic science, DNA can be used as a fingerprinting mechanism that will place a culprit at the scene of the crime through the use of many methods such as Blood Typing, Restrictio Fragment Length Polymorphism (RFLP), PCR analysis, Y-Chromosome Analysis, Mitochondrial DNA Analysis, STR Analysis, and Agarose Gel Electrophoresis. This possibility of concisely placing a person at the scene of a crime has to do with the fact that only about one tenth of a single percent of person's DNA (about 3 million bases) differs from that of another
(viii)
. As an introduction to DNA Analysis, the focus will be to explain DNA Typing and PCR Analysis.
DNA Typing:
DNA Typing is done through the use of complimentary DNA markers that when combined to an unknown sample, will bind to specific regions and provide an image that can be later analyzed. When typing, if two DNA samples are similar in four or five regions, it can be deduced that the samples come from the sample individual
(ix)
. In order to increase the accuracy of the test, it would be possible to use more markers, but using more markers would also mean that more time and money would need to be invested into the assay. As with many tests, there has been no concrete evidence to invalidate the use of only four or five regions as a method of identification.
PCR Analysis:
PCR (polymerase chain reaction) is used to make millions of exact copies of DNA from a biological sample. A carefully developed process that incorporates heating and cooling allows for the denaturing of the DNA sample to produce single strands. As the single strands get exposed to an environment containing a forward and reverse primer, taq polymerase, and excess nucleotide bases, the DNA is in optimal conditions to produce hundreds to thousands and sometimes millions of identical replicas. DNA amplification with PCR allows DNA analysis on biological samples as small as an epidermal cell. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. In the preparation of samples, aseptic techniques must be employed to maintain the viability of the sample as well as the validity of the test.
Analysis of Spectra:
H-NMR:
The principle of Nuclear Magnetic Resonance Spectroscopy relies profoundly on the spin, and hence magnetic moments of nuclei contained within the compound of interest.
When these nuclei are exposed to a strong external magnetic field energy levels will split, thereby creating an opportunity for the absorption of electromagnetic radiation in the radio-frequency (
rf
) region (4-600 MHz). Furthermore, the absorption of
rf
by the nuclei is influenced by it's molecular environment, (i.e., local electrons and neighboring nuclei) therefore, the resulting NMR spectra will be a representation of molecular structure. Note: Spectra are obtained using a suitable NMR spectrometer that either attenuates the
rf
or magnetic field strength in the presence of a rapidly spinning sample.
NMR spectra are displayed on an x-y plane, typically with field frequency (v) on the x-axis, and absorption intensity on the y-axis. Scanning an unknown compound will produce a spectrum with peaks occurring at various frequencies with varying absorption intensities. All spectra are gathered with respect to a tetramethylsilane (TMS) reference peak that is set to 0 Hz.
To interpret NMR spectra the chemist must be familiar with the concepts of
chemical shift
: small differences in absorption frequency due to the chemical group to which the nuclei is bonded, and
splitting
or
spin-spin coupling
: magnetic moment of nucleus interacting with another magnetic moment of immediately adjacent nuclei. Examining spectra in light of these two concepts will enable the deduction of the unknown compound's molecular structure.
Consider the compound ethanol H-O-CH2-CH3 The Hydrogen atom bonded to oxygen will experience electron withdrawal as a result of oxygen's high electronegativity, it is said to be least shielded with respect to the external magnetic field. The electrons circulating around the Hs of CH2 are the next most withdrawn while the Hs of CH3 are the least, and said to be most shielded. Lowly shielded nuclei will yield peaks at high frequencies (large
chemical shift-
up field), while highly shielded nuclei will yield peaks at low frequency (low
chemical shift
-down field, approaching the direction of TMS peak). Therefore peak positions serve as an indication of molecular environment or what atom is adjacent, as electronegative atoms like oxygen or fluorine within a compound will give spectra that contain peaks that are more shifted with respect to TMS as compared to compounds containing less electronegative atoms, i.e. carbon or phosphorous.
Next, in the examination of the spectrum the chemist will have to consider splitting. Because of the several possible spin-spin orientations in neighboring nuclei, a singular peak corresponding to an individual H atom will actually appear as several peaks. For instance, atomic spins of the two H atoms in CH2 may align in one direction either against or with the external magnetic field, thereby weakening the field or strengthening it, respectively. A weakening will give one peak at a larger shift, while the strengthening will give one peak at a lower shift. Furthermore, there are two orientations where spins are opposite and cancel having a net zero effect on the field. This peak will appear between the two other peaks producing a triplet. Note: this triplet will correspond to CH3, while CH2 will appear as a quartet corresponding to the net effect of CH3 on it.
NMR spectral interpretation is very important, however with the proliferation of large chemical reference libraries; this task is facilitated by comparison of an unknown compound to a reference standard.
Raman:
Raman spectroscopy is a technique that provides information, both qualitative and quantitative, on molecular media via inelastic scattering of monochromatic radiation. The wavelength of the scattered radiation is shifted from that of the incident radiation, in fact, these shifts correspond to the same type of quantized vibrational changes that are associated with infrared absorption. For this reason Raman and IR spectra of identical compounds appear very similar in structure and tend to share scattering/absorption bands. Therefore Raman spectroscopy is an ideal candidate for chemical identification and quantification techniques, particularly in aqueous media where IR and NIR techniques are predisposed to complication in gathering practicable data.
Raman spectrometers use a powerful laser source to irradiate a sample of interest. The radiation scatters back from the chemical in the sample at various wavelengths corresponding to the natural frequencies of the molecule. The detector collects the scattered radiation and via a series of computer algorithms, converts the signal into a spectrum much like that seen in IR or UV spectroscopy. Since the observed frequencies correspond to the molecular structure, the chemist may consult a table of known frequencies and eventually deduce the structure or something close to the structure of the compound of interest. Alternatively, the chemist may compare the spectrum against a library of reference standards. This is very similar to the investigative process that a chemist would use when employing FTIR. However, Raman is a very expensive alternative for identification purposes, but it does have another advantage.
Raman spectroscopy can be utilized as a tool for quantitative analysis because molecules elicit a particular Raman shift whose intensity is directly proportional to concentration, via a variant of the Beer-Lambert equation:
I = KVCI0, where,
I is the Intensity of the Raman Band, K is a constant for each band, C is the concentration, and I0 is the intensity of the laser. Much akin to Beer's law and quantitative UV methods (reference HPLC section), this fundamental relationship enables the prediction of chemical concentration in situ.
Raman techniques offer a practical advantage over preparative spectroscopic techniques as simple fiber optics and hand-held probes can be used in the field to obtain spectra of many types of substances without laboratory preparation. Furthermore, Raman probes may be inserted into a solution or solid sample and quantitative information about the component of interest may be obtained with the aid of powerful software and without involved preparative methods.
Entomology:
Forensic Entomology is the use of the insects, and their arthropod relatives that inhabit decomposing remains to aid legal investigations. The broad field of forensic entomology is commonly broken down into three general areas: medicolegal, urban, and stored product pests. For purposes of this curriculum unit and those examples that have been portrayed in forensic science shows, the medicolegal area will be the general focus of review. The medicolegal section focuses on the criminal component of the legal system and deals with the necrophagous (or carrion) feeding insects that typically infest human remains.
(x)
Insects not only aid in the analysis of the crime scene but they may also hinder the investigation. For that reason, trained entomologists are required to assist in the investigation so as to analyze the possible track through pooled and spattered blood, and the feeding and defecation of possible areas where evidence may be obtained from. Therefore it is important to recognize and properly document the natural artifacts that may occur from the presence, feeding, and defecation of roaches, flies, and fleas.
Determination of Unknown:
When samples are obtained from a crime scene, many tests are run because the sample does not come pre-labeled. Therefore, in identifying unknown substances present in a fabric, hair, urine, or other inanimate sample, many tests can be utilized for identification of unknowns. Some samples can be identified using many of the testing that has been discussed in this overview.