Separation Technique Sorts by: Notes

Thin Layer Polarity Substances travel via capillary action up a ( typically

Chromatography polar Silica) stationary phase. Non-polar solutes will
travel further:

[Rf = substance’s distance/ solvent’s distance]

Result: Nonpolar substances elute first.

Column/ Flash layer Polarity Much like TLC, except substances are added from the
Chromatography top of a column and are driven by gravity instead of
capillary action

Result: Nonpolar substances elute first.

Ion exchange Charge Substance is fed through a container containing a

Chromatography charged resin. ( either + or - ). Components of a like
charge will be repelled by the resin and exit ( elute) first.
Neutrally charged components will elute second, and
differently charged components will elute last.

Result: Liked charged elements elute first.

High Performance Affinity for either mobile phase is forced at high pressure through the
Chromatography stationary/mobile phase stationary phase increasing speed and efficiency of
separation.

Result: substances with the least affinity elute first

Size Exclusion Size Mobile phase is dropped through a column containing

Chromatography porous beads. Elements of the mobile phase that are
small enough to fit through these pores, will travel
convoluted, slow-going routes through the beads’
pores, whereas larger elements will bypass the beads,
and travel around them.

Result: Larger elements will elute out the bottom first.

Affinity Chromatography Affinity Commonly used to purify nucleic acids

Makes use of specific binding interactions between
macromolecules often with the aid of antibodies or
affinity tags and magnets.

1. Cell lysates are collected

2. Antibody for protein of interest is added

3. Beads are added that bind to antibodies

4. Beads ( and complexes) are collected via centrifuge

Result: elements that bind to tags/beads can be

collected via centrifuge

Gas Chromatography Volatility Mobile gas phase is sent through a stationary liquid
phase. The sample is injected and vaporized multiple
times. As the sample vaporizes and condenses, the
differences in boiling point will cause different elements
to take different amounts of energy vaporize.

Result: Components with the lowest vaporization point/

highest volatility will elute first.
Separation Technique Sorts by: Notes

Gel Electrophoresis Charges at different pH Used to separate different macromolecules based on

the charges they gain at different pH. (On the mcat, it
will likely be in a passage related to Amino Acids. ).

Sample is injected into a gel with positive and negative

electrodes at either end of the tray. Positively charged
molecules will travel towards the negatively charged
terminal and vice versa.

Charge and pH:

Recall: Acidic and Basic amino acids
have R chains that will either gain or lose
protons based on the pH of the
environment. The Amino/Carboxyl
groups present on all amino acids will do
the same.

Generally speaking, as the environment

gets more and more acidic ( more willing
to donate protons), molecules will pick up these protons and gain a positive charge. In the
same token, as the environment gets more basic ( more willing to accept protons), the
molecules will give up their protons, and gain a negative charge.

pH vs pI Charge Movement Notes

pH> pI Negative Towards the + terminal The environment is

basic, meaning it wants
to take protons from
the substituent, thereby
deprotonating it and
giving it a negative
charge

pH< pI Positive Towards the - terminal The environment is

acidic, meaning it is
eager to give protons
to the substituent,
thereby giving it a
positive charge

pH=pI Neutral No movement The environment and

molecule are at the
Isoelectric point (pI)
making the molecule
neutral. ( zwitterion)
*Only acidic/basic R groups will gain or lose change depending on pH. HOWEVER, amino
groups and Carboxyl groups can gain and lose charge and are present on every amino acid.

Method:

1. Average the pka’s of the substituents in question. This is your pI.

2. Apply the rules above to determine the charge.

Eg:

Glycine contains an amino group ( pka 2.34) , a carboxyl group ( pka 9.6) and a neutral R chain.
At physiological ph, what is glycine’s charge?

1. Average the Pka’s to give you a pI:

(2.34) + (9.6)/2 = 5.97

pI Glycine = 5.97

pH = 7.4

2. Apply the Ph vs PI rule:

Ph (7.4)> pI (5.97)

At physiological pH, Glycine is negatively charged.

Substituent Pka pI

Carboyl Group ( COOH) ~9 1.82-2.18

Amino Group (NH3) ~2 8.95-10.6

Apargic Acid’s R group ~4 8.9

Glutamic Acid’s R group ~4 4.3

Histidine’s R group ~6 6

Lysine’s R group ~10 10.8

Arganine’s R group ~12 12.48

a High Pka value indicates a weak acid. A low pka indicates a strong acid.