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My MCAT > Biochem Analytical Techniques > Flashcards

Biochem Analytical Techniques Flashcards

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MCAT flashcards
1
Q

Purifcation technique that intends to produce a significant quantity of purified proteins for subsequent use

A

prepaative purifications

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2
Q

Purifcation technique that produces a smaller amount of a protein intended for analytical purposes i.e identification, quantification, and functional studies

A

analytical purifification

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3
Q

series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.

A

protein purification

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4
Q

to extract proteins from a sample, ____ must be lysed (if the proteins are not secreted by the cells in the surroundign slution)

A

cellular membranes

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5
Q

name at least three ways in which cellular membranes can be lysed for protein extraction

A

freezing and thawing, mechanical agitation, homogenization, treatment with organic solvents, detergent that disrupts integrity of membrane

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6
Q

what is used to prevent proteases and other enzymes from breaking down proteins during the lysing process?

A

protease inhibitiors

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7
Q

why are protease inhibitors needed during the lysing process of protein purification?

A

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

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8
Q

a technique that helps to separate mixtures in solution by mass and density by applying centrifugal force

A

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

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9
Q

the effect where increasing the ionic strength of a solution increases the solubility of a solute, such as a protein

A

salting in

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10
Q

compare and contract salting in vs salting out

A

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”.

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11
Q

a set of techniques used to separate mixtures by passing them through a medium in which different components travel at different speeds.

A

chrmatography

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12
Q

the phase that doesn’t move in chromatography

A

stationary phase

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13
Q

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.

A

mobile phase

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14
Q

What should you remember when confronted with unfamiliar chromatography problems?

A

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

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15
Q

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

A

paper chromatography

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16
Q

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.

A

thin layer chromatography (TLC)

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17
Q

TLC or paper chromatography?

Which is faster, more precise, and more versatile?

A

TLC

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18
Q

the distance a given solute has migrated up the stationary phase divided by the maxiumum distacne traveled by the mobile phase

A

retention factor (Rf)

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19
Q

how far a solute moves up the stationary phase can be approximated by which equation?

A

retention factor (Rf)

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20
Q

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

A

True

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21
Q

Lower Rf values are assocated with ____ compounds, whereas higher Rf values are associated with ____ compounds

A

polar, nonpolar

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22
Q

Describe the advantages and disadvantages of TLC

A

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

23
Q

This image describes what type of chromatography?

A

column chromatography

24
Q

in column chromatography, the substance which interacts the least with the stationary phase elutes ____ (first or last)

A

First, allowing it to be isolated

25
Q

Name one benefit of column chromatography over TLC

A

Packing the column with stationary phase allows more solute interactions, which improves seperations. Also, lengthening the column can allow for clearer seperations

26
Q

What is the most selective type of column chromatography, and why?

A

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.

27
Q

what HPLC has longer retention times for more polar compounds, and has nonpolar compounds elute more rapidly? and why?

A

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

28
Q

what HPLC has polar molecules elute first, and why?

A

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

29
Q

Name the advantages and disadvantages of HPLC over TLC

A

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

30
Q

chromatography involving injecting a gaseous sample into a mobile phase, typically called the carrier gas, and passing the gas through a liquid stationary phase

A

gas-liquid chromatography

aka gas chromatography

31
Q

which seperation technique is ideal for analyzing volatile compounds with high boiling points?

A

gas chromatography

32
Q

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.

A

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.

33
Q

Describe the pros and cons of** gas liquid chromatography**

A

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

34
Q

in size-exclusion chromatography, ____ (larger/smaller) molecules will elute more quickly

A

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)

35
Q

True or False

size-column chromotagraphy is fast, and can seperate compounds of similar size

A

False, it is only effective (and fast) when it is seperating compounds of different sizes

36
Q

chromatography used to separate charged biological molecules such as proteins, peptides, amino acids, or nucleotides.

A

ion-change chromatography

37
Q

uses a positively charged ion exchange resin with an affinity for molecules having negative charges

A

anion-exchange chromatography

name referes to the type of ions the column is supposed to attract

38
Q

uses a negatively charged ion exchange resin with an affinity for molecules having positive charges

A

cation-exchange chromatography

name referes to the type of ions the column is supposed to attract

39
Q

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

A

electrophresis

40
Q

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.

A

gel electrophoresis

41
Q

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

A

True

42
Q

How can we seperate proteins according to size alone in electrophoresis?

A
  • 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)
43
Q

type of electrophoresis that seperates proteins according to size

A

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

44
Q

the pH at which a protein has a net neutral charge

A

isoelectric point (pI)

45
Q

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

A

isoelectric focusing

46
Q

proteins are first separated by their pI value and then further separated by molecular weight through SDS-PAGE.

A

two-dimensional gel electrophoresis

47
Q

used to quantify the amount of protein in a sample based on its absorption of light at a chaacteristic wavelength

A

spectroscopy

48
Q

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

A

Beer-Lambert Law

49
Q

Name immunochemistry techniques

A
  • ELISA
  • Western blotting
  • radioimmunoassay
50
Q

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.

A

immunochemistry techniques

ELISA, western blotting, ELISA, etc

51
Q

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.

A

Western blotting

usually after electrophoresis

52
Q

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

A

radioimmunoassay

53
Q

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.

A

enzume-linked immunoabsorbent assay (ELISA)

54
Q

purification technique used to seperate molecules based on boiling point

A

distillation

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