Small-molecule chromatography Flashcards
Thin-layer chromatography
A qualitative method of separation
Separation is achieved using the relative affinity of each compound for the solid phase
TLC plate
An aluminium plate sprayed with a layer of silica (the solid phase)
TLC method
The sample is spotted onto the TLC plate and placed in a chromatography tank with the end of the plate submerged in the mobile phase
The solvent moves up the plate and each component of the mixture moves up the plate at a different rate, allowing separation
Rf value
migration distance of sample/ migration distance of solvent
TLC applications
- A cheap and easy way to separate classes of compounds based on their polarity
- Used to test optimum separation conditions in preparation for more high-tech chromatography
- Spots on the TLC plate can be extracted and used
HPLC
High-performance liquid chromatography
A quantitative method of separation
Separates relatively small compounds (<200Da)
Small beads are used as the stationary phase, each compound will have a different interaction with beads and hence will be retained in the column for a different length of time
Types of HPLC
- Analytical HPLC = identifies compounds
- Preparative HPLC = purifies mixtures
HPLC elution
Compounds are eluted in order of polarity in HPLC
This is done using a solvent gradient
HPLC setup
Analytical column = 0.5cm in diameter, 30cm in length
Autosampler = injects very precise volumes into the column
Injection valve = a flat-point needle is used to load the sample
Mobile phase reservoirs = provide the system with solvent
Pump = mixes very precise ratios of samples before loading
Guard column = protects the main column from degradation
Spectrophotometer
Mobile phase reservoirs
As well as containing the solvent, these reservoirs also contain TFA to acidify the solution and make the sample less polar
The reservoirs are also filled with nitrogen or argon to displace any oxygen or carbon dioxide from the system
Guard column
The guard column is a smaller column that sites before the main column
Any very high affinity molecules will bind here, protecting the main column from degradation
HPLC stationary phase
Usually silica-based
The silica is often modified based on the specific separation being carried out
HPLC solid phase
Can be silica, octadecyl, octyl or aminopropyl
Reverse-phase chromatography
Compounds are separated based on their hydrophobicity and are eluted with a relatively non-polar solvent
Normal-phase chromatography
In normal phase chromatography the solid phase is polar and the polarity of the solvent is gradually increased, leading to elution of compounds
