Separations and Purifications

Question 1

Extractions

Answer 1

The transfer of a dissolved compound (the desired product) from a starting solvent into a solvent in which the product is more soluble.
-Concept that like dissolves like
-Two solvents are immiscible, they form two layers that do not mix

Question 2

Like dissolves like

Answer 2

-Polar dissolves polar
-Nonpolar dissolves nonpolar

Question 3

Immiscible

Answer 3

Two solvents that do not mix and form water and oil.

Question 4

Aqueous Phase (layer)

Answer 4

Water layer

Question 5

Organic Phase (layer)

Answer 5

The nonpolar layer

Question 6

Separatory Funnel

Answer 6

Used to separate organic and aqueous layer. Gravity causes denser layer to sink to the bottom of the funnel where it can then be removed by turning the stockpot. (more common for organic layer to be on top but opposite can occur)
-Position of the layers is determined by their relative densities.

Question 7

Multiple Extractions

Answer 7

More will make it more effective rather than a single time

Question 8

Using water to extract aqueous layer.

Answer 8

Solvents that can exhibit hydrogen bonding will be congregate in aqueous layer.

Question 9

Wash

Answer 9

Small amount of solute is used to extract and remove impurities, rather than the compound of interest.

Question 10

Filtration

Answer 10

Isolates a solid from a aliquid. In lab one pours liquid-solid mixture onto a paper filter that allows only the solvent to pass through, like a coffee filter.
-At the end one is left with solid called residue.
-And the flask full of liquid that passed through the filter known as filtrate.

Question 11

Gravity Filtration

Answer 11

In which solvents own weight pulls it though the filter is more commonly used when the product of interest is the filtrate. (the coffee)

Question 12

Vacuum Filtration

Answer 12

In which the solvent is forced through the filter by a vacuum connected to the flask, is more often used when the solid (residue) is the desired product. (The grounds)

Question 13

Recrystallization

Answer 13

Method for further purifying crystals in solution. In this process, we dissolve our product in a minimum amount of hot solvent and let it recrystallize as it cools. The solvent chosen for this process should be one in which the product is soluble only at high temperatures.
-Thus when solution cool, only the desired product will recrystallize out of solution, excluding the impurities.

Question 14

Distillation

Answer 14

Takes advantage of differences in boiling point to separate two liquids by evaporation and condensation.
-Liquid with lower boiling point will vaporize first. The vapors rise up the distillation column to condense in a water-cooled condenser. The condensate then drops down into the vessel. The end product is called distillate.
-Heating temp is kept low so that the liquid with the higher boiling point will not be able to boil and therefore remain a liquid in the initial container.

Question 15

Simple distillation

Answer 15

Technique should only be used to separate liquid below 150C and have at least 25C difference in boiling points. Normal equipment setup.

Question 16

Vacuum Distillation

Answer 16

Whenever we want to distill a liquid with a boiling point over 150C. By using vacuum we can lower ambient pressure thereby decreasing the temperature that a liquid much reach in order to have sufficient vapor pressure to boil.
-Allows to distill compounds with higher boiling points at lower temps so that we don’t have to worry degrading the product

Question 17

Fractional Distillation

Answer 17

Separate two liquids with similar boiling points (less than 25C apart), we use fractional distillation.
-Fractional column connects the distillation flask to the condenser. The fractional column is a column in which surface area is increased by the inclusion of inert objects like glass beads or steel wool. As the vapor rises up the column, it condenses on these surfaces and refluxes back down until rising heat causes it to evaporate again, only to condense at a higher column. Each time the condensate evaporates, the vapor consists for a higher proportion of the compound with the lower boiling point.

Question 18

Chromatography

Answer 18

In all concepts of chromatography the concept is identical.
The more similar a compound is to its surroundings, (whether by polarity, charge, or other characteristics), the more it will stick to and move slowly through its surroundings.

Question 19

Stationary Phase or absorbent

Answer 19

Placing sample on this phase.

Question 20

Mobile Phase

Answer 20

We run this phase, usually a liquid (or gas in Gas Chromatography) through the stationary phase.

Question 21

Process of chromatography

Answer 21

1). Place sample on Stationary Phase.
2) Run mobile phase, usually a liquid (or gas in gas chromatography)
3) This will displace (elute) the sample and carry it through the stationary phase.
4) Depending on characteristics of substances in the sample and the polarity of the mobile phase. it will adhere the stationary phase with different strengths, causing the different substances to migrate at different speeds.
5) Partitioning occurs and it represents an equilibrium between the two phases. Different compounds will have have different partitioning coefficients that will elute at different rates.

Question 22

Difference between Thin Layer Chromatography (TLC) and Paper chromatography.

Answer 22

Similar techniques, varying only in the medium used for the stationary phase.
-Thin layer: of silica gel or alumina adherent to an an inert carrier sheet.
-Paper chromatography: Medium used is paper which is composed of cellulose.
-BOTH OF WHICH ARE VERY POLAR.

Question 23

TLC and Paper Chromatography

Answer 23

1). For these techniques, the sample that we want to separate is placed directly onto the adsorbent itself; this is called SPOTTING because we apply a small, well defined spot on the sample directly onto silica or paper plate.
2) Plate is then developed which involves placing the adsorbent upright in a developing chamber, usually a beaker with a lid or a wide-mouthed jar. At the bottom is a shallow pool of solvent, called ELUENT, which usually a organic solvent of Weak to moderate polarity so it doesn’t bind to silica gel. Samples must be above level of solvent or else they will dissolve into the pool of solvent rather than running up the plate.
3) Solvent will creep up plate by capillary action carrying various compounds in the sample with varying rate. Nonpolar compounds will dissolve in the organic solvent and move quickly as the solvent moves up the plate.
4) When eluent reaches near the top the plate is taken out and dried.
5) Spots are usually white, so either UV light. Or iodine, phosphomolybdic acid or vanillin but this will destroy the compounds so they cannot be recovered.
6) Retardation factor (Rf) is calculated.
Rf=Distance spot moved/Distance solvent moved.

Question 24

Where do polar and non polar end up on TLC?

Answer 24

The MORE non polar the sample is, the further up the plate it will move.
-↑Nonpolar=↑Up plate=↑Rf
-↓Nonpolar=↓Up plate=↓Rf

Question 25

Reverse-Phase Chromatography

Answer 25

Technique is same, however the stationary phase used is nonpolar, and the mobile phase is polar.

Question 26

Where do polar and non polar end up on Reverse-phase chromatography?

Answer 26

The more polar the substance is the farther it will move up the plate.
-↑Polar=↑Up plate=↑Rf
-↓Polar=↓Up plate=↓Rf

Question 27

Retardation factor

Answer 27

This value is relatively constant and can be used to identify unknown compounds. Most frequently performed on a small scale to identify unknown compounds
-Less polar Eluent will less likely to displace polar compounds absorbed to silica gel. (Lower Rf)
-More polar eluent will more likely to displace polar compounds absorbed to silica gel. (Higher Rf)

Question 28

Preparative TLC

Answer 28

Larger scale technique. As the large plate develops the larger spot of sample splits into bands of individual compounds which can then be scraped off and washed to yield pure compounds.

Question 29

Column Chromatography

Answer 29

-Uses and entire column filled with entirely with polar silica or aluminum beads as absorbent, allowing for much greater separation.
-Uses gravity to move the solvent and compounds down the column.
-To speed up process one can force solvent through column using gas pressure called flash column chromatography.
-Solvent polarity can be changed to help elute desired compound.
-Eventually solvent drips out of column, and the different fractions that leave to column can be collected over time.
-After collection solvent can be evaporated leaving behind compounds of interest.
-Can be used to separate and collect macromolecules such as proteins and nucleic acids

Question 30

Ion-Exchange Chromatography

Answer 30

1) Beads in column are coated with charged substances so they attract or bind compounds that have an opposite charge.
2) Positively charged compound will attract and hold negatively charged backbone of DNA or protein as it passes through the column.
3) Salt gradient is used to elute the charged molecules that have stuck to the column.

Question 31

Size-Exclusion Chromatography

Answer 31

1) Beads used in column contain
tiny pores of various sizes.
2) Pores allow small compounds to enter the beads, thus slowing them down. Large compounds cannot fit into pores, so they will move around them and travel through column FASTER.
3).Size of the pores may be carried so that molecules with different molecular weights can be fractionated.
(Common approach in protein purification is to use ion exchange column followed by a size exclusion column.

Question 32

Affinity Chromatography

Answer 32

1) A Protein of interest are bound by creating a column with high affinity for that protein.
2) Coating beads with receptor that binds to that protein or a specific antibody for that protein, in either case the protein is retained in that column.
3) Common stationary phase molecules include nickel, which is used in separation of genetically engineered proteins with histidine tags, antibodies or antigens, and enzyme substrate analogs of interests.
4) Once protein is in column it can be eluted by washing column with a free receptor (or target or antibody), which will compete with the bead bound receptor and ultimately free the protein from the column. Elutants can also be created with varying pH or salinity level that disrupts bonds between the ligand and protein of interest
Drawback: of the elution step is that the recovered substance can be bound to the eluent .
For example: the eluent was an inhibitor of an enzyme, it could be difficult to remove.

Question 33

Gas Chromatography or Vapor pressure Chromatography

Answer 33

Main difference is the eluent is a gas, usually helium or nitrogen, instead of a liquid.
1) The adosrbent is crushed metal or polymer inside a 30 ft column
2) Column is coiled and kept inside oven control temp
3) Mixture is injected to colcumn and vaporized
4) compounds will travel through compounds at different rates because they adhere to the adsorbent in the column at different degrees and will separate in space by the time they reach the end of the column.
5) The injected compounds must be volatile: low melting point, sublimable solids or vaporizable liquids.
6) It is common to use GC to separate molecules using GC and then inject the pure molecules into a mass spectrometer for molecular weight determination.

Question 34

Mass spectroscopy

Answer 34

Involves ionization and fragmentation of compounds; these fragments are then run through a magnetic field, which separates them by mass-to-charge ratio. The total molecular weight can then be determined, or the relative concentrations of the different fragments can be calculated and compared against reference values to identify the compound.

Question 35

High Performance Liquid Chromatography

Answer 35

Eluent is liquid and travels through a column of a defined composition. There are a variety of stationary phases that can be chosen depending on target molecule and the quantity of the material that needs to be purified. Like GC but uses liquid under pressure instead of gas. Hence higher performance over column chromatography

Question 36

Extract acetaldehyde from aqueous solution?

Answer 36

Use ether to create organic layer, and acetaldehyde will remain in aqueous solution.
(PURPOSE IS TO CREATE LAYERS)

Question 37

Silica Gel

Answer 37

KNOW IT IS POLAR, REGARDLESS IF ETHER IS ON IT.