Lab Techniques- Chromatography

What are the basic features present within all types of chomatography?

1. All are use to separate mixtures of compounds
2. Involves a mobile phase and a stationary phase

There are 7 chromatography techniques that are on the MCAT

1. Thin layer (TLC)
2. Column/Flash
3. Ion Exchange
4. High Performance Liquid (HPLC)
5. Size Exclusion
6. Affinity
7. Gas

Analyte

the substance of interest that will be separated by chromatography

Eluent

a solvent that carries the analyte

What is TLC typically used for?

Used for separating compounds based on differing polarities

Why is TLC frequently used in OChem labs?

Fast separation

Used for separating very small amounts of material

What is the mobile and stationary phase of TLC?

Mobile: liquid (compound)

Stationary: absorbent- typically silica

How does TLC work?

\-silica is coated on a glass plate and the plate is placed upright in a sealed container with a shallow layer of solvent

\-sample is spotted near the base of the plate (1cm from the bottom)

\-capillary action draws the solvent up the plate

* more polar molecules travel slower than less polar

\-distance of molecules traveled is measured through Rf values (original spot location/distance traveled by individual component)

What is Column/Flash Chromatography used for?

Used to isolate bulk compounds

How does Flash/Column work?

A column is filled with a silica gel and is saturated with chosen organic solvents and mixture of compounds at the top of the column

Gravity brings the mixture down, more polar molecules travel slower than less polar

Compounds expected to leave in order of least polar to most polar

What are the mobile and stationary phases of Flash?

Stationary: silica beads (polar)

Mobile: organic solvent/compound mixture

What is Ion Exchange used for?

separation of materials based on varying charge states, typically in the separation of protein mixtures (cause proteins can either be protonated or deprotonated at specific pH)

2 types of Ion Exchange

Cation Exchange: uses negatively charged groups to attract positive charges

Anion Exchange: uses positively charged groups to attract negative charges

What is the mobile and stationary phase of Ion Exchange?

Stationary: solid polymeric resin with either positive or negatively charged moieties

Mobile: liquid solvent/mixture

How does Ion Exchange work?

Solvent is fed through stationary phase

Neutral charged groups and groups with the SAME charge as the resin pass through freely

Groups with charges opposite of the moieties in resin move slower

What is High Performance Liquid Chromatography (HPLC) used for?

Separating bulk compounds

Why is HPLC an improvement over Flash?

HPLC forces mobile phase under high pressures, which increases speed and efficiency of separation

Why is HPLC also called reverse phase HPLC?

HPLC contains a nonpolar stationary phase, which means polar molecules elute first instead of last

How does HPLC work?

Under high pressures, mobile phase is separated by different affinities of various compounds

Sample is injected and separates within an HPLC column based on affinities and is detected through a data processing program and a detector attached to the apparatus once eluent exits the column

eluent is collected after detected and components can be isolated if desired

more polar molecules elute first compared to lesser polar/non-polar molecules

Size Chromatography is used for what?

To separated bulk materials based on molecular size

How does Size Exclusion work?

A stationary phase employs chemically inert porous polymer of beads that allows for permeation of small molecules

Beads are packed within a column and solvent is added to the column

Larger molecules elute first because they pass through between beads

Smaller molecules elute last because they travel through the porous beads, thus slowing them down

What is Affinity Chromatography used for?

To purify proteins or nucleic acids from biochemical mixtures (i.e. growth media, blood, cell lysates)

Works on highly specific interactions between macromolecules (antibody, affinity tags, or magnetic bead interactions)

How does Affinity Chromatography work?

With Antibody interactions:


1. Sample is mixed with antibodies that are specific to be against the protein of interest
2. Once antibodies bind to the protein of interest, protein beads are added that are specific to the antibody to create a bead,antibody,protein complex
3. Separation is achieved through centrifuging and the supernatant is removed to isolate the complex

With magnetic beads


1. magnetic beads can bind to target protein
2. magnet is used to collect bead and target protein complex (doesn’t need a centrifuge)

Affinity tags can be used instead of antibodies (affinity tags are created through recombinant technology- popular tag is His a.a tag)

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What affects GC?

Intramolecular hydrogen bonding and hydrocarbon length