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Summary - Advanced Biochemical Analysis of Food
1.1 General principles of chromatography
What is the definition of chromatography?Chromatography is any technique aimed at separating compounds (for analytical or preparative purposes) through competition between a stationary phase and a mobile phase moving in contact with it in a definite direction.
What are the 2 types of chromatography?
- Gas chromatography: mobile phase is a gas
- Liquid chromatography: mobile phase is a liquid
What is the retention time?The tame it takes for a compound to elute. This goes according to there affinity for the stationary and mobile phase.
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 is the resolution?The ability to separate compounds.
A resolution of 1.5 or hihger ensures a perfect peak seperation.
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
What are the general requirements for the detector?
- to have the highest possible sensitivity, i.e. ensuring a strong response to the analyte presence, which allows the detection also of very small amounts of the analytes;
- to show a wide linear range spanning over several order of magnitude of amounts injected;
- to show a stable response, ensuring a constant response in presence of the same amounts of compounds;
- when needed, to be specific, i.e. able to specific detect only the analytes of interest in a complex mixture
- when needed, to be broad, i.e. able to detect the highest possible number of compounds present in a complex mixture, in an ideal case (which is never achieved) all of them.
Technical constraints limit the choice of detectors: typically LC and GC have different detection systems.
1.2 Sample preparation
What are the most common methods for sample preparation in solid samples?
- Solid-liquid extraction: sample is placed in closed container and solvent is added that dissolves/extracts/leaches the analyte of interest; solution is separated from solid by filtration.
- Soxhlet Extraction: sample is placed in disposable porous container (thimble); constantly refluxing fresh solvent flows through the thimble and dissolves analytes that are continuously collected in a boiling flask.
- Homogenization: sample is placed in a blender or a mechanical homogenizer, solvent is added, and sample is homogenized to a finely divided state; solvent is removed for further workup.
- Sonication: use of ultrasound to create vigorous agitation at the surface of a finely divided solid material;
- Dissolution: sample is treated with dissolving solvent and taken directly into solution with or without chemical change
What are the most common methods for sample preparation in liquid samples/suspensions?
- Solid Phase Extraction (SPE): sample is applied to, and liquid is passed through, a column packed solid phase that selectively removes analyte (or interferences)
- Liquid-Liquid Extraction: sample is partitioned between two immiscible phases which are chosen to maximize differences in solubility; interference-free analytes are then recovered from one of the two phases
- Dilution: sample is diluted with solvent which is compatible with HPLC mobile phase or GC stationary phase
- Evaporation: liquid is removed by gentle heating at atmospheric pressure with flowing air or inert gas
- Lyophilization: aqueous sample is frozen and water removed by sublimation under vacuum.
- Distillation: sample is heated to boiling point of solvent and volatile analytes in the vapor phase are condensed and collected.
- Filtration: liquid is passed through paper or membrane filter or SPE cartridge/disk to remove suspended particulates.
- Centrifugation: sample is placed in tapered centrifuge tube and spun at high force (thousands to hundreds of thousands times gravity); supernatant liquid is decanted.
- Sedimentation: sample is allowed to settle when left undisturbed in a sedimentation tank
What are the most commen methods used for sample preparation for gaseous samples?
- Solid or Liquid Phase Trapping: Gaseous sample passed through tube packed with adsorbent (e.g. silica gel, activated carbon)and the eluted with a solvent or directly trapped in a solvent.
- Headspace Sampling: The sample is placed in a closed, thermostated glass vial until equilibrium is established; at equilibrium, analytes partition themselves between a gas phase and the solid (or liquid) phase at a constant ratio; gas phase is sampled and injected into GC for analysis.
- Purge and Trap (Dynamic Headspace): the sample is placed in closed, thermostated container and the headspace vapors are continually removed by means of inert gas flow with subsequent trapping of sample components then thermally desorbed into GC injection port.
- Solid Phase Microextraction (SPME): Fused silica fiber coated with polymeric stationary phase is placed in headspace above sample or directly into liquid sample; analytes diffuse and partition/adsorb onto stationary phase; analytes are thermally desorbed by placing fiber into GC injection port or displaced by means of a liquid to a column for HPLC analysis.
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