FSC311 - Forensic Chemistry

Selectivity Factor (alpha)

(alpha) = k'B/k'A - Describes the separation of two analytes on a column. - Always greater than 1 because A elutes faster than B. - Describes separation of band CENTRES; does not take into account peak widths.

Resolution in Chromatography

- "performance parameter". - Resolution is a measurement used to quantify peak spacing in a liquid chromatography (LC) separation. - The ability to distinguish two signals/peaks that are adjacent to each other (not limited to chromatography). - Unitless dimension. - Does not account for broadening. - You can lengthen column to improve resolution by increasing N (number of plates).

UV-Vis Spectroscopy

- 190-900 nm; photon energies associated within the UV-vis range have enough energy to cause electronic transitions (promotion of electrons from the ground state to an excited state).

Energy-Frequency Relation

- As the wavelength increases, the energy is decreasing (inversely proportional). - As the frequency increases, the energy increases (directly proportional).

Organic Compounds

- Compounds composed of carbon and hydrogen and usually other elements such as oxygen, nitrogen, chlorine, phosphorus, or other elements. - Exceptions: Carbonates, cyanides, carbides (because they lack C-C bonds).

Identification

- Determination of physical and chemical identification of a substance with near absolute certainty as analytical technique will permit. - Degree of certainty will depend on degree of sensitivity and specificity of the analytical technique.

Comparison

- Q vs. K; process of ascertaining whether two or more objects have a common origin. - Specimen vs. standard/reference sample.

IR spectroscopy: FTIR

- The gold standard. - A mathematical algorithm that transform algorithm that transform signals initially acquired in the time domain to the frequency domain. - Signal processing technique that results in greater sensitivity (S/N), better resolution, and fast processing time. - Acquiring data as a function of time and Fourier transforming this into a frequency domain. - BENEFIT = Faster processing time.

IR Spectroscopy: Organic Analysis

- The portion of the infrared region most useful for analysis of organic compounds is often called the mid-infrared region and spans a wavelength range from 2500 to 25,000 nm (2.5 to 25 um) --> Only significant enough to produce vibrational excitation. - Photon energies associated with this part of the infrared are not large enough to excite electrons, but may induce vibrational excitation of covalently bonded atoms and groups. - The covalent bonds in molecules are not rigid sticks or rods, such as found in molecular model kits, but are more like stiff springs that can be stretched and bent.

UV/Vis

- When light/energy is absorbed by a molecule valence (outer shell) electrons are excited from a lower energy state to an excited (△o). - Degree of conjugation determines the magnitude of excitation (△o). - The higher degree of conjugation the less energy is required to excite. - E.g.: Chlorophyll (highly conjugated system) in plant absorb red (low energy), and reflect green (higher energy) --> Huge electron cloud --> Only needs a small amount of energy to get excited.

What is the frequency of the graph?

4Hz

Conjugated System

Alternating double bonds, single bond, double bond.

Frequency (of a wave)

The number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second or Hz

Chromatogram

A visual representation of the chromatography; graph showing the quantity (area) of a substance leaving a chromatography column as a function of time (t); ideally want peaks that are narrow, separate bands

Transition Metals

An element whose atom has a partially filled d sub-shell, or which can give rise to cations with an incomplete d sub-shell.

Physical Property

Describes the characteristic (state) of a substance without any change in composition (Ex. mass, density, colour, volume, BP, MP).

Chemical Property

Describes the characteristic of a substance when it undergoes a change in composition; one or more kinds of matter combine to form a new matter with a different composition (Ex. flammability, chemical reactions).

Partition coefficient (K)

Equilibrium constant K = Cs/Cm Cs = concentration in stationary Cm = concentration in mobile - Molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. - A high K value means we have a lot of concentration of the analyte in the stationary phase (greater affinity for the stationary phase and will take longer for it to elute out of the column) and low concentration in the mobile phase. Vice versa.

Why are organic compounds important?

Forensic chemists need to be able to categorize compounds into organic and inorganic --> Once they are able to do so, they can determine the type of tools that are at their disposal in the analysis of forensic evidence.

Van Deemter Equation

HETP = A + B/v + Cv A = eddy diffusion B = longitudinal diffusion C = mass transfer effect v = rate of elution - Summarizes the effects of different processes on height of plates (and therefore breadth of bands).

HPLC-UV-Vis

High Performance (Pressure) Liquid Chromatography: - Mobile phase is liquid/solvent (combination of solvents). - Stationary phase (non-polar) is beads coated with unique chemical/physical characteristics. - High pressure = high flow rate (Upper pressure limit range: 5800 - 7000 psi (400 - 500 bar)).

Conjugation Effect

Increasing conjugation means... 1. The compound can be excited by less energy (i.e. higher wavelength). 2. The spectra gets shifted toward higher wavelength. 3. The compound has more of an electron cloud. **In the diagram, the degree of conjugation increases from left to right.

IR Bending

Involves a change in angle between two bonds.

IR Stretching

Involves a continuous change in the interatomic distance along the axis of the bond between two atoms.

Until the middle of the 20th century, analytical separations were carried out by...

precipitation, distillation and extraction.

Overall, what does a detector do?

Measures the magnitude/abundance of ionized compound/fragment and amplifies signal (takes the ion and makes it into an electrical signal, amplifies it, and records it).

LC-DAD for Drug Screening

Photodiode Array Detectors (PDAD). - Useful in recording an entire spectrum. - Large number of small detectors. - Based on semiconductor technology. - Light hits photodiode --> Produce current proportional to the intensity of the incident radiation. - Look at the full spectrum at any given time instead of a monochromator. The light will hit the photodiode and there will be a response that is proportional to the intensity of that light

Chemistry

The branch of science that deal with... - Identification of substances. - Characterization: Properties/composition/structure of these substances, how they interact, combine, and change to form new substances.

Wavelength

The distance between two adjacent peaks (or troughs) of a wave.

Amplitude

The distance from the rest position (zero) to the peak or trough of the wave; it is related to the intensity of the electromagnetic radiation.

Ultraviolet Range

λ ∼10-400 nm.

Infrared Range

λ ∼2,500-16,000 nm.

Mass Spectrometry is a technique used for...

The identification and quantitation of compounds.

Wavenumber Calculation

The reciprocal or wavenumber (cm-1) is the wavelength in cm.

Visible Light

- 400 nm - 700 nm. - Energy trend: Red (low energy) < Orange < Yellow < Green < Blue < Violet (high energy).

IR Active

- A molecule must be IR active to be seen in IR spectroscopy. - To be IR active (absorb infrared radiation), it must undergo a net change in dipole moment as a consequence of its vibrational motion. - Only under these circumstances can the alternating electric field of the radiation (IR energy) interact with the molecule and cause changes in the amplitude of one of its motions. - If the frequency of the IR radiation exactly matches a natural vibrational frequency of the molecule, absorption of the radiation takes place that produces a change in the amplitude of the molecular vibration.

Compound

- A substance formed from two or more elements. - Can be subdivided into organic and inorganic.

Resolution (R)

- Ability to separate/resolve two adjacent peaks; measures "sharpness" of a peak. - Higher resolution = greater confidence in what we're identifying. - Can lengthen the column to improve resolution by increasing the # of plates (N) - Want an R of at least 1.5 (0.3% overlap).

Rate Theory

- Account for dynamics of separation. - A more realistic description of the processes at work inside a column --> Takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast). - Van Deemter equation. - Looking at what is actually happening while separation occurs. - Resulting band shape of a chromatographic peak therefore affected by rate of elution and paths available for analyte to travel through (function of particles in stationary phase)

Chromatography

- Analytical technique used to separate mixtures in order to isolate compounds of interest. - A separation process achieved by distributing the substances to be separated between a mobile phase and a stationary phase. - Mobile (usually gas) --> Drives the sample through the column. - Stationary (solid, liquid) --> Used to attract compounds that have similar physio-chemical properties --> Components that are not similar will move through the column faster/less of an affinity for the column/stationary phase. - Separated based on how quickly they travel through the mobile phase.

Matter

- Anything made up of atoms and molecules. - Takes up space and has a mass

Solvent Effect

- As solvent interaction increases, the bands of rotation and vibrational states start to overlap and peaks broaden. - pH value and polarity of the solvent do affect the absorption capability of the organic compound. - Solvent should not absorb in the same region as the substance of interest ("cut off"). - Avoid polar solvents (H-bonds form, lose fine structure)

Examples of Physical Evidence

- Blood/Semen/Saliva: Liquid or dried that may suggest a relation to the offence, detection of possible existence of drugs or poisons. - Drugs: Any substances seized in violation of laws regulating the sale, manufacturer, distribution and use of drugs. - Firearms/ammunition: Evidence involved in criminal offence. - Explosives: Device containing explosive discharge. - Glass: Transfer of glass particle/fragment from person/object. - Paint: Transfer, liquid or dried, from one surface of an object to another during commission of a crime. - Toolmarks: Impression made by a tool in a crime. - Hair and fibre: May assist in establishing transfers between individuals or objects.

Spectroscopy: Sample

- Bombarding a sample with light energy or electromagnetic energy à The system absorbs this energy, a process takes place, and we measure this process as a signal to get information about the physical and chemical characteristics. - Molecules in an organic compound have the tendency to absorb specific frequencies/wavelengths according to their structural characteristics.

Eddy Diffusion (A)

- Broadening is due in part to the multitude of pathways a molecule can migrate through a packed column. - Path 1 is shorter than 2, causes solute molecules of the same species to arrive at the end of the column over time interval, band broadening. - Multiple path by diffusion is sometimes called Eddy diffusion. - In capillary columns (with no packing, A=0). - Overall: Band broadening due to the multiple pathways taken/different retention times... Even though it is the same compound.

Mass Transfer Effect (C)

- Broadening of peaks is a function of mobile phase velocity (mobile phase molecules moving faster than those in stationary phase). - If we start pushing a fast flow rate and we are not allowing sufficient time for equilibration to occur, then thee mobile phase will get pushed forward (t3) à some of the analyte is still left behind/does not want to leave the stationary phase. When it comes out the end of the column, there will be a time difference between a pack of compounds à some comes out with mobile phase, whereas some equilibrate with the stationary phase = BAND BROADENING. - Overall: Too fast = mass transfer effect and too slow = longitudinal diffusion.

Lorentz Force Law

- Charged particle in electric and magnetic fields is given by this force. - Tells us the force imparted on an ion (how will it control the trajectory of the ion so it successfully gets detected). - In the equation, all the variables are a force of which an ion experiences. - How we determine the type of force we have. F = q(E + v x B) F = force q = charge E = energy v = velocity of particles B = magnetic field vector

Scott Test

- Cobalt thiocyanate. - Tests for the presence of cocaine. - Produces transition metal complex. - Positive result = Blue.

Matrix Effect

- Combined effect of all components within a sample, other than the analyte of interest. - Components detected along with your analyte of interest causing either an enhancement in signal or suppression in signal.

Mass Analyzer: Magnetic Sector

- Common mass analyzer. - Guides an ion in the presence of a magnetic field. - Ion source that accelerates an ion into the magnetic sector. - In the presence of the magnetic field, the ion takes a circular trajectory (Two forces: forces caused by the magnetic field and because it is coming in at a particular velocity it also experiences centripetal forces). - When the force of the kinetic energy is equivalent to the force that the ion experiences in the presence of the magnetic field, only these ions will successfully make it through the curvature and get detected (other ones hit the sides of the magnet and get destroyed). - Sorts each fragment based on its mass to charge ratio.

Spectroscopy: Excitation

- Consequence of radiative excitation. - IR: Not enough energy to excite, but enough to vibrate. - UV-VIS: Enough energy in the system to cause excitation of valence shell electrons (electrons in the ground state being promoted to a higher excited state; upon relaxation it releases light and heat and the delta E (absorbance) can be measured). - X-Ray: Kicking our core electrons that are close to the nucleus; the element/compound becomes unstable and electrons in the outer shell want to come down and fill the whole = Releases X-rays that we measure.

Plate Theory

- Describes the efficiency of the chromatography/how efficient it is at separation. - Has contributed significantly in understanding the formation of bands and band broadening. - Chromatographic column contains a large number of separate layers called theoretical plates; the concept is that these are equilibrium units for K = Cs/Cm. - Every time a sample hits the plate, equilibrium occurs. - The more theoretical plates, the more equilibration events occurs. We want the opposite of this. - You want the distance between the plates to be as small as possible. The smaller it is, the more plates we can fit it the length of a column.

UV-Vis Spectroscopy: Quantitative Spectroscopy

- Different solvent systems (vapour, non-polar, polar). - Absorption of UV and visible radiation occurs in one or more electronic absorption bands, resulting in closely packed lines. - Each line results in the transition of an electron from ground state to excited state. Each excited state has many vibrational and rotational energy states associated with it. Figure - By being in a different solvent system, we can get different shapes. - In the absence of the solvent, we are looking at the molecule in isolation, without influence of the solvent around, so each of the peaks represent a vibrational/rotational energy state... As we move to a non-polar system or polar system, we have other types of interactions (solvent and analyte).

Longitudinal Diffusion (B)

- Diffusion refers to migration form high to low concentration; or away from center of band in both directions. - At very low flow rates of mobile phase, longitudinal diffusion (B) becomes very significant.

MS: EI vs. CI

- EI gives extensive fragmentation (70eV), but has an extensive database for fingerprinting structures and trained users can often determine the structure (ex: benzene ring present). - CI is more suitable for MW determination, but structural info is limited. - Alternate EI/CI can combine advantages - Major limitation: MW < 1000 Da, volatility must be high; not suitable for most biological compounds.

Energy and Wavelength Equation

- Electromagnetic energy interacts with matter. - The lower the wavelength, the higher the energy.

Results of IR

- Functional group analysis. - The types of bonds present in a compound can be found by the absorption of IR radiations. - Absorption takes place when the IR frequency is equal to the frequency of the bond --> Analysis of these absorption characteristics reveals details about the molecular structure of the sample. - Software often has libraries that can correlate unknown spectra to those in the library.

HPLC-UV-Vis Applications

- HPLC is used in drug analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics. - For forensic applications, one of the most commonly used types of HPLC is referred to as "reversed phase" (e.g., C18 column - long chain hydrocarbon)

Hard Ionization

- Input excessive internal energy --> fragmentation --> Structural information/mass fingerprint. - Electron impact.

Soft Ionization

- Input little excessive internal energy --> molecular ion --> molecular weight information. - Electrospray ionization (ESI) and Chemical Ionization (CI).

Mass Spectrometry

- MS is a technique used to identify compounds based on their mass-to-charge ratio (m/z). - m/z are fragments (ions = charge particles) derived from a compound, such that a collection of fragments can be used to uniquely identify a compound. Process: 1. Ionization - Add energy to compound to cause fragmentation generating an ionized compound/fragment. 2. Mass Analyzer - Used to separate gaseous ions based on m/z. 3. Detector - Measure magnitude/abundance of ionized compound/fragment and amplifies signal; can quantify by plotting signal response vs. concentration of standard.

When does a compound absorb light?

- More complex molecules, having several multiple - Complex absorbs light when it is a conjugated system. - Degree of conjugation affects the colour of the compound.

MS: Soft Ionization - Electrospray ionization (ESI)

- Most common ionization technique used in forensic analysis. - We have our sample diluted in some sort of mobile phase often with an acid (formic acid at low concentration - lots of positive ions). As it comes out of the column it enters a spray needle (tip of the spray needle to the inlet of the MS has a charge). When a voltage is applied across the spray needle and inlet, a Taylor cone develops from the solvent itself. At some point, Taylor cone will have a spray of tiny little droplets as a results of the voltage being applied (in the liquid itself is where your analyte of interest resides). As the sample travels from one end to the next, the solvent begins to volatize. As the droplets get smaller and the positive charges start to become closer, it hits the Rayleigh limit where a coulombic explosion occurs and you get tiny droplets (analyte ions with positive charges imparted onto the analyte ionic cells). - Excellent for biological compounds with high MW (ex: proteins), but can also be used for smaller compounds. - Analyte does not need to be volatile.

UV-Vis Instrumentation

- Needs a lamp of some source (light source to provide energy). - A monochromator à Picks a specific wavelength. - Shine incident light into the sample. - Amplifier is meant to amplify the signal. - Readout is usually a computer.

Mass Analyzer

- Once a sample is ionized to fragments, we need a mechanism to sort these fragments. - The ions that are formed are separated in an electric and/or magnetic field according to their m/z. - Fundamentals: Lorentz force.

Marquis Test

- Presumptive test for the presence of alkaloids, such as heroin, morphine, amphetamine and most of their derivatives. - When marquis reagent is used with alkaloids, it creates many colours because they are conjugated differently. - Production of a highly conjugated system. - Several expectant colour products that may be observed indicating a positive result, as these drugs conjugate differently. However, when the marquis reagent is reacted with morphine a purple colour is expected and when it is reacted with amphetamines an orange colour is expected.

HPLC-UV-Vis Instrumentation

- Pump = Sucks up solvent. Pumps can change pressure. Pushes the mobile phase through the column. - Injector = How the sample of interest gets into the system. - Column = Where separation occurs (compounds come out at different retention times). - Peaks represent different compounds and the amount of them at different times.

Matrix

- Refers to components of the sample other than your analyte of interest. - E.g.: You want to determine caffeine concentration in blood? Analyte = Caffeine & Matrix = Blood - The matrix has significant effect on your analysis and quality of results.

Limitations of IR

- Sample needs to be IR active. - Sample needs to be pure --> If there are multiple components you cannot determine which signal is coming from what. - Limited to qualitative analysis --> Does not tell us how much there is, only what functional groups are present or we are comparing it to a reference library to tell us what it is.

UV-Vis Limitations

- Sample needs to be relatively pure. - Sample needs to be able to absorb UV-Vis radiation. - Solvent effect. - Little structural information can be extracted, because signals tend to be broad and overlap (ex: limited qualitative value).

Spectroscopy

- Science that deals with the interaction of various types of radiation with matter --> When we bombard energy or electromagnetic radiation at it we can get some information about the compound, like it physical or chemical characteristics. - Use light (IR, UV-vis, X-ray) to interrogate physical evidence and retrieve information (qualitative & quantitative). - Most familiar with the visible range of the electromagnetic spectrum (red square) --> 400nm (blue) to 700nm (red).

Chemical Spot Tests

- Screening tool used to tentatively identify a drug/drug class/poisons. - Many drugs yield characteristic colours when reacted with specific chemical reagents.

MS: Soft Ionization - Chemical Ionization (CI)

- Source: reagent gas (CH5+) - Mostly used for molecular weight determination.

Mass Analyzer: Quadrupole

- Still dependent on the Lorentz force. - Have four rods in close proximity. Each pair of rods has alternating AC and DC voltage. - When there is a particular AC to DC ratio a particular m/z charge ratio will travel through the quadrupole and make it to the detector (takes a circular path). - Manipulating the trajectory of ions so that we can select for a specific m/z ratio.

Mandelin Test

- Tests for basic drugs, primarily ketamine and PMA. - It reacts primarily with alkaloids to produce transition metal complexes. - Several colours = Some include reddish/brown (PMA) or reddish/orange (ketamine).

Duquenois-Levine Test

- Tests for marijuana. - It reacts with cannabinoids to produce a highly conjugated system (adjacent double bonds). - Positive result = Purple.

Ferric Chloride

- Tests for salicylates. - It reacts with phenolic compounds. - Positive result = Blue, violet, purple, red-brown, or green.

Zwikker Test

- Tests primarily for barbiturates and CNS depressants. - Positive result = Purple or yellow/green.

Chromatography was first developed by...

- The Russian botanist Mikhail Tsvet in 1903. - He produced a colourful separation of plant pigments by passing them through a column of powered limestone (calcium carbonate). - Using solvents to extract pigments from plants. - Solid phase = Calcium carbonate. - Mikhail Tsvet named the technique chromatography because of the different colored (Chroma) bands (graphein) observed.

Forensic Chemistry

- The application of scientific methods and principles that are used in chemistry to matters related to the law (analytical, physical, organic, inorganic). - Largely analytical in nature: 1. Deals with the qualitative and quantitative information about the chemical and structural composition of matter. 2. Matter in this case is physical evidence obtained from crime scenes.

MS: Hard Ionization - Electron Impact (EI)

- When we don't know what are sample is, we use this to fragment the ions in a predictable manor. - Source: Electrically heated filament --> Releases electrons. - Sample introduced as a gas and then high energy electrons (70 eV) bombard compounds, resulting in ionization - Rxn: M + e- --> M+ + 2e-. - More suitable for volatile compounds that are thermally stable. - Gives structural information + fingerprint mass spectrum. - Fragments generated from experiment can be compared to the database to determine the compound.

Chromatography is widely used in the forensic and scientific community for...

The separation, isolation, and pre-concentration of chemical analytes.

Retention Time (Rf)

The time required for the compound to travel the length of the column (injection to exit)

Column efficiency can be described in terms of...

Van Deemter Equation in terms of N or HETP.

How do we measure resolution?

W = 4𝜎 - If R = 1.0, then peaks touch with about 2.5% overlap (too big to tolerate). - If R = 1.5, gives a complete separation (0.3% overlap = more acceptable) = baseline resolved.

Electromagnetic radiation such as visible light is commonly treated as...

a wave

Today, separations are carried out by...

chromatography and electrophoresis.

When transition metal coordinate (bonded to) with ligands __________________ occurs because certain geometrical arrangements are more favourable than others.

d orbital splitting

Retention/Capacity Factor (k'A)

k'A = (tr - tm)/tm - How well does it retain the compound? - Migration rate of an analyte A on a column. - Retention factor for an analyte is between one and five is ideal. - If the retention factor was too high, it would take forever for the component to exit the column. If the retention factor was too low, the compound of interest may coelute with the mobile phase.

IR spectroscopy is used for...

qualitative analysis

UV-Vis is used for...

quantitative analysis

Visible Light Range

λ ∼400-700 nm.

Analysis of Physical Evidence

1. Identification. 2. Comparison (Q vs. K).

Properties of Matter

1. Matter. 2. Physical Property. 3. Chemical Property.

Two fundamental concepts of chromatography...

1. Plate theory. 2. Rate theory.

UV-Vis: Standard Approach - Calibration Curve

1. Prepare standards of drug of known concentrations. 2. Measure at its max absorbance wavelength. 3. Plot absorbance as a function of concentration to generate a calibration curve; use equation of line to find concentration of unknown.

Chemical Spot Tests - Cause of Colour Change

1. Production of a highly conjugated system (Ex. Marquis reagent). 2. Formation of transition metal complexes (Ex. cobalt thiocyanate).

IR inactive

No net change in dipole moment occurs during the vibration of homonuclear species like O2, N2, or Cl2. These compounds cannot absorb IR radiation.