1.1 Outline precautions necessary to ensure accuracy and prevent contamination of samples for analysis
Source of Contamination
- Addition of extraneous material before testing – during collection/transport, soil, dirty air, may contaminate sample.
- Laboratory contamination – Lack of clean lab/equipment can cause contamination by paint, dust, or other matter from bench, walls, floor, air or utensils,
- Contamination by analyst – Lack of PPE can cause contamination by dust, dandruff, sweat.
- Isolate incident scene – limiting entry to authorised personnel prevents contamination.
- Wear PPE – eg. pocketless body suits, gloves, to prevent contamination by analyst.
- Seal/label/store samples with sterile forceps and tamper-proof bags/tins/jars – Label with time/date to avoid mishandling, seal to avoid contamination, store to prevent deterioration/tampering.
- Take duplicate samples – for a back-up in case of contamination/for reliability/for the defence counsel to conduct independent analyses
- Follow the chain of custody – Makes witnesses/custodies accountable for problems that may arise with the sample and prevents tampering/ substitution.
- Clean equipment and benches – Sterilize apparatus/benches esp. for DNA analysis.
Need for the Chain of Custody
C.O.C is the documentation of passage of sample from collection point to lab. Requires signing off of witnesses to the: collection, sealing, breaking of seals and performance of analysis to preventing tampering/substitution and thus ensures validity & accuracy.
- Carbon compounds produced by living things. Often contains H. Excludes carbonates, cyanides, CO and CO2.
- Combusts easily in excess O2 to produce no residue.
- Compounds without carbon. Not produced by living things.
- Combusts in excess O2 to produce residue/ Melts or decomposes if volatile.
1.8 Perform first-hand investigations to determine tests to distinguish between organic and inorganic compounds
- Bunsen flame can cause skin burns. Þ Adjust to yellow flame when not in use/ shut off gas after use.
- Heat 50mL of C2H5OH in a spirit burner. Check for residue.
- Heat spatula of NaCl in a crucible over Bunsen burner. Check for residue.
- Repeat step 2 for C4H6O6 (tartaric acid) and Na2CO3.
1.3 Explain that there are different classes of carbon compounds, which can be identified by distinguishing tests
1.9 Summarise a series of distinguishing tests to separate: the groups of hydrocarbons, acids, bases & neutral salts, in the school/forensic laboratory
School Laboratory Tests For Distinguishing Inorganic Compounds – Indicators And pH Meters
- Method for Salts:
- Salt in solution: i) Take a sample, ii) Use pH meter
- Soluble salt in solid form: i) Take a sample, ii) Dissolve in water, iii) Use pH meter
- Insoluble salt in solid form: i) Add Na2CO3 and heat ii) Check for effervescence (Effervescence= acid, no effervescence=go to next step), iii) Add HNO3 and heat, iv) Check for reaction. (Reaction=acid. No reaction= base)
Forensic Laboratory Tests For Distinguishing Organic Compounds – Infrared Spectroscopy
- Place sample in an IR spectrometer
- Scan with different f’s of IR radiation.
- Allow the amount of radiation passing through to be measured and allow it to produce as an infra-red spectrum.
- Compare the measured IR spectrum with standard spectra OR to a table with wavelengths (or wave #s) that specific bonds absorb.
o Each bond absorbs a specific frequency (often influenced by nearby bonds) and hence gives a signature vibrational
frequency. Thus each compound produces a unique signature or spectra
o The amount of radiation passed through the sample detected indicates the % incident radiation absorbed which can be recorded graph as a function of wavelength of the incident radiation.
o Quicker and more accurate
o More sensitive and thus only require traces amounts
o More expensive than school lab tests
Forensic Laboratory Tests For Distinguishing Inorganic Compounds – Indicators, pH Meters And Infrared Spectroscopy
- Put a drop of the solution onto pH paper (dipping paper into sample= contamination) or dip a pH probe into sample.
- Use infrared spectroscopy if insoluble acid (to identify the carboxylic acid (—COOH) functional group).
1.4 Explain that the inorganic chemical properties of soils and other materials may be useful evidence
Reason for forensic analysis of soil
- Soil is mixture of inorganic matter, decaying organic matter and microorganisms (~5% organic, 95% inorganic matter)
- Each soil is unique and consequently, forensic soil analysis can provide useful clues about its history/formation/location of origin:
o Analysis of soil found at a crime scene may show where the culprit has been.
o Analysis of soil attached to shoes, tyres or the clothing of suspects may show if there were recently at the crime scene if it matches the soil from a crime scene.
Useful Properties Of Soil
- Particles size distribution/Colour – Particle size distribution depends on proportion of clay, silt and colour depends on proportion of organic and inorganic matter and varies from place to place. Clay < silt < sand < gravel. Black= organic (peats), red= iron/ aluminium oxides, white= silicates and lime. Soil particles can be sorted by size by sieving samples through wire sieves of various porosity.
- Minerals – Presence of certain minerals can be characteristic of regions. Eg. Soils in eastern Australia often contain high
proportions of quartz. Minerals are identified using a polarising petrographic microscope (Shows unique colour/appearance; Clay minerals are identified using x-ray diffraction/transmittance e– microscopy).
- pH – Some soils are more acidic than others. Eg. Oxidation of sulfide minerals=acidic soil. Limestone weathering=slightly basic
Useful Properties of Other Materials
- Glass fragments – Each glass has its own characteristic density, refractive index and chemical composition. Glass from a broken headlight can be traced back to a vehicle that was at the crime scene. The refractive index (RI) of the glass is measured using laser beams and the frequency of occurrence of the measured RI can be determined using a frequency data table. Glass of rare refractive index is significant evidence to place the suspect at the crime scene if the glass from the crime scene matched glass from their car. The glass composition can also be determined using plasma emission spectroscopy and be matched to a particular make and model of car.
- Paint scrapings – This allows one to trace the paint manufacturer and/or locations where it is used. Samples can be microscopically examined and compared with known samples.
- Pollen – Pollen from various plants may be unique to an area. Pollen species present can be identified using light microscopy or scanning electron microscopy to identify each.
- Metals – This can be analysed by dissolving them in acid and using AAS to test for the ions in them, which can used to determine the origins of the sample.
- Explosive substances and accelerants – Explosive residues can be identified using chromatography to find out the origin of
Steps For Forensic Analysis
- Undertake a macroscopic observation and a low-power stereoscopic examination to look for unusual matter such as paint chips, broken glass and fibres mixed in with the soil. Seal in tamper-proof bag/container for analysis.
- Take representative sample of soil and screen for colour and particle size distribution (sieve samples through wire sieves of various porosity).
- Use a polarising petrographic microscope to identify minerals in soil sample. Minerals show unique variations in colour and appearance when viewed with polarised light.
- Use light microscopy or scanning electron microscopy to identify pollen species present.
1.5 Discuss, using a recent example, how progress in analytical chemistry and changes in technology can alter the outcome of a forensic investigation
1.6 Solve problems and use available evidence to discuss the importance of accuracy in forensic chemistry
Consequences of Inaccurate Forensic Analysis
- Conviction of an innocent person – such that one could spend many years in jail. Eg. Linder Chamberlain spent 4 years in jail before being released when so-called foetal blood found in the family car was found to be adult blood contaminated with rust.
- Premature fines on a person or company – Eg. London bombings. Four random people were convicted because there were false positive readings due of use of outdated tests and lack of reliability. They were fined a large sum.
- Release of a guilty person – The lack of verification of accuracy via chain of custody can allow the defence counsel to discredit the accuracy of the evidence. Eg. O.J Simpson case.
1.7 Solve problems and use available evidence to discuss ethical issues that may need to be addressed during an analytical investigation
- Analyse accurately, completely, objectively. Ensure completeness by analysing samples from all suspects
- Report objectively. Not exaggerate the accuracy/reliability of one piece of evidence over another. Eg. Murray Kear case. ICAC ignored evidence which lead to a biased result where Kear was charged for all legal costs.
- Disclose all evidence, even contradictory evidence. Not hide or cover up evidence. Contradictory evidence may help the accused.
- Report clearly in language that non-chemists can understand. Use of jargon could confuse/mislead jurors.
2.1 Identify that carbohydrates are composed of carbon, hydrogen and oxygen according to the formula: Cx(H2O)y
- Carbohydrates – Compounds of C, H and O with general formula Cx(H2O) Characteristic carbonyl (-C=O) & hydroxyl (-OH) group. Used as energy, structure and genetic control of growth. Eg. Animals use plant carbs as glucose in respiration & store excess as glycogen.
- Monosaccharide – Simplest sugar that cannot be hydrolysed with formula Cx(H2O) Serve as monomers in condensation reactions. Identified using chromatography with suitable solvents.
- Disaccharide – Carbohydrate formed from the condensation reaction of two simple sugars. Can be hydrolysed by heating it
with HCl or enzymes. Eg. Sucrose: C12H22O11(aq) + H2O(l) → 2C6H12O6(aq)
- Polysaccharide – Long polymer chain formed from condensation reaction of many monosaccharides. Can be hydrolysed.
2.2 Identify glucose as a monomer and describe the condensation reactions which produce: -sucrose as an example of a dissacharide; -polysaccharides including glycogen, starch and cellulose
2.4 Distinguish between plant and animal carbohydrates’ composition in terms of the presence of: cellulose, starch, glycogen
2.5 Perform a first-hand investigation to carry out a series of distinguishing tests for the carbohydrates: -reducing and non-reducing sugars, starch
2.6 Perform first-hand investigations using molecular model kits, computer simulations or other multimedia resources to compare the structures of organic compounds including: -monosaccharides, starch
- Use your molecular model kit to construct
- The open-chain form of glucose
- The ring form of glucose
- The ring form of fructose
- A six-monomer section of amylose starch
- A six-monomer section of amylopectin starch , a-1,4- and a-1,6- glycosidic bonds
- Visualise the different forms of monosaccharides, a-b
- Draw diagrams of the models.
- isomerism, glyosidic bonding, effect of position of –OH and CH2OH on bonding.
- Simplifies the process, making it easier to understand.
- Oversimplified, suggesting that the monosaccharides rings are flat hexagons rather than a zigzag shape (for lowest energy configuration).
- Unrealistic sizes/distances b/w atoms and no mobile e–
Fibrous Proteins (Structural Proteins)
- Provide structure to support other bodily tissues.
- Tough and insoluble in water and most solvents.
- keratin (of skin, nails), collagen (of tendons, cartilage), and fibroin (of silk).
Globular Proteins (Functional Proteins)
- Perform metabolic processes eg. transporting nutrient, fighting disease, catalysing biochemical reactions, etc.
- Roughly spherical shape and soluble in water.
- Insulin (controls glucose metabolism), haemoglobin (transport oxygen), antibodies (fight disease), enzymes (catalyse reactions), etc.
- Amino acid – Compound that has two functional groups – an amine group (-NH2) and a carboxylic acid (-COOH) – and a side group. There are 20 standard amino acids used to synthesise proteins. Eg. Glycine, which has simplest ‘R’ group of H.
- a-amino acids – Compound that has the amino group attached to the C atom next to the -COOH group.
3.3 Describe the composition and general formula for amino acids and explain that proteins are chains of amino acids
Composition & Properties of Amino Acids
- Contains an amine group and a carboxylic acid – These groups acts as a weak base (proton acceptor) and a weak acid (proton donor) respectively.
- Contains a side group – There are 4 type of side groups:
- Non polar aliphatic (hydrocarbon based eg. valine and alanine) or aromatic (benzene ring based eg. phenylalanine)
- Polar neutral (eg. tyrosine and serine)
- Polar acidic (eg. aspartic acid and glutamic acid),
- Polar basic (eg. arginine)
Note: Non polar=hydrophobic, polar=hydrophilic
- Amphiprotic – Due to presence of both acidic and basic groups which enables amino acids to react with acids and bases.
- Zwitter ion form in solid state – Most amino acids found in proteins exist as zwitter ions in the solid state as well as in solution at isoelectric point (pI). The pI value for most amino acids tend to be ~5-6.
- Crystalline with high melting points – Instead of the weaker hydrogen bonds and other intermolecular forces, there are much stronger ionic attractions between acid molecules.
- Soluble in water and insoluble in non-polar substances – This reflects the presence of zwitterions. In water, the ionic attractions between the ions in the solid amino acid are replaced by strong attractions between polar water molecules and
the zwitterions, as per dissolution of any other ionic substance in water.
General Formula for Amino Acids
Forms of Amino Acids
- Zwitter ion – a dipolar ion with both positive and negative electrical charges, but a zero net charge. Most amino acids exist primarily as zwitterions because the acidic –COOH group loses a proton to the basic –NH2
- Isoelectric point (pI) – pH at which the amino acid has no net charge i.e. the zwitterion form is dominant. This means it is the pH at which there is no net migration in an electric field. The pI varies depending on the influence of the ‘R’ group. Amino acids with non-polar R groups have isoelectric points close to 6.0. An acidic R group needs a lower pH is to suppress the ionisation of the acid group in R, hence the isoelectric point is much lower than 6. A basic R group needs a higher pH to suppress the ionisation of this basic group, such amino acids have an isoelectric point much greater than 6.
- Primary structure – its amino acid sequence. Each protein has a unique amino acid sequence, which can help forensic chemists to identify the protein.
- Secondary structure – its ability of the polypeptide chains to fold into stable structures (spirals, helices or pleated sheets). Stabilised by H-bonding between adjacent amino acids.
- Tertiary structure – the 3D folding of the secondary structures to form a specific shape. Stabilised by as
disulphide bonds (between sulfur containing amino acids eg. cysteines), ionic bonds (between COO– and NH3+), H-bonds, dispersion forces and dipole–dipole forces.
- Quaternary structure – the way in which protein molecules combine to form a functioning protein cluster. Stabilised by as disulphide bonds, ionic bonds, H-bonds, dispersion
forces and dipole–dipole forces.
Determination of Protein Structure
- Chemist needs to determine
o All the amino acids present
o Relative molar amounts of amino acids present
- Sequence in which the amino acids are linked together
- Automated Edman Degradation Analysis:
- Add phenyl isocyanate (PIC) – Reagent reacts with amino group on terminal amino acid to give PTH amino acid.
- Add acid – Bond breaks to release terminal amino acid.
- Run chromatography (HPLC) on PTH– cleaved amino acid complex to identify the amino acid.
- Repeat steps 1 and 2 until all amino acids have been cleaved from the chain in turn. In this way, a sequence of 50 amino acids can be determined in around an hour.
3.4 Describe the nature of the peptide bond and explain that proteins can be broken at different lengths in the chain by choice of enzyme
Nature of the Peptide Bond
- Peptide bond (-CO-NH-) – Covalent bond linking two consecutive amino acid monomers formed by condensation reaction. Due to the structure of the amino acids, two different molecules can form when a peptide bond is made.
Hydrolysis of Proteins
- Peptide bonds can be broken down to produce smaller proteins or amino acids by hydrolysis, addition of water.
- Hydrolysis methods:
|1.||Reflux protein with 6 M HCl for about 24 hours at 110°C. Eg. Glycylcysteine + water →||glycine + cysteine|
|2.||Heat protein with NaOH(aq) at 100oC.|
|3.||Add peptidases enzymes.|
Enzyme Hydrolysis of Proteins into Different Lengths
- Each enzyme only cleaves specific peptide bonds between specific amino acids, so a protein can be broken down into different lengths by using different enzymes. Eg. Thrombin preferentially cleaves at Arg-Gly, Trypsin cleaves at Arg-Lys.
- The specificity of enzymes is explained by the lock and key model:
- Each enzyme has an active site of specific size and shape. Hence, it can only fit a specific substrate (protein) of complementary shape.
- Similarly, proteases are highly specific and only cleave substrates with a certain sequence.
- Denatured proteins lose its shape and thus cannot bind to enzymes.
3.5 Compare the processes of chromatography and electrophoresis and identify the properties of mixtures that allow them to be separated by either of these processes
- Chromatography – Technique that physically separates substances based on differing degree of solubility/absorption which allows a differential distribution between a moving and a stationary phase.
- Polarity of each phase – Generally, a polar mobile phase and non-polar stationary phase. However, sometimes in reverse phase chromatography, a non-polar solvent may be used, resulting in a non-polar moving phase and polar stationary phase. The most useful types of chromatography for forensics are LLC and GLC.
- Mobile phase – A liquid/gas that dissolves and carries components in a mixture through the stationary phase. Each component has a certain solubility and moves up at a characteristic speed, causing separation. Highly soluble substances dissolve into the solvent and move with it.
- Stationary phase – A solid/liquid to which components in a mixture bind or absorb. Less soluble substances cannot dissolve and move with the solvent.
- Basis of separation – Moves through the medium at different rates due to their different polarity and thus different solubility in the two phases. Also due to its degree of adsorption on the medium. The more polar components will attach to the more polar stationary phase more readily and so move through the paper at a lower speed. Less polar amino acids dissolve more readily in the less polar mobile phase and so move through the paper more rapidly.
- Electrophoresis – Technique that physically separates charged substances based on their charge, shape and size (molecular weight).
- Basis of separation – the separation of amino acids is based on the size of the charge and the size of the amino acids. The more charged, the faster the migration. The heavier and larger the amino acid, the more frictional resistance experienced, thus the slower the migration. The direction of the migration is based in the different sign of the charge of amino acids in different pHs relative to the isoelectric point. Positive charges move towards the negative electrode and negative charges move towards the positive electrode.
3. 6 Discuss the role of electrophoresis in identifying the origins of protein and explain how this could assist the forensic chemist
Identifying Proteins by Electrophoresis
- Method 1:
- Denature by SDS (sodium dodecyl sulfate) or anionic detergent so that they all have a negative charge and behave as globular structures
- Inject buffered solution into wells.
- Apply high voltage.
- Allow pores of gel act as a sieve and separate the protein – SDS anions according to size.
- Run standards of known molecular weight (called ‘markers’)
- Compare the distanced travelled by sample with the known standards.
- Stain with Coomassie Brilliant Blue or other developing agent and shine UV light for fluorescence.
- Method 2:
- Acid hydrolyse protein sample
- Run electrophoresis on analyte in different buffers
- Run electrophoresis on control standards
- Compare analyte with control standards to identify the amino acids making up the protein to be identified.
Role of Protein Electrophoresis to Forensic Chemistry
- Separation and identification of DNA fragments for forensic analysis (‘DNA fingerprinting’)
- Gene mapping of populations (Human Genome Project)
- Determining genetic differences and establishing evolutionary relationships in various species by comparing variations in their proteins as these are controlled by genes
- Protein identification in body fluids. Globular proteins (such as those found in semen and blood) are readily identified when stained and separated. Eg. PMG enzyme has ten major inherited variants. Analysis of PMG variant in conjunction with blood type analysis (A, B, O and Rh system), can be used as forensic evidence.
- Diagnosis of disease. The levels of different blood proteins rise and fall due to diseases eg. cancer, kidney disease and liver dysfunction. Eg. Gamma globulins are antibody proteins that maintain our immunity. Changes in these proteins levels indicate problems with the immune system.
3.7 Perform first-hand investigations using molecular model kits, computer simulations or other multimedia resources to present information which describes the composition and generalised structure of proteins
- General structure of a-amino acids in their non-zwitter ion form: R—CH(NH2)—COOH
- Use your model kit to construct models of the following a-amino acids. Draw each model.
- Alanine: R=CH3
- Valine: R=(CH3)2—CH
- Serine: R=HO—CH2
- Glycine: R=H
- Construct the dipeptide, ala—val. Draw diagrams of each. (Note: –NH2 of 2nd binds to the –COOH of 1st.)
- Construct the tripeptide, gly—val—ser. Draw diagram. (Note: The last amino acid is the carboxyl terminal amino acid.)
- Visualise the variation in composition of amino acids in terms of their side group, and peptide formation.
- Simplifies the process, making it easier to understand.
- Oversimplified – does not account for the mRNA, tRNA, and ribosome involved in the formation of amino acid formation and does not account for the other protein structures.
- Unrealistic sizes/distances b/w atoms and no mobile e–
3.8 Perform a first-hand investigation and gather first-hand information about a distinguishing test for proteins
3.9 Perform a first-hand investigation to carry out chromatography to separate a mixture of organic materials such as the pigments in plants
- The pigments are carried at different rates because they are not equally soluble in the solvent and because they are attracted, to different degrees, to the fibres of the paper through formation of intermolecular bonds.
- Leaves contain pigments chlorophyll a, chlorophyll b (yellow green to olive green), anthocyanin (red, blue, violet), xanthophylls (yellow) and carotenoids (oranges)Since chlorophyll is such a dominant pigment it hides the colours of the anthocyanins, xanthophylls and carotenoids.
- Rf value of pigment differs in different solvents.
- Isopropyl alcohol (2-propanol) is irritating in case of eye contact, ingestion, inhalation. Slightly irritating in case of skin contact. Þ Keep container tightly closed, Keep away from sources of ignition, seal vessel with watch glass.
- Ethanol is highly flammable, harmful by inhalation and ingestion, irritating to eyes and skin. Þ Keep container tightly closed, keep away from sources of ignition, seal vessel with watch glass.
- Hotplate and heated apparatus can cause skin burns. Þ Allow to cool before taking off hotplate.
- Part A – Extracting the Plant Pigments
- Grind up spinach
- Prepare water bath by heating 200mL water in a 0.5L beaker to ~60o Stir and check temperature using a thermometer.
- Place ground spinach in a test tube and add 20mL of ethanol.
- Place test tube in the water bath, stir the contents and allow it to simmer until the colour of solution becomes concentrated. (Concentrate the colour by evaporating some of the solution.)
- Part B – Separating the Pigments
- Draw a line 1cm from the base.
- Place a small drop of plant solution on the pencil line using a capillary tube. (Optional: Let the solvent evaporate and the paper dry. Repeat 10 times to make thin green line of leaf extract 6 mm wide.)
- Add 5mL of ethanol to a test tube and add paper so that immerse the bottom of paper. Ensure the ethanol is below the spot of pigment by attaching the top of the paper to a string that lies on the diameter of the testtube.
- Allow the solvent to move up paper. Remove the paper once it almost reaches the top.
- Allow it to dry and mark the bottom of each pigment band.
- Measure migration distance of each pigment and solvent migration distance (from base). Record.
- Repeat 1-7 for isopropyl-alcohol
3.10 Perform a first-hand investigation to identify the range of solvents that may be used for chromatography and suggest mixtures that may be separated and identified by the use of these solvents
- Examples of common solvent/solvent mixtures: phenol, collidine, butanol/acetic acid, acetone/water
- Note: See Part A and part B of method in ‘3.9’.
- Part C – Varying the solvent
- Repeat the experiment with different solvents, using a fresh chromatography paper for each run.
- Solvent A: 10% acetone, 90% hexane
- Solvent B: 20% acetone, 80% hexane
- Solvent C: 50% acetone, 50% hexane
- Identify the pigment bands on each chromatogram. Label each chromatogram and the solvent used.
3.11 Perform a first-hand investigation to carry out the electrophoresis of an appropriate mixture and use available evidence to identify the characteristics of the mixture which allow it to be separated by this process
- Hook up the power supply and set it to 240V, max 50 Amps, and about a 30 minute timer
- Connect the cell lid to the power supply
- Place the cell on a dark piece of card. (This allows you to see the well better)
- Fill the cell with 0.1% NaHCO3 Buffer solution until it just covers the surface of the gel by a few mm
- Use the micro pipette to collect a 10ul aliquot of a dye
- Wipe the pipette tip on a piece of blotting paper
- Submerge the pipette tip below the surface of the buffer just above the well. Be careful not to piece the gel with the tip
- Fully depress the micro pipette to expel dye into the well. Withdraw the tip before releasing the plunger
- Repeat filling wells until one of each dye has been created, ensuring a new micro pipette tip is used for each dye
- Once the well are full, place the lid of the cell on carefully
- Hit the “run” button on the power supply to commence the chromatography
- Stop the power once the fastest dye is about ¾ along the plate
Structure and Composition of DNA
- ‘Deoxyribonucleic acid’:
o ‘deoxyribo’ because it also contains the sugar deoxyribose o ‘nucleic’ because the molecule occurs in the nuclei of cells
- ‘acid’ because of many phosphoric acid groups
- DNA is a large condensation polymer consisting of 10 million – 100 million nucleotide monomers. Each nucleotide consists of a phosphate, a deoxyribose (5 carbon sugar), one of four nitrogen base.
- DNA occurs as a double helix in which the two helixes are held together by hydrogen bonds.
- The hydrogen bonds holding the two helixes together easily break and reform.
- Consists of a sugar molecule (deoxyribose in DNA) attached to a phosphate unit and to a base.
- Four bases are used in DNA: Adenine, Guanine, Cytosine, Thymine
- The bases form complementary pairs in the DNA molecule:
o Adenine-thymine – this pair is formed by 2 hydrogen bonds
- Cytosine-guanine – this pair is formed by 3 hydrogen bonds
DNA and its Characteristics Allowing Identification of Individuals
- Each person’s DNA is organised into 46 chromosomes, 23 from each parent.
- Each chromosome contains thousands of genes.
- Genes are sections of DNA molecules that determine the sequence of amino acids (primary structure) in the protein that the gene controls the synthesis of.
- Exons are the coding sequences within these genes. They make up of 2% of our DNA. These code for the same proteins in each person, thus the sizes and sequences of exons remain the same for each individual.
- Introns are the non-coding sequences that make up the rest of DNA ie. ‘spam DNA’. They make up of 98% of our DNA.
- Introns are comprised of short, repeating sequences, that is, variable number tandem repeats (VNTRs). The size and length of these VNTRs varies from person to person, and it is for this reason, DNA analysis uses these sections of the DNA molecule to identify an individual from their DNA.
- Every person shares half of their VNTRs with their immediate family (parents and siblings), a quarter with their cousins and grandparents. Only identical twins will have identical DNA sequence.
- Children inherit half their DNA from each parent and consequently share half of their VNTRs with their parent and siblings.
- The characteristics of DNA that make it useful in forensic analysis, particularly for the identification of individuals is summarised:
4.3 Describe the process used to analyse DNA and account for its use in: identifying relationships between people, identifying individuals
DNA Fingerprinting/Profiling – STR/VNTR Analysis
- Sample collection and DNA isolation – Collect sample. Dissolve the DNA from the cellular material (eg. semen, saliva, hair, etc) by adding phenol and water. The DNA is dissolved in the water layer where it then can be extracted and purified.
- DNA amplification – For small amounts or partially degraded DNA, copies of the DNA must be made in order to increase the amount of DNA available for testing by PCR.
- Denaturing – Heat to 95oC to break the hydrogen bonds and separate strands
- Annealing – Cool to 60oC to attach primers
- Extension – Warm to 72oC to initiate PCR
- Repetition – Repeat process many times using more primers. Each cycle doubles the number of stands.
- Separation by gel electrophoresis – Separate according to chain length (thus weight) using gel electrophoresis using polyacrylamide-agarose gel. Run control samples containing polynucleotide samples of known molecular weight. DNA is negatively charged, so they migrate towards the positive electrode. The smaller tandem repeat fragments migrate faster than the heavier fragments.
- Development of the fragment pattern and identification
a) Irradiate gel with laser UV light – UV light causes each of the fluorescent tags attached on the primers to emit light of different colours. A detector identifies each band by the colour of light emitted. The molecular weight of each fragment in each sample can be identified using control standards. OR
b) Tag with radioactive probes – The DNA fragments are denatured in alkali to produce single strands. The DNA pattern on the gel is transferred to a nylon membrane, and the strands are hybridised with radio-labelled probes consisting of a short stretch of DNA of a known sequence that is complementary to the intron regions ie. the VNTRs (repeat sequences). In turn, each fragment/band becomes radioactive. The nylon membrane is exposed to an X-ray film so that the radiation emitted from each band darkens the film to make a DNA fingerprint. Compare control sample to the DNA profile (the number of STRs on each DNA molecule for 10 different segments from identical positions). OR c) Tag with fluorescent probes – The strands are hybridised with nucleotides with fluorescent probes that are complementary to the sequence of different VNTRs. Different probes complement with different VNTRs and emit light of different colours. These colours can then be used to identify the size of the DNA fragments.
- The difference in length in the introns can give the forensic chemist some idea of the likelihood of the sample being shared by two or more people. The certainty of an analysis can be increased by looking at several different fragments. As a result, the probability of a secondary match decreases with each additional fragment chosen. Thus, even with common loci (specific regions), the probability of a match can reach fairly high level relatively quickly as each loci can be considered independent of each other.
- ↑ Samples= ↑comparison= ↑positive identification
4.4 Analyse information to discuss the range of uses of DNA analysis in forensic chemistry and use available evidence in discussing the issues associated with its use in terms of the ethics of maintenance of data banks of DNA
Uses for DNA Analysis in Forensic Chemistry
- Criminal activity – used to identify the person who produced a biological sample at a crime scene.
- Paternity testing – used to identify the father of a child in disputed paternity cases.
- Relationship testing – used to establish familial links.
- Identity testing – used to establish the identity of a person by comparing DNA to living relatives. This may be used after a disaster /catastrophe whereby a person’s body may not be in any condition for a visual identification.
Uses for DNA Analysis in Scientific Research
- Testing for predisposition for or the actual presence of genetic disease – Eg. Gene mutation can change the chain size which changes the genetic coding which can subtract important protein and add protein which could increase risk of disease. Eg. Huntingtin disease.
- Classification of organisms – by comparing DNA sequences to other related organisms
- Dating evolutionary changes and dating migration paths – of human and hominoid ancestors
- Breach of privacy – It can reveals information about inherited genetic diseases, paternity and possible future susceptibility to various health problems. Life insurers, medical insurers and employers could abuse DNA profile information in a discriminatory way. Eg. Employers/insurers may deny applicants/people whose DNA show that they represent a higher risk to a certain disease/health problems due to the existence of mutations in their genes.
- Goes against ‘innocent until proven guilty’ – Often suspects are pressured to provide DNA samples in order to prove their innocence. Also, DNA evidence is commonly mistaken as infallible/strong evidence, however, it can be easily contaminated. Hence, innocent people can be wrongfully convicted based on a DNA sample.
5.1 Explain what is meant by the destructive testing of material and explain why this may be a problem in forensic investigations
- Testing that involves techniques that result in the irreversible consumption or destruction of the sample
- Examples of when destructive testing is disadvantageous: o Identifying the authenticity of artworks
o Establishing the authenticity of historical artefacts
o Proving the authenticity of use of precious metal in jewellery
5.2 Identify, outline and assess the value of the following techniques in the analysis of small samples:- gas-liquid chromatography, high performance liquid chromatography
5.4 Analyse and present information from secondary sources to discuss the ways in which analytical techniques may provide evidence about samples
6.2 Identify that the emission of quanta of energy as electrons move to lower energy levels may be detected by humans as a specific colour
- Electrons in an atom normally exist in their stable ground state in well-defined energy levels. However, if the electrons absorb energy either through heating or applying a potential difference, they become excited.
- Their energy increases so the electrons jump to a higher energy level that corresponds to the amount of energy it possesses. Without the external source of energy, these excited electrons release the absorbed energy and fall back to their ground state. The energy emitted by excited electrons is quantized, that is, energy is emitted as discrete packets.
- Note: The mathematical relationship is expressed as E=hf. Visible light range= 400-750nm.
Since electron orbit shells have well defined energy levels, it means that electrons cannot possess energy that lies in between two levels. Thus, electrons can only absorb or emit an exact amount of energy that will allow them to make a full jump to a different energy level.
- Since energy is inversely proportional to wavelength, atoms in the gas phase can only emit or absorb certain wavelengths.
- Each element has a different no. of electrons. Thus, each element has a unique electron configuration and therefore different energy levels. Since the electrons can only have the same amount of energy as these quantised energy levels, the energy they can absorb/emit are also different. Therefore, each element has a unique set of wavelength it emits called its emission spectrum. This is not only confined to the visible spectrum, as emission can also lie in the infra-red, ultraviolet and x-ray ranges. As long as one wavelength on the emission spectra is different, the element is different. However, sometimes the resolution of the equipment may not be sufficient to differentiate between very fine spectral lines.
- When an excited electron falls back to its ground state, it doesn’t have to fall the entire distance in one go. It can take many different paths to reach its ground state. This accounts for the different spectral lines in an element’s emission spectrum, as each different jump releases radiation of a certain wavelength.
Basis of Identification For AES
- Every element has its own unique emission spectrum, thus the emission spectrum of a sample can analysed to identify the elements present.
- AES apparatus on the right:
Atomic Emission Spectroscopy
- The sample is vaporised and placed in flame or a carbon arc. A carbon arc consists of two carbon electrodes that are close together. The flame or voltage between the carbon arcs excites the electrons in the sample which emits radiation as they fall to ground state.
- The radiation is focused through a lens and then passes through a monochromators. Monochromators are prisms that can separate an incoming radiation into its component wavelengths
- These wavelengths can be detected by:
- A photographic plate or film, in which case the instrument is known as a spectrograph.
- A photomultiplier, in which case the instrument is known as a spectrometer. A photomultiplier is a sensitive device that converts light into a current proportional to the intensity of the light. A computer then produces the emission spectrum by graphing the intensity of the light as a function of wavelength
- An eyepiece, in which the instrument is known as a spectroscope. The emission spectrum can be direction observed through the eyepiece.
Pros/Cons of AES
- The emission spectra of a sample can used for quantitative analysis but AAS is much more accurate, as measuring the amount of radiation absorbed can be used to measure the concentration of elements present. However, AES can analyse samples with multiple elements and distinguish between them whereas AAS requires a different hollow cathode lamp for each element. Thus, AES is more commonly used for qualitative analysis, i.e. identification of elements in a mixture.
Common Uses of AES
- Monitoring the composition of steel to ensure its quality (Note: This is one of earliest uses of AES)
- Monitoring water quality by measuring concentrations of various metal ions. (Although AAS can measure concentrations more accurately, AES is preferred for its versatility (many different elements can be monitored in a water body.)
- Determining the lead content in soil, water or air to prevent or detect lead poisoning.
6.6 Identify data, choose equipment, plan, and perform a first-hand investigation using flame tests and/or spectroscope analysis as appropriate to identify and gather first-hand information to describe the emission spectrum of a range of elements including Na and Hg
Method – Flame Test
- Place some crystals of sodium chloride in a Petri dish and add drops of hydrochloric acid to wet the crystals.
- Clean a platinum wire by dipping it in conc hydrochloric acid. Hold it in the blue Bunsen flame till all colour is lost.
- Dip the wire into the wet crystals of salt and hold it in the Bunsen flame. Observe and record flame colour.
- Repeat steps 1-2 and
- Prepare acid slurry of strontium chloride in another Petri dish.
- Repeat steps 1-4 for the slurry. Observe and record flame colour.
6.7 Analyse and identify individual elements present in a mixed emission spectrum and use available evidence to explain how such information can assist analysis of the origins of a mixture
Use of AES in Determining the Origins of a Mixture
- The ability to identify elements through their unique emission spectrum is particularly useful for forensics in determining the origins of a mixture. The use of AES in forensic chemistry has greatly increased due to the development of faster and more
Applications of AES in Forensic Chemistry
- Detecting lead poison by analysing the victim’s kidney
- Determining the origin of chips of paints at a crime scene by analysing its composition and then comparing it to the paint at different locations
- Determining the origin of small pieces of glass, metals or alloys at a crime scene by analysing its composition and then comparing its spectra with those of likely sources
- Determining the origin of a soil sample by identifying and measuring its concentrations of less common elements and then comparing it to the soil composition of various locations
- Identifying forgeries of paintings by famous artists by analysing a sample of its paint and then comparing the result to the result of a sample of paint of a known work by the artist.
Limitations of AES Analysis
- It is far less accurate than AAS for quantitative analysis
- It is almost useless in analysing organic compounds as there is such a huge variety of hydrocarbons and their isomers which all contain carbon, hydrogen, oxygen and etc.
- The biggest disadvantage for AES in terms of forensic analysis is that it is a form of destructive analysis. Since the sample must
be vaporised into gases, it could be problematic if the tests need to be repeated. Often the sample available at a crime scene is very tiny and scarce.
- New improvements in AES technology include the use of plasma to excite electrons. Plasmas are highly ionised gas that has extremely high temperature (~5000°C). Its temperature allows it to excite more electrons in comparison to a carbon arc, thus making the instrument more sensitive.