Forensic Toxicology

FSCI 4500

Analytical Workflow

1. Evidence receipt and documentation

2. Examination and sampling

3. Screening/ Presumptive Testing (Color test)

4. Instrumental Analysis

5. Data Interpretation and reporting

Presumptive testing VS. Vibrational Spectroscopy

PS: quickly screens an unknown substance

Narrows down the ID

Helps determine the next steps

Provides limited structural information

Vibrational Spectroscopy: What can the molecule itself tell us?

How molecular bonds vibrate

Provides info about functional groups and molecular structures

Produces a characteristic spectral pattern

More specific information about the substance.

Limitations of color test

Cross-Reactivity: Different substances can react with the same reagent-same color

False Positives

Mixture Interference: Other compound may mask, weaken or change the expected color

Subjective interpretation: Color can look different depending on the analyst, lighting, concentration or strength of the reaction

Positive presumptive test not equal to identification

What info provided by FTIR, RAMAN, GC, LC, MS and GC-MS / LC-MS

FTIR: How molecular bonds absorb infrared radiation provides functional groups and molecular fingerprint

Raman: How molecular bonds interact with laser light provides vibrational fingerprint

GC: Separate volatile components of a mixture

LC: Separate components in the liquid phase - especially useful for nonvolatile or heat-sensitive compounds

MS: Measures mass-to-charge ratio (M/Z) provide molecular and fragmentation info

GC-MS / LC-MS: Separates and identifies components of complex mixture

Vibrational Spectroscopy

FTIR: Energy that molecule absorbed

Exposes the sample to Infrared radiation

measures with frequencies of IR are absorbed

Absorption occurs when IR energy interacts with molecular vibrations

Produces a characteristic IR spectrum

Raman: Molecule change the scattered light

Exposes the sample to laser light

measures changes in the energy of scattered light

These energy changes are related to molecular vibrations

Produces a characteristic Raman spectrum

Spectroscopy- how electromagnetic radiation interacts with matter (3 ways)

Radiation encounters a substance:

Absorption: Matter takes in energy

Radiation is absorbed at specific energies; The absorbed energy can cause changes within the molecule; FTIR relies on absorption

Emission: Matter releases energy

An excited material releases energy as it returns to a lower-energy state. The energy is emitted as electromagnetic radiation.

Scattering: Radiation is redirected

Light interacts with matter and change direction some times changes energy too.

Different energy leads to molecular interaction

Microvalves lead to molecular rotation

Infrared lead to molecular vibration

UV/Visible lead to electronic excitation

IR interact with molecule

IR radiation has the right range of energy to interact with molecular vibrations

Different bonds vibrate at characteristic frequencies, which allow us to learn about molecular bonds and structures

Molecular motions (3 Types)

Stretching: Bonds lengthen and shorten

Bending: Bond angles change as atoms move

More complex: Molecules can rock, wag and twist

Different bonds and molecular structures vibrate at characteristic frequencies - like a molecular fingerprint

Why different bonds appear at different positions in an FTIR spectrum

Different positions in an FTIR is depending on vibration frequenct and that depends on

Bond strength: Stronger bond causes stiffer spring, which causes higher vibrational frequency

Eg: C-C < C=C ….

Atomic Mass: Lighter atoms causes faster vibration and lead to higher frequency.

Eg: O-H with H often appear at higher wavenumber and relatively high frequency

C-Cl with heavier atoms appear at lower wavenumbers

Stretching vibrations

Stretching occurs when the distance between bonded atoms changes (Changes bond length)

Symmetric stretching: Bonds move together

Asymmetric Stretching: Bonds move differently

Bending Vibrations

Scissoring: towards and away

Rocking: Move together in the same direction

Wagging: Tougher out of the molecular plane

Twisting: Move out of the molecular plane in opposite direction

Molecule vibrational pattern

Atoms Present (Mass)

Bond Strengths (Single, double or triple)

Functional Groups (OH, NH)

Molecular Geometry

Infrared Spectroscopy

IR measures how molecules absorb infrared radiation.

1. Radiate the sample

2. Molecule absorbs IR energy

3. Molecular Vibrations are Excited

4. Absorption is measured

Infrared Region

IR can be divided into three main areas based on wavelength and energy

Near IR (14000-4000)

Higher -energy IR region used for some specialized analytical applications

Mid-IR (4000-400)

Corresponds to many fundamental molecular vib

Provides info about bonds and functional groups

Primary region we use for FTIR

Far IR (400-10)

Lower energy vibrations, more specialized applications

Wavenumber - on the x-axis

Wavenumber is related to the frequency and energy of the molecular vibration

The relationship: Higher wavenumber means higher frequency and higher energy (True for the opposite)

Typical FTIR range (4000 cm^-1 to 400 cm^-1 ) higher wavenumber on the left and lower on the right

When vibration appear in an FTIR spectrum

Molecules are always vibrating- but FTIR can only detect vib that change the dipole moment

1. molecular vibration (bond length changes during vibration)

2. Change distribution (electron distribution shifts)

3. Dipole Moment Changes

4. IR energy can be absorbed (IR radiation matches the energy of the vibration)

5. Peak Appears in the Spectrum

Polar Bonds

Polar bonds are likely to produce strong IR absorptions. Polar bonds shared between atoms with different electronegativities and electrons are shared unequally.

O-H Stretching (3200-3600) Very polar bond and typically a strong absorption

N-H Stretching (3300-3500) Polar bond and produces a characteristic absorption to identify amines and amides

C=O (1650-1800) Highly polar bond, strong prominent absorption

Greater change in dipole moment during vibration causes a stronger IR absorption

TWO major regions of an FTIR Spectrum

1. Functional Group region (4000-1500), This region contains absorptions associated with many recognizable functional groups (OH, NH, CH, C=O) Those peaks can provide clues about the types of chemical groups present

2. Fingerprint Region (1500-400) This region contains a complex pattern of vibrations that can be highly characteristic of a specific compound. It is especially useful when comparing an unknown spectrum to a known reference or spectral library.

Functional group we care most in Drug Chemistry

Nonpolar molecule vs polar molecule

Nonpolar molecules do not have dipole moment.

They share electrons equally and change evenly during distributed

Polar molecule has a dipole moment.

Atoms are different so one atom pulls more strongly on the shared electrons

Electrons are pulled closer to the more electronegative atom and created a partial charge

Read an FTIR Spectrum

Left Side: High number with high energy that common stretching vib (OH,NH and CH)

Right Side: Lower wave number and lower energy, more complex vibration and much of the fingerprint region

Transmittance VS. Absorbance

Transmittance (%T) measures how much IR gets through. <inverse variation>

Less IR pass through- more IR absorbed- lower %T and Absorption peaks point DOWN

Absorbance (A) measures ow much IR is absorbed

More IR absorbed- higher absorbance; Stronger absorption- Larger Peak and absorption Peaks point UP

Peak Shape

Broad Peak:

Some absorptions occurs over a wide range of wavenumbers, such as O-H groups because their vibrations are strongly affected by hydrogen bonding

Sharp Peaks:

Other bonds produce narrower more defined peaks. C=O groups commonly produce strong relatively sharp absorptions.

Peak intensity:

Peak may also vary in strength, Strong peak indicate greater IR absorption

The Fingerprint Region

Range between 1500 and 400 cm^-1 contains many overlapping molecular vibrations so evaluate the overall fingerprint pattern rather than ID every peak

It shows what types of chemical groups are present and pattern match a particular compound