Gel Filtration Chromatography Flashcards
______________ is a very powerful technique for the physical separation of molecules on the basis of size.
Size exclusion chromatography
What is a molecular disease of hemoglobin?
Sickle cell anemia
Hemoglobin is made up of ______________ which is associated to form a large, globular protein.
4 polypeptides (small proteins)
Hemoglobin has a color _____ and has a molecular weight of ________.
brown; 65000 daltons
The mass of beads within the column is often referred to as the _________.
column bed
This kit contains 8 columns which are prefilled with beads that effectively ___________ molecules that are below 60,000 daltons.
separate (“fractionate”)
__________ allows the separation of individual components from complex mixtures.
Chromatography
What is used in the stationary phase for paper chromatography?
paper
What is the mobile phase?
the solvent and the molecules to be separated
In step 1 of the procedure, the last two tubes will be labeled as _________ and __________.
Waste; Column Buffer
The liquid used to dissolve the biomolecules to make a mobile phase is called a _____.
buffer
Hemoglobin has a higher affinity for _____ than oxygen.
carbon monoxide
What kind of chromatography separates molecules based on charge?
Ion-exchange chromatography
Vitamin B12 is _____ and has a molecular weight of _______.
pink; 1350 daltons
At high altitudes, where the amount of oxygen is decreased, the body responds by increasing the number of _______ produced.
red blood cells
Larger molecules are ____ from the beads.
“excluded”
How is chromatography commonly used in biotechnology?
* purifying biological molecules like proteins;
* for medicine;
In ____________, a biomolecule (often an antibody) will bind to the protein to be purified and is attached to the beads.
affinity chromatography
In simple terms, what does chromatography accomplish?
It allows the separation of individual components from complex mixtures.
In step 2, how many ml of column buffer should be pipetted into the tube labeled column buffer?
4 mL
In ion exchange chromatography, if the beads is positive, it will bind negatively charged ions. This technique is called _________.
anion exchange
In step 8, when all of the liquid has been drained from the column, what should be added to the top of the column?
3ml of column buffer
_______. a protein found in red blood cells, functions to transport ______ to the tissues of the body.
Hemoglobin; oxygen
________ and ___________ are the two biomolecules in your sample.
Hemoglobin; vitamin B12
The mixture of biomolecules dissolved in the buffer is called a
sample
Microscopic porous spheres
beads
What happens in Fraction 3?
The larger protein molecules emerge from the column first.
In affinity chromatography, the elution buffer often contains ________.
high concentrations of salt or acid
Sources rich in vitamin B12 include
eggs, dairy products and meats
When the contaminant is separated from the protein of interest, a ____________ is used to get the desired protein to elute from the column.
high salt buffer
In step 7, when all the liquid has been drained from the column, what should be added?
Another 250 ul of column buffer
The side effects of sickle cell anemia can be alleviated by ____________ from people who have normal hemoglobin and red blood cells.
frequent blood transfusions
The beads act as ______ or _____ and function to filter small molecules which become temporarily trapped within the pores.
“traps”; “sieves”
The hemoglobin used in this experiment is _______.
bovin hemoglobin
What is used in the stationary phase for column chromatography?
glass beads called resin
The larger molecules which pass quickly through the column will end up in the early tubes or _____.
“fractions”
_________ is a vitamin essential to humans and other vertebrates.
Vitamin B12
True or false? Vitamin B12 is not found in plants and vegetable foods.
True
Hemoglobin can bind to ______ and _______.
oxygen; carbon monoxide
Chromatography consists of a _____ phase and a ______ phase.
mobile; stationary
In step 4, why should the column not be jammed tightly into the collection tubes?
The column will not flow
Molecules travel through the stationary phase at different rates because of their ______.
chemistry
What is the main component of red blood cells?
Hemoglobin
Gel filtration chromatography is commonly referred to as…
size exclusion chromatography.
A mixture of large and small ____ is applied to a column of porous beads.
proteins
How does the Vitamin B12 complex pass through the intestine and into the blood stream?
It binds to the carrier protein
As the buffer flows down the column, the small _____ molecules penetrate into the ____ and are ____.
protein; beads; slowed
The ___________ emerge from the column first.
larger protein molecules
In ion exchange chromatography, when the contaminant is separated from the protein of interest, a _______ is used to get the desired protein to elute from the column.
high salt buffer
in Fraction 1, what is happening?
A mixture of large and small proteins are applied to a column of porous beads.
In step 6, after adding the protein mix, what should be added next to the top of the column?
250 ul of column buffer
One function of vitamin B12 is ______________.
the breakdown of fats
In step 9, when _____ have been collected into tube 2, transfer the column onto ____.
5 drops of buffer; tube 3
Molecules greater than 60,000 pass around the beads and are excluded from the column — also referred as to as the _________ of a column.
exclusion limit
Why are muscles that are very active and require a lot of oxygen dark in color?
High myoglobin content
What is responsible for the red-brown color of hemoglobin?
iron-containing heme group
As the liquid flows through the column, molecules below 60,000 daltons enter the beads and pass through the column more _____.
slowly
In step 5, what should be dropped onto the column bed?
one drop of protein mix
What kind of chromatography separates molecules based on size?
-Gel filtration/Size exclusion chromatography -Molecular sieve chromatography
In this lab, what will be investigated?
Size exclusion chromatography.
What happens in Fraction 2?
As the buffer flows down the column, the small protein molecules penetrate into the beads and are slowed.
People who have a strict vegetarian diets are often deficient in what?
Vitamin B12.
Once the fractions have been collected, they should be covered with ________.
parafilm
In __________, if the beads are negatively charged, they bind to positively charged molecules.
cation exchange
In ______________, microscopic beads which contain tiny holes are packed into a column.
gel filtration chromatography
Vitamin B12 are required in large quantities. True or false?
No, they are only required in minute quantities
In step 10, what should be done to the column when finishing collecting drops?
Cap it
In affinity chromatography, what is the buffer used for?
To disrupt the bond between the protein of interest and the antibody
In ion exchange chromatography, what is the purpose of the high salt buffer?
To get the desired protein to elute from the column
As the liquid flows down the column, what happens to the molecules below 60000 daltons?
Molecules enter the beads and pass through the column more slowly.
As the liquid flows down the column, what happens to the molecules above 60000 daltons?
Molecules pass around the beads are are excluded from the column.
negatively charged resin; binds to positive
Cation exchanger
positively charged resin; binds to negative
Anion exchanger
