Post Quiz 2 Material Flashcards

Question

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

Answer 1

* 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)

Answer 2

* 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

Answer 3

* 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

Answer 4

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

Answer 5

* 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’**

Answer 6

* **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

Answer 7

* 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

Answer 8

* 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

Answer 9

* **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

Answer 10

Defined in terms of peak FWHM; capable of very high resolution FWHM = full-width at half-max

Answer 11

* 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

Answer 12

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

Answer 13

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.

Answer 14

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.**

Answer 15

* **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)

Answer 16

* 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, R_f * **Applications** → separation of small molecules (dyes, amino acids, peptides, sugars, purines)

Answer 17

Relative mobility

Answer 18

* Similar principle to paper chromatography * Developed to replace paper chromatography

Answer 19

* Better resolution * Faster * More reproducible

Answer 20

* Screen corn and peanuts for mycotoxins before processing * Lipids, carbohydrates, vitamins, amino acids, natural pigments

Answer 21

* 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**

Answer 22

* **Modes of interaction** * Adsorption * Partition * Hydrophobic interaction * Ion exchange * Affinity * Size exclusion * \>1 mode of interaction can occur in a given separation

Answer 23

* 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

Answer 24

* 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

Answer 25

* Water molecules organize themselves around non-polar groups → driven by increased entropy (lower energy)

Answer 26

* 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.

Answer 27

* **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

Answer 28

* **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

Answer 29

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

Answer 30

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

Answer 31

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

Answer 32

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

Answer 33

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

Answer 34

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

Answer 35

* **Normal** → polar stationary; non-polar mobile; most non-polar solutes elute first * **Reverse** → non-polar stationary; polar mobile; most polar solutes elute first

Answer 36

* **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)

Answer 37

* 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.

Answer 38

* **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

Answer 39

* 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

Answer 40

* 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)

Answer 41

* 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

Answer 42

* 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

Answer 43

* 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

Answer 44

* 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)

Answer 45

* **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)

Answer 46

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

Answer 47

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

Answer 48

* UV-Visible Absorption * Fluorescence * Refractive Index * Amperometric * Conductivity

Answer 49

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**

Answer 50

Selective More sensitive than UV-Vis Absorbance

Answer 51

* 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

Answer 52

Measure electrochemical oxidation-reduction of analyte Sensitive Selective e.g., used for phenolics, CHO

Answer 53

Measure change in conductivity of eluent Sensitive **Non-selective** e.g., used for ions

Answer 54

Collect fractions as a function of time or volume Further analysis of separated components

Answer 55

* \>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

Answer 56

* Gradient elution → increase polarity of mobile phase, e.g., hexane + CH₂Cl₂

Answer 57

* retention volume, V_R * retention time, t_R * peak width, w * peak height, h * Note t₀ = dead-volume

Answer 58

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

Answer 59

It corrects for differences in system

Answer 60

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

Answer 61

Site of binding/unbinding of solute from stationary phase

Answer 62

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

Answer 63

* 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)**

Answer 64

Reduce effects by → narrow column; smaller particle size

Answer 65

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 R_s)

Answer 66

* Refers to the separation between two peaks * A function of the stationary phase and mobile phase

Answer 67

* 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)

Answer 68

* **Identify unknown compounds (peaks) by:** * (1) Compare t_R or V_R 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

Answer 69

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

Answer 70

**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

Answer 71

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

Answer 72

**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

Answer 73

**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

Answer 74

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.

Answer 75

* 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

Answer 76

Positive charge

Answer 77

Negative charge

Answer 78

**Lower** pH(buffer) \> pH(protein) → binds to anion exchanger pH(buffer) \< pH(protein) → binds to cation exchanger

Answer 79

* 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

Answer 80

**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.

Answer 81

* 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.

Answer 82

* 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**

Answer 83

* 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**

Answer 84

* 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.

Answer 85

* 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.

Answer 86

* Reduces HETP when flow rate is optimized, to provide for proper equilibrium but minimize longitudinal diffusion.

Answer 87

* Increases the L (and the N), assuming HETP remains unchanged → leads to better resolution

Answer 88

* Reduces the Eddy diffusion, to reduce the HETP.

Answer 89

* Reduces the Eddy diffusion, to reduce the HETP.

Answer 90

* 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

Answer 91

* Use the peak height or peak area to plot against concentration

Answer 92

* 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

Answer 93

* 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.

Answer 94

* Speed * Improved resolution * Greater sensitivity * Reusable columns * Ideal for ionic species and large molecules * Easy sample recovery

Answer 95

* 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.

Answer 96

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

Answer 97

* 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.

Answer 98

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.

Answer 99

* **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**

Answer 100

* **Thermally unstable** (otw \> degradation products) * **Non-volatile** * Sugars, polyphenolics, fatty acids, amino acids, peptides, proteins, polysaccharides, vitamins

Answer 101

* 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

Answer 102

* Sample preparation * Component isolation * Sample concentration * Sample derivatization

Answer 103

* Headspace sampling * Distillation * Solvent extraction * Solid-phase extraction * Direct injection

Answer 104

**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

Answer 105

* **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

Answer 106

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

Answer 107

* **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

Answer 108

* 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)

Answer 109

* 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 :(

Answer 110

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

Answer 111

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

Answer 112

* Sample * Injection * Capillary column * Packed column * Temperatures * Carrier gas * Detector

Answer 113

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

Answer 114

* **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

Answer 115

* 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

Answer 116

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

Answer 117

* 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

Answer 118

* 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

Answer 119

* 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)

Answer 120

* 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

Answer 121

* 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**

Answer 122

* 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

Answer 123

False. They allow switching.

Answer 124

Determination of migrants from polystyrene packaging materials into yoghurt.

Answer 125

* 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

Answer 126

**Cation:** Charge on column → negative; nature of compounds bound → cations **Anion**: charge on column → positive; nature of compounds bound → anions

Answer 127

**HETP:** height equivalent of a theoretical plate **N:** number of theoretical plates **L**: length of column

Answer 128

* 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

Answer 129

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

Answer 130

**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

Answer 131

**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

Answer 132

* 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)

Answer 133

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

Answer 134

**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

Answer 135

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

Answer 136

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

Answer 137

Thermal conductivity detector, TCD

Answer 138

Electron capture detector, ECD

Answer 139

Thermal conductivity detector, TCD

Answer 140

Flame photometric detector, FID Photoionization detector, PID

Answer 141

Thermal conductivity detector, TCD

Answer 142

Electron capture detector, ECD

Answer 143

Flame photometric detector, FPD

Answer 144

* 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

Answer 145

* 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.

Answer 146

* Measure peak height or area (area is more accurate)

Answer 147

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

Answer 148

* 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.

Answer 149

* **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

Answer 150

**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

Answer 151

* **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.

Answer 152

* **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

Answer 153

* **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

Answer 154

* 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.

Answer 155

**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

Answer 156

* 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.

Answer 157

* To minimize the nonspecific reactions and also protect the adsorbed antigen or antibody from surface denaturation.

Answer 158

* 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.

Answer 159

**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

Answer 160

Bound hapten format → uses less antibody reagent Bound antibody format → more sensitive

Answer 161

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

Answer 162

**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.

Answer 163

**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.

Answer 164

**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.

Answer 165

**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

Answer 166

**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.

Answer 167

* 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.

Answer 168

* 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.

Answer 169

* **Detecting specific proteins** → allergens; bacteria; viruses; food proteins; species of meat and fish * **Detecting small molecules** → pesticides; drugs

Answer 170

* Any molecule that induces the formation of antibodies * Binds to antibodies * Generally \>5000Da

Answer 171

* 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!)

Answer 172

* 5 major classes → IgA, IgE, IgG, IgM, IgD

Answer 173

* 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

Answer 174

**Epitope** → specific region of antigen that binds to antibody **Linear epitope** → formed by a continuous sequence of amino acid residues

Answer 175

**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

Answer 176

* Collection of different IgG * Bind antigen at different epitopes * Produced from animal serum

Answer 177

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

Answer 178

* A molecule that binds IgG * Induces IgG production

Answer 179

* A small molecule that binds IgG * Does not induce IgG production

Answer 180

* small molecule covalently linked to carrier protein * Induces IgG production

Answer 181

* Able to _discern_ between free antigen and IgG-antigen complex * Able to _quantify_ IgG-antigen complex at low concentration

Answer 182

* Antibody as capture molecule (to search for target antigen) * Antigen (target) as capture molecule (to trap antibody)

Answer 183

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

Answer 184

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

Answer 185

* **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)

Answer 186

* Antibody that binds the antigen

Answer 187

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

Answer 188

Primary antibody is labelled with enzyme

Answer 189

Secondary antibody is labelled with enzyme.

Answer 190

* 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

Answer 191

* **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

Answer 192

* **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

Answer 193

* **Sample introduction** * Static method (e.g., direct injection) * Dynamic method (e.g., gas or liquid chromatography)

Answer 194

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

Answer 195

* **Mass analyzer** * Separates ions/fragments based on their m/z * Analogous to the dispersion element in optical spectroscopy

Answer 196

* measure ions using electron multipliers * similar to PMT's used in optical spectroscopy

Answer 197

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.

Answer 198

Electronic

Answer 199

Electronic

Answer 200

Vibrational

Answer 201

Electronic \> Vibrational \> Rotational \> Translational

Answer 202

An interferometer

Answer 203

Attenuated Total Reflectance (ATR) Reflectance

Answer 204

Transmission → must first mix with potassium bromide or Nujol oil

Answer 205

Same as the analyte

Answer 206

* Lower detection limit * Measure multiple elements at once * Use of explosive fuel gas not required

Answer 207

The light source

Answer 208

Emission involves transitions between a greater number of possible states

Answer 209

Mass to charge ratio

Answer 210

**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**

Answer 211

* 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. ## Footnote **Radiation source \> wavelength isolator \> sample/reference cell \> wavelength isolator \> detector \> readout**

Answer 212

* 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.

Answer 213

* 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.

Answer 214

* 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.

Answer 215

* 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)

Answer 216

* For food safety * For quality control * To meet government standards * For research

Answer 217

AOAC International

Answer 218

Standard deviation

Answer 219

Sensitivity

Answer 220

Systematic

Answer 221

An inhibitor or pH adjustment