Small-molecule chromatography Flashcards Preview

Clinical Biochemistry > Small-molecule chromatography > Flashcards

Flashcards in Small-molecule chromatography Deck (16)
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1
Q

Thin-layer chromatography

A

A qualitative method of separation

Separation is achieved using the relative affinity of each compound for the solid phase

2
Q

TLC plate

A

An aluminium plate sprayed with a layer of silica (the solid phase)

3
Q

TLC method

A

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

4
Q

Rf value

A

migration distance of sample/ migration distance of solvent

5
Q

TLC applications

A
  • 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
6
Q

HPLC

A

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

7
Q

Types of HPLC

A
  • Analytical HPLC = identifies compounds

- Preparative HPLC = purifies mixtures

8
Q

HPLC elution

A

Compounds are eluted in order of polarity in HPLC

This is done using a solvent gradient

9
Q

HPLC setup

A

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

10
Q

Mobile phase reservoirs

A

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

11
Q

Guard column

A

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

12
Q

HPLC stationary phase

A

Usually silica-based

The silica is often modified based on the specific separation being carried out

13
Q

HPLC solid phase

A

Can be silica, octadecyl, octyl or aminopropyl

14
Q

Reverse-phase chromatography

A

Compounds are separated based on their hydrophobicity and are eluted with a relatively non-polar solvent

15
Q

Normal-phase chromatography

A

In normal phase chromatography the solid phase is polar and the polarity of the solvent is gradually increased, leading to elution of compounds

16
Q

Applications of HPLC

A
  • Proteomics
  • Lipidomics
  • Metabolomics
  • Clinical toxicology