UV and IR Spectroscopy and GC-MS Study Notes

UV Spectroscopy Basics

• Definition: UV spectroscopy involves the absorption of light in the wavelength range of $200 - 400\,nm$. This energy absorption leads to electronic transitions.

• Mechanism: When atoms or molecules absorb light energy, they transition from a state of low energy (ground state) to a state of high energy (excited state). These transitions occur specifically between electronic energy levels within the ultraviolet and visible spectrum.

Electronic Transitions

• Transition Types: In compounds other than alkanes, electrons can undergo several possible transitions depending on their orbital types. The most important transitions, ordered by increasing energy, are:

  • $n \rightarrow \pi^{*}$: Occurs in carbonyl compounds consistently.

  • $n \rightarrow \sigma^{*}$: Occurs in compounds containing oxygen, nitrogen, sulfur, and halogens (lone pair electrons).

  • $\pi \rightarrow \pi^{*}$: Occurs in alkenes, carbonyl compounds, alkynes, and azo compounds.

  • $\sigma \rightarrow \sigma^{*}$: Found in alkanes (saturated hydrocarbons).

• Significance: The transition of the lowest energy is generally the most important for analysis.

• Selection Rules: Not all transitions are possible; they are governed by selection rules.

  • Spin Selection Rule: Changes in the spin quantum number of an electron during transition are not allowed. Such transitions are termed "forbidden."

Transition Probabilities

• $\pi \rightarrow \pi^{*}$: Allowed Transition.

• $n \rightarrow \pi^{*}$: Partially forbidden.

• $n \rightarrow \sigma^{*}$: Very weak (forbidden).

Beer-Lambert Law

• Principle: Explains the relationship between the light absorbed by a solution and its concentration/path length. It states that absorbance is directly proportional to the concentration of the absorbing species and the path length of the sample.

• Equation:     $A = \log\left(\frac{I_0}{I}\right) = \epsilon cl$     $A = \epsilon cl$

• Variables:

  • $A$: Absorbance.

  • $I_0$: Intensity of light incident upon the sample cell.

  • $I$: Intensity of light leaving the sample cell.

  • $c$: Molar concentration of the solute ($mol\,dm^{-3}$ or Molar).

  • $l$: Length of the sample cell ($cm$).

  • $\epsilon$: Molar absorptivity ($L\,mol^{-1}\,cm^{-1}$).

UV Spectroscopy Exercise

• Scenario 1: A solution of benzonitrile in water ($c = 1 \times 10^{-10}\,M$) is examined at $\lambda = 224\,nm$. The absorbance $A = 1.30$ and path length $l = 1\,cm$.

  • Question: What is the molar absorptivity ($\epsilon$)?

  • Calculation: $\epsilon = \frac{A}{cl} = \frac{1.30}{(1 \times 10^{-10}\,mol\,dm^{-3}) \times (1\,cm)} = 1.3 \times 10^{10}\,L\,mol^{-1}\,cm^{-1}$.

• Scenario 2: The same solution is examined at $271\,nm$ where $\epsilon = 1000$.

  • Question: What is the absorbance reading and the intensity ratio $\frac{I_0}{I}$?

  • Absorbance Calculation: $A = 1000 \times (1 \times 10^{-10}) \times 1 = 1 \times 10^{-7}$.

  • Ratio Calculation: Since $A = \log\left(\frac{I_0}{I}\right)$, then $\frac{I_0}{I} = 10^A = 10^{1 \times 10^{-7}}$.

Instrumentation of UV Spectrophotometer

• Components:

  1. Light Source:

     • Deuterium lamp: Emits radiation in the UV region.

     • Tungsten lamp: Used for radiation in the visible region.

  2. Monochromator: Acts as a diffractor to spread the beam into its component wavelengths.

  3. Detector: Records the intensity of transmitted light. Common types include photomultipliers or photodiodes.

  4. Data System: A computer records the plot of absorbance versus wavelength.

Solvent Selection in UV Spectroscopy

• Requirement 1 (Transparency): The solvent must be transparent at the measurement wavelength. Solvents have a "cutoff wavelength," below which they absorb strongly.

  • Rule: Choose a solvent with a cutoff lower than the analyte's $\lambda_{\max}$.

  • Common Solvent Cutoffs:

    • Acetonitrile: $190\,nm$

    • Water: $190\,nm$

    • Cyclohexane: $195\,nm$

    • Isooctane: $195\,nm$

    • n-hexane: $201\,nm$

    • 95% ethanol: $205\,nm$

    • Methanol: $205\,nm$

    • Trimethyl phosphate: $210\,nm$

    • 1,4-dioxane: $215\,nm$

    • Chloroform: $240\,nm$

• Requirement 2 (Solubility): Solid analytes must dissolve completely to form a clear, homogenous solution. Cloudy solutions scatter light and distort results.

• Requirement 3 (Reactivity): The solvent must not react with the analyte.

• Requirement 4 (Volatility): The solvent should not evaporate quickly.

• General Usage Guide:

  • Water: For polar compounds.

  • Ethanol/Methanol: Widely used for various polarities.

  • Hexane: For non-polar compounds.

Chromophores and Auxochromes

• Chromophore: The part of a molecule responsible for absorbing UV or visible light. They contain electrons excitable by UV light.

  • Chromophore Classes and Typical $\lambda_{\max}$:

    • $C=C$ (alkene): $\pi \rightarrow \pi^{*}$ ($175\,nm$)

    • $C\equiv C$ (alkyne): $\pi \rightarrow \pi^{*}$ ($170\,nm$)

    • $C=N$: $n \rightarrow \pi^{*}$ ($160\,nm$)

    • $C=O$: $\pi \rightarrow \pi^{*}$ ($180\,nm$)

    • $CHO$: $\pi \rightarrow \pi^{*}$ ($190\,nm$)

    • Aromatic rings: $\pi \rightarrow \pi^{*}$ ($205\,nm$)

    • $COOH$: $n \rightarrow \pi^{*}$ ($205\,nm$)

    • $COOR$: $n \rightarrow \pi^{*}$ ($205\,nm$)

    • $R-CONH_2$: $n \rightarrow \pi^{*}$ ($210\,nm$)

    • $NO_2$ (nitro): $n \rightarrow \pi^{*}$ ($217\,nm$)

    • $N=N$ (azo): $\pi \rightarrow \pi^{*}$ ($340\,nm$)

    • $OH$: $n \rightarrow \sigma^{*}$ ($180\,nm$)

• Auxochrome: Groups attached to a chromophore that increase the intensity and/or wavelength of absorption. Examples: $-CH_3, -OH, -OCH_3$, halogens, amino groups.

Effects on Absorption Band

1. Bathochromic Shift (Red shift): Shift to a longer wavelength (lower energy).

2. Hypsochromic Shift (Blue shift): Shift to a shorter wavelength (higher energy).

3. Hyperchromic Effect: An increase in absorption intensity.

4. Hypochromic Effect: A decrease in absorption intensity.

Practical Guide for UV Interpretation

1. Low/Medium intensity band < 220 nm: Usually indicates no transitions. Possibilities include amines, alcohols, and thiols.

2. Two low-intensity bands (250 - 360 nm) with no major short-wave absorption: Usually indicates $n \rightarrow \pi^{*}$ transitions (e.g., $C=O, C=N, N=N, NO_2, COOR, COOH, CONH_2$).

3. Two medium-intensity bands ($\lambda_{\max} > 200\,nm$): Generally indicates an aromatic system.

4. High-intensity bands (> 210 nm): Generally represent an $\alpha,\beta$-unsaturated ketone, a diene, or a polyene.

5. Multi-system compounds (e.g., simple ketones): Show two absorptions: an $n \rightarrow \pi^{*}$ transition at longer wavelengths (> 300\,nm, low intensity) and a $\pi \rightarrow \pi^{*}$ transition at shorter wavelengths (< 250\,nm, high intensity).

6. Highly colored compounds: Likely contain a long-chain conjugated system or a polycyclic aromatic chromophore.

UV Spectroscopy Exercises

• Possible Transitions:

  1. Cyclopentane ($C_5H_{10}$): Saturated, only $\sigma$ bonds. Transition: $\sigma \rightarrow \sigma^{*}$.

  2. Acetaldehyde ($CH_3CHO$): Contains $C=O$ and lone pairs on Oxygen. Transitions: $\pi \rightarrow \pi^{*}$, $n \rightarrow \pi^{*}$, and $\sigma \rightarrow \sigma^{*}$.

  3. Dimethyl ether ($CH_3-O-CH_3$): Transition: $n \rightarrow \sigma^{*}$.

  4. Methylvinyl ether ($CH_2=CH-O-CH_3$): $\pi$ bond and lone pairs. Transitions: $\pi \rightarrow \pi^{*}$, $n \rightarrow \pi^{*}$, $n \rightarrow \sigma^{*}$.

  5. Trimethylamine: Transition: $n \rightarrow \sigma^{*}$.

  6. Cyclohexane ($C_6H_{12}$): Saturated. Transition: $\sigma \rightarrow \sigma^{*}$.

• Acetone Spectrum Interpretation:

  • $166\,nm$: $\sigma \rightarrow \sigma^{*}$ transition.

  • $189\,nm$: $\pi \rightarrow \pi^{*}$ transition.

  • $279\,nm$: $n \rightarrow \pi^{*}$ transition.

Infrared Spectroscopy (IR)

• Purpose: Used primarily to identify functional groups in organic compounds.

• EM Spectrum Range: Mid-IR used by chemists ranges from approximately $2.5 \times 10^{-5}\,m$. Standard scale is $4000\,cm^{-1}$ to $400\,cm^{-1}$.

• Mechanism: Measures stretching and bending vibrational frequencies of covalent bonds. Absorption increases the amplitude of vibrational motion.

• Dipole Requirement: Only bonds with a dipole moment can absorb IR radiation. Symmetric bonds (e.g., in symmetrical alkanes/alkenes) are IR inactive.

• Allowed Vibrations:

  1. Symmetric stretching

  2. Antisymmetric stretching (occurs at higher frequencies than symmetric)

  3. In-plane bending (e.g., scissoring, rocking)

  4. Out-of-plane bending (e.g., wagging, twisting)

IR Frequency Regions

• $O-H / N-H$: $4000 - 3300\,cm^{-1}$

• $C-H$: Around $3000\,cm^{-1}$

• $C\equiv N / C\equiv C$: $2400 - 2000\,cm^{-1}$

• $C=O$: $1820 - 1650\,cm^{-1}$

• $C=C / C=N$: $1650 - 1550\,cm^{-1}$

• Fingerprint Region: Below $1500\,cm^{-1}$ (unique to each molecule).

IR Instrumentation and Sample Preparation

• Spectrophotometer Types:

  1. Dispersive: Uses a hot wire beam divided into sample and reference paths. Passed through a monochromator to a thermocouple detector. Records in the frequency domain.

  2. Fourier Transform (FT-IR): Produces an interferogram (time domain) containing all frequencies. A mathematical Fourier Transform converts it to a frequency domain spectrum.

• Sample Holders: Glass and plastic absorb IR, so cells are made of ionic substances like Sodium Chloride ($NaCl$) or Potassium Bromide ($KBr$).

  • $NaCl$: Useful for $4000 - 650\,cm^{-1}$.

  • $KBr$: Useful for $4000 - 400\,cm^{-1}$.

• Methods:

  1. Neat: A drop of liquid pressed between salt plates.

  2. KBr Pellet: Mixing ground solid with KBr powder and pressing under high pressure. (Disadvantage: KBr absorbs water).

  3. Nujol Mull: Grinding solid with mineral oil. (Disadvantage: Nujol bands at $2924, 1462, 1377\,cm^{-1}$ may overlap sample).

  4. Solution: Dissolving in solvents like Carbon Tetrachloride ($CCl_4$). (Disadvantage: $C-Cl$ stretches at $785\,cm^{-1}$ may obscure bands).

Factors Influencing Carbonyl ($C=O$) Frequency

• Typical Ranges:

  • Acid Chloride: $1800\,cm^{-1}$

  • Anhydride: $1810$ and $1760\,cm^{-1}$

  • Ester: $1735\,cm^{-1}$

  • Aldehyde: $1725\,cm^{-1}$

  • Ketone: $1715\,cm^{-1}$

  • Carboxylic Acid: $1710\,cm^{-1}$

  • Amide: $1690\,cm^{-1}$

• Influence Factors:

  1. Conjugation: Adjacent $C=C$ lowers the $C=O$ frequency.

  2. Ring Size: Decreasing ring size increases strain and increases frequency (e.g., six-membered cyclic ketone at $1715\,cm^{-1}$, five-membered at $1745\,cm^{-1}$).

  3. $\alpha$-Substitution: Halogens ($Cl, Br$) on the alpha-carbon shift the band to a higher frequency due to electron withdrawal.

  4. Hydrogen Bonding: Reduces absorption frequency (e.g., Methyl salicylate at $1680\,cm^{-1}$).

Specific Functional Group Absorptions

• Alkanes:

  • $C-H$ stretch: $3000\,cm^{-1}$

  • $CH_2$ bend: $1465\,cm^{-1}$

  • $CH_3$ bend: $1375\,cm^{-1}$

  • Long chain band (n > 4\,CH_2): $720\,cm^{-1}$

• Alkenes:

  • Vinyl $=C-H$ stretch: > 3000\,cm^{-1} ($3095 - 3010\,cm^{-1}$).

  • $C=C$ stretch: $1660 - 1600\,cm^{-1}$. (Trans is weak, Cis is stronger; symmetrical is inactive).

• Alkynes:

  • Terminal $\equiv C-H$: $3300\,cm^{-1}$.

  • $C\equiv C$: $2150\,cm^{-1}$.

• Aromatics:

  • $C-H$ stretch: > 3000\,cm^{-1} ($3050 - 3000\,cm^{-1}$).

  • $C=C$ ring stretch: $1600$ and $1475\,cm^{-1}$.

  • Substitution patterns (Bending $900 - 690\,cm^{-1}$):

    • Monosubstituted: Strong band near $690\,cm^{-1}$.

    • Ortho: One band near $750\,cm^{-1}$.

    • Meta: Three bands at $690, 780$, and $880\,cm^{-1}$.

    • Para: One strong band at $800 - 850\,cm^{-1}$.

Gas Chromatography-Mass Spectrometry (GC-MS)

• Definition: A hyphenated analytical technique combining separation (GC) and identification (MS) of components in a complex gas mixture.

• Importance:

  1. Forensic/Toxicology: Identifying illicit drugs.

  2. Environmental: Detecting VOCs and pesticides in soil/water.

  3. Food: Identifying flavors and contaminants.

  4. Pharmaceutical: Monitoring drug purity and degradation.

• Components of GC:

  1. Carrier Gas: Mobile phase ($He, N_2$, or $H_2$); must be inert.

  2. Injector: Heated port to vaporize the sample.

  3. Column: Houses the stationary phase (polar or non-polar).

  4. Oven: Temperature-controlled chamber for the column.

  5. Detector: Measures eluting components (e.g., FID or MS).

  6. Data System: Computer that generates the chromatogram.

• MS Process:

  1. Ionization: Electron Impact (EI) bombards sample with electrons to create positive fragment ions.

  2. Mass Analysis: Fragments separated by mass-to-charge ($m/z$) ratio via analyzers like Quadrupole or Time-of-Flight (TOF).

  3. Detection: Registers ion quantity and produces a Total Ion Chromatogram (TIC) and Mass Spectrum.

Interpreting GC-MS Data

• GC Chromatogram (TIC):

  • X-axis: Retention Time (RT). Fast-moving (small or less polar) molecules have lower RT.

  • Y-axis: Peak area/height, used to quantify components.

• Mass Spectrum:

  • X-axis: $m/z$ ratio.

  • Y-axis: Relative abundance.

  • Base Peak: The tallest peak, representing the most stable/abundant fragment.

  • Molecular Ion Peak ($M^{+}$): Represents the un-fragmented molecule; reveals the molecular weight.