MCAT Separation Techniques

Thin Layer Chromatography (TLC)

- Separation based on polarity

- Stationary phase: thin strip of silica (polar)

- Mobile phase: nonpolar solvent

- Rf = distance traveled/solvent front

- More polar = smaller Rf

- Used for DIAGNOSTIC purposes

Column (flash) chromatography

- Separation based on polarity

- Stationary phase: silica beads (polar)

- Mobile phase: solvent with mixture

- Retention time: how long you stay in the column before eluting

- More polar = longer retention time

- Used for SEPARATION purposes (for bulk amounts)

Ion Exchange Chromatography

- Separation based on charge via ionic interactions

- Stationary phase: beads with (+) or (-) charged functional groups bound to oppositely charged metal ions

- Mobile phase: solvent with mixture

- When mixture is added, groups with the same charge as the metal ions replace the metal ions, interacting with the charged functional groups of the beads

- Those groups with the same charge as the functional groups on the beads (and neutral groups) elute more quickly (shorter retention time)

- Used for SEPARATION purposes (for bulk amounts)

High performance liquid chromatography (HPLC)

- Separation based on polarity

- Stationary phase: nonpolar

- Mobile phase: polar (THIS IS REVERSE TLC)

- Mobile phase is pressurized by pump, inserted into injector, and taken through stationary phase to detector

- Increased pressure of the mobile phase increases the flow rate and efficiency through the stationary phase (this is better than column chromatography)

- More polar = faster elution

- Used for SEPARATION or ANALYTICAL purposes

Size exclusion chromatography

- Separation based on molecular size

- Stationary phase: inert beads with pores

- Mobile phase: solvent with molecules

- Large particles cannot fit into any pores in the beads and therefore just move around the beads quickly

- Small particles will get caught in the pores of the beads and will move more slowly as it takes a long path

- Increased size = decreased retention time

- Used for SEPARATORY purposes

Affinity chromatography

- Separation based on specific binding

- This is most often used to purify proteins or nucleic acids from complex biochemical mixtures like cell lysates, growth media, or blood

1. Put cell lysate (mixture from cells that you've lysed) into tube

2. Antibody against the protein of interest is added

3. To isolate the antigen-antibody complex, a microbe derived protein covalently linked to a solid support is added (beads)

- This creates antibody/antigen/protein A/bead complex

4. Centrifugation pushes this complex to the bottom (as the pellet) and suspends supernatant on top

5. Supernatant is removed

6. Pellet is resuspended

- You can also use magnetic beads and isolate the complex with a magnet instead of centrifugation

Gas Chromatography

- Separates based on volatility (boiling points) of compounds

- Stationary phase: solid column with liquid adsorbant on the surface

- Mobile phase: sample carried along by stream of inert gas

- Less volatile (higher boiling point) = more time interacting with liquid phase

- Higher boiling point = longer retention time

- Used for ANALYTICAL purposes

How does branching affect boiling point of hydrocarbons?

- Branching interferes with Van der Waals interactions by decreasing the surface area available for intermolecular interaction and thus decreases the boiling point

How does molecular weight affect boiling point of hydrocarbons?

- As molecular weight of hydrocarbons increases, so does the boiling point, because there are more Van der Waals interactions that are hard to break

Inter vs intramolecular hydrogen bonds and their affect on boiling point

- Intermoelcular hydrogen bonds increase boiling point

- Intramolecular hydrogen bonds decrease boiling point because it decreases the amount of intermolecular hydrogen bonding

Distillation

- Raising the temperature of a liquid until it can overcome the intermolecular forces that hold it together in the liquid phase

Simple distillation

- When trace impurities need to be removed from a relatively pure compound, or when a mixture of compounds with significantly different boiling points need to be separated

Fractional distillation

- Separated based on boiling point

- Used for mixtures with components of very similar boiling points

- Fractional distillation column is packed with glass beads to stainless steel sponge

- This subjects the liquid mixture to many vaporization-condensation cycles as it moves up the column towards the condenser

- Lower boiling point liquid boils first and is condensed in the cool vapor column and thus collected first

Mass Spectrometry

- Separation based on mass/charge (m/z) ratio (which detects molecular weight)

- Sample is ionized and then acted upon by a magnetic field

- Peaks can be viewed as molecular mass

- MW = largest peak that is most shifted to the right

- Small peaks next to big peaks = isotopes

- Big peaks that aren't the MW peak = broken fragments created during ionization of sample

UV/Vis Spectroscopy

- Detects absorption of UV or visible light

- Transition metals and long conjugated pi systems (aromatic) can absorb light

- The more extensive the conjugated system is, the longer the wavelength of maximum absorption will be (less energy)

- The color a compound maximally absorbs is complementary to the color it will appear to our eyes (ROYGBV)

Liquid-liquid extraction

1. Water layer (aqueous layer) and diethyl ether layer (organic layer)

2. Extraction of carboxylic acids

- Use NaHCO3 to deprotonate carboxylic acid

- This will enter the aqueous layer while the rest of the compounds remain in the ether layer

3. Extraction of phenols

- Use 10% NaOH to deprotonate the phenol (pKa ~10)

- This will enter the aqueous layer

4. Extraction of amines

- Use 10% HCl to protonate amines (now positively charged)

- This will enter the aqueous layer

- Carried out in a separatory funnel

IR spectroscopy

- Used to detect functional groups

Wavenumber

- The reciprocal of wavelength

- Directly proportional to both frequency and energy

IR stretching frequency of carbonyls

~1700 cm

IR stretching frequency of alkenes

~1650 cm

IR stretching frequency of triple bonded carbon and cyano groups

2260 cm - 2100 cm

IR stretching frequency of alcohols

3600 cm - 3200 cm

- Very broad

IR stretching frequency of amines

3150 cm - 2500 cm

IR stretching frequency of hydrocarbons

3300 cm - 2700 cm

What four pieces of information can you infer from an NMR spectrum?

1. The number of sets of peaks in the spectrum tells you the number of nonequivalent hydrogen sets

2. The splitting pattern of each peak tells you how many protons are interacting with that proton set

3. The mathematical integration tells you how many equivalent protons are in that set

4. The chemical shift values of those set peaks gives information about the environment of the protons in the set

Upfield and downfield

- Upfield: refers to lower ppm values (shift right) due to high electron density (less deshielded)

- Downfield: refers to higher ppm values (shift left) due to low electron density (more deshielded)

Affect on shift when a proton is close to an electronegative atom

- Being close to an electronegative atom (Cl, Br, O, N, etc.) means that the atom will hog all the electrons for itself, make the proton environment less exposed to hydrogens, and shift it downfield (to the left)

Hybridization effects on chemical shift values

- The greater the s-orbital character of a C-H bond, the less electron density on the hydrogen, and the more downfield it is