Separation techniques

Question 1

What characteristics can be used for separation?

Answer 1

1. Solubility: salts
2. Ion charge
3. Polarity: hydrophobic & hydrophilic
4. Molecular size
5. Binding specificity: affinity (Ag & Ab)

Question 2

Give e.g. of procedure/s that use binding specificity

Answer 2

(- use Ab & Ag)

- affinity chrmaotography

Question 3

difference of priciples b/w cation & anion exchange chromatography

Answer 3

• cation: bead binds to +ve proteins

- anion: bead binds to -ve proteins

Question 4

in affinity chromotography, which ligand is appropriate to separate:

a) Ag
b) Ab
c) Enzyme

Answer 4

a) Ag -> Ab
b) Ab -> Ag
c) Enzyme -> substrate

Question 5

a) What is HPLC &

b) what is it used for?

Answer 5

a) HPLC: High Performance Liquid Chromatography: has stationary & mobile phase (pumped through column & detector detects time. require high pressure to move the beads
b)
> Quantify compounds
> Identify compounds
> Separate mixture of compounds
> Purify individual components of the mixture

Question 6

In HPLC, Columns are filled with adsorbents varying in particle (a), and in the (b) of their surface.
• (c) particle size = requires use of (d) pressure (“backpressure”) = typically improves chromatographic (e) • Sorbent particles nature may be: (f) or (g) in nature.

Answer 6

a) size
b) nature
c) smaller
d) higher pressure (bc smaller beads = small space = more force vs large beads = large space = less force)
e) resolution: degree of separation between consecutive analytes emerging from the column
f) hydrophobic
g) polar

Question 7

Give 3-4 examples of columns’ size used in HPLC?

Answer 7

C-2: Ethyl Silyl
C-8: OCtyl Silyl
C-18: Octadecyl Silyl
CN: Cyanopropyl Silyl

Question 8

Normal phase vs reversed phase HPLC

a) nature of solvent
b) nature of stationary
c) type of interaction
d) principle

Answer 8

Normal:
a) solvent = non-polar b) stationary: polar
c) ionic interaction (polar= longer)
d) Adsorption
RVS:
a) solvent= polar b) stationary: non-polar
c) hydrophobic interaction (less polar = longer)
d) Partition (affinity of analyte b/w mobile & stationary phase)

Question 9

What is the right HPLC phase for separation of

a) Oils;
b) biomedical substances
c) Why?

Answer 9

a) Oils: normal HPLC bc hydrophobic solute choose hydrophobic solvent = normal
b) biomedical substances: rvs HPLC bc hydrophilic solute choose hydrophilic solvent = rvs

Question 10

State 3-6 of the wide applications of HPLC in industry?

Answer 10

• Environmental
• Bioscience
• Clinical
• Pharmaceuticals
• consumer products
• Chemical

Question 11

a) mass spectrometry separates molecules based on their …

b) how does MS work?

Answer 11

a) mass/charge potential ratio (M/Z or M/e)
b) [i] charges the molecule w/ electron beam
[ii] fragmentation= ionised molecule broken into fragments = varying sizes w/ varying stability

Question 12

define purification

Answer 12

• characterise a compound

- assumes no interferance w/ impurities

Question 13

define separation

Answer 13

separate closely related molecules like isomers

Question 14

define Identification

Answer 14

compare spectral properties of an unknown compound against compounds with known spectral properties

Question 15

define Quantification

Answer 15

concentration of a compound can be determined by separation of a peak and comparison against standards

Question 16

princilple of hydrophobic interaction chromatography

Answer 16

• stationary phase: resin (hydrophobic)
• mobile phase / solvent: hydrophobic
  => hydrophilic eluted 1st bc can’t bind to resin

Question 17

components of HPLC

Answer 17

A - Mobile Phase Reservoir
B - Pump
C - Sample Injector
D - Column (Stationary Phase)
E - Detector
F - Data Analyser
G - Waste Container

Question 18

Define:

a) retention time (tR)

b) t0

Answer 18

a) tR: Time compounds stays in equilibrium before it gets separated
b) t0: time to elute the solvent (solvent exits first)

Question 19

equation of retention factor

Answer 19

k = (tR - t0) / t0

Question 20

what is the retention factor (a - alpha) & the equation & interpret

Answer 20

a) measures duration (t) spent interacting w/ stationary phase (high = long)
b) a = k2 / k1
c) larger gap in b/w apex of each peaks = better

Question 21

partitioning in the reverse phase is the separation based on the…

Answer 21

analyte’s (affinity/) relative partitioning b/w stationary & mobile phase

Question 22

requirements of samples in mass spectrometry

Answer 22

• samples must be pure & volatile

- (l), (s) or (g) under STP

Question 23

difference b/w 1º, 2º & 3º ion in MS (after fragmentation) & rank their stability

Answer 23

1º: 1 C group bound to charged C
2º: 2 C groups bound to charged C
3º: 3 C groups bound to charged C
Stability: 3º > 2º > 1º

Question 24

in mass spectrometry, what is the parent peak (aka ion peak) and the base peak?

Answer 24

a) parent peak: largest molecule weight (biggest fragment)

b) base peak: tallest peak = most stable