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FNH 302 > Post Quiz 2 Material > Flashcards

Post Quiz 2 Material Flashcards

1
Q

What are the basic components of a MS? [5]

A
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2
Q

What are the unique aspects of data that MS provides?

How is this useful in the analysis of foods?

A
  • Provides for detection and identification of an unknown compound.
  • Useful when you need to identify a specific component of food.
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3
Q

What is EI ionization?

A
  • A fragmentation method
  • Once in the ion source, the compound is exposed to a beam of electrons emitted from a filament composed of rhenium or tungsten metal
  • When a direct current is applied to the filament, it heats and emits electrons that move across the ion chamber toward a positive electrode on other side.
  • As the electrons pass through the source region, they come in close proximity to the sample molecule and extract an electron, forming an ionized molecule
  • Once ionized, the molecules are unstable and through a series of reactions, breaks into smaller molecular fragments.
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4
Q

What is CI ionization?

A
  • A fragmentation method
  • A gas is ionized (e.g., methane) which directly ionizes the molecule
  • ‘soft ionzaton’
  • Only a few fragments are produced
  • Most important use is to determine the molecular ion
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5
Q

What is the base peak on a mass spectrum?

A
  • The fragment that has the highest abundance or intensity.
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6
Q

What is the precursor ion peak?

A
  • Peak that has the highest mass number and represents the positively charged intact molecule with an m/z equal to the molecular mass
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7
Q

What is the difference between nominal mass and mono-isotopic mass?

A
  • Nominal mass (used synchronously with molecular mass) → the sum of the integer mass of the most abundant isotope for each element, i.e., C=12; H=1; N=14; O=16
  • Mono-isotopic mass → the sum of the most abundant isotopic mass for each of the constituent elements, i.e., C=12.0000; H=1.007825; N=14.003074; O=15.994915
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8
Q

What does MALDI stand for and how does it differ from ESI?

A
  • In MALDI (matrix assisted laser desorption/ionization)→ the sample is dissolved in a matrix and ionized using a UV laser. Thus the matrix plays an important role in ionization, acting both as the absorber of the laser energy which causes it to vaporize, and as a proton donor and acceptor to initiate charge transfer to the analyte
  • ESI → consists of a spray nozzle where the mobile phase from the HPLC exists. At the ESI tip a fine spray of highly charged droplets are produced in a nano-spray. Repulsive forces due to the accumulation of ‘like’ charges inside the rapidly reducing micro-droplet volume, creates a charged Taylor cone where ions are emitted as gas phase ions. These are then directly down the mass analyzer for ion separation and eventual detection.
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9
Q

What are the major differences between the quadrupole, ion trap, time of flight, and Fourier transform mass analyzer?

What are the advantages of using each analyzer?

What is especially unique about a Fourier transform-based mass analyzer?

A

TOF → ions leave the ion source with the same kinetic energy (KE) and travel a fixed distance to the detector. KE = ½mv2 (mass, m; velocity, v), so heavier ions have a lower velocity and lighter ions have a faster velocity, so the ions are separated base don their mass (and thus velocity); assuming that all ions have the same charge of +1 → ADVANTAGES: samples can be measured faster; higher sensitivity; higher resolving power

Quadrupole → uses four rods with varying electrical potentials that selectively filter ions very rapidly to scan a range of masses. It is fast and the detector can be made very small which explains its popularity in bench top MS instruments. However, resolution is not very good (about 0.5 mass units)

Ion trap → has been called a 3D quadrupole and is somewhat similar except ALL ions are trapped and then released over time to produce the MS spectra. It is also small in size and fast. Resolution is about the same as quadrupoles. A good resolution ion trap can perform tandem MS experiments (i.e., multiple MS/MS)

Fourier transform → Unique from other mass analyzers because the ions themselves are never resolved in space or time, nor are they detected by impinging upon a detector. Instead, the frequency is measured as a function of the applied electric or magnetic field. This results in sub part-per-million mass accuracy measurements.

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10
Q

What is the working principle behind the MALDI-TOF based microbiology identification?

A
  • In this technique, bacteria or fungi from the culture plates are directly spotted onto the MALDI target plate, sprayed with matrix, and then directly analyzed on the MALDI-TOF instrument.
  • The resulting spectrum, representative of the microorganism’s proteomic fingerprint (charged protein molecules) is matched against a known, verified spectrum in the library, and if there is a positive hit, the bacteria or fungi is rapidly identified.
  • This technique does depend on having known protein fingerprint spectra of the microorganisms in the library.
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11
Q

How does mass spectrometry work?

A

Molecule → ion (ionization) → separated according to m/z (mass analyzer) → (ion fragmentation (more structural information) → detector (count charges)

  • Mass-to-charge ratio (m/z/) =molecular mass/charge
  • Typically transfer positive charge to M → ‘positive ion mode’
  • Mass spectrometry measures the ‘spectrum’ of m/z (charged particles)
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12
Q

What are the applications of mass spectrometry?

A
  • Can accurately (down to 0.0001 Da) measure the mass of small and large molecules
  • Applications:
    • Molecular composition & structure
    • Food safety (toxins); food quality (nutrients); adulteration detection; bioactive compounds: proteomics; metabolomics
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13
Q

Describe mass spectra.

A
  • Base peak → most abundant ion
  • Precursor/parent ion → intact molecule
  • Product ions/daughter ions → fragments featuring successive cleavages
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14
Q

What is a Dalton?

A

1 Da = 1 g/mol = 1 amu

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15
Q

What is nominal mass?

A

Sum of integer mass of the > abundant isotopes; a.k.a. molecular mass

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16
Q

What is monoisotopic mass?

A

Sum of the > abundant isotopes of each element

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17
Q

What is average mass?

A

Sum of the average atomic masses of each element including all isotopes weighted by relative abundance

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18
Q

Summarize mass spectrometry instrumentation.

A
  • Sample introduction
    • Static method (e.g., direct injection)
    • Dynamic method (e.g., gas or liquid chromatography)
  • Ionization
    • Samples are vaporized (converted to gas phase)
    • Converts molecules to ions & fragments, each with a m/z
  • Mass analyzer
    • Separates ions/fragments based on their m/z
    • Analogous to the dispersion element in optical spectroscopy
  • Detector
    • measure ions using electron multipliers
    • similar to PMT’s used in optical spectroscopy
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19
Q

Describe vacuum use in mass spectroscopy.

A
  • All samples enter MS in gas phase
  • Operated under strong vacuum (10-8 - 10-11 atm)
    • Avoid collisions of ions with other molecules
    • For proper operation of instruments (ion lenses, mass analyzers and detectors)
    • For high accuracy and resolution
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20
Q

Describe the static method of sample introduction in MS.

A
  • Can be used for pure samples (that have at least some volatility)
  • Not for a complex mixtures of compounds
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21
Q

Describe the dynamic method of sample introduction in MS.

A
  • Sample must be separated into individual compounds, then analyzed by MS.
  • Required for complex mixtures of several compounds
  • e.g., GC-MS or LC-MS
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22
Q

Briefly list ionization methods in MS. [5]

A
  • Electron Impact ionization (EI) → used for GC-MS, small volatile compounds
  • Chemical ionization (CI) → ‘soft’ ionization = complementary to EI
  • Atmospheric Pressure Chemical Ionization (APCI) → used for LC-MS, small, low polarity compounds
  • Electrospray Ionization (ESI) → used for LC-MS, small and large molecules
  • Matrix-Assisted Laser Desorption Ionization (MALDI) → for large biopolymers
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23
Q

Describe the principle of electron impact ionization in MS.

A
  • Used for GC-MS; small volatile compounds
  • Compound (M) exposed to a beam of electrons emitted from a filament (70eV)
  • Produces positive charged ions/radicals
  • M + e- → M•+ + 2e-
  • M•+ undergo fragmentation
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24
Q

Describe the principle of chemical ionization.

A
  • ‘Soft’ ionization = complementary to EI
  • Similar to EI, but that an excess of reagent gas is mixed with the sample
    • Reagent gas (e.g., CH4) is ionized
    • Reacts with sample (MH) to generate ions, (M-H)+ or (M+H)+
      • (M-H)+ due to hydride loss from MH
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25
Q

Describe the principle of the atmospheric pressure chemical ionization method of MS.

A
  • Used for LC-MS; small, low-polarity compounds
  • Sample enters vapourizer tube
    • N2 carrier gas
    • 400-500°C
    • Voltage applied at exit
    • ‘Corona discharge’ = ionization of gases (N2, H2O, M)
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26
Q

Describe the principle of electrospray ionization in MS.

A
  • Used for LC-MS; small and large molecules
  • Sample exits capillary as a spray (micro- to nano-droplets)
  • N2 carrier gas
  • electrical potential applied
  • charge repulsion builds as droplet size decreases = release of ions into gas
  • Generates positive or negative ions depending on the compound
  • Generates multiply charged ions (low m/z values even for large molecules, e.g., 2-100kDa)
  • ‘Soft’ ionization method as fragmentation is rare
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27
Q

Describe the principle of the MALDI ionization method in MS.

A
  • For large biopolymers
  • Sample dissolved in a matrix (usually with a weak organic acid with strong UV absorbing properties)
    • Strong UV absorption 266-355nm
  • Ionized using a UV laser
  • Matrix (M) absorbs UV and becomes ionized (M+H)+
  • Matrix transfers charge to sample (S+H)+
  • Some instruments allow selection of positive or negative ion mode
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28
Q

Briefly list the types of mass analyzers used in MS.

A
  • Quadrupole (Q)
  • Ion Trap (IT)
  • Time of Flight (TOF)
  • Fourier-transform (FT) based analyzers
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29
Q

Describe a quadrupole (Q) mass analyzer.

A
  • Four rods used to generate two but out-of-phase electric potentials:
    • One is alternating current (AC)
    • Second is direct current (DC)
    • Potentials are varied such that ions are attracted/repulsed by rods
    • Only ions of certain m/z will get through = ‘mass filter’
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30
Q

Compare IT vs Quad mass analyzers.

A
  • Ion trap → stable ions are trapped; unstable ions are ejected and detected
  • Quadrupole → stable ions reach detector; unstable ions hit rods and are pumped away
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31
Q

Describe the principle of an ion trap (IT) mass analyzer.

A
  • Consists of a donut-shaped ring electrode + end-cap electrodes
  • Voltage (variable) is applied to the ring
  • Ions with certain m/z circulate in stable orbit
  • As voltage is increased, lighter m/z ions escape, and heavier ions are trapped
  • Released ions pass through gap to detector
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32
Q

Describe the principle of the Time of Flight (TOF) mass analyzer in MS.

A
  • Separates ions according to time required to reach detector
    • Ions leave source with same kinetic energy (KE)
    • Heavier ions are slower
    • Lighter ions are faster
    • All ions travel a known distance
    • Lighter ions reach the detector first
    • Increasing the path length increases resolution/separation of ions
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33
Q

Describe Fourier-Transform (FT) mass analyzers. [2]

A
  • FT-Ion Cyclotron Resonance (FT-ICR) MS
    • Ion trap based on trapping ions in a magnetic field
    • Measure frequency of ion motion as a function of applied magnetic field
    • Use FT to process data into mass-spectra
  • FT-Orbitrap MS
    • Ion trap based on trapping ion in an electric field
    • Measure frequency of ion motion as a function of applied electric field.
    • Use FT to process data into mass-spectra
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34
Q

Discuss the resolution of MS.

A

Defined in terms of peak FWHM; capable of very high resolution

FWHM = full-width at half-max

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35
Q

What is tandem, MS/MS?

A
  • Connect mass analysers in series
  • (1) Ion m/z separation, (2) fragmentation, (3) ion m/z separation
  • Fragmentation via collision with ions (gases introduced); e.g., collision induced dissociation
  • Allows for more structural information & unambiguous comparison with spectral libraries
  • Image = triple quadrupole
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36
Q

Describe MS application for identification of fatty acids.

A
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37
Q

Describe green tea extract with LC-MS.

A
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38
Q

Describe caffeine f. coffee extract with LC-MS/MS.

A

More fragmentation can provide a more detailed fingerprint (i.e., important information to help discern between molecules of the same mass)

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39
Q

Describe how MS can be used to determine how many different proteins there are in milk.

A
  1. Pre-fractionate using ion-exchange chromatography, IEX
  2. Chop into smaller peptides using proteases (trypsin)
  3. Use LC-MS/MS* to identify small peptide fragments (e.g., ~6 a.a. = 600 Da)
  4. Match peptide fragments against entire gene/protein sequences (databases) to identify protein.
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40
Q

What is chromatography?

A

A method to separate components in a mixture

Chromatography is a general term applied to a wide variety of separation techniques based on partitioning of sample (solute) between moving (mobile) phase and a fixed (stationary) phase.

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41
Q

Describe phases in chromatography and how they allow for separation of a mixture of compounds.

A
  • Mobile phase → gas (GC); liquid (LC); or supercritical fluid (SFC)
  • Stationary phase → liquid or solid
  • Series of equilibrations between mobile and stationary phase
  • Relative interaction of a solute with these two phases is described by the partition coefficient (K) or (D)
  • Achieve separation of compounds having different D values (i.e., different affinities for mobile versus stationary phases)
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42
Q

Briefly list some kinds of chromatography.

A
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43
Q

Describe the principle of paper chromatography.

List a few applications.

A
  • Paper (cellulose) serves as a support for the liquid stationary phase
  • Stationary phase is usually water
  • Mobile phase is an organic solvent
  • Sample is applied as a small spot or streak
  • Suspend paper in a closed container
  • Solvent travels up (ascending) or down (descending) the paper to develop the separation.
  • Components of a mixture are characterized by their relative mobility, Rf
  • Applications → separation of small molecules (dyes, amino acids, peptides, sugars, purines)
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44
Q

What is Rf?

A

Relative mobility

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45
Q

What is the principle of thin layer chromatography?

A
  • Similar principle to paper chromatography
  • Developed to replace paper chromatography
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46
Q

What are advantages of thin layer chromatography over paper chromatography? [3]

A
  • Better resolution
  • Faster
  • More reproducible
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47
Q

What are a few applications of thin layer chromatography?

A
  • Screen corn and peanuts for mycotoxins before processing
  • Lipids, carbohydrates, vitamins, amino acids, natural pigments
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48
Q

What is the most basic set-up for column liquid chromatography?

A
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49
Q

What is a typical set-up for column liquid chromatography?

A
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50
Q

What is the principle of column liquid chromatography?

A
  • Solutes fractionated by differential migration through a tube of stationary phase
    • Mobile phase is liquid;
    • Stationary phase is a solid, or a liquid supported by an inert solid
    • Stationary phase is packed in column and equilibrated with mobile phase
    • Mobile phase moves through column by gravity flow or pump
    • Elution may be isocratic (constant mobile phase composition) or a gradient (changing nature of mobile phase)
    • Solutes are separated based on strength of interaction with stationary phase
    • Column eluate is collected
    • Detector response is recorded as chromatogram
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51
Q

Discuss principles of separation in chromatography.

A
  • Modes of interaction
    • Adsorption
    • Partition
    • Hydrophobic interaction
    • Ion exchange
    • Affinity
    • Size exclusion
      • >1 mode of interaction can occur in a given separation
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52
Q

Discuss non-covalent interactions.

A
  • Ion-ion → very strong
  • Ion-dipole → moderately strong
  • Dipole-dipole → weak
  • Dipole-induced dipole → quite weak
  • Instantaneous dipole-induced dipole → very weak
  • Van der waals
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53
Q

Describe hydrogen bonding.

A
  • H-bond donor must be an electronegative atom (N, O, F)
  • H-bond acceptor must be an electronegative atom (N, O, F)
  • H-bond strength of ~2-8kcal/mol → distance/orientation dependent
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54
Q

Describe the hydrophobic effect.

A
  • Water molecules organize themselves around non-polar groups → driven by increased entropy (lower energy)
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55
Q

Describe adsorption chromatography.

A
  • Liquid-solid chromatography
  • Stationary phase (adsorbent)
    • Finely divided solid (max. surface area)
    • Permit differential interaction with components to be separated
    • Commonly used: silica (acidic), alumina (basic)
  • Mechanism:
    • Solvent and solutes compete for binding sites on the surface
    • Changing the strength of the mobile phase will alter solute-surface interactions
      • e.g., increasing polarity of the mobile phase to elute the solute from a polar surface
  • Intermolecular forces → electrostatic; v.d.w.
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56
Q

Describe normal phase chromatography (a type of partition chromatography)

A
  • Principle → compounds partition between two phases
    • Stationary phase → liquid phase immobilized on inert support material
    • Mobile phase → liquid eluting through the column
  • Normal phase chromatography
    • Polar stationary phase
    • Nonpolar mobile phase
      • Most nonpolar solutes are the first to elute
      • Best for hydrophilic substances e.g., CHO, amino acids, pigments
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57
Q

Describe reverse phase chromatography.

A
  • Principle → compounds partition between two phases
    • Stationary phase → liquid phase immobilized on inert support material
    • Mobile phase → liquid eluting through the column
  • Reverse phase chromatography
    • Non-polar stationary phase
    • Polar mobile phase
      • Most polar solutes are first to elute
      • Best for hydrophobic compounds e.g., lipids, phenolics, peptides
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58
Q

Reverse phase chromatography is best for hydrophilic substances e.g., CHO, amino acids, pigments.

True or False?

A

False.

  • Reverse phase chromatography
    • Non-polar stationary phase
    • Polar mobile phase
      • Most polar solutes are first to elute
      • Best for hydrophobic compounds e.g., lipids, phenolics, peptides
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59
Q

Reverse phase chromatography is best for hydrophobic compounds e.g., lipids, phenolics, peptides

A

True.

  • Reverse phase chromatography
    • Non-polar stationary phase
    • Polar mobile phase
      • Most polar solutes are first to elute
      • Best for hydrophobic compounds e.g., lipids, phenolics, peptides
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60
Q

Normal phase chromatography is best for hydrophilic substances e.g., CHO, amino acids, pigments.

True or False?

A

True.

  • Normal phase chromatography
    • Polar stationary phase
    • Nonpolar mobile phase
      • Most nonpolar solutes are the first to elute
      • Best for hydrophilic substances e.g., CHO, amino acids, pigments
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61
Q

Normal phase chromatography is best for hydrophobic compounds e.g., lipids, phenolics, peptides.

True or False.

A

False.

  • Normal phase chromatography
    • Polar stationary phase
    • Nonpolar mobile phase
      • Most nonpolar solutes are the first to elute
      • Best for hydrophilic substances e.g., CHO, amino acids, pigments
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62
Q

In normal phase chromatography most non-polar solutes are first to elute.

True or False?

A

True.

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63
Q

In normal phase chromatography most polar solutes are first to elute.

True or False?

A

False.

In normal phase chromatography most non-polar solutes are first to elute.

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64
Q

In reverse phase chromatography, most non-polar solutes are first to elute.

True or False?

A

False.

In reverse phase chromatography most polar solutes are first to elute.

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65
Q

In reverse phase chromatography most polar solutes are first to elute.

True or False?

A

True.

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66
Q

Compare and contrast normal and reverse phase chromatography.

A
  • Normal → polar stationary; non-polar mobile; most non-polar solutes elute first
  • Reverse → non-polar stationary; polar mobile; most polar solutes elute first
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67
Q

Describe supports used in partition chromatography.

A
  • Coated supports → liquid coating on a solid matrix
    • Silica gel (hydrated silica)
    • Cellulose
    • Glass beads
      • Disadvantage → liquid stationary phase is often stripped off
  • Bonded supports → liquid stationary phase covalently bonded to a support via a chemical reaction
    • Very common in reversed-phase HPLC (silica bonded to C8 or C18)
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68
Q

Describe the principle of hydrophobic interaction chromatography.

A
  • Biomolecules adsorb to a weak hydrophobic surface at high salt concentration
  • Elute adsorbed molecules by decrease in [salt] in mobile phase over time
  • Takes advantage of hydrophobic moieties on surface of compound
  • Salt screens charges (repulsive interactions)
  • Used to purify proteins, enzymes, antibodies, etc.
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69
Q

Describe the phases in hydrophobic interaction chromatography.

A
  • Stationary phase
    • Hydrophilic support (e.g., cellulose, agarose, silica)
    • Bonded to hydrophobic ligands (e.g., butyl-Sepharose)
  • Mobile phase
    • Start with buffered 1M ammonium sulfate, then decrease salt to 0M
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70
Q

Describe the principle of ion-exchange resins.

A
  • A type of adsorption chromatography
  • Electrostatic interactions between solute and stationary phase
  • Stationary phase contains fixed functional groups, with positive or negative charge
  • Cation exchangers → contain negative groups; will bind cations (positive solutes)
  • Anion exchangers → contain positive groups; will bind cations (negative solutes)
  • Elution → change pH; increase ionic strength (add NaCl) to screen ES interactions wit solute
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71
Q

Describe the principle of affinity chromatography. List a few applications.

A
  • A type of adsorption chromatography
  • Separation based on reversible interaction between a solute and an immobilized ligand
    • Antibodies (specific)
    • Enzyme inhibitors (specific)
    • Lectins (general) → CHO-binding proteins
  • Stationary phase → high surface area, inert (e.g., agarose, cellulose, dextrans); spacer arm = holds ligand away from surface/access solute
  • Applications → purification of pesticides, polysaccharides, mycotoxins, proteins (enzymes, antibodies, anything with a known ligand)
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72
Q

Describe the elution process of affinity chromatography.

A
  • Analyte introduced to ligand
  • Analyte binds ligand
  • Disrupt interaction → nonspecific = change pH, temp, [salt]; specific = add excess of competing ligand
  • Rinse column; ready for next use
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73
Q

Describe the principle of size-exclusion chromatography. List some applications.

A
  • Molecules are separated based on size (hydrodynamic radius)
  • Stationary phase = solid material (agarose; dextrans) with pores ~ size of molecules of interest
  • Separation
    • Molecules too large to enter the pores travel with the mobile phase in the interstitial phase & elute first
    • Smaller molecules get slowed down by entering the pores & elute later
  • Applications → purification of proteins, polysaccharides, large polymers
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74
Q

Describe the pumps used in HPLC.

A
  • Deliver mobile phase through system, at a typical flow rate of ~0.4-1mL/min
  • Reciprocating, piston-type pump, or dual piston pump
  • Sensitive to dust and particulate matter
  • Usually filter mobile phase (0.45 or 0.22 um filters), and degas (to avoid air bubbles in pump and detector
    • Single pump = isocratic flow
    • Binary pump = isocratic flow/gradient flow
    • Quad pump = isocratic flow/gradient flow
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75
Q

Discuss the HPLC injector.

A
  • Valve injectors → separate sample introduction from high-pressure eluent system
  • LOAD position → sample loaded into external, fixed-volume loop
  • INJECT position → loop becomes part of eluent flow stream
  • Fixed-volume loading loops allow different volumes of sample to be injected (usually 10-100ul; could be much more or less)
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76
Q

What is an HPLC autosampler?

A
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77
Q

What are the two types of HPLC columns?

A
  • Pre-columns (guard columns)
    • Installed between injector and analytical column
    • Same media type as analytical column
    • Protect analytical column from adsorbed materials
  • Analytical columns
    • Varying lengths and diameters
    • Small diameter columns (often called ultra-HPLC)
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78
Q

Describe silica-based column packings in HPLC.

A
  • Bonded phases → hydrocarbons covalently bonded to -OH groups on silica particles
  • Pellicular packing material → thin layer deposited on an inert particle
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79
Q

Describe porous polymeric column packings in HPLC.

A
  • Microporous (gel-type) → crosslinked co-polymers
  • Macroporous → highly cross-linked; network of smaller beads joined to form a larger bead
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80
Q

List detectors used in HPLC. [5]

A
  • UV-Visible Absorption
  • Fluorescence
  • Refractive Index
  • Amperometric
  • Conductivity
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81
Q

Describe UV-Visible absorption as a detector in HPLC.

A

Selective

Sensitive

  • Single wavelength → filter used to select
  • Multi/variable wavelength → wavelength selection by monochromator; can monitor several wavelengths/entire spectra (slow
  • Diode Array Detector (DAD)
    • Light is dispersed into a spectrum that falls across an array of photodiodes; DAD scans 200-700nm every 0.1s → continually generates spectra (abs as function of time & wavelength)
    • Obtain a full spectra for each time point in chromatogram
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82
Q

Describe fluorescence detection in HPLC.

A

Selective

More sensitive than UV-Vis Absorbance

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83
Q

Describe refractive index detection in HPLC.

A
  • Measure change in RI of mobile phase due to presence of dissolved analytes
  • Non-selective
  • Sensitive to temperature & flow rate
  • Not used with gradient elution
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84
Q

Describe amperometric detection in HPLC.

A

Measure electrochemical oxidation-reduction of analyte

Sensitive

Selective

e.g., used for phenolics, CHO

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85
Q

Describe conductivity detection in HPLC.

A

Measure change in conductivity of eluent

Sensitive

Non-selective

e.g., used for ions

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86
Q

Describe the fraction collector in HPLC.

A

Collect fractions as a function of time or volume

Further analysis of separated components

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87
Q

Describe reverse phase HPLC.

A
  • >70% of HPLC are reverse phase
  • Partitioning of molecules according to their hydrophobicity & polarity between the stationary phase (column; nonpolar) & the mobile phase (solvent; polar)
  • Nonpolar stationary phase (e.g., C18)
  • Polar mobile phase (e.g., water with methanol; acetonitrile)
  • Gradient elution → increase nonpolarity of mobile phase
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88
Q

Describe normal phase HPLC.

A
  • Gradient elution → increase polarity of mobile phase, e.g., hexane + CH2Cl2
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89
Q

List some applications of HPLC in analysis of food.

A
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90
Q

What parameters define a chromatographic peak? [4]

A
  • retention volume, VR
  • retention time, tR
  • peak width, w
  • peak height, h
  • Note t0 = dead-volume
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91
Q

What factors influence tR?

A

Retention time, tR

Influenced by → nature of stationary/mobile phases; column dimensions, temperature, flow rate, system dead-volume

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92
Q

What is adjusted retention time, t’R?

A

It corrects for differences in system

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93
Q

What defines resolution in HPLC?

A

Resolution is a function of efficiency (a), selectivity (b), and capacity (c ).

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94
Q

What is a theoretical plate?

A

Site of binding/unbinding of solute from stationary phase

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95
Q

What is HETP and what does it depend on?

A

Height equivalent to a theoretical plate = HETP = a.k.a. ‘plate height’, H

  • Smaller H (>N) = minimal spread of solute, sharper peaks
  • HETP depends on (1) particle size, (2) column diameter, and (3) flow rate
  • HETP = L/N
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96
Q

Describe the Van Deemter equation & plot.

A
  • eddy diffusion = multiple flow paths (A)
  • at low flow rate = solutes diffuse from peak centre (high → low concentration) (B/u)
  • at high flow rate = solutes not given enough time to separate (Cu)
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97
Q

How can the effects of eddy diffusion on the efficiency of HPLC be reduced? [2]

A

Reduce effects by → narrow column; smaller particle size

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98
Q

Discuss how column length relates to efficiency of HPLC.

A

L = HETP * N

  • For a given type of column (same H), a longer column will have greater N
  • Greater N improves the resolution (4x increase in N = 2x increase in Rs)
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99
Q

What does selectivity refer to in HPLC?

A
  • Refers to the separation between two peaks
  • A function of the stationary phase and mobile phase
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100
Q

What does capacity refer to in HPLC?

A
  • A measure of the amount of time the solute spends in/on the stationary phase, relative to the mobile phase
  • Small k’ = solute spends little time on the column (poor separation)
  • Large k’ = solute spends >>time on the column (good separation; but, broader peaks)
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101
Q

Describe qualitative analysis in HPLC.

A
  • Identify unknown compounds (peaks) by:
    • (1) Compare tR or VR to standarsd, run on the same system
    • (2) compare t’R if using different systems/columns
    • (3) spike sample with standard - observe which peak height/area increases
  • Different compounds may have identical retention times! Use other methods:
    • (4) compare UV-Vis spectra of sample & standard (e.g., PDAD)
    • (5) compare ratio of absorption/fluorescence signal (unique for different compounds)
    • (6) collect peak and run using another spearation mode
    • (7) use another analytical technique, e.g., mass spectrometry, to ID compounds
  • Also, chromatogram can be used to show absence of compounds in a sample
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102
Q

Describe quantitative analysis in HPLC.

A
  • External standard
    • Prepare calibration standards & analyze
    • Plot calibration curve: Astd vs [standard]
    • Measure unknown sample (Aunk) and use calibration curve to determine [unknown]
  • Internal standard
    • Prepare calibration standards that also contain internal standard
    • Plot calibration curve: Astd/AIS versus [standard]
    • Add internal standard to known sample
    • Measure Aunk/AIS and use calibration curve to determine [unknown]
    • IS = similar but distinct from analyte
    • Advantages = nullifies errors due to loading & instrument response
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103
Q

Compare adsorption vs. partition chromatography.

Nature of stationary phase

Nature of mobile phase

How solute interacts with phases

A

Adsorption: stationary phase → solid; partition → liquid or gas; interactions → v.d.w. forces; electrostatic interactions; hydrogen bonding; hydrophobic interactions

Partition → stationary phase → liquid; partition → liquid or gas; interactions → solute partitions between liquid and stationary phases according to partition coefficient

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104
Q

Compare normal-phase vs. reverse-phase chromatography.

Nature of the stationary phase

Nature of the mobile phase

What elutes last

A

Normal-phase: Stationary → polar; mobile → nonpolar; elutes last → most polar compounds

Reverse-phase → stationary → nonpolar; mobile → polar; elutes last → most nonpolar compounds

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105
Q

Compare internal and external standards.

Nature of standards

How standards are handled in relation to sample

What is plotted on the standard curve

A

Internal: nature → not present in sample; added in constant amount to sample and standard; plot ratio of peak height or area of internal standard vs. other standard compounds (y-axis) vs. concentration of standard compounds (x-axis)

External: nature → same as present in sample; prepare standard of different concentrations and inject separate from sample; plot peak height or area of each standard vs. concentration of standards

106
Q

Compare TLC vs. column liquid chromatography.

Nature and location of the stationary phase

Nature and location of the mobile phase

How the sample is applied

Identification of solutes separated

A

Thin-layer C: stationary phase → solid (or liquid) in thin layer on plate or sheet; mobile phase → liquid at bottom (and/or top) of chamber); sample applied as dot on stationary phase at bottom of plate/sheet; visualize bands by chemical reaction or fluorescence

Column LC: stationary phase → liquid (or solid) inside column; mobile phase → liquid passes through column; apply sample to top of column; use detectors

107
Q

What is the advantage of bonded supports over coated supports for partition chromatography?

A

With bonded supports, the stationary phase may be covalently attached to the support material, so the stationary phase is not stripped off with continued use of the column.

108
Q

You are performing LC using a stationary phase that contains a polar nonionic functional group. What type of chromatography is this, and what could you do to increase the retention time of an analyte?

A
  • This is normal phase chromatography (polar stationary phase).
  • To increase retention time → decrease the polarity of the mobile phase to make the solute want to interact more with the column and less with the mobile phase
109
Q

You applied a mixture of proteins, in a buffer at pH 8.0, to an anion-exchange column. On the basis of some assays you performed, you know that the protein of interest is adsorbed to the column.

Does the anion-exchange stationary phase have a positive or negative charge?

A

Positive charge

110
Q

You applied a mixture of proteins, in a buffer at pH 8.0, to an anion-exchange column. On the basis of some assays you performed, you know that the protein of interest is adsorbed to the column.

What is the overall charge of the protein of interest that adsorbed to the stationary phase?

A

Negative charge

111
Q

You applied a mixture of proteins, in a buffer at pH 8.0, to an anion-exchange column. On the basis of some assays you performed, you know that the protein of interest is adsorbed to the column.

Is the isoelectric point of the protein of interest (adsorbed to the column) higher or lower than pH 8.0?

A

Lower

pH(buffer) > pH(protein) → binds to anion exchanger

pH(buffer) < pH(protein) → binds to cation exchanger

112
Q

You applied a mixture of proteins, in a buffer at pH 8.0, to an anion-exchange column. On the basis of some assays you performed, you know that the protein of interest is adsorbed to the column.

What are the two most common methods you could use to elute the protein of interest from the anion-exchange column? Explain how each method works.

A
  • change the mobile phase pH, to change the charge on the protein so it will no longer bind to the column
  • increase the ionic strength (e.g., add NaCl) to the mobile phase, to weaken the electrostatic interactions
113
Q

Explain the principle of affinity chromatography, why a spacer arm is used, and how the solute an be eluted.

A

Principle → a type of adsorption chromatography, in which separation is based on the specific, reversible interaction between a solute molecule and a ligand immobilized on the stationary phase.

The spacer arm is used to hold the ligand away from the support surface, enabling it to reach into the binding site of the analyte.

114
Q

What is the gradient elution from a column, and why is it often advantageous over isocratic elution?

A
  • Gradient elution refers to changing the mobile phase (e.g., increasing the ionic strength or pH) to (1) enhance resolution and (2) decrease analysis time.
115
Q

A sample containing compounds A, B, and C is analyzed via LC using a column packed with a silica-based C18 bonded phase. A 1:5 solution of ethanol and H2O was used as the mobile phase. The following chromatogram was obtained. Assuming that the separation of the compounds is based on their polarity:

Is this normal or reversed phase chromatography? Explain your answer.

A
  • The stationary phase is silica-based C18 bonded phase → a nonpolar material
  • The stationary phase is nonpolar and the mobile phase is polar
  • Reverse phase chromatography
116
Q

A sample containing compounds A, B, and C is analyzed via LC using a column packed with a silica-based C18 bonded phase. A 1:5 solution of ethanol and H2O was used as the mobile phase. The following chromatogram was obtained. Assuming that the separation of the compounds is based on their polarity:

Which compound is the most polar?

A
  • In reverse phase chromatography, the last eluted compound will be the most nonpolar
  • A is eluted first → A does not interact with the column, but interacts more with the mobile phase → A is the most polar
  • C elutes last → C interacts more with the nonpolar stationary phase → C is the most nonpolar
117
Q

A sample containing compounds A, B, and C is analyzed via LC using a column packed with a silica-based C18 bonded phase. A 1:5 solution of ethanol and H2O was used as the mobile phase. The following chromatogram was obtained. Assuming that the separation of the compounds is based on their polarity:

How would you change the mobile phase so compound C would elute sooner, without changing the relative positions of compounds A and B? Explain why this would work.

A
  • After ~6mins (after A and B are eluted), change to a less polar (i.e., more nonpolar) solvent (i.e., less water).
  • This would make compounds A and B elute as normal, but then would make the mobile phase to be more like the stationary phase, so compound C, which is more nonpolar, would elute sooner.
118
Q

A sample containing compounds A, B, and C is analyzed via LC using a column packed with a silica-based C18 bonded phase. A 1:5 solution of ethanol and H2O was used as the mobile phase. The following chromatogram was obtained. Assuming that the separation of the compounds is based on their polarity:

What could possibly happen if you maintained an isocratic elution model at low solvent strength?

A
  • Compound C might not elute because the mobile phase is too polar so that there is a chance that compound C might not interact with the mobile phase at all.
119
Q

Using the Van Deemter equation, HETP, and N, as appropriate, explain why the following changes may increase the efficiency of separation in column chromatography:

Changing the flow rate of the mobile phase.

A
  • Reduces HETP when flow rate is optimized, to provide for proper equilibrium but minimize longitudinal diffusion.
120
Q

Using the Van Deemter equation, HETP, and N, as appropriate, explain why the following changes may increase the efficiency of separation in column chromatography:

Increasing the length of the column

A
  • Increases the L (and the N), assuming HETP remains unchanged → leads to better resolution
121
Q

Using the Van Deemter equation, HETP, and N, as appropriate, explain why the following changes may increase the efficiency of separation in column chromatography:

Reducing the inner diameter of the column

A
  • Reduces the Eddy diffusion, to reduce the HETP.
122
Q

Using the Van Deemter equation, HETP, and N, as appropriate, explain why the following changes may increase the efficiency of separation in column chromatography:

Decreasing the particle size of the packing material

A
  • Reduces the Eddy diffusion, to reduce the HETP.
123
Q

State the factors and conditions that lead to poor resolution of two peaks. [5]

A
  • Lose column efficiency and resolution due to increase in HETP and reduced N.
  • Operating at lower or higher than optimum mobile phase flow rate.
  • Change in mobile phase variables, e.g., strength and temperature
  • Loss of column selectivity and decrease in capacity factor
  • Loss of column packing material or stripping off the functional groups of the stationary phase
124
Q

How can chromatographic data be used to quantify sample components?

A
  • Use the peak height or peak area to plot against concentration
125
Q

Why would you choose to use an internal standard rather than an external standard?

Describe how you would select an internal standard for use.

A
  • Use of internal standards can minimize errors due to sample preparation, apparatus noise, and operator technique (including injection volume). Injection volumes need not be accurately measured, and detector response need not remain constant.
  • Internal standard must not interfere chromatographically with components of interest in the sample. It must not be naturally present in the sample, but it should have a chemical structure similar to the compounds of interest
126
Q

To describe how using internal standards works, answer the following:

What specifically will you do with the standards?

What do you actually measure and plot?

How do you use the plot?

A
  • Add standard in constant amount to sample and standard solutions.
  • Peak height, area, or mass is measured. Ratios of peak height, area, or mass (compound of interest/internal standard) are calculated and plotted against concentration of standard to obtain calibration curves. A separate response curve must be plotted for each sample component to be quantified.
  • Peak height, area, or mass ratios (compound of interest/internal standard) are calculated and used to read concentration of each relevant component from the appropriate calibration curve plotted.
127
Q

Why might you choose to use HPLC rather than traditional low-pressure column chromatography? [6]

A
  • Speed
  • Improved resolution
  • Greater sensitivity
  • Reusable columns
  • Ideal for ionic species and large molecules
  • Easy sample recovery
128
Q

What is a guard column and why is it used?

A
  • A guard column is a short, expendable column used to protect the analytical column from strongly adsorbed sample components.
  • It is installed between the injector and the analytical column.
  • It contains the same packing material as the analytical column, but the particle size may be larger.
129
Q

What is the primary function of an HPLC detector (regardless of type)?

A

Primary function → translates concentration changes in the HPLC column effluent into electrical signals

130
Q

You are performing HPLC using a stationary phase that contains a nonpolar functional group.

What type of chromatography is this, and what could you do to increase the retention time of an analyte?

A
  • Stationary phase → nonpolar → reverse phase chromatography
  • To increase retention time → increase the polarity of the mobile phase, which will make the solute want to interact more with the column and less with the mobile phase.
131
Q

What is the principle of gas chromatography?

A

GC separation is based on partition between the mobile phase and either nonpolar or polar stationary liquid phase (affinity for stationary phase); also based on boiling point of compounds (volatility; lower BP = more volatile)

This is unique to LC where partition/affinity is the only factor.

132
Q

What analytes are suited for GC?

A
  • Thermally stable (< degradation products)
  • Volatile (i.e., enter vapour phase; don’t burn up in injector)
  • e.g., aromatics (flavour/aroma compounds), contaminants, pesticides
  • sugars, polyphenolics, fatty acids, amino acids → after derivatization
  • GC provides really good separation of complex mixtures
133
Q

What analytes are suited for HPLC?

A
  • Thermally unstable (otw > degradation products)
  • Non-volatile
  • Sugars, polyphenolics, fatty acids, amino acids, peptides, proteins, polysaccharides, vitamins
134
Q

Describe a gas chromatographic system.

A
  • Sample injected (T = ~250 degrees Celsius)
  • Vaporized
  • > samples can’t be injected directly → get degradation products from non-volatiles which would add ‘noise; extra peaks’
  • No pump as there is in LC → pressurized gas cylinder
  • Coiled column
135
Q

What is likely required prior to GC analysis? [4]

A
  • Sample preparation
  • Component isolation
  • Sample concentration
  • Sample derivatization
136
Q

What are volatile isolation methods in GC? [5]

A
  • Headspace sampling
  • Distillation
  • Solvent extraction
  • Solid-phase extraction
  • Direct injection
137
Q

What are headspace methods in GC?

A

Direct injection of headspace vapours above a food product

  • Direct (static) headspace sampling → headspace of sample is taken using a gas-tight syringe (e.g., yoghurt headspace to check for polystyrene contaminants)
  • Dynamic headspace sampling (purge and trap) → involves passing large volume of headspace vapours through a cryogenic trap or an adsorbent trap; Volatiles captured; Released (desorption) by heating or solvent
  • e.g., hexanal from lipid oxidation
138
Q

What are distillation methods in GC?

A
  • Distill then extract → co-distill sample with steam; collect distillate and extract with solvent; concentrate volatiles
  • Distil + extract → simultaneous distillation and extraction (SDE)
  • e.g., extraction of hop aromas
139
Q

What is solvent extraction in GC?

A
  • Liquid-liquid extraction
  • Two immiscible phases
  • e.g., water + organic solvent
  • Batch or continuous extraction
  • Followed by distillation to isolate volatiles from non-volatiles
140
Q

Compare solid phase extraction (SPE) to solvent extraction in GC.

A
  • Compared to solvent extraction, SPE:
    • Less solvent, glassware, and time required; efficient method
    • Better precision and accuracy
    • Readily automated
    • e.g., wine, bread, fruit, vegetables
  • Different phases → reverse (traps nonpolar compounds), normal (traps polar compounds), IEX (trapping ionic compounds), affinity
  • Process → load sample (analyte binds); wash off unbound; elute analyte
141
Q

Describe solid phase microextraction (SPME).

A
  • Stationary phase is bound onto a fine fused silica filament
  • Fiber is immersed in the sample, or in the headspace above the sample
  • After equilibration, fiber is removed from sample
  • Inserted into GC.
  • Adsorbed volatiles are thermally desorbed from fiber
  • Fibres are reusable/inexpensive
  • e.g., wide range of samples (environmental, biological, food, pharmaceuticals, fragrances)
142
Q

Describe sample derivatization.

A
  • Some compounds must be derivatized (chemically modified) before GC analysis:
    • are thermally unstable
    • are too low in volatility (e.g., sugars, amino acids)
  • Note in the image → not FAMEs → what are they?
    • They are ethers and ketones; the textbook drawing is incorrect :(
143
Q

Describe the analysis of sugars by GC.

A
  • Converted to sugar alcohols
  • Esterified to make them volatile
  • Without this preparation sugars may caramelize or degrade at the high temperatures required for GC
144
Q

Describe GC instrumentation.

A
  • Carrier, inert gas = mobile phase
  • Fraction collectors may be present; uncommon due to small sample sizes in GC
145
Q

What are important parameters to be recorded for each separation in GC instrumentation?

A
  • Sample
  • Injection
  • Capillary column
  • Packed column
  • Temperatures
  • Carrier gas
  • Detector
146
Q

What are the functions of the injection port in GC? [3]

A
  • Sample introduction
  • Sample vaporization → injector port is usually 20 degrees Celsius hotter than the column oven maximum temperature
  • Sample dilution and splitting
147
Q

Describe injection port volumes and types.

A
  • Small volumes
    • Inject 0.1-3ul sample (may be further diluted with a split port type)
    • Source of error → relative errors are large when measuring small volumes
    • Internal standards common with GC compared to HPLC for quantitative processes
  • Injection port types:
    • Split → sample is diluted 1:50-1:100
    • Splitless → all analyte is loaded
  • Direct on-column
  • Thermal desorption → sample heated - volatiles carried to split/splitless injector; e.g., for samples extracted with SPME
148
Q

Describe oven use in GC.

A
  • GC column housed in an ‘oven’
  • Temperature is essential to separation
  • Analytes with BP < oven temp will condense on the stationary phase
  • As oven temp increases, the analytes are >> in mobile phase
  • Isothermal
  • Temperature programming → program the temperature so that the temperature ‘ramps up’ over time to allow better separation of peaks
149
Q

Describe packed columns in GC.

A
  • Stainless steel or glass
  • 2-4mm I.D. & 0.5-5m long
  • Solid packed coated with liquid stationary phase
150
Q

Describe capillary columns in GC.

A
  • Hollow fused silica glass
  • 0.1-0.5mm I.D. & 5-100m long (allows for more theoretical plates → better resolution)
  • Stationary phase → liquid coating chemically bonded to glass walls of column, and internally cross-linked
  • 0.1-5um thickness
  • Thicker = better for more volatile compounds
  • Thinner = better for larger, less volatile compounds
  • Thicker film = more bleeding
151
Q

Describe the thermal conductivity detector (TCD) in GC.

A
  • Carrier gas passes over & cools a tungsten filament
  • Filament temperature is proportional to electrical resistance
  • Presence of analyte reduces cooling, increases temperature, increases resistance
  • Non-sensitive → least sensitive compared to other detectors; but useful for pairing with MS
  • Non-destructive
  • General → can detect almost anything
152
Q

Describe the flame ionization detector (FID) in GC.

A
  • Eluate passes through & burned in a hydrogen flame
  • Potential (300V) applied across the flame
  • Current flow is proportional to #ions in flame
  • Works only for organic compounds (C-C, C-H bonds)
  • > sensitive than thermal conductivity detector
  • Destructive (burning sample; cannot connect with MS)
  • Specific to organics (proteins, sugars, fatty acids)
153
Q

Describe electron capture detector (ECD).

A
  • Flow of electrons between Ni63 (radioactive) and anode
  • Sample passes through current & captures electrons
  • Reduction in current proportional to [analyte]
    • more sensitive to halogen-containing compounds
  • much more sensitive
  • Non-destructive
  • Specific to halogens (F, Cl, Br, I; & conjugated pi bonds); e.g., pesticides
154
Q

Describe flame photometric detector (FPD).

A
  • Eluate is burned in a flame
  • S, P-containing groups give rise to chemiluminescence (emission of photons)
    • Amount of emission is proportional to the amount of S or P-containing groups present.
  • much more sensitive
  • Destructive
  • Specific to S, P containing compounds
155
Q

Describe photoionization detector (PID)

A
  • Eluate is ionized by a UV lamp
  • Current generated is proportional to [analytes] → higher current = greater [analyte]
  • Lamp power can be tuned to selectively ionize certain groups
  • much more Sensitive
  • Less destructive than being in a flame; but molecules do become ionized.
  • ~general
156
Q

GC instruments may allow switching between several different detectors.

True or False?

A

True.

157
Q

GC instruments cannot allow switching between several different detectors.

True or False?

A

False.

They allow switching.

158
Q

Describe this application of GC.

A

Determination of migrants from polystyrene packaging materials into yoghurt.

159
Q

Describe GC as an official method for nutrition labelling of fat content.

A
  • Solvent extraction + quantify lipids using GC
  • Procedure:
    • Add internal standard (e.g., known amount of specific fatty acid)
    • Add antioxidant (to limit lipid oxidation)
    • Acid/alkaline hydrolysis procedure
    • Extract lipids with ethyl ether, petroleum ether
    • Derivative fatty acids (add methyl group) = Fatty Acid Methyl Esters (FAME)
    • Separate & quantify using GC (i.e., areas under peaks)
    • Total fat = sum of individual FAMEs
160
Q

Compare cation vs. anion exchangers.

Charge on column

Nature of compounds bound

A

Cation: Charge on column → negative; nature of compounds bound → cations

Anion: charge on column → positive; nature of compounds bound → anions

161
Q

Compare HETP vs. N vs. L

(from the equation HETP = L/N)

A

HETP: height equivalent of a theoretical plate

N: number of theoretical plates

L: length of column

162
Q

What factors would you consider in choosing an HPLC detector? [8]

A
  • Solute type and concentration
  • Sensitivity
  • Linear range
  • Selectivity
  • Compatibility with the solvent
  • Effect of temperature or flow rate changes
  • Possibility of use with gradient elution
  • Initial and operating costs
163
Q

Describe UV-Vis absorption detector in HPLC.

A

UV-Vis absorption detector → measures absorption of radiation in UV and visible wavelength range, according to Beer’s law → selective detector.

164
Q

Describe fluorescence detectors in HPLC.

A

Fluorescence detector → measures the emission of electromagnetic radiation by molecules that fluoresce (i.e., absorb at one wavelength, emit at another) → more sensitive and selective than UV detector

165
Q

Describe refractive index detectors in HPLC.

A

Refractive index detector → measures change in refractive index of the mobile phase due to presence of solutes → universal detector, since it responds to all solutes → sensitive to changes in temperature and flow rates → cannot be used with gradient elution

166
Q

Describe amperometric detectors in HPLC.

A
  • Measures the change in current as analyte is oxidized or reduced by the application of voltage across electrodes of the flow cell
  • Sensitivity and selectivity improved by adding pulse techniques (i.e., pulsed amperometric detection)
167
Q

Describe conductivity detectors in HPLC.

A
  • Responds to presence of ions eluting from the column (e.g., ion chromatography) → not very selective
168
Q

What is solid-phase extraction and why is it advantageous [6] over traditional liquid-liquid extractions?

A

In solid-phase extraction → a liquid sample is passed through a column filled with chromatographic packing; solutes with affinity for the packing are retained and others elute; then rinse packing with an eluant that will remove solute of interest.

ADVANTAGES

  • less solvent required
  • less glassware needed
  • faster
  • better precision and accuracy
  • minimal solvent evaporation for GC analysis
  • readily automated
169
Q

Why must sugars and fatty acids be derivatized before GC analysis, while pesticides and aroma compounds need not be derivatized?

A
  • Compounds that must be derivatized are those that are:
    • Thermally unstable
    • Too low in volatility
    • Yield poor chromatographic separation due to polarity
170
Q

What is the principle of the TCD in GC.

A

Thermal conductivity detector

  • As carrier gas passes over a hot filament (tungsten), it cools the filament at a certain rate depending on the carrier gas velocity and composition.
  • The temperature of the filament determines its resistance to electrical current.
  • When a compound elutes with the carrier gas, it has less of a cooling effect, so temperature increases and resistance increases
171
Q

Which GC detector is the least sensitive?

A

Thermal conductivity detector, TCD

172
Q

Which GC detector is the most sensitive?

A

Electron capture detector, ECD

173
Q

Which GC detector is the least specific?

A

Thermal conductivity detector, TCD

174
Q

Which GC detector has the greatest linear range?

A

Flame photometric detector, FID

Photoionization detector, PID

175
Q

Which GC detector is nondestructive to samples?

A

Thermal conductivity detector, TCD

176
Q

Which GC detector is commonly used for pesticides?

A

Electron capture detector, ECD

177
Q

Which GC detector is commonly used for volatile sulfur compounds?

A

Flame photometric detector, FPD

178
Q

You plan to use GC to achieve good chromatographic separation of compounds A, B, and C in your food sample. You plan to use an internal standard to quantitate each compound.

How do you choose the internal standard for your application? [3]

A
  • Not a compound present in the samples to be tested
  • Has the same characteristics as compounds to be quantitated
  • Elutes at time different from compounds of interest
179
Q

You plan to use GC to achieve good chromatographic separation of compounds A, B, and C in your food sample. You plan to use an internal standard to quantitate each compound.

What do you do with the internal standard, relative to the standard solutions for Compounds A, B, and C and relative to the food sample. Be specific.

A
  • Add a constant amount of internal standard to food sample and to solutions of mixed standard compounds.
  • Inject constant volume of sample of each type.
180
Q

You plan to use GC to achieve good chromatographic separation of compounds A, B, and C in your food sample. You plan to use an internal standard to quantitate each compound.

What do you measure?

A
  • Measure peak height or area (area is more accurate)
181
Q

You plan to use GC to achieve good chromatographic separation of compounds A, B, and C in your food sample. You plan to use an internal standard to quantitate each compound.

If you were to prepare a standard curve, who would you plot?

A
  • ([peak area of standards A, B, and C] /[peak area of internal standard]) vs. (concentration of standards A, B, C)
182
Q

You plan to use GC to achieve good chromatographic separation of compounds A, B, and C in your food sample. You plan to use an internal standard to quantitate each compound.

Why are internal standards commonly used for GC?

A
  • Because the sample volumes injected for GC are very small, so it is difficult to always inject the exact same volume.
  • This helps to monitor the process and normalize the response to the internal standard so the result is more reliable and comparable.
183
Q

A fellow lab worker is familiar with HPLC for food analysis but not with GC. As you consider each component of a typical chromatographic system (and specifically the components and conditions for GC and HPLC systems), explain GC to the fellow worker by comparing and contrasting it to HPLC based on the listed parameters.

A
  • Parameter → GC → HPLC
  • Mobile phase → inert gas → liquid
  • Stationary phase → liquid or solid → liquid or solid
  • Column length → 0.5-100m → 10-25cm
  • Phase of analytes, injected → gas or liquid → liquid
  • Phase of analytes, detected → gas → liquid
  • Temperature of column → 100-300 degrees C → ambient-85 degrees C
  • Detectors → FID, TCD, ECD, PFPD, PID, MS → UV-Viis, RI, fluorescence, amperometric, conductivity, MS
  • Gradient → temperature → mobile phase
  • Nature of sample → volatile → not volatile
  • Identification of peaks → same → same
  • Quantification of peaks → same → same
  • Description of column efficiency → same → same
184
Q

State in general terms the differences among the types of samples appropriate for analysis by GC vs. HPLC and give several examples of food constituents appropriate for analysis by each.

A

GC applications (generally volatile compounds) → fatty acids; triglycerides; cholesterol; pesticides; herbicides; polychlorinated biphenyls; drugs; flavour compounds; volatiles in packaging materials; separation of stereoisomers; headspace analysis

HPLC applications → amino acids; antioxidants; aspartame; caffeine, carbohydrates; dyes; folates; lectins; organic acids; phenolic compounds; pigments; proteins; fat-soluble and water-soluble vitamins

185
Q

What is the relationship between an antigen and an antibody?

A
  • An antigen is any molecule that induces the formation of antibodies and can bind to these antibodies.
  • Antibodies are immunoglobulin proteins proteins by animals in response to an antigen.
186
Q

What is an epitope?

What are the two different types?

A
  • Epitope → the specific region on the surface of the antigen molecule bound by a single antibody binding site is known as an epitope.
  • Two types of epitopes → a linear epitope or a conformational epitope
187
Q

What is the difference between monoclonal and polyclonal antibodies?

A
  • Monoclonal → identical antibodies with only one type of binding site, derived from a single hybridoma (fused cancer cells) that can be cultured.
  • Polyclonal → collection of different antibodies that bind different epitopes on the antigen
188
Q

All immunoassays have two conditions that they must satisfy; what are they?

A
  • There must be some method to separate or differentiate free antigen from bound antigen.
  • Antigen or antibody must be quantifiable at low concentrations for maximum intensity.
189
Q

What is a hapten and what is a conjugate antigen?

A

Hapten → small molecule (or some derivative of it) that must be linked to a large carrier protein before it can induce antibody formation. A hapten by itself cannot induce an immune response even though it can bind to the antibody.

Conjugate antigen → a carrier protein-linked hapten

190
Q

What are five general steps for an ELISA procedure?

A
  • Coating of antibody or antigen on a solid phase.
  • Blocking the remaining uncoated surface on the solid phase with a blocking buffer containing non-specific protein such as bovine serum albumin.
  • Incubating with different immunoassay reagents at a specified temperature and time.
  • Washing the coated surface to separate free, unbound molecules found bound molecules.
  • Detecting the colour developed from the assay.
191
Q

What is the rationale for the blocking step in ELISA protocols?

A
  • To minimize the nonspecific reactions and also protect the adsorbed antigen or antibody from surface denaturation.
192
Q

What is the difference between direct and indirect immunoassays?

A
  • Direct or indirect immunoassays refers to their detection method of the assay signal through the use of a label directly or indirectly.
  • Direct → detecting molecules are linked to a label (such as an enzyme, radioisotope, or fluorescent compound) to directly measure the amount of antibody or antigen.
  • Indirect → amount of antibody or antigen is measured indirectly most often with a labelled anti-species antibody.
193
Q

Two common immunoassays are the non-competitive sandwich assay and the competitive assay. Which molecules are best detected by each? Why?

A

Non-competitive sandwich assay → large molecules with at least two epitopes are better detected with the sandwich assay

Competitive assay → small molecules (one epitope) are better detected with the competitive assay → because small molecules (one epitope) just physically don’t have the surface area to ind to two IgG at once

194
Q

What are two types of competitive ELISA procedures?

A

Bound hapten format → uses less antibody reagent

Bound antibody format → more sensitive

195
Q

Give four common applications of immunoassays in food analysis.

A
  • Chemical residue analysis
  • Identification of bacteria and viruses
  • Determination of allergens
  • Meat and fish species authentication
  • Detection of genetically modified organisms in food
196
Q

What is the principle of FID in GC.

A

Flame ionization detector

  • As compounds elute from the column, they are burned in a hydrogen flame.
  • A potential is applied across the flame.
  • The flame will carry a current across the potential that is proportional to the organic ions present in the flame from the burning of an organic compound.
  • The current flowing across the flame is applied and recorded.
197
Q

What is the principle of ECD in GC?

A

Electron capture detector

  • The ECD contains a radioactive foil coating that emits electrons as it undergoes decay.
  • The electrons are collected on an anode, and the standing current is monitored by instrument electronics.
  • As an analyte elutes from the column, it passes between the radioactive foil and the anode.
  • Compounds that capture electrons reduce the standing current and gives a measurable response.
198
Q

What is the principle of FPD in GC?

A

Flame photometric detector

  • Works by burning all analytes eluting from the analytical column
  • And then measuring specific wavelengths of light that are emitted from the flame using a filter and photometer
  • The wavelengths of light that are suitable in terms of intensity and uniqueness are characteristic of sulfur and phosphorus.
199
Q

What is the principle of PID of GC?

A

Photoionization detector

  • Uses UV radiation to ionize analytes eluting from the analytical column.
  • The ions are accelerated by a polarizing electrode to a collecting electrode
  • The small current formed is magnified by the electrometer of the GC to provide a measurable signal
200
Q

What are advantages and disadvantages associated with each type of immunoassay format (e.g., direct vs. indirect)?

A

Direct → simpler but needs more purified immunoreagents

Indirect → adds a step but there is no need to link the primary antibody with a label, such as an enzyme; anti-species antibodies come with a variety of label options that offer advantages in specific applications and gives increased sensitivity; anti-species antibodies can be prepared in a selective manner, so antibody classes can be differentiated.

201
Q

Describe the bound hapten format of ELISA assay.

A
  • The protein-bound hapten is immobilized onto a solid surface
  • After washing, a competition is created between the protein-bound hapten and the free small antigen molecule in a food extract for the binding to the limited sites on the antibody labelled with an enzyme.
202
Q

Describe the bound antibody format of the ELISA assay.

A
  • A limited amount of antibody is immobilized onto the solid phase and creates a competition between enzyme labelled conjugate antigen and free small antigen molecules in the food extract
  • This format is somewhat more sensitive than the bound hapten format, although it may use more antibody reagent.
203
Q

What are the applications of enzyme-linked immunosorbent assays? (ELISA)

A
  • Detecting specific proteins → allergens; bacteria; viruses; food proteins; species of meat and fish
  • Detecting small molecules → pesticides; drugs
204
Q

What is an antigen?

A
  • Any molecule that induces the formation of antibodies
  • Binds to antibodies
  • Generally >5000Da
205
Q

What is an antibody?

A
  • Immunoglobulins (Ig)
  • Proteins produced by animal B cells in response to antigen
  • B cells produce 10-100s of millions of different Ig
  • Particular Ig up-regulated in response to antigen
  • (i.e., the Ig already exist, lying in wait!)
206
Q

What are the 5 major classes of immunoglobulins?

A
  • 5 major classes → IgA, IgE, IgG, IgM, IgD
207
Q

What is the structure of IgG?

A
  • 2 heavy chains (orange)
  • 2 light chains (blue)
  • Connected by S-S bonds
  • Mw 150,000 Da (150kDa)
  • Antibody binding → at top; N-terminals of HC and LC
  • Domains
    • Fragment antigen binding (FAB) → more variable in sequence
    • Fragment crystallizable (FC) → less variable, ‘constant’ among multitudes of IgG
208
Q

What is a linear epitope?

A

Epitope → specific region of antigen that binds to antibody

Linear epitope → formed by a continuous sequence of amino acid residues

209
Q

What is a conformational epitope?

A

Epitope → specific region of antigen that binds to antibody

Conformational epitope → formed by noncontagious amino acid sequences folded into close proximity from neighbouring or overlapping peptide chain on antigen surface

210
Q

What are polyclonal antibodies?

A
  • Collection of different IgG
  • Bind antigen at different epitopes
  • Produced from animal serum
211
Q

What are monoclonal antibodies?

A
  • Specific IgG
  • Binds single epitope of antigen
  • Produced from cultured cells
  • ‘Hybridomas’ = cross of melanoma and B-cells
212
Q

What is an antigen?

A
  • A molecule that binds IgG
  • Induces IgG production
213
Q

What is a hapten?

A
  • A small molecule that binds IgG
  • Does not induce IgG production
214
Q

What is a conjugate antigen?

A
  • small molecule covalently linked to carrier protein
  • Induces IgG production
215
Q

What are the basic requirements of an immunoassay?

A
  • Able to discern between free antigen and IgG-antigen complex
  • Able to quantify IgG-antigen complex at low concentration
216
Q

Describe capture molecule in ELISA.

A
  • Antibody as capture molecule (to search for target antigen)
  • Antigen (target) as capture molecule (to trap antibody)
217
Q

Describe surface-based assays.

A
  • The capture molecule (antigen or antibody) is adsorbed to a hydrophobic surface (e.g., 96-well plate made of plastic
218
Q

What is ELISA?

A

Use enzyme (covalently linked to one of the reacting partners) as the means of detection; converts substrate (colourless) → product (colour)

219
Q

What is the general protocol for ELISA?

A
  • Coat antigen (or antibody) onto surface, e.g., plastic 96-well plate
  • Block surface with BSA; covers any remaining plastic surface
  • Incubate with antibody (or antigen); binds sample added in step 1
  • Wash off unbound antibody (or antigen)
  • Detect antigen-antibody complex; visually (quantitative), spectrophotometer (quantitative)
220
Q

What is a primary antibody?

A
  • Antibody that binds the antigen
221
Q

What is a secondary antigen?

A
  • Antibody that binds the primary antibody
  • ‘detection antibody’
  • binds to FC region
  • from a different species (e.g., goat anti-rabbit)
222
Q

What is direct detection in ELISA?

A

Primary antibody is labelled with enzyme

223
Q

What is indirect detection in ELISA?

A

Secondary antibody is labelled with enzyme.

224
Q

What is the advantage of the indirect detection method in ELISA?

A
  • Can be difficult to label primary antibody (low amounts) with enzyme
  • Easier to use a dedicated, commercially prepared detection antibody
  • Indirect method is more common
225
Q

What is sandwich ELISA?

A
  • Capture antibody & detection antibody → bind to different epitopes on the same antigen → best for large antigens
  • Indirect detection → amplify signal via binding multiple E using avidin-biotin
226
Q

Compare noncompetitive with competitive ELISA.

A
  • Noncompetitive assay → amount of enzyme is directly linked to amount of antibody-antigen complex; colour intensity is proportional to amount of target molecule
  • Competitive assay → antigen in sample competes with an added antigen for IgG; sample antigen-IgG complex is washed away; added antigen-IgG-E complex gives colour; colour intensity is proportional to (amount of target molecule)-1
227
Q

Describe how ELISA can be used to detect nut allergens.

A
228
Q

Summarize sample introduction in MS. [2]

A
  • Sample introduction
    • Static method (e.g., direct injection)
    • Dynamic method (e.g., gas or liquid chromatography)
229
Q

Summarize ionization in MS. [2]

A
  • Samples are vaporized (converted to gas phase)
  • Converts molecules to ions & fragments, each with a m/z
230
Q

Summarize the mass analyzer in MS. [2]

A
  • Mass analyzer
    • Separates ions/fragments based on their m/z
    • Analogous to the dispersion element in optical spectroscopy
231
Q

Summarize the detector in MS. [2]

A
  • measure ions using electron multipliers
  • similar to PMT’s used in optical spectroscopy
232
Q

Light has both wave and particle-like properties. When considering how light is dispersed through a prism, light is best treated as a […].

A

Wave

233
Q

When light is absorbed by a molecule, it is best treated as a […].

A

Particle

234
Q

Which of the following molecules is most likely to absorb in the VIS region?

A

All except 4

Having ‘some’ conjugated bonds isn’t necessarily enough; but conjugation is necessary (~7 or more).

Tryptophan does not absorb in the visible region.

235
Q

Absorption & emission of light by atoms involves which transitions?E

A

Electronic

236
Q

Molecular absorption of radiation in the VIS range results in transitions between what types of energy levels?

A

Electronic

237
Q

Molecular absorption of radiation in the IR range results in transitions between what types of energy levels?

A

Vibrational

238
Q

Energy levels of atoms and molecules are quantized, meaning that only light of a certain frequency can be used to excite a particular transition.

True or False?

A

True.

239
Q

What is the correct rank order of transitions from highest to lowest?

A

Electronic > Vibrational > Rotational > Translational

240
Q

Fundamental absorption is associated with which frequency of IR light?

A

Mid-IR

241
Q

Compared to dispersion-type spectrometers, a unique component of an FTIR spectrometer is […].

A

An interferometer

242
Q

With near and mid-IR spectrometers, solid samples can be directly measured using which mode?

A

Attenuated Total Reflectance (ATR)

Reflectance

243
Q

With near and mid-IR spectrometers, solid samples can be indirectly measured using which mode?

A

Transmission → must first mix with potassium bromide or Nujol oil

244
Q

What element does the cathode consist of in a Hollow Cathode Lamp?

A

Same as the analyte

245
Q

What are advantages of ICP-OES over flame AAS for elemental analysis? [3]

A
  • Lower detection limit
  • Measure multiple elements at once
  • Use of explosive fuel gas not required
246
Q

AAS and AES require the same instrumental components except for the […].

A

The light source

247
Q

With atomic spectroscopy, why are there more emission lines than excitation lines?

A

Emission involves transitions between a greater number of possible states

248
Q

If a photon has twice as much energy as required to excite an atom from energy level 0 to energy level 1, then the atom would absorb half the photon’s energy.

A

False.

249
Q

While a monochromator separates light according to wavelength, a mass analyzer separates molecules based on their […].

A

Mass to charge ratio

250
Q

Describe the basic components of a UV-VIS absorption spectrometer (i.e., list each component and give a short description of what it does).

A

Radiation source → generally a lamp which provides the electromagnetic radiation for the analyte of interest to absorb

Wavelength isolator → generally a diffraction grating monochromator which consists of entrance and exit slits, concave mirrors, and a dispersing element. It disperses radiation so that a specific monochromatic wavelength can be made to exit the monochromator and be direct to the sample. This is necessary because Beer’s law is only useful for monochromatic radiation

Sample/reference cell holder → holds sample or reference for analyzing

Detector → often a phototube or a photomultiplier tube. The intensity generated is proportional to the number of photons arriving from the radiation beam passing through the sample/reference. The photomultiplier is preferred because it will amplify the signal to allow for detection of lesser intensity

Readout device → displays results

A linear set-up from Radiation source > wavelength isolator > sample > detector > readout

251
Q

What changes to a UV-VIS spectrometer instrument would be needed to make this into a Fluorescence spectrophotometer?

A
  • To make a UV-VIS spectrometer into a fluorescence spectrophotometer two wavelength isolators will be required, one for the excitation radiation and one for the emitted radiation.
  • Further, the detector for will be placed at a 90 degree angle with regard to the light source, so the set-up will not be linear as it was for the UV-VIS set-up.

Radiation source > wavelength isolator > sample/reference cell > wavelength isolator > detector > readout

252
Q

In fluorescence spectroscopy, how does the wavelength of the emitted radiation differ from the wavelength of the absorbed radiation? Why?

A
  • The emitted radiation will have a longer wavelength (and lower energy) than the absorbed radiation.
  • This is because a fraction of the energy difference between the excited and ground states is dissipated as heat during vibrational relaxation.
  • Excited species undergo vibrational relaxation to the lowest vibrational energy level within the excited electronic state, and then a transition to the ground state with the emission of a photon.
  • The emitted photon will have an energy equal to the energy difference between the lowest vibrational level of the excited electronic state and the ground electronic state.
  • To reiterate, some of the energy associated with the absorbed light is dissipated as heat during vibrational relaxation, so the emitted light has lower energy and longer wavelength.
253
Q

Explain the basics of how absorption IR light can be used to identify and quantify a particular compound.

A
  • Quantitative analysis using IR spectroscopy relies on instrument calibration with known standards (or identical food products), and measurements at multiple wavelengths because of overlapping absorption bands.
  • Multivariate statistical techniques are used to relate the data to concentration, and multivariate regression is used to calibrate the instrument (by comparing data to conventional methods data).
  • A molecule will absorb IR radiation if it vibrates such that its electric dipole moment changes.
  • Absorption is proportional to the concentration of the analyte.
  • IR absorption is unique for each group of atoms because different bond strengths and different masses give rise to discrete energy levels.
  • Different functional groups will have characteristic absorption spectra, which can be identified by shining IR light through the analyte of interest, and then measuring the transmitted light.
  • Frequencies of absorptions can be used to identify specific groups within molecules.
254
Q

For AAS, give 5 examples of potential sources of error from sample preparation.

A
  • Not using distilled and/or deionized water
  • Not using reagent grade chemicals
  • Not using sufficiently cleaned labware
  • Using glass rather than plastic labware
  • Not diluting sample properly.
255
Q

For AAS, give 6 examples of potential sources of error from measurement.

A
  • Spectral overlap
  • Variation in sample transportation to atomizer
  • Suppression of solute volatization
  • Formation of thermostable oxides
  • Ionization of element (need to add suppressors)
  • Background shift (for ICP-OES specifically)
256
Q

Why is analysis of foods carried out? [4]

A
  • For food safety
  • For quality control
  • To meet government standards
  • For research
257
Q

Which organization is a source of official methods of analysis of foods?

A

AOAC International

258
Q

Which metric represents precision?

A

Standard deviation

259
Q

For a method of analysis, the magnitude of change in signal with change in concentration of a compound represents what?

A

Sensitivity

260
Q

In performing a gravimetric analysis, an improperly calibrated balance would likely give what kind of error?

A

Systematic

261
Q

It is easier to eliminate random error from an analysis than to eliminate systematic error?

True or False?

A

False.

262
Q

How could undesirable enzyme activity be limited during sample handling? [2]

A

An inhibitor or pH adjustment

FNH 302 (3 decks)

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