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Liquid chromatography

29 important questions on Liquid chromatography

What are the principles for chromatography?

- You have a column with a loading phase
- You load your sample
- You add the mobile phase
- You collect each compound separately
- In this way the separation is based on differences in retention between components.

What are the different types of chromatography?

- gas chromatography
- liquid chromatography

For a very volatile compound you better use a gas chromatography while for nonvolatile compounds you better use liquid chromatography.

Why do you use liquid chromatography?

It is ideal for most applications in the bioanalysis because it is:
- highly versatile
- high sensitivity
- high selectivity
- fast
- robust

How does the direct injection into the auto sample go?

- You have aj needle that is inside the sample vial.
- This one is connected to the measuring pump.
- Then, suddenly the rotor turns.
- This means that the sample that is sucked up into the tube will now go to the column the other way around because the needle is collected to the column.

What kind of stationary phases do you have?

- glass
- peek
- stainless steel
A column is filled with particles around 3,5 um.

How does the UV detector work?

With this detector, all compounds absorb light and can be measured.

What happens inside the HPLC column?

The mostly used separation mechanism is hydrophobic interaction.
- in this case the stationary phase is silica with c18 chain attached.
- The mobile phase is water with a modifier (e.g. methanol or acetonitrile)
- The modifier intermediates between water and the non polar surface.

How does the hydrophobic interaction occur?

- polar compounds have more affinity for the (polar) mobile phase and will elute early / in high water content
- non-polar compounds have more affinity for the (non-polar) stationary phase and will elute late/ need more organic solvent.

What is a gradient elution?

Here the mobile phase composition is varied during the separation.
- This compresses the chromatogram.
- However it also means a decrease resolution

How works the LLE model?

The liquid liquid extraction model works as follows:
- The column is divided into separate regions (plates).
- Each plate consists of a mobile phase and a solid phase and in each region an equilibrium is obtained just like in a LLE separation funnel.
- After equilibrium, the mobile phase, shifts one region further.

What is the selectivity factor?

It is the ratio of K' between 2 compounds

What are remarks about the LLE model?

- migration of analytes depends on K'.
- initial block shape results in a Gaussian shape
- the equilibrium has to be complete
- The difference in K" leads to a separation
- The LLE tubes corresponds to theoretical plates. This means that more extractions over the same volume means a narrower peak and more theoretical plates means a narrower peak as well.

How the plate number can be calculated?

(retention time/standard deviation at 50%)square root

How can the retention time be calculated?

t0 + tr.
In this way the capacity factor can be calculated as well according to :
Tr-t0/t0

What does the capacity factor mean?

K'= 0 -> no retention, so no affinity for stationary phase.
K'is infinity -> no elution (trapped on the stationary phase
Ideal is that K' lies between 2 and 10.

How do you calculate the resolution factor?

R = (t2-t1)/0,5(w1+w2)
The higher the resolution the better it is. normally at r = 1,2 baseline separation is achieve. However, r = 1,5 is often taken as a minimum for a workable separation.

How do you calcuate the number of plates and the heights of the peaks?

number of plates: you need to know the tr and the width (50%).
Formula: 5,54*(tr/width)^2
or length/ height

heights: column length (mm)/ number of plates.

What is the effect of particle size?

The smaller the particle the better the column because there is a bigger area on the particles. 1,7 um is the smallest.

How a high N can be obtained?

- longer columns
- lower values for the height (H)

The minimal H is obtained with
- better packings
- smaller stationary phase particles
- optimized mobile phase flow

The pressure increases with (particle diameter)^2

What are the typical values for LC?

L = 5-15 cm
N = 5000-15000
H = 5-15 um

What kind of different stationary phases are there?

- silica or polymer based
- aliphatic chains
- aromatic group
- end capping groups
- many modifications of the above
- hydrophobic interaction is the primary mechanism.
- There are dipole interactions and pipi as selective tools.

What kind of mobile phases are there?

1) modifier type:
- acetonitrile: dipole momentum (induction)
- methanol: H-bonding acceptor and donor
- tetrahydrofurane H-bonding acceptor
2) percentage of the modifier
3) ionic strength
- higher buffer concentration
4) pH
- changes charge state
5) ion pairing agents

What is the silica pH range and what is the Hybrid Particle pH range?

silica: pH 1-8
hybrid particle pH range: 1-12

What is ion pairing?

In this way charged compounds can become neutral by pairing. This makes it suitable for a reverse phase chromatography.
No ion pairing is very old-fashioned.
This can be done by adding a ion pair reagent. This reagent can interact both with the stationary phase and with the analyte.
- It depends on the concentration of the ion pair reagent and the concentration of the modifier.

What are examples of ion pairing?

- sodium dodecyl sulphate
- tetra octyl ammonium bromide

What if you use an ion exchange?

- Here the stationary phase contains a charge and the compounds will have an electrostatic interaction.
The interaction is influenced by:
- the charge
- type of counter/ competition ions
- ionic strength
1) is the ionic strength (in the solution) low: there will be more retention
2) is the ionic strength (in the solution) high: then there will be less retention

You can add sodium ions for example. This will compete with the analyte for the negative charge.

What is the difference between anionic and cationic?

anionic: this is a negatively charged particle that is attracted by a positive anode.
cationic: This is a positively charged particle that is attracted by a negative cathode.

How can chiral recognition occur?

Chiral recognition can occur when the chiral stationary phase is able to interact differently with each enantiomer to form transient-dieastereomeric complexes. This requires a minimum of 3 interactions through:
- H-bonding
- pipi interactions
- dipole stacking
- inclusiong complexing
- steric bulk

How occurs the LC in the bioanalytical practice?

- the sample should not contain any particles, proteins or strongly retained compounds. This means that sample preparation is necessary.
- This means that the sample extract should be compatible with the initial mobile phase.

You need to consider the
- pH
- modifier type and content
- also separation from undetected compounds that could affect the ionization in the LC-MS.

The question on the page originate from the summary of the following study material:

introduction

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