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7MRFFS01: Analytical Chemistry > Liquid chromatography > Flashcards

Liquid chromatography Flashcards

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1
Q

LO

A
  • To understand the importance of (U)HPLC, HILIC and IC
  • To understand how and why they work
  • Be aware of the various modes of separation to help identify which might be best suited to a particular separation
  • Be able to give practical examples of LC at work
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2
Q

What is liquid chromatography and what are the common forms of the MP, SP and separation systems?

A
  • Separation technique for compound mixtures dissolved, or extracted into, liquids

* Mobile phase is a liquid
* Stationary phase is usually in a particle-packed column, or on a planar surface
* Separation system can be **preparative or analytical **(scales)

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3
Q

How does GC compare with LC?

A
  • GC, interaction of compounds of interest with stationary phase only as mobile is inert (H2 and He)
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4
Q

How does LC interaction with the stationary and mobile phase differ to that of GC?

A

o More complex interactions to consider/ optimise
o Permits many modes of separation
o Flexibility

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5
Q

Different names for the MP depending on application

A

o TLC- ‘system solvent’, ‘developer’
o (U)HPLC- ‘mobile phase’, ‘eluent’
o IC- ‘eluent’
o GC- ‘carrier gas’
o CE- ‘buffer electrolyte’

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6
Q

Acronyms used

A
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7
Q

For NPLC whats the polarity of the MP and the SP.
Some examples
What order the solutes elute in
MP examples

A
  • A non-polar MP
  • A polar SP
  • E.g., silica, alumina, amino, phenyl as polar/induced polarity groups
  • Solutes elute in the order of increasing polarity (adsorption based)
  • Mobile phases: hexane, dichloromethane, toluene
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8
Q

What different groups can attach to the Silica surface (Si-OH) in NPLC?

A
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9
Q

What are silanols?

A

A silanol is a functional group in silicon chemistry with the connectivity Si-O-H and is related to the hydroxy functional group (C-O-H)

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10
Q

With RPLC:
* Polarity of the MP and SP
* Order the solutes elute in
* SP examples
* MP examples

A
  • A polar MP
    non-polar SP
  • Solutes elute in order of increasing non-polarity (by adsorption/ partition)
  • SP:
    o E.g., silica bonded with C1-C18 non-polar chains
    o E.g., polystyrene divinylbenzene (PS-DVB)
  • MP:
    o E.g., methanol/ water
    o Acetonitrile/ water
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11
Q

What is phase collapse in regard to RPLC?

A

Phase collapse is when >95% causes C18 chains to collapse. Adding amide groups adds polarity to stop this from happening (or other functional groups which are polar)

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12
Q

What is end-capping?

A
  • Non-polar functional groups (e.g.,C18) can be bulky, and at manufacture can get ‘residual silanol’ groups, which make the SP more polar
  • Cause ‘secondary interactions’, e.g., tailing peaks
  • Want to get as many functional groups on Si as possible
  • To use a secondary functionalisation with smaller non-polar group to reach residual Si to avoid 2˚ unwanted interactions
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13
Q

With ion chromatography:
* What does it separate ions based on?
* What used as an eluting species?
* What is used during gradient elutions and why?
* What is present on the SP surface?
* Examples
* Applications

A
  • Separates ions based on their size and charge
  • Uses a competing ion (e-) in the MP as an eluting species
  • For gradient elution, incremental concentrations of this species are used to elute large mixtures
  • SP surface is grafted with oppositely charge groups to electrostatically attract analyte ions
  • Examples
    o 20mM NaOH in H20 as an MP on alkanol quaternary ammonium as a SP (for anion separations)
    o 20mM methansulfonic acid in H2O as MP on alkylsulphonate SP (for cations M+)
  • Applications:
    o Inorganic anions (Cl-, NO3-,SO42-)
    o Alkali, alkaline earth and transition metals
    o AA,peptides and other charged biomolecules
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14
Q

Ion exchange equilibria

A
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15
Q

With HILIC:
* What is the polarity of the MP and SP?
* What is HILIC
* Order it elutes solutes
* examples

A
  • Polar MP,
    Polar zwitterionic SP
  • An intermediate between RPLC and NPLC where there is small % water in a predominantly polar organic water-miscible solvent
  • Solutes elute in the order of increasing polarity
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16
Q

What are some MP/SP interactions?

A
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17
Q

How is the SP selected?

A
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18
Q

Tell me the following about mobile phases:
* What is it often mixtures of?
* Considerations when choosing the MP
* Common modifiers
* Common solvents

A
  • Often mixtures of solvents and additives (‘modifiers’)
  • Considerations
    o Physical properties: high purity (especially for MS), no particulates, no gas, green, etc
    o Strength: need to elute analytes, polarity (water vs hexane (NP vs RP), ion concentration (IC)
    o** Selectivity:** interactions (change MP parameters e.g., acetate –> carbonate)
    o Miscibility: components dissolved in different reservoirs must be miscible

Common modifiers:
* Buffers: maintain pH
** Acids/bases: **Ionise/ de-ionise analytes
** Ionic strength
* Ion-pair reagents **
o Association complexes between analyte/modifier, to aid retention in RP-LC
* **Amines: **reduce tailing for basic analytes in RP-LC
* Remember: must be volatile for MS

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19
Q

What are some types of stationary phases?

A
  1. **Columnar **(uses particles, monolith, and capillary scale column GC)

2.** Planar** (TLC)

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20
Q

What are some common solid substrates for TLC and what are they coated with?

What are some examples of MP used for TLC?

A
  • Solid substrate (glass, plastic, Al) coated with SP (silica, alumina, polyamide)
  • MP: toluene, methanol, acetonitrile (+buffers)
    o Generally isocratic
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21
Q

What is the equation for calculating the retardation factor in TLC?

A
22
Q

What are the types of TLC?

A

1D and 2D

23
Q

What are some common detection methods for TLC?

A

o Visual (dyes)
o UV (drugs, light box)
o Fluorescence
o Radioactive labelling
o ‘Developers’,E.g.., ninhydrin, hexachloroplatinate
o Analysis of spots by LC-MS/GC-MS
o MALDI

24
Q

What are the pros and cons to TLC?

A

*** Pros **
o Cheap
o Low volume MP
o Small sample volume
o Multi-dimension (2D)

* Cons
o Poor efficiency
o Non-confirmatory
o Limited for quantification
o Not easy to automate (HP-TLC)

25
Q

Columnar chromatography:
* type of column
* Column material
* Preparative scale
* Analytical scale

A
  • Particle-packed, or monolithic column, rather than spread on a surface
    o **Particles: **spherical particles, ideally with narrow size distribution (more common)
    o Monolithic: single piece of material with flow-through pores
  • Columns typically stainless steel, though also fused silica, plastic, titanium, etc.

*** Preparative scale **
o Large-scale (up to tons of material)
- Purification, extraction, preparation of standards/ reagents, etc

*** Analytical scale **
o Small scale: typically, 30-250mm columns
o Analysis only- nL to µl injection volume

26
Q

Compare HPLC vs (U)HPLC

A

HPLC vs (U)HPLC
o Small particles (HPLC 3-5 microns, UHPLC <2 microns)
o High-pressure (UHPLC very high pressure, specialist pumps)
o 4.6mm column id- ‘standard bore’
o 2.1mm i.d. or less- ‘narrow bone’

27
Q

Draw the general features of the LC instrumentation

A
28
Q

With the LC instrumentation there is a gradient pump and a degasser. Why are these present?

A

Mobile phase reservoir/ degasser
o Houses mobile phase(s) (100-1,000mL)
o Gradient pump- multiple reservoirs and ‘solvent selector’
o Degasser: used to remove air from liquids when they mix on-line
o (inert gas headspace for IC)

29
Q

Whats good practice for preparing MPs?

A

o Separately measure components
o Mix and degas
o Add buffers/ pH adjust as needed
o Filter
o Rinse glassware (10-20mL)
o Fill reservoir and prime pump(s)

30
Q

What does the gradient elution change?

A
31
Q

Whats required for the analytical pump in the LC?

A
  • Analytical pump- want to avoiding pumping in system

o Need pulseless flow (typically 0.05-5.00 mL/min)
- Capillary/ nano-flow &laquo_space;micro-flow &laquo_space;standard flow

o High pressure

o Corrosion-proof (typically stainless steel, PEEK with ruby/ sapphire/ titanium)

32
Q

Why are there generally two-pump heads in LC?

A

One fills, whilst the other is emptying- continuous, pulseless flow)

Ideally small internal volume (‘dead’ volume, ‘delay’ volume)

33
Q

Why is priming required in LC?

A

o Important step prior to analysi
o Open a tap on the outlet, to divert flow to waste
o MP(s) pumped from reservoirs at high flowrate for 5-10 mins (can do this as removed column)
o Check for air

34
Q

How does an auto-injector/ auto-sampler work?

A

o Inject sample as discrete slug onto column
**o Uses flow-switching valve(s) **
o Reproducible sample volume injection- use sample ‘loops’
o Include loop/needle washes (carryover)
o Can include on-line sample preparation steps

35
Q

Why are column ovens fitted?

A

o Columns fitted with 1/32” or 1/16” fitting, using a nut and ferrule
o Make sure all MP are primed through
o Ensure no leaks

36
Q

Why are guard columns often used on columns?

A
  • Increase col. lifetime
  • Save money
  • Can use for some separations (do at low pressure and quick if good enough for some separations)
  • Generally, use 3-5 guard columns: 1 column
37
Q

With the column over, what are the general temperature control ranges and the types of ovens used?

A

o Temperature control to within **+/- 0.5 to 1.0˚c **

o Pre-column MP heating ideal/useful

o Types of oven:
Fan oven
 Water baths
 Peltier ovens- uses semi-conductors

38
Q

What is the van’t Hoff equation? What it is a useful parameter for and what can it affect?

A

Temperature and HPLC
o check Alters thermodynamics, hence interaction of SP and MP (Van’t Hoff equation: −ΔG∘=RTlogeKp))
∆G= Gibbs free energy
K= equilibrium constant
T= temperature

o Useful parameter to influence/adjust selectivity

o Can affect:
* Retention time
* Selectivity (peak resolution/ separation, and order)
* Peak shape
* Back-pressure
* Detector signal

39
Q

MP pre-heating…

A
40
Q

Column packing architectures

A
41
Q

What is the chromatographic plate theory and its assumptions?

A
42
Q

Chromatographic plate theory

A
43
Q

What does efficiency, H,depend on? and when plotted what does it look like

A
44
Q

The efficiency curve is comprised of three componenets, what does each of these components represent?

A
45
Q

Tell me about Term A for efficiency curve

A
46
Q

Tell me about Term B for efficiency curve

A
47
Q

Tell me about Term C for efficiency curve

A
48
Q
A
49
Q

What are common LC detectors?

A

o UV/Vis
o Fluorescence
o Conductivity
o Evaporate light scattering
o Electrochemical
o Refractive index
o Mass spectrometry

50
Q

What is peak fronting and peak tailing and the solutions for each?

A
51
Q

What is peak splitting and the solution for it?

A
52
Q
A

Black: gradient profile
* Over is RPC
* Start at 5% water to keep C18 ready to interact with amide
* Strength of elution is good for separation

Blue: sample

Red: pressure trace for injection
* Reproducible pressure trace
* Can show blockage(shoots up), leaks (drops off), bubble (not smooth trace), pressure trace looks different if acetonitrile used instead or if wrong way round
* Methanol less viscous than water which causes pressure drop
* Good diagnostic tool especially if peaks absent

7MRFFS01: Analytical Chemistry (13 decks)

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