Chromatography - Liquid Chromatography (LC)
8 important questions on Chromatography - Liquid Chromatography (LC)
How do you get a better resolution in liquid chromatography?
What is the differnece between isocratic or gradient elution in LC?
Isocratic elution performs badly if in the mixture there are very diverse compounds, making difficult to find a solvent composition which is optimal for every compound.
In this latter case, which is actually the most common one in complex mixtures like food, gradient elution is more indicated, being able to efficiently separate mixtures of many different compounds.
Is the order of the peaks changed in LC when you change the solvent composition?
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What are the additives in LC eluents and why are they used?
- Buffers (if soluble in the eluents): to keep the pH at a defined level, Used for separation of molecules containing acidic or basic groups, in order to fix their ionization state.
- Acids (in silica based columns): to keep the silanol groups protonated. Deprotonated silanols induce excessive tretention and peak distortion.
- Ion pairing agents (acids, bases or salts): used for ionic analytes. A lipophilic counterion is added, ablebto be retained by the reverse phase and at the same time forming a tight ion pair with the ionic analytes, ensuring that the analytes are also retained
What is ion exchange chromatography?
Ionic analytes are exchanged and thus retained.
Elution usually is done by increasing NaCl concentrations.
Retention depends form pH, ionic strength, number of charges, charge density
What is hydrophobic interaction (HI) chromatography?
- Mostly for proteins
- Stationary phase is made by short alkyl chains (methyl-butyl, phenyl) in order to avoid excessive interaction
- A chromatographic matrix containing hydrophobic groups, binds proteins from aqueous solutions to different extents depending on the protein structures and a range of controllable factors including concentrations of salts, pH, temperature and organic solvents as illustrated in the figure below.
- During HIC, sample components bind to the column in high ionic strength buffer, typically 1 to 2 M ammonium sulfate or 3 M NaCl. Elution is usually performed by decreasing the salt concentration, stepwise or using a gradient.
What is hydrophilic interaction liquid chromatogrpahy (HILIC)?
- HILIC separations are based on the use of polar stationary phases (silanol, amino, amide, ionic) eluted with hydrophobic mobile phases, generally acetonitrile, containing a small percentage of water.
- Polar stationary phases retain water strongly on their surface.
- In presence of low water percentages, the polar compounds stick to the stationary phase; when water is increased they elute in the mobile phase.
What are the detectors used in LC?
- UV-Vis (Ultraviolet/Visible detection), sensitive but limited to compounds showing absorbance (for example proteins, phenolics)
- Fluorescence, very sensitive, but limited to fluorescent compounds, also after derivatization with a fluorophoric group (amino acids)
- ELSD (Evaporative Light Scattering Detection), can be applied to every compound, but not very sensitive and often used for compounds not being detectable by any other method (saponins)
- ADl (Amperometric Detection): sensitive, but limited to charged molecules or molecules easily oxidized (salts, mono- and oligosaccharides, amino acids)
- MS (mass spectrometry), general, if the right ionization conditions are found.
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