Exercise 7 - separating organic compounds

column chromatography uses:

- consists of a chromatography column, matrix and buffer. - separates molecules based on size and shape - large, rod-shaped molecules move through the column fastest.

Paper chromatography uses:

- consists of a mobile phase and a stationary phase - separates molecules based on solubility and polarity. - Highly soluble, non-polar molecules will move with the solvent more and stick to the paper less causing them to move farther up the paper. - Rf = distance moved by sample/distance from origin to solvent front.

Gel Electrophoresis uses:

- consists of an agarose gel, buffer and electrical power supply. - separates molecules based on charge, shape and size. - positively charged molecules move toward cathode (-) - negatively charged molecules move toward anode (+) - Small, sperical molecules move through gel faster.

In column chromatography, molecules are separated based on which of the following? a. shape and size b. solubility and polarity c. charge and size d. shape and shape

A. Shape and size Small molecules get slowed down in the mazes of the beads while the large molecules move quickly around the beads. Large molecules will thus move through faster.

In paper chromatography, which molecules will be more attracted to the paper? a. polar b. non-polar c. large d. small

A. polar the polarity causes molecules to be more attracted to the paper.

In gel electrophoresis, the buffer does which of the following? a. regulates pH and conducts electricity b. regulates pH and keeps the matrix from drying out c. moves up the stationary phase, taking soluble molecules with it. d. all of the above

A. regulates pH and conducts electricity Extremes in pH can change the shape of molecules, thus affecting the separation results. A buffer helps prevent this from happening. The buffer also conducts electricity.

How can we determine the contents of an unknown solution using paper chromatography? a. run the paper chromatography using several known solutions and your unknown solution. Compare the resulting Rf values. b. run the paper chromatography using only your unknown and analyze the color spectrum. c. run the paper chromatography using only your unknown and observe whether it moved to the positive or negative electrode. d. none of the above.

A. run the paper chromatography using several known solutions and your unknown solution. Compare the resulting Rf values. Each molecule has a distinctive Rf. By comparing the Rf of your unknown to the Rf of several known solutions, you can determine its identity.

If you were to use column chromatpgraphy to separate a solution containing starch (large) and glucose (small), which molecule would move through the column faster? a. starch because it is larger b. glucose because it is smaller c. starch because it is polar d. glucose because it is polar

A. starch because it is larger in column chromatography, polarity does not influence the rate significantly. The larger molecules move faster through the matrix by going around the beads.

In paper chromatography, what can we predict would happen to the Rf if we allowed the procedure to run twice as long? a. we would have a higher Rf value b. we would have a lower Rf value c. There would be no difference in the Rf value

A. we would have a higher Rf value as the molecules are given more time to move up the paper, the Rf value increases.

In gel electrophoresis, what can we predict would happen if we decrease the voltage? a. the molecules would take less time to separate b. the molecules would take more time to separate c. there would be no difference in the rate of separation

B. The molecules would take more time to separate the higher the voltage is, the faster the molecules travel through the gel. Thus, when the voltage is decreased, the molecules will take longer to reach a clear separation.

If you had a solution that contained starch (large) and glucose (small), which procedure done in class would be the best way to separate them into solutions? a. paper chromatography b. column chromatography c. gel electrophoresis d. non of the procedures would work

B. column chromatography separates molecules based on size and shape into solutions.

When separating DNA fragments, they all move toward the anode, what is the charge of DNA? a. positive b. negative c. some segments are positive, some are negative d. DNA is not charged

B. negative molecules that a negative charge are attracted to the anode (positive electrode)

In column chromatography, the buffer does which of the following? a. regulates pH and conducts electricity b. regulates pH and keeps the matrix from drying out c. moves up the stationary phase, taking soluble molecules with it. d. all of the above

B. regulates pH and keeps the matrix from drying out extremes in pH can change the shape of molecules, thus affecting the separation results. A buffer helps prevent this from happening. A buffer also helps prevent the matrix from drying out (which can keep it from working properly).

In paper chromatography, molecules are separated based on: a. shape and size b. solubility and polarity c. charge and size d. shape and shape

B. solubility and polarity non-polar molecules will move with solvent, farther up the paper whereas insoluble, polar molecules will be more attracted to the paper and move less.

When separating organic compounds using paper chromatography, which molecules move the farthest up the paper? a. soluble polar molecules b. soluble non-polar molecules c. insoluble polar molecules d. insoluble non-polar molecules

B. soluble, non-polar molecules polar molecules stick to the paper more and non-polar, soluble molecules move with the solvent more.

If you were to use gel electrphoresis to separate molecules of similar charge and shape but different molecular weights, which one will move through the gel more quickly? a. the larger molecule b. the smaller molecule

B. the smaller molecule the gel slows larger molecules down.

In column chromatography, what can we predict would happen if we used a shorter column? a. there would be a greater distance of separation between molecules. b. there would be a smaller distance of separation between molecules. c. there would be no difference in the separation of molecule.

B. there would be a smaller distance of separation between molecules. the molecules would not have as much time to separate with a shorter column

In gel electrophoresis, molecules are separated based on which of the following? a. shape and size b. solubility and polarity c. charge and size d. shape and shape

C. Charge and size A current through the gel causes a separation of positive and negative molecules. Large molecules move more slowly through the gel so they will also be separated based on size.

Which of the following procedures would be best for sequencing DNA? a. paper chromatography b. column chromatography c. gel electrophoresis d. non of the procedures would work

C. Gel electrophoresis separates molecules by size and gives a visual representation. It can thus show the correct order of bases for a spliced DNA molecule.

In paper chromatography, the mobile phase: a. regulates pH and conducts electricity b. regulates pH and keeps the matrix from drying out c. moves up the stationary phase, taking soluble molecules with it. d. all of the above

C. moves up the stationary phase, taking soluble molecules with it. Molecules that are highly soluble in the mobile phase will move farthest up the paper.

column chromatography

Molecules can move through through the column of beads in two ways. Fast route between the beads, or slow route through the tiny pores of the beads. Big molecules too big to fit in beads pores move through the column quickly, whereas smaller molecules enter the beads' pores and move through the column more slowly.

Power supply (gel electrophoresis)

Provides a direct current across the gel. Negatively charged molecules move through the gel toward the positive electrode (anode). Possitively charged molecules move through the gel toward the negative electrode (cathode). The greater the voltage, the faster the molecules move.

stationary phase

The part of a chromatography experiment that does not move during the experiment, either paper or gel.

Agarose gel

a jelly-like slab used to separate molecules on the basis of molecular weight preferred gel for DNA electrophoresis

buffer (gel electrophoresis)

conducts electricity and helps control the pH. The pH affects the stability and charge of the samples

Rf

distance moved by sample ------------------------------------------------- (divided by) distance from origin front

Buffer (column chromatography)

helps control the pH of the sample and helps carry the sample though the matrix which separates the chemicals mixed in the sample.

mobile phase

is the solvent that moves along the paper (solubility: polarity)

Matrix

material in the column that fractionates or separates the chemicals mixed in the sample.

paper chromatography

method of separating a mixture of different colors. The liquid soaks through the paper and carries the mixture with it. Some substances are carried faster than others so the substances are separated along the paper

Gel electrophoresis

separates molecules according to their charge, shape and size

column chromatography

separates molecules according to their size and shape.

Column

tube having a frit and a spout at its bottom. the frit is a membrane or or porous disk that supports and keeps the matrix in the column but allows water and solutes to pass.