Summary Advanced Biochemical Analysis of Food

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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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What is the difference between enzyme specificity and enzyme selectivity?
Enzyme specificity is the AA aftehr which the enzyme can cleave the peptide bond.
Enzyme selectivity is the rate of hydrolysis of each cleavage site. Compared to the total sum of rates of hydrolysis of all cleavage sites.
So specificity is about where the cutting of the peptide bond can happen and slectivity is about how fast it can go
On what two things are analysis based?
  • Direct analysis - Molecular composition 
  • Fractionation - Solubility (e.g. in polar or a-polar solvents)
Why do people genetically modify cells for a assay?
They change the properties in such a way that the results of the toxin is a easily measured effect
What are the is the integrative, specificity, high throughput, ethical considerations for organismal, tissue, cell, enzyme and receptor bioassays?
See sceme.
What is a single marker approach based on?
Looking if certain DNA is present or not. Looking if you have a certain compound proving there is fraud. Mainly looking at one thing. However geographic origin, farming management systems or the application of special processes can not be detected int his way.
What are targeted methods/single marker approach?
Methods that target 1 thing for instance:
  • Moisture content 
  • Melamine testing 
  • Stabile isotope measurements 
What is the problem with the little spectroscopes?
The situation is not controlled. Manny factors that can influence the measurement and give a false result.
What is a new trent in food safety?
Little spectroscopes gives a IR spectrum telling you if the product is real or not.
What was the result of the egg experiment?
Biological eggs are all in the same range and normal eggs have a big variation.
What group of egg sampels were taken and what was doen with them
They polled the eggs in order to get the mean of the farm.