Separation techniques Flashcards
What characteristics can be used for separation?
- Solubility: salts
- Ion charge
- Polarity: hydrophobic & hydrophilic
- Molecular size
- Binding specificity: affinity (Ag & Ab)
Give e.g. of procedure/s that use binding specificity
(- use Ab & Ag)
- affinity chrmaotography
difference of priciples b/w cation & anion exchange chromatography
- cation: bead binds to +ve proteins
- anion: bead binds to -ve proteins
in affinity chromotography, which ligand is appropriate to separate:
a) Ag
b) Ab
c) Enzyme
a) Ag -> Ab
b) Ab -> Ag
c) Enzyme -> substrate
a) What is HPLC &
b) what is it used for?
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
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.
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
Give 3-4 examples of columns’ size used in HPLC?
C-2: Ethyl Silyl
C-8: OCtyl Silyl
C-18: Octadecyl Silyl
CN: Cyanopropyl Silyl
Normal phase vs reversed phase HPLC
a) nature of solvent
b) nature of stationary
c) type of interaction
d) principle
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)
What is the right HPLC phase for separation of
a) Oils;
b) biomedical substances
c) Why?
a) Oils: normal HPLC bc hydrophobic solute choose hydrophobic solvent = normal
b) biomedical substances: rvs HPLC bc hydrophilic solute choose hydrophilic solvent = rvs
State 3-6 of the wide applications of HPLC in industry?
- Environmental
- Bioscience
- Clinical
- Pharmaceuticals
- consumer products
- Chemical
a) mass spectrometry separates molecules based on their …
b) how does MS work?
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
define purification
- characterise a compound
- assumes no interferance w/ impurities
define separation
separate closely related molecules like isomers
define Identification
compare spectral properties of an unknown compound against compounds with known spectral properties
define Quantification
concentration of a compound can be determined by separation of a peak and comparison against standards
princilple of hydrophobic interaction chromatography
- stationary phase: resin (hydrophobic)
- mobile phase / solvent: hydrophobic
=> hydrophilic eluted 1st bc can’t bind to resin
components of HPLC
A - Mobile Phase Reservoir B - Pump C - Sample Injector D - Column (Stationary Phase) E - Detector F - Data Analyser G - Waste Container
Define:
a) retention time (tR)
b) t0
a) tR: Time compounds stays in equilibrium before it gets separated
b) t0: time to elute the solvent (solvent exits first)
equation of retention factor
k = (tR - t0) / t0
what is the retention factor (a - alpha) & the equation & interpret
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
partitioning in the reverse phase is the separation based on the…
analyte’s (affinity/) relative partitioning b/w stationary & mobile phase
requirements of samples in mass spectrometry
- samples must be pure & volatile
- (l), (s) or (g) under STP
difference b/w 1º, 2º & 3º ion in MS (after fragmentation) & rank their stability
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
in mass spectrometry, what is the parent peak (aka ion peak) and the base peak?
a) parent peak: largest molecule weight (biggest fragment)
b) base peak: tallest peak = most stable
