chromatography

Question 1

what is chromatography

Answer 1

method of separation, components separated are distributed between 2 phases (stationary and mobile)

Question 2

general concepts- how are samples loaded

Answer 2

sample introduced in small volume (at inlet of column or another carrier of the stationary phase)

Question 3

general concepts- elution
-what is it

Answer 3

mobile phase (eluent) transports sample component in contact with stationary phase, due to different interactions between sample and stationary phase the sample migrates at different speeds and elute from the system at different retention times

Question 4

general concepts- detection
-how are compounds visualised

Answer 4

compounds visualised when they elute out of system, visualisation done by UV-vis, fluorescence, refractive index, evaporative light scattering detectors

Question 5

what causes different retention time

Answer 5

different interactions between sample components and stationary phase

Question 6

types of chromatography

Answer 6

TLC, size-exclusion, reverse-phase, ion exchange, gas chromatography

Question 7

what is TLC ,its typical stationary phase ,its plate backing support and how is the sample loaded

Answer 7

thin layer chromatography

typical stationary phase- SiO2, Al2O3
typical TLC plate backing support- glass, aluminium, plastic

spot sample on line with capillary tube

(the one done in a beaker and lid)

Question 8

how to calculate Rf value in TLC

Answer 8

Rf=distance travelled by sample/distance travelled by solvent front

Question 9

how are samples detected in TLC

Answer 9

under UV or visible light after elution, may need staining

Question 10

name 3 universal stains

Answer 10

KMnO4, cerium ammonium nitrate, iodine

Question 11

name 2 functional group specific stains

Answer 11

ninhydrin- primary/secondary amines

dinitrophenylhydrazine (DNP)- aldehydes, ketones

Question 12

what stain is specific for primary and secondary amines

Answer 12

ninhydrin

Question 13

what stain is specific for aldehydes and ketones

Answer 13

dinitrophenylhydrazine (DNP)

Question 14

describe the basic mechanism of chromatography

Answer 14

mobile carrier phase flows over supported stationary phase, solute molecules move back and forth between stationary and mobile, presence in stationary phase decreases the rate which they are carried by mobile phase

Question 15

in SiO2 chromatography what are the common interactions between analyte and stationary phase

Answer 15

hydrogen bonds, dipole dipole, VDW

Question 16

compare the interactions with the stationary phase between polar and non polar compounds

Answer 16

polar compounds interact more strongly with stationary phase so it moves less

nonpolar interacts more weakly, moves further

Question 17

how to compare TLC with column chromatography

Answer 17

same stationary phase= same principles
polar compounds interact stronger than non polar, polar move less quickly through the column, the product that runs faster in TLC (non polar) will be eluted from column first

Question 18

what increases Rf value

Answer 18

increased polarity of mobile phase

Question 19

how does a mixture of products get separated into components in chromatography

Answer 19

sample has some components that interact stronger so move slower, components that interact weakly will move faster, different rates cause separation

Question 20

advantages and disadvantages of TLC

Answer 20

advantages- quick, simple, low tech, cheap, little training needed

disadvantages- chemical nature of compounds must be confirmed with other techniques, qualitative

Question 21

how does flash chromatography work

Answer 21

uses compressed gas (eg. nitrogen) to push solvent through column, uses finer particle size for stationary phase (eg. silica gel)

(kind of like column ig)

Question 22

what does HPLC stand for, what is it and how does it work

Answer 22

high performance liquid chromatography

highly improved form of column chromatography, solvent forced through under high pressure

Question 23

how can results be detected

Answer 23

UV-vis, fluorescence, MS, refractive index, evaporative light scattering

Question 24

what is retention time
(tR)

Answer 24

time taken for solute to pass through a chromatography column, calculated as time from injection to detection

Question 25

what is total retention volume (VR)

Answer 25

volume of mobile phase entering column between sample injection and emergence of peak maximum of sample component of interest

Question 26

how to calculate retention time

Answer 26

tR=VR/Fc Fc=mobile phase flow rate tR=retention time VR=total retention volume

Question 27

what is the number theoretical plates (N)

Answer 27

indicates how efficiently a column can separate a mixture of products into its individual components

Question 28

how to calculate number of theroretical plates from a graph

Answer 28

N=16(tR/Wb)^2 retention time= measured from 0 to intersection of tangents width of triangle base= measured where tangent intersect with x axis (pic on one note)

Question 29

describe the results of the number of theoretical plates (N)

Answer 29

narrower base=more efficient=greater N value wider base=less efficient=smaller N value

Question 30

what is size exclusion chromatography work and what is it used for

Answer 30

separates different compounds according to the size in solution used for analysis/purification of macromolecules

Question 31

when the stationary phase of a size exclusion chromatography is a swollen gel what is it called

Answer 31

gel permeation chromatography

Question 32

what is gel permeation chromatography

Answer 32

size exclusion chromatography but with swollen gel as the stationary phase

Question 33

describe the stationary phase in size exclusion chromatography

Answer 33

stationary phase are porous beads

Question 34

how does size exclusion chromatography work

Answer 34

size of molecules in sample determine whether they enter pores of poruous beads, larger molecules (higher MW) will not enter the pores so it will have a shorter elution path and be the first eluted from the column, smaller molecules will follow the shape of the pores so it has a longer path and therefore be eluted last

Question 35

what is reversed phase chromatography and how does it work

Answer 35

stationary phase has hydrophobic groups instead of polar ones (also in a column) separation of molecules based on hydrophobicity, hydrophobic compounds interact more strongly with stationary phase than polar compounds (nonpolar compounds go slower i think)

Question 36

uses of reversed phase chromatography

Answer 36

sample analysis and purify large amounts of compounds

Question 37

why is normal phase chromatography often preferred

Answer 37

analytes to hydrophobic/phillic for reversed phase, for purification of products on a lab scale

Question 38

difference between normal phase and reversed phase chromatography

Answer 38

normal phase- stationary phase is polar and mobile is non polar -polar molecules interact strongly so move less reverse phase- stationary phase is non polar, mobile phase is polar -nonpolar molecules travel further

Question 39

what is ion exchange chromatography

Answer 39

separates compounds based on overall charge, limited to purification of ionisable molecules

Question 40

how does ion exchange chromatography work

Answer 40

charged molecule binds to a oppositely charged stationary phase stronger charge will bind more strongly so it will elute last, weaker charge elute first

Question 41

types of ion echangers

Answer 41

anion exchangers quaternary ammonium- strong protonated tertiary amines- weak cation exchangers sulfonic acid salts- strong carboxylates- weak

Question 42

what is gas chromatography

Answer 42

separation technique carried out in a column where the mobile phase is gas, used to isolate volatile components

Question 43

how does gas chromatography work, what mobile phase and stationary phase is used, how it it detected

Answer 43

analytes vapourised to be injected mobile phase- inert gas carrier (He,N2, Ar) stationary phase- solid adsorbent or liquid on inert support detection- flame ionisation detectors/ thermal conductivity detector/ mass spectrometer