Chromatography Flashcards
name two different types of column chromatography techniques
affinity chromatography , ion-exchange chromatography and size exclusion chromatography are common types of chromatographic techniques
using one of the chromatographic techniques that you mentioned above , can you think of an example in which two molecules could be purified using liquid phase chromatography
examples of affinity chromatography would include the purification of an antibody over a solid support containing the antigen (antibody-antigen interactions would be a good background lecture topic) . Also , the purification of an enzyme over a solid support containing the substrate would be another example of affinity chromatography .
examples of ion-exchange chromatography would include the separation of two proteins with very different isoelectric points - for example at PH 7.2, lysozyme is (+) charged , whereas albumin is (-) charged
what type of chromatography is being used in this lab activity
size exclusion chromatography
briefly explain how the chromatography in this activity functions
molecules can be separated on the basis of size by passing a mobile phase of the mixture over a column of porous beads which act to filter a mixture . this filtering causes smaller molecules to pass through the column more slowly than large molecules . Thus , large molecules exit the column first and are present in early fractions , whereas smaller molecules exit the column later and are present in later fractions.
if the following mix of molecules was purified using size exclusion chromatography , what would be the order in which the molecules pass through the opening in the bottom of the column ? mixture contains : haemoglobin (65,000 daltons) , myoglobin (17,000 daltons) , myosin (180,000 daltons)
1st - myosin
2nd- haemoglobin
3rd- myoglobin
if a size exclusion chromatography column is said to have an exclusion limit of 40,000 daltons , would haemoglobin (60,000 daltons) be fractionated or excluded from the column
haemoglobin would be excluded from the column . It is larger than the pores of the beads ; thus it is excluded from the pores and would pass through the column quickly
would vitamin B12 (1,350 daltons) be fractionated or excluded from the column
vitamin B12 would be fractionated by the column - its molecular weight is much lower than the exclusion limit causing it to be filtered through the pores
what gives hemogloblin and myoglobin their distinctive reddish-brown colours
its the heme or the iron-containing group of both molecules that give them their distinctive red colour. Iron is naturally coloured red - think of iron pills or red iron containing soil
if athletes wanted to increase their endurance by increasing their oxygen-carrying capacity , how might they accomplish this increase
RBC’s , which contain haemoglobin are responsible for delivering oxygen to tissues. An athlete would increase oxygen capacity by increasing RBC number , thereby increasing the overall number of haemoglobin molecules in the body . Athletes accomplish this by training at high altitudes where the body responds to the decrease of oxygen in the air by producing more RBCS. additionally some athletes resort to blood-doping , which involves infusing RBCS directly into the blood stream. this raises the RBC concentration , raising the oxygen capacity . However , blood doping can be extremely dangerous since too high of a concentration of RBCS can thicken the blood resulting in strokes or heart attacks
would you expect a flightless bird such as an ostrich or turkey to have a preponderance of dark meat in its wings or legs
the preponderance of dark meat would be in the legs . active muscles require high myoglobin contents to supply oxygen to the working muscles.
would you expect a bird of flight to have a preponderance of dark meat in its wings or legs
the preponderance of dark meat would be in the wings. in this case the wings are the more active muscles and would require higher myoglobin contents
how could gene therapy where a normal gene is substituted for a defective copy of a gene help individuals inflicted with sickle-cell anaemia
gene therapy involves the placement of a normal gene into the body of an individual inflicted with a genetic disorder. Because patients with sickle-cell anaemia have a defective copy of the gene for haemoglobin , the substitution of the defective gene with a normal gene could , in theory cure the sickle cell individual .It should be mentioned that the normal gene is contained in the individual as a supplemental gene usually carried by a virus and does not physically replace the defective gene
think about the colour of the protein mix that was applied to the column - why was the mixture reddish-brown
the iron containing heme group of haemoglobin contributes the majority of red-brown colour to the protein mix .vitamin b12 is dark pink. the mixture of both produces a dark reddish-brown solution
why do you think the column needed to be dry when the protein mix was loaded
slight diffusion naturally occurs as the molecules pass through the sec column . for this reason , it is desirable to apply highly concentrated solutions of biomolecules to the column. Having the top of the column of the bed dry when the protein mix is loaded decreases the overall amount of diffusion that occurs during the purification process
why did u need to add more buffer after the protein mixture was loaded onto the column (step 7 and 8 of the protocol)
in order to keep the mixture of hemogloblin and vitamin b12 in the fluid phase , additional buffer needs to be added to the column after the protein mixture was applied. the buffer insures that the fluid phase will move through the column resulting in completion of the chromatographic separation procedure
