Chromatography and its reading methods

Background
- In toxicology, it is used to determine the molecules of interest.
- Chormatography is the method to separate the molecules --> you read the results of chromatograme (time on the x-axis)
- In general types of column:
   - gas: long, with membrane, oven to heat the molecules.
   - liquid: short, granules inside, pressure needed to pass the molecules in the column (ultra high pressure, high pressure), can be used directly.
- After the separation, several methods can be used to 'read' the molecules

'Reading' methods
- Flame Ionized Detection (FID)
  - in our lab, we used it to detect ethanol.
  - the molecule should appear at the same timepoint as moleucles on the databank on the x-as.
  - the height of the peak (and the correlated area under the curve) say about the concentration of the molecule.

Diode Array Detection (DAD)
- Measures the light.

Mass spectrometry (MS)
- It fragments the molecules that already passed through the chromatography column and give them charges.
- The charged molecules will pass through the MS column and sort them based on the molecular weight.
- m/z: mass over the charge. If the charged molecule is only one, than m/z is m.
- can be combined with other MS.

How to read the results
- the combination of chromatographs and the mass spectrometer.
- the computer compares the peak with the database: area under the curve (larger than higher concentration), retention index (longer than later) and the match.
- check whether all high peaks on chromatogram are represented.