Biochem Analytical Techniques Flashcards
Purifcation technique that intends to produce a significant quantity of purified proteins for subsequent use
prepaative purifications
Purifcation technique that produces a smaller amount of a protein intended for analytical purposes i.e identification, quantification, and functional studies
analytical purifification
series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.
protein purification
to extract proteins from a sample, ____ must be lysed (if the proteins are not secreted by the cells in the surroundign slution)
cellular membranes
name at least three ways in which cellular membranes can be lysed for protein extraction
freezing and thawing, mechanical agitation, homogenization, treatment with organic solvents, detergent that disrupts integrity of membrane
what is used to prevent proteases and other enzymes from breaking down proteins during the lysing process?
protease inhibitiors
why are protease inhibitors needed during the lysing process of protein purification?
Because proteases (and other enzymes) that are usually contained in lysosomes and other compartments are let loose when the cell membrane is broken, and these enzymes can wreak havoc on the proteins that scientist want to analyze
a technique that helps to separate mixtures in solution by mass and density by applying centrifugal force
centrifugation
in protein purification (after protease inhibition) centrifugation seperates proteins from denser organelles fragments, and other cell debris. However, this is not a very detailed/selective process
the effect where increasing the ionic strength of a solution increases the solubility of a solute, such as a protein
salting in
compare and contract salting in vs salting out
The solubility of proteins usually increases slightly in the presence of salt, referred to as “salting in”. However, at high concentrations of salt, the solubility of the proteins drop sharply and proteins can precipitate out, referred to as “salting out”.
a set of techniques used to separate mixtures by passing them through a medium in which different components travel at different speeds.
chrmatography
the phase that doesn’t move in chromatography
stationary phase
the phase in chromatography that does move. It moves through the stationary phase picking up the compounds to be tested. As this phase continues to travel through the stationary phase it takes the compounds with it.
mobile phase
What should you remember when confronted with unfamiliar chromatography problems?
Identify the stationary phase and figure out which type of molecule (nonpolar/polar, small/large, positive/negative, etc) will interact with it most strongly. that kind of molecule will elute last
chromatography in whichstationaryphase is a piece of filter paper and the mobile phase is a liquid solvent that carries the solutes in the sample up the filter paper via capillary action
paper chromatography
chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose.
thin layer chromatography (TLC)
TLC or paper chromatography?
Which is faster, more precise, and more versatile?
TLC
the distance a given solute has migrated up the stationary phase divided by the maxiumum distacne traveled by the mobile phase
retention factor (Rf)
how far a solute moves up the stationary phase can be approximated by which equation?
retention factor (Rf)
True or False
The retention factor (Rf) can range from zero to one, but no greater. So if an Rf value on the MCAT is greater than 1 you can immediately strike it from your answer choices
True
Lower Rf values are assocated with ____ compounds, whereas higher Rf values are associated with ____ compounds
polar, nonpolar
Describe the advantages and disadvantages of TLC
Pros:
* More precise, fast, and versatile than paper chomatography
* non-destructive
* components are easily recovered by removing sections of gel & solubilizing the component followed by filtration
Cons:
* only an analytical filtration
* fairly imprecise
* generally slow technique
* requires a lot of solvent
* compounds with similar polarities cannot be seperated
This image describes what type of chromatography?
column chromatography
in column chromatography, the substance which interacts the least with the stationary phase elutes ____ (first or last)
First, allowing it to be isolated
Name one benefit of column chromatography over TLC
Packing the column with stationary phase allows more solute interactions, which improves seperations. Also, lengthening the column can allow for clearer seperations
What is the most selective type of column chromatography, and why?
High-performance liquid chromatography (HPLC)
HPLC works like regular column chromatography, except the mobile phase is passed through an absorbent at bery high pressures. The high pressures allow it to take place more quickly while still yielding very good resolution.
what HPLC has longer retention times for more polar compounds, and has nonpolar compounds elute more rapidly? and why?
normal-phase HPLC
Normal-phase HPLC has a nonpolar mobile phase & a polar stationary phase
Thus, polar compunds have longer retention times bc a.) polar compounds will interact more intensely with the polar stationary phase and b.) they’re not optimally soluble i the nonpolar solvent compromising the mobile phase
what HPLC has polar molecules elute first, and why?
reverse-phase HPLC
This HPLC has a **polar mobile phase **and a **nonpolar stationary phase
Thus, nonpolar compunds have longer retention times bc a.) nonpolar compounds will interact more intensely with the nonpolar stationary phase and b.) they’re not optimally soluble in the polar solvent compromising the mobile phase
Name the advantages and disadvantages of HPLC over TLC
Pros:
* rapid due to high pressures involved
* more surface interactions –> better separations bc of smaller particle size for packing material
* less solvent is needed
* non-destructive
Cons
* conditions must be carefully controlled
* detection techniques may need to be varied with each compound based on its sensitivity
chromatography involving injecting a gaseous sample into a mobile phase, typically called the carrier gas, and passing the gas through a liquid stationary phase
gas-liquid chromatography
aka gas chromatography
which seperation technique is ideal for analyzing volatile compounds with high boiling points?
gas chromatography
in gas chromatography, ____ (low/high) boiling points with vaporize ____(first/last) and elute more ____ (slowly/quickly) through the column. ____ (low/high) boiling points also correlate with ____ (low/high) polarity, so if the stationary phase is polar, then a nonpolar compound with a ____(low/high) boiling point will elute even more rapidly.
in gas chromatography, low boiling points with vaporize first and elute more quickly through the column. Low boiling points also correlate with **low **polarity, so if the stationary phase is polar, then a nonpolar compound with a low boiling point will elute even more rapidly.
Describe the pros and cons of** gas liquid chromatography**
Pros:
* fast
* can effect many seperations bc of small particle sizes in stationary phase
* temperature conditions can be changed and controlled over a wide range
Cons:
* if boiling point of a substance is higher than the temp of the column, the substance can condense on the stationary phase
* substances can dissolve in the liquid
* substances can remain in gas phase
* substances can be destroyed at higher tempratures
* conditions must be carefully controlled
in size-exclusion chromatography, ____ (larger/smaller) molecules will elute more quickly
larger molecules will elute more quickly
because small particles get stuck in pores of the beads
the column retains small particles that interact with the stationary phase (beads)
True or False
size-column chromotagraphy is fast, and can seperate compounds of similar size
False, it is only effective (and fast) when it is seperating compounds of different sizes
chromatography used to separate charged biological molecules such as proteins, peptides, amino acids, or nucleotides.
ion-change chromatography
uses a positively charged ion exchange resin with an affinity for molecules having negative charges
anion-exchange chromatography
name referes to the type of ions the column is supposed to attract
uses a negatively charged ion exchange resin with an affinity for molecules having positive charges
cation-exchange chromatography
name referes to the type of ions the column is supposed to attract
technique used to separate DNA, RNA or protein molecules based on their size and electrical charge. An electric current is used to move the molecules through a gel or other matrix
electrophresis
technique used to separate DNA, RNA or protein molecules based on their size and electrical charge. The molecules to be separated are pushed by an electrical field through a gel that contains small pores.
gel electrophoresis
True or False
gel electrophoresis is an electrolytic cell, in which the current is applied to drive an otherwise nonspontanteous source. Thus, the anode is negative and the cathode is positive
True
How can we seperate proteins according to size alone in electrophoresis?
- neutralize the charge across all proteins in a sample
- applying an anionic detergent to the sample to apply a uniform negative charge to all sample proteins (i.e SDS)
type of electrophoresis that seperates proteins according to size
SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
the pH at which a protein has a net neutral charge
isoelectric point (pI)
the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI value; allowing the seperation of proteins with different charge states based on the relative number of acidic and basic residues
isoelectric focusing
proteins are first separated by their pI value and then further separated by molecular weight through SDS-PAGE.
two-dimensional gel electrophoresis
used to quantify the amount of protein in a sample based on its absorption of light at a chaacteristic wavelength
spectroscopy
states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance
used in spectroscopy
Beer-Lambert Law
Name immunochemistry techniques
- ELISA
- Western blotting
- radioimmunoassay
Name the (general) technique
based on the selective, reversible and non-covalent binding of antigens by antibodies. These methods are employed to detect or quantify either antigens or antibodies.
immunochemistry techniques
ELISA, western blotting, ELISA, etc
used to detect a specific protein in a blood or tissue sample. The method involves using gel electrophoresis to separate the sample’s proteins. The separated proteins are transferred out of the gel to the surface of a membrane.
Western blotting
usually after electrophoresis
immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. Used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies
radioimmunoassay
assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Relies on antibodies to detect a target antigen using highly specific antibody-antigen interactions.
enzume-linked immunoabsorbent assay (ELISA)
purification technique used to seperate molecules based on boiling point
distillation
