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

Rf=0 start and Rf=1 solvent front

- More polar = smaller Rf (more attracted to polar)

Non polar (alkenes or aromatics etc.) = High Rf

Polar (ketones, esters, alkyl halides, aldehydes, ethers, etc,)

Highly polar (alcohols, amines, carboxylic acidss)= Low Rf

“Polar is Lower and Slower”

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

-Non polar elutes first!

- 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

-Cation exchange (+) is retained

- 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)

-Really good/effective!

-Separation based on polarity

- Stationary phase: nonpolar

- Mobile phase: polar (THIS IS REVERSE TLC)

- More polar = faster elution

Regular:

-Stationary phase is polar so most non polar elutes first

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, quicker to elute

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

-Lock/key interactions

Stationary phase: Small particles of resin linked to antibody linked to elute target protein

-Add competitive antibody binding chromatography

Gas Chromatography

- Separates based on volatility (boiling points) of compounds

-Volatility: How easy to convert to a gas

-More volatile (lower BP)

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

- Higher boiling point = longer retention time

-Low BP elute first more volatile!

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

- Significantly different boiling points need to be separated > 30 degrees C difference

Only constitutional isomers can be distinguished

Fractional distillation

- Really good/effective!

- Used for mixtures with components of very similar boiling points

- Diastereoemers can be separated!

- 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

- Conjugated pi systems

C=O vs C=C

Liquid-liquid extraction

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

2. Extraction of carboxylic acids

- Use NaHCO3 (weak base) to deprotonate carboxylic acid (strong 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 (strong base) to deprotonate the phenol (weak acid)

- This will enter the aqueous layer

4. Extraction of amines

- Use 10% HCl (strong acid) to protonate amines (base)

- This will enter the aqueous layer

IR spectroscopy

- Used to detect functional groups

Wavenumber

- The reciprocal of wavelength

- Directly proportional to both frequency and energy

IR stretching frequency of carbonyls C=O

~1700 cm

IR stretching frequency of alkenes C=C

~1650 cm

IR stretching frequency of triple bonded carbon and cyano groups C=-N/C=-C triple bonds

2260 cm - 2100 cm

• Primary Amines (R-NH\({}_{2}\)): Show two distinct N-H stretching bands (symmetric and asymmetric).

• Secondary Amines (R\({}_{2}\)NH): Show one sharp, weaker N-H stretching band.

• Tertiary Amines (R\({}_{3}\)N): Show no N-H stretching bands because they have no N-H bonds. [1, 2, 3]

IR stretching frequency of alcohols

3600 cm - 3200 cm

- Very broad

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 (n + 1 rule) neighbors=n

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

Shifts:

-H>-CH>-CH2>-CH3. Carboxylic acids>aldehydes way greater than -H