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A snapshot of the summary - Advanced Biochemical Analysis of Food
1.1 General principles of chromatography
What is the LOD, LOQ, LOL and dynamic/linear range?LOD: limit of detection, The minimum amount producing a signal significantly different from the background noise
LOQ: Limit of quantification, The minimum amount from which the area under the eluting peaks can be measured reliably
LOQ is always higher than LOD
LOL: Limit of Linearity, The upper limit for getting a linear response
dynamic /linear range: The range from the amount corresponding to the LOQ and the one corresponding to the LOL
With what part of the chromatographic system can you do a quantification?Quantification (with proper calibration) can be properly made only in the dynamic range
What are the factors contributing to a good resolution?
- Efficiency: number of theoretical plates (number of equilibria between mobile and stationary phase) for unit of space, resulting in narrow peaks
- Retention: the ability of the stationary phase to “retain” an analyte, resulting in higher retention times
- Selectivity: the differential interactions of the different analytes with the stationary phase
What is the difference between the peaks shown here?First 2: Same retention, same selectivity, improved efficiency of first one then you get better resolution
Last 2: Efficiency is increased, but at the same time retention is decreased, while selectivity is mostly unchanged: identical resolution
What are the requirements for gas chromatography and liquid chromatography?For gas chromatography: analytes have to be volatiles (at the temperatures used)
For liquid chromatography: analytes have to be soluble (in the solvents used)
The compounds should always go into the mobile phase.
It should also have some affinity both for the stationary and the mobile phase.
With what are you going to analyse aroma compounds, proteins and insoluble dietary fibers?Aroma compounds : gas chromatography
Proteins: liquid chromatography
Insoluble dietary fiber (as such) : chromatography not possible if no changes are made on the fiber
What happens if the compound has no affinity for either the mobile ore the stationary phase?
- If they have no affinity for the stationary phase, they will stay in the mobile phase all the time and they all elute immediately: no separation
- If they have no affinity for the mobile phase, they will be stuck in the column and never seen again: no separation
1.3 Gas Chromatography (GC)
What are the principles of separation in GC?
- High temperatures used (in the range 50C – 350C) to increase volatility of compounds
- The most volatile compounds (lower boiling points) elute first
- For compounds of similar volatility, the ones with polarity most similar to the stationary phase will elute later (more retained)
How is the coating of the columns done in GC?A silica tube with coatings of derivatized polysiloxane. This gives different polarity to the stationary phase.
Most common is 5% phenyl-95% methyl-siloxane
How do you get the best separation in your gc?Best separation is achieved by choosing stationary phases with polarity similar to that of the analytes to be separated