46- insulin ii general chromatography Flashcards
how does Zn+2 promote crystallization
- Insulin forms hexamer crystals in presence of Zn+2
- six units of insulin
- Partly purified insulin is dissolved and crystalized by adding Zn+2
- Crystals of insulin
- Crystals are then recrystallized
- Much higher purity
- very effective technique that requires you starting with a pretty pure substance
- Main impurity still proinsulin (lower amounts)
- Much lower rates of allergy
- Longer onset for intolerance of species-derived insulin
outline the development of chromatography in the late 1950s
- Ion exchange
- Separates proteins with different ionization
- Gel filtration (size exclusion)
- Separates proteins based on size
- sort them into small medium large to clean up proteins
- HPLC methods made biggest impact
- high pressure liquid chromatography
how does extraction work
look at diagram
how does purity depend on solubility properties
- different solvents that are imiscble with each other → one water and one organic
- cannot use the water/organic technique for biological drugs becayse they will become denatured
- look at notes
how does chromatography partition molecules between a solvent and a solid
notes <3
explain how the liquid in chromatography moves through small particles of solid
look at notes.
explain how the moving solutions are always in equilibrium
- always an equilibrium between the drug in the liquid and the drug stuck to the solid
- equilibrium has to reestablish when the liquid moves down
- as you move down, the mixture gets separated
- if you have a material that is in the liquid, then it will move very quickly down the column as the liquid goes through it
- if you have a material that gets stuck to the side, then it will move very slowly down the column as the liquid goes through it
how does the column function like a series of repeated extractions
- Molecules continually exchange between solution and adsorption as liquid moves through the column
- Different molecules exchange at different rates
- Different equilibrium for each molecule type
- using this info we know how to separate the components in our mixture
- Controls how quickly molecules move through the column
what does low adsorption mean for speed. what does high adsorption mean for speed
- Low adsorption = fast passage
- High adsorption = slow passage
how does the column operate
- Mixture (in solution) is loaded at the top of the column
- Max concentration
- Solvent is added at top and removed from bottom
- Continuous flow of solvent through column
- Mixture separates as it moves through column
- Collect different molecules as they exit
how can you isolate each component in the mixture
look at the picture
how do columns work in a laboratory columns
- Collect a set of fractions of approximately the same volume
- set the container under the column for a certain amount of time
- in this way we collect an equal volume of material coming off the column
- Test each fraction to find the component you are looking for
- look at the picture i guess
how do columns work in a production setting
- Use detector at the column outflow
- dectector will change the collection vessel every time something new comes out
- Change fractions when detect molecules exiting
- look at the picture i guess
