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5C: Separation and Purification Methods

Extraction, distillation, every flavor of chromatography and electrophoresis — how a single pure compound or protein is pulled out of a mixture.

16 topics · 61 questions

Each topic is a path, not a menu: work down it in order and every question builds on the one above. Click any of them to watch the answer draw itself.

5C. Extraction and the Distribution of a Solute Between Immiscible Solvents

  1. How it worksHow does a separatory funnel split a solute between water and ether by its distribution coefficient?
  2. Worked exampleFind how much solute stays in the water after two 50 mL ether extractions when K equals 4.
  3. Common mistakeWhy isn't one large extraction as effective as three small ones using the same total solvent?
  4. Going furtherWhat changes about which layer sits on top when you swap ether for dichloromethane?
Your turnmedium1 of 3
479 mg of a solute sits in 82 mL of water. The solution is shaken once with 72 mL of ether, in which the solute's distribution coefficient K = [ether]/[water] is 4. How many mg stay in the water?

5C. Acid-Base Extraction

  1. How it worksHow does deprotonating benzoic acid turn a molecule that lives in ether into one that lives in water?
  2. Worked exampleWalk me through pulling benzoic acid, aniline and naphthalene apart using acid and base washes.
  3. Common mistakeWhy doesn't a neutral compound move between the layers no matter how much acid you pour in?
  4. Ties togetherHow does the pH that steers an acid-base extraction connect back to the buffer equation for a weak acid?
Your turnhard1 of 3
Benzoic acid has a pKa of 4.77. A chemist washes the ether layer with a buffer at pH 4 rather than with strong base. What percentage of the benzoic acid is present as the benzoate anion in that aqueous wash?

5C. Distillation: Simple, Fractional and Vacuum

  1. How it worksHow does a fractional column give a mixture dozens of vaporize-and-condense cycles in one pass?
  2. Worked exampleWhich distillation handles liquids boiling at 80 and 110 degrees C, and which handles 80 and 82?
  3. Going furtherWhat changes about the boiling point when you pull a vacuum on a compound that decomposes when hot?
  4. Common mistakeWhy doesn't an azeotrope ever separate cleanly no matter how tall you build the column?
Your turneasy1 of 3
One mixture's components boil at 82 and 125 degrees C; a second mixture's boil at 80 and 83 degrees C. Which technique suits each?

5C. Recrystallization

  1. How it worksHow does a solvent whose dissolving power swings hard with temperature separate product from junk?
  2. Worked exampleFind what went wrong when a recrystallization returns 95 percent yield with a broad melting range.
  3. Going furtherWhat happens to crystal purity when you crash-cool the flask in ice instead of cooling it slowly?
Your turnhard1 of 3
A compound dissolves at 24 g per 100 mL in the hot solvent and 3 g per 100 mL in the cold. 11 g of it is dissolved in the minimum volume of hot solvent and cooled. How many grams can be recovered as crystals?

5C. The Principle Behind Every Chromatography

  1. How it worksHow does a compound's tug of war between a moving phase and a stuck phase set its travel speed?
  2. Common mistakeWhy isn't chromatography just an elaborate way of filtering molecules by size?
  3. Worked exampleCompare how a polar and a nonpolar compound move through polar silica flushed with nonpolar solvent.
  4. Going furtherWhat changes about every separation once the stationary phase is nonpolar and the mobile phase polar?
Your turneasy1 of 3
A polar compound and a nonpolar compound are run on polar silica with an eluent of 22 percent ethyl acetate in hexane. Which moves faster, and why?

5C. Column Chromatography

  1. How it worksHow does solvent draining through a silica bed carry weakly bound compounds ahead of tightly bound ones?
  2. Worked exampleFind the elution order of an alkane, a ketone and an alcohol on silica run with hexane and ethyl acetate.
  3. Common mistakeWhy doesn't running the column faster give you the same clean separation in less time?
  4. Going furtherWhat changes about the separation when you raise the eluent polarity partway down the column?
Your turneasy1 of 3
An alkane, a ketone and an alcohol are loaded together onto a silica column run with 24 percent ethyl acetate in hexane. In what order do they come off?

5C. Thin-Layer Chromatography and Rf

  1. How it worksHow does capillary action haul solvent up a silica plate and drag dissolved compounds partway along?
  2. Worked exampleFind the Rf of a spot that traveled 2.4 cm when the solvent front reached 6.0 cm.
  3. Worked exampleFind which compound is more polar when one spot reads Rf 0.20 and the other reads Rf 0.80 on silica.
  4. Common mistakeWhy isn't a high Rf a sign that the compound is more polar on an ordinary silica plate?
Your turneasy1 of 3
On a TLC plate a spot travels 3 cm from the baseline while the solvent front reaches 8 cm. What is the spot's Rf?

5C. Gas-Liquid Chromatography and HPLC

  1. How it worksHow does a heated capillary coated in liquid sort compounds by boiling point and polarity at once?
  2. How it worksHow does shoving solvent through tightly packed particles at high pressure sharpen every peak?
  3. Worked exampleFind which of three compounds leaves a gas chromatograph first at boiling points 36, 69 and 126 degrees C.
  4. Going furtherWhat changes about retention time when you steepen the oven's temperature ramp?
Your turneasy1 of 3
Three compounds of similar polarity boil at 37, 70 and 129 degrees C and are injected together onto a gas chromatograph. Which leaves the column first, and why?

5C. Paper Chromatography

  1. How it worksHow does water trapped inside cellulose act as a stationary phase while solvent climbs past it?
  2. Worked exampleFind which of two amino acids on a paper chromatogram is the more hydrophobic one.
  3. Going furtherWhat happens to the separation when you run a second solvent at right angles to the first?
Your turneasy1 of 3
On a paper chromatogram, alanine travels 2 cm from the baseline and leucine travels 5.12 cm, with the solvent front at 9.33 cm. What is leucine's Rf?

5C. Size-Exclusion Chromatography

  1. How it worksHow do porous beads send small molecules on long detours while large ones sail straight down?
  2. Common mistakeWhy isn't size-exclusion chromatography a sieve that holds the large molecules back?
  3. Worked exampleFind the elution order of a 15 kDa, a 70 kDa and a 450 kDa protein on a size-exclusion column.
  4. Going furtherWhat changes about the separation when every protein is smaller than the bead pore size?
Your turneasy1 of 3
Proteins of 20 kDa, 79 kDa and 486 kDa are loaded together onto a gel-filtration column. In what order do they elute?

5C. Ion-Exchange Chromatography

  1. How it worksHow does a negatively charged resin capture positively charged proteins and let salt pry them loose?
  2. Worked exampleFind which of two proteins with pI 4.5 and pI 9.0 sticks to a cation exchanger run at pH 7.
  3. Going furtherWhat happens to a bound protein when you push the buffer pH past its isoelectric point?
  4. Ties togetherHow does the charge logic behind ion exchange match the charge logic behind isoelectric focusing?
Your turnmedium1 of 3
A protein has an isoelectric point of 7.4 and sits in a buffer at pH 8. Enter +1 if its net charge is positive there and -1 if its net charge is negative.

5C. Affinity Chromatography

  1. How it worksHow does a ligand bolted to the resin capture only the protein that binds it and nothing else?
  2. Worked exampleWalk me through purifying a histidine-tagged protein on a nickel column and eluting it with imidazole.
  3. Common mistakeWhy doesn't an affinity column guarantee a pure product after a single pass?
Your turneasy1 of 3
A histidine-tagged protein is bound to a nickel column and then eluted with 239 mM imidazole. How does the imidazole get it off?

5C. Gel Electrophoresis and SDS-PAGE

  1. How it worksHow does SDS coat every protein with the same charge-to-mass ratio so that only size still matters?
  2. Worked exampleFind the approximate mass of a band running between 25 kDa and 50 kDa markers on a log scale.
  3. Common mistakeWhy isn't the fastest band on an SDS gel the one with the most negative native charge?
  4. Going furtherWhat changes about the banding pattern when the reducing agent is left out of the sample buffer?
Your turnhard1 of 3
On an SDS gel the 54 kDa marker runs 3 cm from the well and the 26 kDa marker runs 5 cm. Migration distance is linear in the logarithm of mass. A band runs 3.56 cm. What is its mass in kDa?

5C. Isoelectric Focusing and Native Gels

  1. How it worksHow does a protein drift along a pH gradient until its net charge hits zero and the pull vanishes?
  2. Worked exampleFind where a protein of pI 6.2 comes to rest in a gel running from pH 3 at one end to pH 10 at the other.
  3. Going furtherWhat changes about a native gel result compared with a denaturing one for a four-subunit protein?
  4. Ties togetherHow does stacking isoelectric focusing with an SDS gel produce a two-dimensional map of a proteome?
Your turnmedium1 of 3
An isoelectric focusing strip 15 cm long carries a linear pH gradient from pH 4 at the anode end to pH 11 at the cathode end. How far from the anode end does a protein of pI 6 come to rest?

5C. Quantitative Analysis of an Isolated Protein

  1. How it worksHow does absorbance at 280 nm turn a protein's aromatic side chains into a concentration reading?
  2. Worked exampleFind the protein concentration when a 1 cm cuvette reads absorbance 0.42 with an extinction coefficient of 21000.
  3. Common mistakeWhy isn't a colorimetric protein assay a measure of how active the enzyme actually is?
  4. Going furtherWhat happens to a 280 nm reading when nucleic acid contamination rides along with the protein?
Your turnmedium1 of 3
A protein of molar extinction coefficient 35893 per M per cm is read in a 1.47 cm cuvette at 280 nm, giving an absorbance of 0.896. What is its concentration in micromolar?

5C. Separating Enantiomers From a Racemic Mixture

  1. How it worksHow does reacting a racemic mixture with one pure enantiomer convert the pair into separable diastereomers?
  2. Worked exampleWalk me through resolving racemic lactic acid with a single enantiomer of an amine as resolving agent.
  3. Going furtherWhat changes when the stationary phase itself is chiral instead of adding a resolving agent?
  4. Ties togetherHow does the separability of diastereomers explain why a drug can be sold as one pure enantiomer?
Your turneasy1 of 3
A racemic mixture is run on 5 different achiral columns and gives a single peak every time. Why?

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