Separations, Purification and Spectroscopy (AAMC 5C)
Chemists and biochemists spend much of their time separating mixtures and then proving what they isolated. This page covers the separation methods the MCAT expects you to reason about (extraction, distillation, recrystallization, chromatography and electrophoresis) and the spectroscopic tools used to identify molecules (IR, UV-Vis, 1H NMR and mass spectrometry). Experimental passages lean on this material heavily: you will often have to decide which technique separates two compounds or which spectrum belongs to which product.
Extraction
Liquid-liquid extraction separates compounds by their different solubilities in two immiscible solvents, usually water and an organic solvent such as diethyl ether, ethyl acetate or dichloromethane. "Like dissolves like": polar and charged species prefer water, nonpolar species prefer the organic layer. The mixture is shaken in a separatory funnel and the layers are drained separately. The denser solvent is the bottom layer: dichloromethane (about 1.3 g/mL) sinks below water, while ether and ethyl acetate float on top.
Convert a neutral organic compound into a charged salt to move it into water, then reverse the reaction to recover it.
· Aqueous NaHCO3 (weak base) deprotonates carboxylic acids only.
· Aqueous NaOH (strong base) deprotonates carboxylic acids and phenols.
· Aqueous HCl protonates amines, making water-soluble ammonium salts.
Neutral compounds stay in the organic layer throughout. Acidifying the basic aqueous layer regenerates the acid, which then precipitates or can be extracted back.
Several extractions with smaller volumes of solvent recover more product than one extraction with the same total volume. After extraction, a drying agent (anhydrous MgSO4 or Na2SO4) removes traces of water from the organic layer.
Filtration and recrystallization
Filtration separates a solid from a liquid: gravity filtration when the filtrate is wanted, vacuum filtration when the solid is wanted. Recrystallization purifies a solid by dissolving it in a minimum of hot solvent in which it is soluble hot but poorly soluble cold. On slow cooling, pure crystals form and impurities stay dissolved. A pure crystalline solid has a sharp melting point; impurities lower and broaden the melting range.
Distillation
Distillation separates liquids by boiling point. The liquid with higher vapor pressure (weaker intermolecular forces) boils first and is condensed and collected as the distillate.
- Simple distillation: for liquids whose boiling points differ by more than about 25°C, or to separate a liquid from a nonvolatile solid.
- Fractional distillation: a fractionating column packed with glass beads or metal gives many vaporization-condensation cycles, so liquids with closer boiling points can be separated.
- Vacuum distillation: lowering the external pressure lowers boiling points, protecting compounds that would decompose at their normal boiling point.
Chromatography
All chromatography relies on two phases: a stationary phase that stays put and a mobile phase that moves through it. Each compound partitions between the two. A compound with higher affinity for the stationary phase moves more slowly; one with higher affinity for the mobile phase moves faster and elutes first.
Thin-layer chromatography (TLC)
A plate coated with polar silica gel (the stationary phase) is spotted with sample and placed in a shallow pool of a less polar solvent such as a hexane/ethyl acetate mixture. The solvent creeps up by capillary action. Polar compounds bind silica strongly and travel a short distance; nonpolar compounds travel farther.
Rf = (distance traveled by the spot) / (distance traveled by the solvent front). Rf is always between 0 and 1. On normal-phase silica, a less polar compound has a larger Rf. Making the solvent more polar raises the Rf of every spot. In reverse-phase TLC (nonpolar stationary phase), the order flips and polar compounds run farthest.
Column, gas and high-performance liquid chromatography
- Column chromatography: the same principle as TLC on a preparative scale. Silica or alumina is packed in a column, and the mobile phase carries compounds down; fractions are collected as they elute. Nonpolar compounds come off first on silica.
- Gas chromatography (GC): the sample is vaporized and carried by an inert gas (helium or nitrogen) through a long coated column. Separation depends mainly on volatility (boiling point) and interaction with the coating. Only volatile, heat-stable compounds work. GC is often coupled to a mass spectrometer (GC-MS).
- HPLC: liquid mobile phase pumped at high pressure through a tightly packed column of tiny particles, giving fast, high-resolution separations of nonvolatile compounds, including many drugs and biomolecules. Reverse-phase HPLC with a nonpolar C18 column is the most common format.
Protein chromatography
| Method | Separates by | Key point |
|---|---|---|
| Size-exclusion (gel filtration) | Size | Large proteins elute first: they cannot enter the bead pores and take the shorter path |
| Ion-exchange | Charge | Anion exchangers have positive beads and hold negative proteins; cation exchangers do the reverse. Elute with rising salt or changing pH |
| Affinity | Specific binding | Beads carry a ligand, antibody or metal (e.g., Ni2+ for His-tagged proteins); elute with free ligand or competitor |
Electrophoresis and Isoelectric Focusing
In electrophoresis, charged molecules migrate through a gel under an electric field. Migration speed increases with charge and field strength and decreases with size and friction (v = Ez/f). Negative molecules move toward the positive electrode (anode).
- Agarose gel electrophoresis: separates DNA and RNA fragments. Nucleic acids carry a uniform negative charge per nucleotide from their phosphate backbone, so they separate by length; smaller fragments travel farther.
- Native PAGE: polyacrylamide gel without denaturant. Proteins keep their shape and charge, so they separate by a combination of size, shape and charge, and can remain active.
- SDS-PAGE: sodium dodecyl sulfate denatures proteins and coats them with negative charge roughly proportional to length. Charge and shape differences are erased, so proteins separate by mass alone. Adding a reducing agent such as β-mercaptoethanol breaks disulfide bonds so subunits run separately.
- Isoelectric focusing: proteins move through a gel with a fixed pH gradient. A protein in a region where pH < pI is positive and moves toward the cathode; where pH > pI it is negative and moves toward the anode. It stops where pH = pI and its net charge is zero.
- Two-dimensional electrophoresis: isoelectric focusing in one direction, then SDS-PAGE at right angles, separating proteins by pI and by mass.
Infrared (IR) Spectroscopy
IR radiation makes bonds vibrate (stretch and bend). A bond absorbs IR only if the vibration changes the molecule's dipole moment, so symmetric bonds such as the C=C of a symmetric alkene may be weak or absent. Peaks are reported in wavenumbers (cm−1); higher wavenumber means higher energy. Stronger bonds and lighter atoms vibrate at higher wavenumbers. The region below about 1,500 cm−1 is the fingerprint region, unique to each molecule but rarely interpreted on the MCAT.
| Bond / group | Approximate wavenumber (cm−1) | Appearance |
|---|---|---|
| O–H (alcohol) | 3,200 to 3,600 | Broad, strong (hydrogen bonding) |
| O–H (carboxylic acid) | 2,500 to 3,300 | Very broad, overlaps C–H |
| N–H | 3,300 to 3,500 | Sharper; primary amines show two spikes, secondary one |
| C–H (sp3) | 2,850 to 3,000 | Present in almost all organics |
| C≡N, C≡C | 2,100 to 2,260 | Sharp, medium |
| C=O (carbonyl) | 1,650 to 1,750 (ketone about 1,715) | Sharp, very strong |
| C=C | 1,600 to 1,680 | Medium to weak |
UV-Visible Spectroscopy
UV-Vis light promotes electrons from the highest occupied molecular orbital (HOMO) to the lowest unoccupied one (LUMO). Conjugation narrows the HOMO-LUMO gap, so more extended conjugated systems absorb longer wavelengths. With enough conjugation (as in β-carotene) absorption moves into the visible range, and the compound appears the complementary color of the light it absorbs: a compound absorbing blue-violet light looks yellow-orange.
A = εlc, where A is absorbance (unitless), ε the molar absorptivity, l the path length and c the concentration. Absorbance is proportional to concentration, which is how a spectrophotometer measures protein (absorbance at 280 nm from aromatic residues) or nucleic acid (260 nm) concentrations.
Proton (1H) NMR Spectroscopy
In a strong magnetic field, hydrogen nuclei align with or against the field. Radio-frequency energy flips them between these spin states, and the exact frequency depends on each proton's electronic environment. Results are plotted as chemical shift (δ, in ppm) relative to the reference tetramethylsilane (TMS, set to 0 ppm). A spectrum gives four pieces of information:
- Number of signals: the number of chemically distinct sets of protons.
- Chemical shift: electron-withdrawing neighbors pull electron density away (deshielding), moving a signal downfield (to the left, higher ppm).
- Integration: the area under each signal is proportional to the number of protons it represents.
- Splitting: by the n + 1 rule, a signal is split into n + 1 peaks by n equivalent protons on adjacent carbons. Protons on O or N often appear as broad singlets because they exchange.
| Proton type | δ (ppm) |
|---|---|
| Alkyl C–H | 0.9 to 1.7 |
| H on carbon next to C=O | 2.0 to 2.6 |
| H on carbon bonded to O (alcohols, ethers, esters) | 3.3 to 4.5 |
| Vinylic C=C–H | 4.5 to 6.5 |
| Aromatic | 6.5 to 8.0 |
| Aldehyde | 9 to 10 |
| Carboxylic acid | 10.5 to 12 |
In ethanol (CH3CH2OH), the CH3 has 2 neighbors and appears as a triplet (integration 3) near 1.2 ppm. The CH2 has 3 neighbors and appears as a quartet (integration 2) near 3.7 ppm, downfield because it is bonded to oxygen. The OH is a broad singlet. A triplet-quartet pair with a 3:2 ratio is the signature of an ethyl group.
Mass Spectrometry
Mass spectrometry ionizes molecules (often by knocking out an electron to form a radical cation), separates the ions by mass-to-charge ratio (m/z) and counts them. It destroys the sample but needs only a tiny amount.
- Molecular ion peak (M+): the intact ionized molecule; gives the molecular mass.
- Base peak: the tallest peak, set to 100% abundance. It is the most stable fragment, which is not necessarily the molecular ion.
- Isotope patterns: chlorine gives M and M+2 peaks in about a 3:1 ratio (35Cl and 37Cl); bromine gives M and M+2 in about a 1:1 ratio.
Worked MCQs
Five MCQs on the patterns the exam returns to most often. Read every explanation, including the ones you got right.
Q1. A mixture of benzoic acid and naphthalene is dissolved in diethyl ether and shaken with aqueous NaHCO3. Where does each compound end up?
Bicarbonate is basic enough to deprotonate a carboxylic acid, forming the ionic sodium benzoate, which dissolves in water. Naphthalene is a neutral hydrocarbon with no acidic proton, so it stays in the organic layer.
Q2. Four compounds are run on a silica gel TLC plate with a hexane/ethyl acetate eluent. Which is expected to have the highest Rf?
Silica is polar, so polar compounds that can hydrogen bond (acids, alcohols, amides) stick to it and travel less. Toluene is the least polar, interacts least with the stationary phase, and travels farthest, giving the highest Rf.
Q3. An unknown shows a strong, sharp IR absorption near 1,715 cm−1 and no broad absorption between 2,500 and 3,600 cm−1. The compound is most likely:
A sharp, strong peak near 1,715 cm−1 is a C=O stretch. A carboxylic acid would also show a very broad O–H band from 2,500 to 3,300, and an alcohol a broad band near 3,200 to 3,600. An amine would show N–H peaks and no carbonyl. A ketone fits best.
Q4. In the 1H NMR spectrum of ethyl acetate (CH3COOCH2CH3), the signal for the O–CH2 protons is expected to appear as:
The CH2 has three neighboring protons on the CH3, so n + 1 = 4 peaks: a quartet. Being bonded to the ester oxygen deshields it to about 4.1 ppm. The singlet near 2.0 ppm is the acetyl CH3, and the triplet near 1.2 ppm is the ethyl CH3.
Q5. SDS-PAGE separates proteins primarily on the basis of:
SDS denatures proteins and coats them with negative charge roughly proportional to their length, so every protein has a similar charge-to-mass ratio and a similar rod-like shape. Migration then depends mainly on mass, with smaller proteins moving farther. Separation by pI is isoelectric focusing.
Quick Recap
- Extraction: like dissolves like; NaHCO3 pulls out carboxylic acids, NaOH pulls out acids and phenols, HCl pulls out amines.
- Distillation: simple for > 25°C difference, fractional for closer boiling points, vacuum for heat-sensitive compounds.
- Chromatography: stronger attraction to the stationary phase means slower movement; on silica, nonpolar compounds have high Rf.
- Size-exclusion: big proteins elute first. Ion-exchange: by charge. Affinity: by specific binding.
- SDS-PAGE separates by mass; native PAGE by size, shape and charge; isoelectric focusing by pI.
- IR: broad O–H 3,200 to 3,600; very broad acid O–H 2,500 to 3,300; sharp C=O about 1,700.
- UV-Vis: more conjugation, longer wavelength; A = εlc.
- NMR: shift (environment), integration (number of H), n + 1 splitting (neighbors). Mass spec: M+ gives mass; Cl 3:1 and Br 1:1 at M+2.