Separation Techniques, Spectroscopy, and Analytical Methods

Gas Liquid Chromatography

Separates molecules based on boiling point. Intermolecular forces, molecular weight, and branching contribute to a molecule’s boiling point. For compounds with the same functional group but different carbon counts, a. higher molecular weight indicates a higher boiling point and so. a longer retention time.

Distillation

A technique that separates molecules based on their boiling points.The molecule with the lowest boiling point distills before the molecules with higher boiling points. The mixture must be heated slowly to ensure that a lower boiling compound evaporates before a higher boiling compound and that the molecules are separated.

Simple Distillation

Ideal for compounds with boiling points > 25 Celsius apart and less than 150 Celsius

Fractional Distillation

Ideal for compounds with boiling points < 25 Celsius apart and less than 150 Celsius

Vacuum Distillation

Ideal for compounds with boiling points > 150 Celsius

Vacuum Distillation

Vacuum distillation is performed under reduced pressure, lowering a compound’s boiling point relative to its boiling point at atmospheric pressure. Vacuum distillation is ideal for compounds with boiling points > 150 Celsius to prevent degradation.

Boiling Chips and Stir Bar

Boiling chips are porous materials used in simple and fractional distillation that introduce nucleation sites where small bubbles form, overcoming the surface tension and allowing even boiling (prevents superheating). Stir bars are used in place of boiling chips in vacuum distillation to prevent superheating.

Branching (Multiple Substituents to the Atom)

Decreases surface area and decreases boiling point

No Branching (No Substituents to the Atom)

Increases surface area and increases boiling point

Hydrogen Bonds:

Increases the boiling point

H NMR

Detects hydrogen atoms in a molecule as an external magnetic field and radio frequency are applied to the sample. Hydrogen atoms in different magnetic environments within 3 bonds of each other cause spin spin splitting of peaks in the spectrum. The splitting pattern can be determined

n + 1 rules

Number of peaks = n + 1

n is the number of neighboring hydrogens

No coupled Hydrogens = Singlet

One coupled Hydrogens = Doublet

Two coupled Hydrogens = Triplet

Three coupled Hydrogens = Quarter

Thin Layer Chromatography (Purity)

TLC is a technique used to evaluate a compound’s purity. The components of a mixture are separated based on polarity, and a single spot on a TLC plate is indicative of a compound’s purity.

Size Exclusion Chromatography

Separates compounds by size and compounds similar in size elute together and appear in one peak.

Gas-Liquid Chromatography

A technique that separates compounds in a mixture based on boiling point. The mobile phase is an inert gas, and the stationary phase is the liquid that coats the column in the heated oven. Molecules with lower boiling points reach the detector before molecules with higher boiling points.

Separation of a Mixture Using TLC occurs because:

The mixture components travel up the TLC plate at different rates depending on the strength of the intermolecular interactions between the mixture component and the stationary and mobile phases.

ore Hydrogen Bonds =

Stronger affinity for stationary/polar phase

Nonpolar Solvents:

Decrease a compound’s affinity for the mobile phase.

Polar Solvents:

Decrease a compound’s affinity for the stationary phase.

Simple Distillations vs Vacuum Distillations

Simple distillations are done at atmospheric pressure, whereas vacuum distillation is done at reduced pressure, decreasing the compound’s boiling point relative to the boiling point at atmospheric pressure. so Vacuum BP is less than Simpler BP

IR Spectrum

An infrared spectrum displays absorption signal characteristics of the functional group in a particular molecule based on the IR frequencies absorbed by those functional groups. Functional group bands appear in the same region of the IR spectrum regardless of the overall structure of the molecule.

Constitutional Isomers (IMFs)

Constitutional isomers can experience different intermolecular forces, contributing to the differences in their boiling points. The isomer that experiences increased intermolecular hydrogen bonding has a higher boiling point compared to the isomer that experiences increased intramolecular bonding.

Superheating

When a liquid is heated above its boiling point but it does not boil. Surface tension causes the vapor pressure inside the bubbles to increase as they form, causing them to explode at the surface. Addition of boiling chips gives the bubbles a surface to form on as liquid is heated and even allows for boiling.

Mass Spectromertry

A sample is ionized by high energy electrons but the electrons do not change energy levels.

NMR Spectroscopy

An external magnetic field is applied to a sample. Radio waves, which are low in energy, are used to detect hydrogen atoms and excite them from the a spin state to the B spin state.

TLC with UV Light

UV light can be used to visualize the results of TLC. if the reactions can absorb UV light (photons). UV light carries a large amount of energy that can excite electrons of UV chromophores to a higher energy state.

UV Chromophores

Double and Triple bond carbonyls (C=O)

Rf Value

The ratio of the distance up the plate a compound travels to the distance the solvent travels. A polar compound will have a smaller Rf value than a nonpolar compound.

Absorption (Functional Groups)

Functional groups show absorption in the infrared spectrum at different frequencies depending on the bond type in the functional group. Functional group absorptions: (3650-3200 cm^-1 for Alcohols O-H and phenol stretch) (3550-3060 cm^-1 for amide N-H stretch) (3100 for C-H sp² stretch) (3000-2875 cm^-1 for C-H sp³ stretch) (2260-2100 cm^-1 triple bonds) and (1850-1650 ^-1 for C=O stretch)

Mass Spectrometry

A technique that measures the molecular weight. Molecules in a sample are bombarded with a beam of electrons, producing positively charged ions and fragments of the molecule.The ionized fragments are detected and a mass spectrum is generated. The y axis represents ion abundance and the x axis represents the mass to charge (m/z) ratio. The peals observed in the mass spectrum are ionized fragments of the sample.

Extraction

Extraction is a technique that that uses an immiscible organic solvent (an organic solvent and an aqueous solution that do not dissolve each other) as a partition to separate molecules in a mixture based on solubility. Molecules dissolve best in solvents with similar polarity (like dissolves like). Hydrophobic molecules have a greater affinity for the organic layer because the organic layer is less polar than the aqueous layer. The denser layer is on the bottom.

Separation of Carboxylic Acids and Amides:

Can be done via Extraction. Carboxylic acid with long hydrocarbon chains can be induced to enter aqueous layer if they are converted to an ionic salt via deprotonation by either a strong base or a weak base. The amide remains in the organic layer because it cannot be deprotonataed by a weak base. Amide hydrolysis requires a strong base or strong acid and heat(HCO3 is strong acid).

Strong BAse

X(OH) X(OH)2

Weak Bases

Water, ammonia, ammonium hydroxife, pyridine, amine

Acid Base Extraction

A mixture of organic compounds with acidic or basic functional groups can be separated by acid base extraction. The basic or acidic molecules enter the aqueous layer as ionic salts by extraction with an acid or base, respectively. Amines are weak bases that require strong acids (H2SO4) to be protonated. Phenols are weak acids that require strong bases (NaOH) to be deprotonated. They must be added in the sequence asked.

Dilution Factor

The dilution factor of each step in a serial dilution is calculated by dividing the final volume Vf of the dilute solution by the volume of the solute solution transferred Vt. Multiplying together the dilution factors from each dilution step yields he overall DF of the dilution sequence.

Resonance Energy

The difference in energy between the alpha and beta spin states. It is proportional to the effective magnetic field (B, eff) experienced by the hydrogen nucleus. Electrons generate magnetic fields (B,ind) that shield a nucleus from an external magnetic field.

B,eff = B,0 - B,ind

H NMR Signal

Hydrogen atoms on a molecule that display rotational or planar symmetry are in identical magnetic environments. Equivalent hydrogens have identical chemical shifts and are represented as a single signal on the H NMR. The relative integration of a H NMR signal is proportional to the number of protons (H’s in the molecule = signal amount) in a magnetic environment.

n + 1 rule

The splitting pattern of a given proton can be determined by the N + 1 rule where N = the number of equivalent protons on neighboring adjacent atoms.

A C-H bonded to 4 C’s is a singlet (no coupled hydrogens)

A C-H bonded to 3 C’s is a doublet (1 coupled hydrogens)

A C-H bonded to 2 C’s is a triplet (2 coupled hydrogens)

A C-H bonded to 1 C is a quarter (3 coupled hydrogens)

High Performance Liquid Chromatography

A purification technique that separates compounds based on polarity and consists of mobile phase, stationary phase, detector, and computer for data acquisition. The mobile phase is a solvent or mixture of solvents that are pumped through the system under pressure, and the stationary phase is a column made of either polar or nonpolar material.

Extraction

A technique that is used to separate molecules based on their solubility in aqueous (polar) or organic (nonpolar) phases. The mixture components will dissolve in a solvent of the same polarity (like dissolves like). Hydrophobic (nonpolar) molecules will dissolve in a nonpolar organic solvent and hydrophilic molecules will dissolve in aqueous solvent.

Rf Value

The Rf value is the unitless ratio of how far a compound travelled up the TLC plate compared to the solvent.

Ultraviolet Light (UV) Light

Electromagnetic radiation that corresponds to wavelengths between 200nm and 400nm, UV absorption causes an electron transition from the ground state to a higher energy level. In the ground state, pi electrons from double bonds are in the pi bonding molecular orbital, and nonbonding electrons are in the n nonbonding molecular orbital. These electrons can be excited to the pi* antibonding orbital via pi → pi* or n → pi* transitions.

Molecules with UV chromophores (double and triple bonds, carbonyls conjugated systems):

Can be visualized with UV light.

When UV chromophores (double and triple bonds, carbonyls conjugated systems) absorb UV light:

Energy from the UV light excites electrons to a higher energy excited state.

Heteroxyclic

The atom (X) is a part of the ring

High Performance Liquid Chromatography (Normal Phase and Reverse Phase)

In HPLC there are two types of columns, normal and reverse phase depending on the polarity of the compounds being separated. NP-HPLC uses a polar stationary phase and a nonpolar mobile phase while RP-HPLC uses a non polar stationary phase and a polar mobile phase

Polar Solvents:

Acetone, methanol, and water.

Nonpolar Solvents

Hexane

Dilution Factor Equation

DF = Vf1 / Vt1 x Vf2 / Vt2

Dilutions are performed to decrease the concentration of a solution when a small amount is needed and cannot accurately be measured. A small volume of concentrated solution (Vt) is transferred to a certain volume of solvent (Vs) and the final volume (Vf) is the sum of Vt and Vs.

1:20 = DF = 20mL

Normal Phase HPLC (Graph)

Time is the x axis and absorbance is the y axis, Nonpolar molecules interacts more with the mobile phase and have a shorter retention time. Polar interacts more with the stationary phase and a longer retention time.

Racemic Mixture

A 50:50 mixture of enantiomers, which have the same chemical and physical properties and cannot be directly separated. So, the separation of the enantiomers requires the addition of a resolving agent to change their physical properties by creating a pair of diastereomers. The resolving agent is removed once the diastereomers are separated to yield the original molecules as single enantiomers.

A mixture of organic molecules with acidic or basic functional groups can be separated by an acid-base extraction.

The acidic or basic molecule can enter the aqueous layer when extracted by an acid or base, respectively.

Bronsted Lowry Bases

Contain electronegative atoms that can accept an H+ proton from acids. Electronegative atoms (F, O, and N) make a molecule polar. Atoms with only hydrocarbons are nonpolar.

IR Absorption Range

IR Frequency Functional Group Frequency Range

1. Single Bonds. Alkanes C-H: 3000, Alkenes C=H: 3100, Alkynes 3300, Hydroxyl O-H: 3650-3200, Carboxylic Acids: 3000-2500, Amines N-H: 3600-3200, Thiols 2600-2500

2. Double Bonds. Alkenes: 1630, Aromatic Ring: 1600-1475, Imines and Oximes C=N: 1650-1500, Ester: 1745, Aldehyde: 1725, Ketone: 1715, Carboxylic Acid: 1710, Amide: 1650,