Separation Science Flashcards
(89 cards)
1
Q
why is separation required in analytical chemistry?
A
so that compounds in mixtures/solutions can be separated and analyzed
- can also test purity of a compound
2
Q
are LC/GC qualitative or quantitative?
A
Both approaches can be qualitative and quantitative
3
Q
how are compounds separated?
A
they are separated by their different affinities to the column (stationary phase)
- compounds w lower affinities will elute sooner & vice versa
4
Q
what is the fundamental equation for resolution?
A
Where:
N = number of theoretical plates
α = selectivity or separation factor
k = retention factor
5
Q
what does each part of the following equation relate to?
A
6
Q
which parameter has the highest influence on resolution?
A
Selectivity - small changes in selectivity result in big changes in resolution
- dictating compound affinity to column
7
Q
when does retention have significant influence on resolution?
A
retention has a significant influence on resolution at small k-values
8
Q
what does efficiency mean?
A
efficiency describes the separation power of the column
9
Q
what effect does using smaller particles in stationary phase have?
A
smaller stationary phase particle size means a greater number of theoretical plates - mildly improving resolution
10
Q
what does resolution describe?
A
resolution describes the ability of a column to separate peaks of interest (no units)
11
Q
what is the minimum resolution value for measurable separation and quantitation?
A
1
12
Q
what is the optimal resolution value?
A
1.6 is considered a baseline separation & ensures the most accurate quantitative result
13
Q
what does the retention factor k consider?
A
the retention factor is the ratio between the time a solute spends in the stationary and mobile phases
- calculated by dividing the retention time by the time for an unretained peak (tM)
14
Q
what factors affect the retention factor?
A
- stationary phase
- mobile phase (both HPLC & GC)
- temperature
15
Q
what does selectivity mean?
A
selectivity is a measure of time or distance between two peaks
16
Q
what does it mean if α = 1?
A
then the 2 peaks have the same retention time and hence co-elute
17
Q
what factors affect selectivity?
A
- stationary phase
- mobile phase (HPLC but not GC); no interaction with inert gas in GC
- temperature
18
Q
what are theoretical plates?
A
a theoretical plate is the hypothetical stage in which two phases of a substance form an equilibrium
19
Q
why is column efficiency measured?
A
column efficiency is used to compare the performance of different columns - expressed as the number of theoretical plates, N
20
Q
what does more theoretical plates mean for the column?
A
a column with high N will lead to narrower peaks at a given retention time than a column with lower N
- columns with high plate numbers are more efficient
21
Q
how can resolution be increased?
A
resolution can be improved by improving selectivity, retention & efficiency
22
Q
what factors affect the resolving power and cause peak broadening in a column?
A
23
Q
what equation relates resolving power of the chromatographic column to the various flow and kinetic parameters
A
The Van Deemter Equation
- lower h is best
24
Q
describe the shape of eddy diffusion, self-diffusion (axial) & resistance to mass transfer curves against flow rate. how does the overall Van Deemter curve appear?
A
25
how do H2, He & N2 compare as carrier gases in GC
H2 is best as minimum is at highest flow rate - is also cheap thru electrolysis of water
- safety hazard
He is great as its minimum is broad, meaning resolution is not vastly affected by changes in flow rate - tho is expensive
N2 is okay as it is readily available but small changes in flow rate result in large changes in resolution
26
are linear velocities and flow rates temperature dependent?
Yes
At constant head pressure, linear velocities decrease as temperature increases
27
why are smaller particle sizes preferred?
because smaller particles lead to lower heights of theoretical plates
- therefore higher separation efficiency
- separation efficiency also suffers less when increasing flow rate when using smaller particles
28
what factors affect column efficiency?
- column length; increasing length increases efficiency
- particle size; decreasing size increases efficiency
29
how does increasing efficiency, retention factor, k, & selectivity independently affect resolution?
- increasing efficiency gives linear increase to resolution
- increasing k gives small increase to resolution
- increasing selectivity provides biggest improvement on resolution
These are macro-factors, temperature & flow rate are micro-factors
30
how can hydrogen be made safe for GC?
by using hydrogen generator that hydrolyses water - allowing for minimum amount of H2 to be generated for GC
- very environmentally friendly as only by-product is oxygen
31
whats the issue with using 1.8 um particle size in HPLC?
it creates very high back pressure, needing higher pressure to push compounds through - UHPLC
- 3.5 um is used normally as golden medium
32
how may types of equilibrium are there in GC?
1 - affinity of compound in mobile phase and stationary phase
33
how may types of equilibrium are there in LC?
3
- dissolution/dissociation
- solvation; ions are surrounded by solvent
- partially solvated compound and stationary phase
- compound dissociates from stationary phase with/without solvation
34
why is it harder to control LC than GC?
because there is a higher number of equilibria in LC
- making it much more difficult to control
- but allows for more flexibility as there are more parameters to play with
35
what properties must a compound have to be suitable for GC?
they must be sufficiently volatile between 200-400 degrees that do not decompose
- GC can separate polar & non-polar compounds
36
what are the applications of GC?
- food & flavour analysis
- environmental analysis
- industrial chemical analysis
- petroleum industry analysis
37
what doesa gas chromatograph consist of?
- gas source
- inlet (& autosampler); also acting as a vaporizer
- a column
- a detector
38
why is it important to use a pure carrier gas? (>99.9995%)
because contaminants may react with the sample / column, create spurious peaks & overload the detector
39
what is also recommended for GC?
traps for water, hydrocarbons and oxygen is recommended
40
why, if the analyte is in water, can it not be directly injected into GC inlet? What must be done alternatively?
no direct injection as it creates steam
water must be purged, dried, and trapped before introduction to inlet for vaporization
41
how are headspace samples taken?
gaseous phase of sample is taken from vial
- equilibrium is pushed using heat or shaking vial
- allows for clean injection
42
how is a sample introduced into a GC machine?
most common inlets are injection ports and sampling values
- often heated (200-400 degrees) to vaporize sample
43
what inlet types are there for GC?
Split/Splitless
Cool-on-Column
Programmable Temperature
44
why is the column placed in a well-controlled oven in GC?
because most separations are highly temperature dependent
45
when would the oven temperature be increased in GC?
to elute all compounds
46
what is a GC column composed of?
narrow tubing with a internal thin polymer coating
47
what does selecting the right column depend on?
depends on;
- selectivity
- polarity
- phenyl content
48
what macro-factor does column diameter affect?
it influences;
- efficiency
- solute retention
- head pressure
- carrier gas flow rate
49
what does column length affect?
- efficiency
- solute retention
- head pressure
- bleeding & costs
50
what does the detector do in GC?
- produces stable electronic signal when pure carrier gas is in detector
- produce different signal when component is passing through detector
51
what are the common detectors available for GC?
Thermal conductivity detector
Flame ionization detector
Electron capture detector
Nitrogen-Phosphorus detector
Flame photometric detector
Atomic emission detector
Mass selective detector
52
which detector is the only non-destructive detector for GC?
Thermal conductivity detector
- can be used on small molecules
53
which detector is the most and least sensitive in GC? Which detector is the most versatile?
- electron capture detector is most sensitive
- thermal conductivity detector is least sensitive
- flame ionisation detector dynamic range is V.broad & versatile
54
what is the difference between serial and parallel arrangements of detectors in GC?
serial arrangement must have TCD before destructive detector
- maximum sensitivity; no conc. loss
- has problems with standardization
parallel arrangement allows column effluent to different detectors, controlled by valve
- lower sensitivity
- can control independently
- most common
55
why do later peaks broaden and why does the baseline curve upwards at the end of the run?
peaks broaden due to Van Deemter equation - non-perfect stationary phase, resistance to mass transfer...
run is consistent with ramped temperature run
- heating causes release of impurities from stationary phase, raising the baseline linearly as baseline signal is stronger from impurities
- density of mobile phase also decreases, making compound elution harder; requiring higher pressure
- more thermal noise in electronics as heat increases
56
how can changes in density of gas and temperature be compensated in GC?
Constant pressure
- heating causes slower elution
- hard to push heated compound with high bp through column
- causing peak broadening
Constant flow
- applies variable pressure to maintain flow
- pressure increased if temperature increased to aid elution
57
why is constant flow preferable to constant pressure when increasing temperature?
this is because Van Deemter equation contains flow in it
- if flow is maintained, VD eqn now has 2 constants in it
- operate at minimal point (carrier gas) for desired flow
58
when is constant pressure preferred?
when analysing mixtures with compounds of similar bp (e.g. 2 hexane isomers)
- if you compensate to maintain flow, peaks will broaden and merge
- will lose resolution
- constant pressure allows for natural separation of compounds by boiling point (small range of bp)
59
when is constant flow preferred?
when you have very complex mixtures with compounds of very different boiling points
- always compensating for changes in temp and pressure to maintain flow
- broadening of peaks does not matter so much as compounds elute at different times due to boiling points of compounds in mixture being very different
60
what types of compounds is HPLC used to separate?
polar/non-polar non-volatile compouunds
61
whatre the typical applications of HPLC?
- pharmaceuticals
- majority of proteins
- polymers
- pesticides, vitamins & food additives
- natural products
- thermally unstable compounds; trinitrotoluene, enzymes
62
what limitations does HPLC have?
it has lower separation compared to GC
63
what components make up a HPLC system?
- mobile phase compartment
- pump
- injector
- column
- detector
64
whatre typical flow rates for HPLC?
1-2 mL/min
65
up to what pressure can HPLC and UHPLC pumps deliver?
standard LC pumps - 400 bar
UHPLC pumps - 600 - 1500 bar
66
whatre the five main differences between HPLC & UHPLC?
- UHPLC have smaller particle sizes
- UHPLC has smaller internal column diameter
- UHPLC has much slower flowrates (0.2 - 0.7 mL/min) - due to mass transfer w small particles
- UHPLC has much higher backpressure; resistance to mass transfer
- UHPLC requires detector with higher sampling rate due to narrower peaks
67
why is HPLC generally preferred over UHPLC?
because it is cheaper and more predictable
- UHPLC sees common use in R&D
68
when are isocratic and gradient elution preferred respectively?
isocratic - best for simple separations
gradient - best for analyzing complex samples
- pushing late eluting compound through column for earlier elution
- linear gradients are most common
69
whatre typical HPLC sample volumes?
0.1 - 20 μL
70
what is the role of the injector in HPLC?
to introduce the dissolved sample into the flow stream of the mobile phase
71
what types of columns are used in HPLC?
- analytical; most common
- preparative
- capillary
- nano; components in cells/living organisms
72
what 4 modes are used to separate most compounds?
- normal-phase; polar stationary phase
- reversed-phase; non-polar stationary phase - most common
- ion-exchange
- size-elution
73
what compounds can be separated by reversed-phase chromatography?
can be used for non-polar, polar, ionizable & ionic molecules
74
how is reversed-phase chromatography usually done?
mobile phase is primarily water-based to begin with - adding organic solvents as a function of time
75
why is water-miscible organic solvent gradually added in reversed-phase chromatography?
organic solvent increases the solvent strength and elutes compounds that are strongly retained on stationary phase
76
what compounds are best separated using normal-phase chromatography?
- water-sensitive compounds
- geometric isomers
- cis-trans isomers
- class separations
- chiral compounds
77
what compounds are best separated using ion-exchange chromatography?
- inorganic and organic ions in aq. solution
- ionic dyes
- amino acids
- proteins
78
what is the mobile/stationary phase of ion-exchange chromatography?
stationary phase - ionic groups
- sulfonic, tetraalkyl-ammonium
mobile phase - aqueous buffer
- phosphate, formate
79
what is size-exclusion chromatography mainly used for?
polymer characterisation & proteins
80
what two modes are used in size-exclusion chromatography?
non-aqueous - gel permeation chromat.
aqueous - gel filtration chromat.
81
what is significant to note about size-exclusion chromatography?
that in either aqueous/non-aqueous mode, there should be no interaction between sample compounds and the column packing
- instead, molecules diffuse into pores of porous medium
82
how are molecules separated in size-exclusion chromatography?
molecules are separated depending on their size relative to the pore size
83
whatre the most common types of detectors?
- spectroscopic detection - UV-VIS; most expensive
- refractive index detection - RI
- fluorescence detection - FD
- mass spectrometry - MS; hyphenated systems
- electrochemical detection - ECD
84
how do UV-VIS detectors work in HPLC?
- UV light is shone through flow cell, sensor measures light passing through
- eluted compounds that absorb this light energy change amount of light falling on sensor
- resulting change in electrical signal is amplified & directed to recorder/data system
- UV spectrum is sometimes also obtained
85
how do refractive index detectors work?
measures compound's ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase in a reference flow cell
- amount of deflection is proportional to analyte concentration
86
what are 2 limitations of refractive index detectors?
- considered universal detector but is not very sensitive
- can only be used for isocratic analysis
87
how do fluorescence detectors differ to UV-VIS detectors?
- fluorescence detectors offer higher sensitivity & selectivity
- allows for quantification & identification of compounds & impurities in complex matrices at trace concentrations
- can only detect compounds that fluoresce
88
how do electrochemical detectors work?
electrochemically active substances separated on column undergo an electrochemical reaction, resulting in an electrical current that is recorded
89
why are electrochemical detectors useful?
ECD is an extremely selective and sensitive technique
- allows detection of halide ions
