Perdita Barran

Question 1

How is separation achieved in column chromatography?

Answer 1

Through partitioning of solutes between the mobile phase (a liquid or a gas) and the stationary phase

Question 2

Give the Van Deemter equation and briefly describe each of the terms.

Answer 2

H ~ A + B/ux + C*ux

H = Height equivalent to theoretical plate (want this term to be reduced)

ux = linear flow rate

A = multiple paths term

B = longitudinal/eddy diffusion. Band broadening as solute diffuses from areas of high concentration to areas of low concentration (fast flow rate -> less time for diffusion to occur and less band broadening)

C = mass transfer term. Band broadening due to finite time it takes for solute to partition between stationary and liquid phases. While some solute is stuck in stationary phase, the ‘rest of the band’ moves on thus leading to band broadening. Slower flow allows more time for solute to partition back into mobile phase

Question 3

What are superficially porous particles?

Answer 3

Particles which have a nonporous core and a ~0.25 micrometre thick porous silica layer. The stationary phase is bonded throughout this porous outer layer. Mass transfer is more efficient into a 0.25 micrometre layer than into fully porous particles, thus reducing the mass transfer term. This leads to higher efficiency at a faster flow rate, especially in the case of large macromolecules (e.g. proteins) which diffuse more slowly than small molecules

Question 4

Name 2 types of chromatography and briefly explain the principle behind their separation mechanism.

Answer 4

1. Molecular exclusion - large particles are eluted faster than small ones. In this form of chromatography, the column is packed with porous silica beads. Small particles can be retained by the beads and thus spend more time on the column, whereas larger particles are excluded.
2. Affinity chromatography - this is the most selective type of chromatography as it depends on specific interactions between the stationary phase and analyte. For example, the stationary phase may be some type of enzyme which only reacts with one protein in the sample.

Question 5

What is the most common bonded phase?

Answer 5

Octadecylsilane (C18, CDS).

Question 6

How is separation of enantiomers achieved?

Answer 6

Enantiometers have the same physical and chemical properties. Therefore they can only be separated by a chiral stationary phase (optically active bonded phase).. A (S,S) chiral stationary phase will have stronger interactons with the (S)-enantiomer rather than the (R)-enantiomer, thus enabling separation.

Question 7

Define:

i) Normal-phase chromatography

ii) Reversed-phase chromatography

Answer 7

i) A more polar stationary phase paired with a less polar mobile phase
ii) A less polar stationary phase and a more polar mobile phase

Question 8

i) What is the eluent strength of a mobile phase?
ii) How is it increased in normal-phase chromatography?
iii) How is it increased in reversed-phase chromatography?

Answer 8

i) The adsorption energy of a solvent on bare silica (the value of heptane is defined as 0)
ii) Normal-phase chromatography has a more polar stationary phase, therefore the eluent strength of the mobile phase is increased by making it more polar
iii) Reversed-phase chromatography has a less polar stationary phase and so the eluent strength of the mobile phase is increased by making it less polar.

Question 9

Define

i) Isocratic elution
ii) Gradient elution

Answer 9

i) Elution with a constant solvent composition

ii) Elution with a continuous change in solvent composition in order to increase the strength of the mobile phase

Question 10

What is HILIC? Briefly describe how this technique works and what type of molecule(s) it is most suitable for.

Answer 10

Hydrophilic interaction chromatography. HILIC uses a polar stationary phase and a solvent made up of aqueous buffer and an organic modifier (e.g. CH3CN). The eluent strength of the mobile phase is increased by increasing the vol% of aqueous buffer relative to the organic modifier. HILIC is most suitable for small molecules that are too polar to be retained by reversed-phase columns.

Question 11

What can happen if the solvent in which the sample is dissolved in has a greater mobile phase strength than the mobile phase?

Answer 11

This can lead to doubled peaks or altered retention times

Question 12

What is the most common type of detector in HPLC? Briefly describe this detector.

Answer 12

A UV detector as many solutes absorb UV light. This type of detector is generally coupled with a photodiode array which is used to record the spectrum as each analyte is eluted. UV detectors have a linear response to solute concentration over 5 orders of magnitude (i.e. Beer-Lambert’s law is obeyed over this range).

Question 13

What is the most sensitive type of detector in HPLC? Explain the principles behind its operation and give a reason as to why they are not widely used.

Answer 13

A fluorescence detector (detection limits ~0.001 - 0.01 ng). Analytes are excited with a laser, causing them to emit fluorescent (atom and transition-specific) radiation which is then measured. Although these detectors are very sensitive, they only respond to the (few) analytes that fluoresce. Their utility can be increased via derivitisation.

Question 14

Describe the principles behind an evaporative light-scattering detector.

Answer 14

This type of detector responds to any analyte that is significantly less volatile than the mobile phase. Eluate is passed through a nebulizer, forming uniform droplets. After passing these droplets through a heated drift tube, solvent evaporates leaving behind fine mist of solid particles to enter the detection zone. Particles are detected by the light they scatter from a diode laser to a photodiode array.

Question 15

Describe a charged aerosol detector. What are the benefits to this type of detector?

Answer 15

Positive charge is transferred to aerosol particles flowing from the charging chamber through a small-ion trap. Charged plates are used to attract small ions, whereas aerosol particles are too large to be deflected and pass through the trap into the detector. Total charge reaching the collector is measured by an electrometer.
This type of detector is sensitive and almost universal.

Question 16

Describe the principle behind a refractive index detector. Give one advantage and one disadvantage of this type of detector.

Answer 16

In a refractive index detector, there are two triangular compartments through which pure solvent or eluate passes. Collimated visible light passes through the cell with pure solvent in both compartments and is directed to a photodiode array. When solute with a different refractive index passes through the compartments, light is deflected thus irradiating different pixels of the array.
Advantage: almost universal
Disadvantage: useless with gradient elution as it is impossible to match up the reference cell when the solvent composition is constantly changing.

Question 17

How would you develop a gradient separation?

Answer 17

Run a scouting gradient (i.e. a wide range of ~9-95% organic modifier for a time tG = 40-60 min). Let the difference in retention times between the first and last peaks be equal to Δt. If Δt/tG > 0.25, gradient elution should be used. If Δt/tG < 0.25, isocratic elution may be preferable.
If gradient elution is chosen, can try eliminating portions of the gradient before the first peak and following the last peak and running again. If the separation is still acceptable, may try reducing gradient time.

Question 18

What variables are there when it comes to method development in HPLC?

Answer 18

i) Column temperature (elevated temperatures -> faster elution)
ii) Polarity of the solvent
iii) Stationary phase
iv) Changing column pH (neutral forms are better retained by reversed-phase columns, for example)

Question 19

Define
i) Dwell volume
ii) Dwell time
and state the relationship between the two.

Answer 19

i) The dwell volume is the volume between when the solvent is mixed, and when it meets the column. Dwell volume ranges from 0.5 - 10 mL in different systems
ii) The dwell time is the time taken for solvent to reach the column.

We can calculate the dwell time, tD via:
tD = dwell volume (mL) / flow rate (mL min-1)

Question 20

What is an AUC?

Answer 20

An analytical ultracentrifuge. This consists of a preparative ultracentrifuge and an optical detection system to measure the sample concentration inside the centrifuge during or after sedimentation.

Question 21

Give 5 examples of AUC applications

Answer 21

i) Determination of sample purity
ii) Characterisation of assembly and disassembly mechanisms of biomolecular complexes
iii) Determination of subunit stoichiometries
iv) Detection and characterisation of macromolecular conformational changes
v) The measurement of equilibrium constants and thermodynamic parameters for self and hetero-associating systems.

Question 22

Name 3 types of detectors used in AUC and briefly explain the principle behind their operation.

Answer 22

i) Schlieren optical system - light is passed through a specific point in the cell. The concentration is changing at this point due to sedimentation, resulting in varying degrees of refraction. The change in radial distance is measured by the displacement of an image on a camera which is plotted against time.
ii) Rayleigh optical system - light is passed through the solvent and sample and the diffraction pattern created is recorded. The data is plotted as refraction index against radius in the cell. A greater refractive index at the far end of the cell is expected due to sedimentation.
iii) Absorption optical system - absorbance against radius in the cell is measured - the higher the concentration, the stronger the absorbance. Good for analytes which absorb in the UV range.

Question 23

What happens at high rotor speeds?

Answer 23

Sedimentation dominates over diffusion.

Question 24

Describe a problem with the analysis of heterogeneous solutions and explain how this can be overcome.

Answer 24

At high rotor speeds, large molecules sediment out of solution too quickly and may not be observed. At low rotor speeds, small molecules sediment out of solution too slowly to create a useful gradient (or may not sediment out at all).
This problem is overcome by ratcheting up the rotor speed after a set time step.

Question 25

What is IM-MS? Describe the principles behind how it works.

Answer 25

Ion-mobility mass spectrometry. Allows for gas-phase separation of isobaric species on the basis of their mobility, i.e. the ease in which they move under an applied electric field whilst their motion is impeded by collisions with a small, inert gas (e.g. N2). Their drift time is converted to a collisional cross section (CCS).

Question 26

Give an equation for the ion mobility, K.

Answer 26

K = v/E = L/tE

v = ion velocity
E = applied electric field
L = length
t = time

Question 27

What is IM-MS most useful for?

Answer 27

The study of proteins. Native conditions -> compact proteins, denaturing conditions -> elongated structures (with intermediate charge states in between the two extremes, all with different conformations)

Question 28

How can resolution be increased in IM-MS?

Answer 28

Increase by increasing the applied voltage, Vd, or by lowering temperature. N.b. increasing the voltage begins to align molecules in the field.

Question 29

How is transmission increased in drift-tube IM-MS instruments?

Answer 29

Through the use of an ion funnel. These are used to collimate the ions at the end of the drift tube.

Question 30

What is TWIM-MS, and how does this technique achieve separation?

Answer 30

Travelling wave ion-mobility mass spectrometry. This technique uses an RF field to hold ions in place with a superimposed DC component that ‘pushes’ ions through. Ions of high mobility K ‘roll over’ the wave less often than ions of low K thus allowing them to be separated.

Question 31

Give one advantage and one disadvantage of TWIM-MS

Answer 31

Advantage: higher resolution than drift-tube instruments
Disadvantage: no direct CCS information due to the use of non-uniform electric fields. Calibration is required.