Analytical Techniques in Chemistry: Chromatography, Electrophoresis, and Spectroscopy

Unit 4, Topic 1: Learning Objectives and Analytical Overview

• Amino Acid Separation and Identification: Students must explain how amino acids are separated using paper or Thin-Layer Chromatography (TLC). This involves understanding intermolecular forces, solubility in mobile and stationary phases, and the use of retention factor ($R_f$) values.

• Electrophoresis Analysis: Students must explain the separation of amino acids by electrophoresis, focusing on the pH of the buffer, isoelectric points ($pI$), and the movement of charged ions toward electrodes.

• Data Analysis (Chromatography): Analyze paper/TLC chromatograms and electrophoresis data to identify amino acids and calculate retention factors using the formula:

$R_f = \frac{\text{distance travelled by the sample from its origin}}{\text{distance of the solvent front from the sample origin}}$

• Spectroscopic Analysis: Analyze data from mass spectroscopy and infrared (IR) spectroscopy to determine the identity and chemical structure of organic molecules.

General Principles of Chromatography

• Definition: Chromatography is an analytical technique used for separating and analyzing complex mixtures of organic molecules. It can be performed on very small samples and at low concentrations.

• The Two Phases:

  • Stationary Phase: A solid or liquid onto which substances are absorbed.

  • Mobile Phase: A liquid or gas (the solvent) that carries the substances through the stationary phase.

• Mechanism of Separation: Separation is based on the way particles absorb onto the stationary phase surface and desorb into the mobile phase solvent.

  • Particles strongly attracted to the stationary phase move the slowest.

  • Particles strongly attracted to the solvent move the fastest.

• Solvent Polarity: If water is used as the solvent, polar substances move more quickly than less polar components because they have a higher affinity for the polar mobile phase.

Thin-Layer Chromatography (TLC)

• Setup: A TLC plate consists of a thin layer (approximately $0.1\,mm$) of absorbent material, such as silica gel, coated on a glass or aluminum backing.

  • Stationary Phase: The absorbent material (e.g., silica gel).

  • Mobile Phase: The liquid solvent.

• Qualitative Analysis: TLC and paper chromatography are useful for identifying which chemicals are present in a mixture by comparing them to standards (reference chemicals).

• Chemical Properties of Silica: Silica is highly polar and features $-OH$ groups. Therefore, it interacts via hydrogen bonding with polar substances.

• Retention Factor ($R_f$):

  • Identifies components by comparing calculated values to known standards under identical conditions.

  • The value must always be between $0$ and $1$.

  • Calculation Example: If a blue component moves $3.5\,cm$ and the solvent front moves $10\,cm$, the calculation is:      $R_f = \frac{3.5}{10} = 0.35$

Separation of Amino Acids via TLC

• Amino Acid Components:

  • Alpha-carbon (center).

  • Amino functional group (weakly basic).

  • Carboxylic acid functional group (weakly acidic).

  • R-group (unique side chain for the 20 amino acids).

• Predicting TLC Behavior:

  • Non-polar R-groups (e.g., Isoleucine, Phenylalanine) will interact differently with the stationary phase than polar ones.

  • A non-polar amino acid will travel further (higher $R_f$) if the solvent is non-polar, or move slowly if the stationary phase is non-polar.

• Case Study: Aspartame: Aspartame is composed of aspartic acid and phenylalanine.

  • Identification: A spot (Spot A) that travels a similar distance to a known non-polar standard (like Isoleucine) can be identified as phenylalanine (also non-polar).

Electrophoresis Principles

• Mechanism: Separation of nucleic acids and amino acids based on their charge and size by applying an electric charge (voltage) to a gel.

  • Negatively charged molecules migrate toward the positive electrode (Anode).

  • Positively charged molecules migrate toward the negative electrode (Cathode).

• Separation Parameters:

  • Charge: This is the primary driver for amino acid separation since they are of similar size.

  • Size: The gel acts as a matrix; smaller molecules move faster through the spaces than larger ones.

• Visualization: Gels are often stained (e.g., Ethidium Bromide for DNA, which fluoresces under UV light).

Isoelectric Point ($pI$) and Electrophoresis

• Isoelectric Point ($pI$): The specific pH value at which an amino acid has no net charge.

  • Each amino acid has a different $pI$ based on its functional groups.

  • At pH > $pI$: The amino acid forms an anion (negative charge).

  • At pH < $pI$: The amino acid forms a cation (positive charge).

• Examples at pH 7.0:

  • Alanine ($pI = 6.1$): At pH 7.0 (pH > pI), alanine is negatively charged and migrates toward the anode.

  • Lysine ($pI = 9.7$): At pH 7.0 (pH < pI), lysine gains a proton, is positively charged ($+2$), and migrates toward the cathode.

  • Aspartic Acid ($pI = 3.2$): At pH 7.0 (pH > pI), both carboxyl groups lose protons, resulting in a $-2$ charge; it moves toward the anode.

Infrared (IR) Spectroscopy

• Technique: Uses electromagnetic radiation to change the vibration of chemical bonds. IR light does not have enough energy to promote electrons to higher energy levels but can induce bond bending and stretching.

• Dipole Moment Requirement: For a molecule to absorb IR radiation, the vibration (stretching or bending) must change the overall dipole of the molecule. Diatomic molecules like $N_2$ and $O_2$ do not absorb IR.

• Vibration Types:

  • Diatomic: Simple stretch.

  • Triatomic: Symmetrical stretch, asymmetrical stretch, bending (scissoring), wagging, twisting, and rocking.

• Spectrum Units:

  • Transmittance: The amount of light passing through the sample (not absorbed). Measured in percentage (%).

  • Wavenumber ($cm^{-1}$): The unit for the x-axis, representing waves per unit distance.

Interpreting IR Spectra

• Bond Strength and Mass:

  • Stronger bonds (e.g., $C\equiv C$) absorb higher frequencies than weaker bonds (e.g., $C-C$).

  • Higher mass atoms absorb lower frequencies of IR radiation.

    • $C-H \approx 3000\,cm^{-1}$

    • $C-Cl \approx 750\,cm^{-1}$

    • $C-Br \approx 600\,cm^{-1}$

• Spectral Regions:

  • Functional Group Region: Above $1400\,cm^{-1}$, used to identify specific functional groups.

  • Fingerprint Region: Below $1400\,cm^{-1}$. These absorption bands are unique to specific compounds and can indicate purity.

• Key Absorption Bands (Stretching):

  • Alcohol ($O-H$): $3200-3600\,cm^{-1}$ (strong, broad due to hydrogen bonding).

  • Carboxylic Acid ($O-H$): $2500-3000\,cm^{-1}$ (very broad).

  • Carbonyl ($C=O$): $1700-1750\,cm^{-1}$ (strong, sharp).

  • Alkanes/Alkenes ($C-H$): $2720-3100\,cm^{-1}$.

  • Amines ($N-H$): $3300-3500\,cm^{-1}$ (narrower than $O-H$).

  • Alkenes ($C=C$): $1620-1680\,cm^{-1}$.

Mass Spectrometry (MS)

• Applications: Determining protein structures, drug detection, cancer markers, and planetary atmosphere analysis (Mars/Saturn moons).

• Process:

  1. Ionization: A high-energy electron stream knocks an electron off the molecule, creating a parent molecular ion ($M^+$).

  2. Acceleration: Ions are accelerated through an electric field.

  3. Deflection: A magnet deflects ions based on their mass-to-charge ($m/z$) ratio. Smaller mass or higher charge causes greater deflection.

  4. Detection: Ions hit a plate, creating a burst of current.

• Fragmentation: Continual bombardment causes the cation to break into a smaller cation and a neutral free radical (the free radical is not detected).

• Spectrum Features:

  • Molecular Ion Peak ($M^+$): Reflects the molar mass of the original molecule.

  • Base Peak: The tallest peak (highest relative abundance), set at $100\%$.

  • Rule of 13: For hydrocarbons ($C_xH_y$), divide the $m/z$ by $13$. The quotient is the number of carbons, and the sum of the quotient and the remainder is the number of hydrogens.

    • Example: $m/z = 86$. $86 / 13 = 6$ remainder $8$. Formula: $C_6H_{14}$.

Fragmentation and Isotopes in MS

• Common Fragments:

  • $-15$: Loss of a methyl group ($CH_3$).

  • $-17$: Loss of a hydroxyl group ($OH$).

  • $-29$: Loss of an ethyl group ($C_2H_5$) or formyl group ($CHO$).

  • $-45$: Loss of a carboxyl group ($COOH$) or ethoxy group ($CH_3CH_2O$).

• Nitrogen Rule: Molecules with an odd molecular ion ($M^+$) usually contain an odd number of Nitrogen atoms.

• Isotope Effects:

  • Chlorine: Naturally occurs as $^{35}Cl$ and $^{37}Cl$, leading to two molecular ion peaks separated by $2\,m/z$ units.

  • Bromine: Naturally occurs as $^{79}Br$ and $^{81}Br$ in nearly equal amounts, resulting in two peaks of similar height at $M$ and $M+2$.

Integration of Analytical Techniques

Structural determination usually requires combining multiple datasets:

• Elemental Analysis/Percentage: Determines the empirical formula.

• Infrared Spectroscopy (IR): Identifies functional groups (e.g., presence of $C=O$ or $OH$).

• Mass Spectrometry (MS): Confirms the molar mass and identifies structural fragments.

• Example Integration: A molecule with $m/z = 74$, peaks at $29, 45, 57$, and IR bands at $1700\,cm^{-1}$ and $2500\,cm^{-1}$ is likely propanoic acid ($C_3H_6O_2$).