Biochem Analytical Techniques

Question 1

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

Answer 1

prepaative purifications

Question 2

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

Answer 2

analytical purifification

Question 3

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

Answer 3

protein purification

Question 4

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

Answer 4

cellular membranes

Question 5

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

Answer 5

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

Question 6

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

Answer 6

protease inhibitiors

Question 7

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

Answer 7

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

Question 8

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

Answer 8

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

Question 9

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

Answer 9

salting in

Question 10

compare and contract salting in vs salting out

Answer 10

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”.

Question 11

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

Answer 11

chrmatography

Question 12

the phase that doesn’t move in chromatography

Answer 12

stationary phase

Question 13

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.

Answer 13

mobile phase

Question 14

What should you remember when confronted with unfamiliar chromatography problems?

Answer 14

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

Question 15

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

Answer 15

paper chromatography

Question 16

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.

Answer 16

thin layer chromatography (TLC)

Question 17

TLC or paper chromatography?

Which is faster, more precise, and more versatile?

Answer 17

TLC

Question 18

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

Answer 18

retention factor (R_f)

Question 19

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

Answer 19

retention factor (R_f)

Question 20

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

Answer 20

True

Question 21

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

Answer 21

polar, nonpolar

Question 22

Describe the advantages and disadvantages of TLC

Answer 22

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

Question 23

This image describes what type of chromatography?

Answer 23

column chromatography

Question 24

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

Answer 24

First, allowing it to be isolated

Question 25

Name one benefit of column chromatography over TLC

Answer 25

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

Question 26

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

Answer 26

High-performance liquid chromatography (HPLC) ## Footnote 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.

Question 27

what HPLC has *longer* retention times for *more polar* compounds, and has _nonpolar compounds_ elute _more rapidly_? and why?

Answer 27

normal-phase HPLC | Normal-phase HPLC has a nonpolar mobile phase & a polar stationary phase ## Footnote 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

Question 28

what HPLC has *polar* molecules *elute first*, and why?

Answer 28

reverse-phase HPLC | This HPLC has a **polar mobile phase **and a **nonpolar stationary phase ## Footnote 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

Question 29

Name the advantages and disadvantages of HPLC over TLC

Answer 29

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

Question 30

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

Answer 30

gas-liquid chromatography | aka gas chromatography

Question 31

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

Answer 31

gas chromatography

Question 32

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.

Answer 32

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.

Question 33

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

Answer 33

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 temp**ratures * conditions must be carefully controlled

Question 34

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

Answer 34

*larger* molecules will elute *more quickly* | because small particles get stuck in pores of the beads ## Footnote the column retains small particles that interact with the stationary phase (beads)

Question 35

# True or False size-column chromotagraphy is fast, and can seperate compounds of similar size

Answer 35

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

Question 36

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

Answer 36

ion-change chromatography

Question 37

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

Answer 37

anion-exchange chromatography | name referes to the type of ions the column is supposed to attract

Question 38

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

Answer 38

cation-exchange chromatography | name referes to the type of ions the column is supposed to attract

Question 39

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

Answer 39

electrophresis

Question 40

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.

Answer 40

gel electrophoresis

Question 41

# 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

Answer 41

True

Question 42

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

Answer 42

* 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)

Question 43

type of electrophoresis that seperates proteins according to size

Answer 43

SDS-PAGE | sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Question 44

the pH at which a protein has a net neutral charge

Answer 44

isoelectric point (pI)

Question 45

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

Answer 45

isoelectric focusing

Question 46

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

Answer 46

two-dimensional gel electrophoresis

Question 47

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

Answer 47

spectroscopy

Question 48

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**

Answer 48

Beer-Lambert Law

Question 49

Name immunochemistry techniques

Answer 49

* ELISA * Western blotting * radioimmunoassay

Question 50

# 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.

Answer 50

immunochemistry techniques | ELISA, western blotting, ELISA, etc

Question 51

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.

Answer 51

Western blotting | usually after electrophoresis

Question 52

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

Answer 52

radioimmunoassay

Question 53

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.

Answer 53

enzume-linked immunoabsorbent assay (ELISA)

Question 54

purification technique used to seperate molecules based on boiling point

Answer 54

distillation