fdsc2100 study questions II

Atomic absorption Spectroscopy principle

Measures how much energy the sample absorbs
Absorption: excitation of electrons in the ground state

Uses absorption of light (UV or visible) to measure concentration of gas-phase atoms → atoms absorb light and make transitions to higher electronic energy levels)
Spectrum looks like a rainbow with a few lines taken out of it

Since samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace

Then determine concentration via standard curve after calibrating instrument with standards of known concentration

Atomic absorption spectrophotometer

Light → lens → atomized sample → lens → monochromator → detector → amplifier → readout

Light source: Usually hollow-cathode lamp of element being measured

Atomizer: atoms of interest must be in atomic state and well separated → undergo desolvation and vaporization via flame or graphite furnace (high temp)
Sample solutions usually aspirated into nebulizing/mixing chamber to form small droplets before entering flame

Monochromator: isolates absorption line from background light due to interferences

Detector: photomultiplier

Atomic emission spectroscopy

Source of radiation measured comes from excited atoms in the sample
Emission: electrons in excited state fall to lower energy levels (Light!)

1. Energy is applied to sample
2. Atoms are excited to higher energy levels
3. wavelengths characteristic of the individual elements are measured when they move back to ground state
→ ratio of excited atoms to ground-state occurring in plasma (E Source)
Spectrum looks like a couple colored lines on a dark background

Ratio of number of excited atoms to ground-state atoms occurring in the plasma (energy source) is described by Maxwell-Boltzmann equation

ICP-OES in atomic emission spectroscopy

The instrument used for atomic emission spectroscopy → contains yttrium, has a bunch of sensors to sense different wavelengths of light

The yttrium acts as a standard reference to improve precision and accuracy

Uses argon plasma as an excitation source → super hot
Argon provides an inert condition, minimizing interference from oxides
Nearly complete atomization minimizes chemical interference
Uniform temperatures in a plasma allow for broader concentration ranges to be measured

Small machine go very hot (plasma is like the sun) → add vaporized acid with minerals in it → allows you to take advantage of boltzmann equation → excited to ground state separation

both AAS and AES

For analysis of minerals
Requires elements of interest to be in atomic state and physically separated → using high temps or put in highly oxidized environment (like acid)

Spectra are complementary to each other

Sample preparation considerations for mineral analysis

Make sure no contamination from comminution, glass ware, reagents → do not let them contact metal!!

Use grinding equipment that does not contain mineral of interest

Acid wash glassware 

Use purest reagents available

Reagent blanks (controls) to know how much contamination/interference?

Also see ICP-OES → oxides and chemical interferences

Why is it important to measure mineral content of foods and other aspects of food production?

Processing impacts mineral content:

- acidified cottage cheese; polishing of rice and grains; leaching due to cooking in water

• Fortification → must quantify minerals

• NaCl: flavor; protein solubility; preservation

• Phosphates: increase water holding capacity of meats; texture of

processed cheese

• Calcium: gelation of proteins and gums

• Water quality and safety

• Mineral build up in processing equipment, a.k.a. “scale” →  clean to make sure no leftover minerals, measure mineral content 

Antigens and antibodies

• PROTEIN → immunoglobulins

  • Synth by body, specific binding to antigen via antigen binding site → Epitope → 3 amino acid sequence that is recognized and identify the protein → will bind via hydrogen bonding

    • Can use antigens to make antibody attach to something and detect it

      • Linear epitote on antigen → the amino acids are next to each other so if its denatured you can still bind it → so for hot samples you should use these

      • Conformational epitote on antigen → the amino acids may only be next to each other in a specific conformation, not when linear → when denatured its no longer bound

Antigens and Haptens and what are immunoassays?

Antigen: substance able to elicit immune response (recognized by Ab) → Protein, molecule, cell or organism

Hapten = small low molecular weight Ag (<1000 Da) → attaches to larger molecule to cause an immune response, but does not cause one on its own

Immunoassays: detection methods that involve an immunoreaction between antigen (Ag, foreign protein) and antibody (Ab)

Immunoassays principle

• Ab recognizes Ag → Ab-Ag reaction → detection/quantification of complex → standard curve (quantitative) OR determination of Presence/absence (+/-) (qualitative)

  • Antigen is attached to sample/bottom of plate, antibody attaches to it, if protein of interest is there it will attach antibody and you see what is stuck → see how light scatters across it 

  • Sometimes need secondary antibody → antibody that recognizes an antibody → you can put a marker on it like a fluorophore

Lateral flow test strips

For detection of proteins, (ex home pregnancy test)

No washing step needed to separate bound and unbound protein

Advantages: simple, low cost, reliable, rapid (aprrox 20 min) → can do detect arent proteins

Relies on colloidal gold or colored latex beads for signal → so we can see the line for the concentration

similar mechanism to the chloride quantab strips we used (except those are not immunoassays because they just rely on capillary action or something not antigens)

Types of immunoassays format

• Direct → Ag bound → add labeled Ab

• Indirect → Ag bound → Add Ab1 → Add labeled Ab2

• Sandwich: Ab bound → Add Ag → Add labeled Ab

• Competitive: Free Ag or Ab added

Chromatography vocabulary

Technique for separating components of mixtures as they are carried by a mobile phase through a stationary phase

Elution: the process of removing a solution from the stationary phase
Eluant: the mobile phase once it is eluted from the column
Adsorbent: stationary phase in chromatography, mainly in adsorption chromatography

Normal vs reverse phase chromatography

Normal phase chromatography: has a more polar stationary phase and less polar mobile phase
→ As described by Michael Tswett
→ Silica base is very polar, mobile phase is less polar
→ Lets say we have compounds from polar to non polar: A, B, C, D
→ So the most polar compounds will stick to the column, non polar compounds will go through
→ A sticks first, then B, then C, D flows through
→ Order of elution: D, B, C, A (falls out last)

Reversed phase chromatography: has a less polar stationary phase and a more polar mobile phase
→ Stationary phase is nonpolar
→ Same compounds
→ Polar compounds will flow through, nonpolar will stick to column
→ D sticks first, then C, then B, A flows through
→ Order of elution: A, B, C, D (falls out last)
→ Order of elution is reverse of normal
→ Better for amino acids and sugars

The three major components of chromatography

The compounds to be separated → interaction with column depends on polarity
→ Compounds will interact with stationary and mobile phase based on polarity → same polarity = they stick
→ More interaction = better separation
→ Choose column based on polarity

Stationary phase → not moving material, supporting material
→ May be a solid support like silica or a moiety bonded or adsorbed on a solid support

Mobile phase → moving material that is passed through the stationary phase
→ In liquid chromatography, the mobile phase is the solvent
→ In gas chromatography, the mobile phase is a gas

How do the three major components of chromatography interact?

Chromatography triangle!!

Partition chromatography

Relies on compounds dissolving and coming out of solution
Almost like solvent extraction

• A liquid is absorbed on a solid matrix such as silica gel to form a liquid stationary phase. This stationary phase liquid is usually polar and the mobile phase is nonpolar. Separation is dependent on relative solubilities of solutes in mobile and stationary phases. Solutes are partitioned between the two phases in the same way as solvent extraction

• Stationary phase: thin film as coated phase on solid supports such as silica, starch, cellulose, or glass bead

Adsorption chromatography

Relies on compounds bonding and debonding
More interactions is better → based on similar polarity

• Stationary → bonding, mobile → debonding

Ion exchange chromatography

adsorption chromatography based on charge to separate protein peptide, amino acids?

ions in a liquid exchange interact with ions on an ionic solid supports → type of adsorption chromatography, reversible process

• Mobile: Polar liquid

• Stationary: ionic solid

• Separation principle: adsorption

• Retention varies with: molecular charge

• Applications: separate ionic from nonionic material 

  • Separate positive (Cationic) from negative (anionic compounds)

  • Separate mixtures of similarly charged ions from each other

Common compounds to be analyzed with adsorption chromatography in order of increasing polarity

• Separation is based on polarity (mainly), volatility, molecular size, and charge

• Nonpolar?

  • Saturated hydrocarbons (like sat fat acids)

  • Unsaturated hydrocarbons → double bonds increase the polarity

    • 1 db → 2db (like omega 6, 18:2) → 3 db (like omega 3, 18:3)

• Intermediate polar

  • Aromatics → especially conjugation increase polarity

    • Like flavonoids! 

  • Halogenated compounds

  • Ethers

  • Nitro compounds, tertiary amines

  • Esters, ketones, aldehydes

• Polar

  • Primary amines (like amino acids?), alcohols

  • Amides

  • Acids 

Adsorption chromatography adsorbents in order of decreasing adsorptivity

• Adsorbent = solid stationary phase

• Silica gel, Alumina, charcoal, magnesium silicate, magnesium oxide, starch, cellulose

  • Charcoal is bad because contamination and oxidation

• Want no water in sample or mobile phase when using things like silica gel because it will mess everything up because it is very polar and will stick to the silica

• Bonded phase: surface of adsorbent is covered with organic groups which are covalently bound to it → such as nonpolar Octyl C8 and octadecyl C18 (most commonly used stationary phase in reverse chromatography because it is super nonpolar)

Mobile phase in adsorption chromatography

• Least to most polar

  • (nonpolar) Pentane → hexane → petroleum ether → (intermediate) ethyl ether → methylene chloride → isopropyl alcohol (skip?)→ tetrahydrofuran (skip?) → chloroform → ethyl acetate → (polar) acetone → ethanol → methanol → acetonitrile → water

  • Ethyl acetate is a very unique solvent for natural products

    • Most polar solvent that is not immiscible with water

    • Good for partially polar compounds like phospholipids or phenolic compounds

  • Most common for chromatography, especially HPLC: acetonitrile

Thin layer chromatography device and stationary phases

• Device

  • TLC paper

    • Sample spotted onto the circles (origin). Insert into TLC place and immerse in solvent (but not passed the line) → solvent drawn up by capillary force and the original mixture will be separated as the solvent is drawn up past the origin → solvent acts as mobile phase

• Stationary phase: silica gel most common in TLC and normal phase chromatography

  • Because it's the most polar

  • So the solvent will be relatively less polar

  • Used to separate lipids, fatty acids, sugars, alkaloids, and amino acids

• Stationary phase: alumina

  • Also good for normal phase chromatography, but less polar than silica

  • Alkaloids and other basic compounds

  • Used to separate food dye, phenols, steroids, and vitamins

Normal vs reverse phase TLC

• Normal phase TLC

  • Silica gel, or alumina

  • Non polar compounds move faster than polar

• Reverse phase TLC

  • Use C-18 (ODS), C-8

  • Polar compounds move faster than non-polar compounds

  • Application: high polarity compounds

What is Rf in TLC and what do we use for detection

• Rf Values = distance a solute moves/distance the solvent moves

  • Should be constant for a given solute/sorbent/solvent

• Detection: 

  • uses UV light with fluorescent indicator

  • Iodine and place iodine crystal in closed chamber

    • React with double bounds and aromatic rings to give a column

Basic HPLC instrumentation

• Solvent (mobile phase) → pump → controller → injector (where you put sample) → column (stationary phase) → detector → recorder

  • In his lab he uses 2 pumps to mix the solvents → use 2 solvents and change polarity and increase separation…

  • Controller to control speed

Polarity ranking of HPLC columns

from polar to nonpolar

• Silica → amino → cyano → Methyl - C1 → Octyl - C8 → Octadecyl - C18 → C30

• C30 for carotenoid analysis, uncommon tho

• Silica (Si) column: unmodified porous silica packing, most popular normal phase chromatography

  • Si-OH

  • Sensitive to H2O because of its strong OH group → DO NOT USE H2O!!! remove H2O from sample for analysis → sodium sulfate!

• Bonded phase columns

  • Methyl (C1)

  • Octyl (C8)

  • Octadecyl (C18) - most popular for reverse phase chromatography because it is nonpolar

What is a guard column and why is it used?

Precolumn or guard column: matches the column of packing materials (matches stationary phase of actual column), but much smaller and cheaper, placed just before actual column for prefiltering of materials to protect actual column

Other considerations for care (not on study questions)
Stability of column → may be sensitive to temp, pH, certain particle size materials → read specifications

• Pressure: want pressure <4000psi

• Changing solvents → make sure they are immiscible when you are changing b/w solvents

  • If not, you may increase pressure and damage the column

• Column reactivation 

• Column cleaning → wash column b/w different solvents

Column specifications

• Particle size: use smaller particle size to increase sensitivity

  • 3um: use 0.2 um membrane to filter mobile phase

  • 5um: most common, use 0.45um membrane to filter mobile phase

  • 10um: preparative column

    • For purification mainly -> when isolating column from natural products

• Column length and column ID

  • Length: 25, 15, 10cm

  • ID

    • Analytical: 4.6, 2.1 (higher sensitivity)

    • Preparative: 10, 21,2, and 50.8mm (purify larger amount of compounds from mixture, not as common)

(not on study questions)

UV cutoff

• Wavelength at which the solvent absorbance in a 1cm path length cell is equal to 1AU (absorbance unit), using water in the reference cell → equivalent to 10% transmittance

• This is the point at which you can’t use the data because the absorbance of the solvent will result in interference and limit sensitivity

• Make sure your solvent’s UV cutoff is below the wavelength range you are looking to measure

• Acetone is bad because it has a high UV cutoff

Isocratic vs gradient elution

Solvent delivery

• Isocratic elution: composition of mobile phase is constant

• Gradient elution: composition of mobile phase changes with time during separation

  • If sample contains a wide range of polarities, the separation can be done by changing the polarity of the solvent mixture during the separation

  • Used for mixtures of polar and non-polar compounds

Sample preparation for HPLC

• Should make a clear solution with the solvent and be particle free

  • Cloudy = not totally dissolved, so you cannot run experiment

• Sample solubility in mobile phase

• Solvent should be miscible with mobile phase

  • Otherwise causes precipitation

  • Maybe use same mobile phase to dissolve sample! convenient

• Polarity of solvent

  • More similar polarity to mobile phase, the better to avoid precipitation

Concerns with mobile phase for HPLC

• Miscible → make sure solvent is miscible 

• Particle Free → filter mobile phase through a 0.45um membrane for 5um column

  • Because any particle larger than that size will get stuck and ruin the column

• De-gas: air bubbles

  • Once pressure drops, air bubbles come up and will give a UV absorption - increase vibration of baseline, reduces sensitivity of assay

HPLC detectors

UV-visible absorption (similar to spectrophotometry), fluorescence (similar to spectrophotometry), Refractive index, electrochemical, conductivity

UV-Vis absorption vs fluorescence detector for HPLC

UV-vis

• Measures absorption of radiation beam of a UV-vis spectrophotometer with a 190-800nm range according to Beer’s Law

• Sensitive to aromatics and conjugated pi bond systems

  • Like alkenes, aromatics, and compounds with multiple bonds b/w C, O, N, or S

• Basically recall spectrophotometer diagram

Fluorescence
Some compounds are capable of absorbing UV radiation and subsequently emit radiation of a longer wavelength instantly (fluorescence)

• Conjugated aromatic rings have fluorescence!

• Higher sensitivity and selectivity than UV detector

For the diagrams of these detectors, refer to the spectrophotometry diagrams

UV Vis spectrophotometer

Schematic of spectrophotometer:
1. Hydrogen or tungsten lamp → release UV or white light
2. pass through monochromator or filter (allows you to choose specific wavelength of light)
3. pass through adjustable slit (determines amount of light) → pass through cell with sample
4. transmitted light passes into photomultiplier → converts light signal to electronic signal in photometer → data shows up on recorder

Fluorescence spectrophotometer

Schematic of fluorometer:
1. Hydrogen or tungsten lamp → passes through light selector
2. Incident light passes through slit 1 to sample → sample is irradiated → sample absorbs light and releases the energy as fluorescence
3. Released light is passed through slit 2 into another light selector → transmitted light goes to photomultiplier → photometer → recorder

Refractive index detector

• RI aka sugar detector

  • Universal detector, detects difference in refractive index between column eluent and reference stream of pure mobile phase

Electrochemical detector

• ECD

  • Measures current associated with the oxidation and reduction of compounds

  • For any compounds with oxidation or reduction potential (like antioxidants vit E, vit C, phenolics, flavonoids)

Conductivity detector

• Conductivity detector

  • Universal, reproducible, high-sensitivity detection of all charged species

  • Quantification of anions, cations, metals, organic acids, and surfactants down to ppb level

Chromatography separations

• Retention time - from injection to peak measurement → Used for identification of compound, each compound has its own specific retention time

• Peak height (h) - preferred for sharper peaks

  • From baseline to top of peak

• Peak area (a) - preferred for wider peaks

• Baseline → bottom of peak how sensitive

• Peak width at half height (W_1/2)

  • You want a narrow peak

• Dead Time (t₀)

  • Time from injection of solvent to just before solvent goes through column

  • Starting point of adjusted retention time

• Adjusted retention time (t’_R) →Used for identification of compound, each compound has its own specific retention time

• Spike - spike standard w/ blank…? to identify compound peak you are analyzing

  • Also use retention time

Used for GC and HPLC

Methods of HPLC analysis

• Qualitative: 

  • Identifying compound from retention time, relative retention time, spike known standard, or purification (collect interested peak)

• Quantitative: calculations using peak height or area 

Cellular antioxidant activity assay principle

• for assessing antioxidant activity

• Basically they treated cells with antioxidants or fruit extracts with a compound (DCFH-DA) that fluoresces when oxidized. → these would enter the cell or bind the membrane because the diester group makes it nonpolar and lipid soluble → after it diffuses into the cell cellular esterases can cleave DA off to get DCFH → now the compound is polar and is trapped in the cell

• They put in a source of radicals (ABAP) to diffuse into cells → this would produce more radicals (to increase sensitivity) and oxidize the fluorescent compound → antioxidants would prevent this formation → so more active antioxidants would mean less oxidation and less fluorescence

Advantages of CAA over traditional chemical antioxidant activity assays

• Other assays use environments that are not as comparable to the conditions of the human body or the relevant substrates we must protect, oxidants encountered.

• Also do not take into account the characteristics of the antioxidants or compounds

• Cellular antioxidant activity assay takes into account the complexity of biological systems → “accounts for some aspects of uptake, metabolism, and location of antioxidant components within cells”

Peroxyl radical scavenging capacity assay

• Used to analyze hydrophilic and lipophilic antioxidants of food extracts → I think they are analyzing how well the antioxidants inhibit the peroxyl radicals

• Median Effective Concentration - “EC50 is the concentration of antioxidant that causes a 50% inhibition of the control reaction and therefore produce a PSC unit of 0.5”

  • EC50 lower = better

• “the PSC unit represents the extent of inhibition of the control reaction by antioxidants”

  • PSC higher = better

• Uses same reaction/principles as CAA, but measures fluorescence of reaction at different concentrations of the antioxidant to make a dose response curve of fluorescence vs reaction time

Sodium borohydride/chloranil-based assay

• for quantifying total flavonoids

  • “used to measure the total flavonoid content of fruits, vegetables, whole grains, herbal products, dietary supplements, and nutraceutical products”

    • accurate, precise, and specific for flavonoids, clearly has wide applications (see above)

• 4-Carbonyl group containing flavonoids (ones with ketone groups) reduced to flavan-4-ols (catechins) using sodium borohydride, catalyzed with aluminum chloride for high yield → flavanols oxidized to anthocyanins by chloranil in acetic acid solution → anthocyanins quantified spectrophotometrically at 490nm after they added vanillin and concentrated HCl for reaction

Major components of a GC system

Injector, Column, column oven, carrier gas, detector, recorder, gas supply system

Stationary phase: column
Mobile phase: carrier gas, very different than a solvent
Compounds you are analyzing
The interactions are mainly between the column and compound

What type of chromatography is gas-liquid chromatograph based on for separation of compounds? What is the principle of separation?

It is more generally used and is based on partition chromatography

• Mobile: gas

• Stationary: liquid

• Separation principle: partition (dissolving/coming out of solution)

  • Interaction of compound with coating material and liquid phase → dissolving or coming out of solution

  • Equilibrium b/w liquid stationary phase and gas mobile phase → They go into stationary phase and then volatilize and move through the column to the next theoretical plate → each theoretical plate = phase change from gas to liquid and then back to gas (1 equilibrium)

• Retention varies with: Polarity, molecular size, volatility → boiling point

  • More volatile will move through quicker

Column selection

• Principle: like dissolves like

  • Non polar column best for analyzing non polar compounds, polar columns best for separating polar compounds

• Special considerations

  • Polarity of an effective stationary phase (selectivity - like dissolves like) → affects separation

  • Efficiency: total # of theoretical plates → more interactions

    • You inject the sample until it comes out the other end?

  • Stationary phase content → thin vs thick layer 

    • Thick = more interaction, thin = more plates

  • Temperature stability of the stationary phase → if temp too high you could damage your column

GC FID Gas supply

• Carrier gas (usually nitrogen, helium, or hydrogen) - 30ml/min

  • Helium is good because smaller molecule = better separation

  • Hydrogen in theory should be better, but its pretty unsafe if it builds up because a bad grad student forgets to turn it off

• Hydrogen - 30 ml/min - helps produce the flame

• Air - 300 ml/min?? - helps produce the flame

• Make-up gas - used for capillary columns because they can only do 2-3 ml/min so the makeup gas compensates (28ml/min) for that in order to keep the 1:10 ratio of carrier gas to O2…?

  • Introduced after carrier gas passes through column, just before it enters the detector

types of columns for gas chromatography

• Silicones, most common

  • Dimethylsiloxane - most common, very stable, resistant to higher temps, relatively low polarity

  • Diphenyl-dimethylsiloxane: 5, 20, 35, and 50% (maximum)

    • Increases polarity because of the double bonds it contains

• Polyethylene glycol (PEG)

  • poly(ethylene glycol) PEG, Carbowax 20M - most polar column in gas chromatography

  • poly(alkylene glycol): less polar than PEG

    • Incorporation of propylene oxide into the polymer backbone of PEG

Polarity of gas chromatography columns

• Non polar to polar

  • Poly(dimethylsioxane) → poly(5%diphenyl/95%dimethylsioxane) → poly (20% diphenyl/80% dimethylsioxane) → poly (35% diphenyl/ 65%dimethylsioxane) → poly (50% diphenyl/50%diemthylsioxane) → Poly alkylene glycol → PEG (carbowax 20M)

Flame ionization detector

• universal for C-containing compounds with C-C or C-H bonds

  • Compound must be volatile → If its not, do derivatization like  methylation (like on fatty acids to make them volatile) → If thats not enough, do silylation

  • Compounds are burned in a H flame (for chemical decomposition) to generate ions and electrons that carry current

  • Gas supply: carrier gas, hydrogen, air

  • No response to H2O, NO2, H2S, anything without C-C or C-H bonds

    • Cause it cannot detect the oxygen

  • Wide linear range and high sensitivity (10--100pg)

Mass spectrometry detector

• Sample is bombarded by energetic e- and the molecule is ionized to lose an e-

• Further bombardment causes the ions to fragment

• Gather “fingerprint” of compound, mainly for identifying structure at first → but now we can use it for analysis with HPLC!

• Sensitivity 0.25-100pg

• Relative abundance vs mass to charge ratio (m/z)

Thermal conductivity detector (TCD)

• Universal detector, can detect almost everything, including H2O, CO, or CO2

  • Mainly for gas analysis 

  • Ex: you won’t see H, O, N on GC FID because they are found in the carrier, flame, or make up gas

  • So this unique for H, O, N

• Resistance change on hot filament

• Sensitive 500pg/mL

methods of GC analysis

• Standards

  • External standards: standards used for calculations, like standard curves or dose response curves → calibration curves using known concentrations

  • Internal standards: like adding C17 fatty acids to check % recovery, because C17 is not naturally made (only even #s)

    • Added into the food sample, but not originally present

    • Used to calculate % recovery → 85% is pretty alright, above 90% is good

    • To improve assay → recall % recovery used to evaluate accuracy

• Identifications

  • Retention time

  • Relative retention time

  • Spike known standard - like if you find a peak for DHA, and then you had more DHA and the peak increases, then you know it is actually DHA

  • Mass spectrometry

• Quantitative GC analysis

  • Peak height or area

Chemical derivatization and when to use it

• volatile or non-polar compounds may be carried out by GC without derivatizing the sample → can be directly analyzed

  • Polar compounds need chemical derivatization, like fatty acids or sugars

  • Derivatization used in cases where it 

    • increases the volatility and decreases polarity of polar compounds (specifically sugars are pretty polar),

    • Stabilizes compounds which are unstable at temperatures required for GC,

      • Like if its not stable at high temperatures, like fatty acids which require lower temps

    • Improves separation of group of compounds on GC by changing polarity

What are the differences in compounds for analysis by GC versus HPLC?

Compounds for GC must be volatile (non polar) and heat stable → may need derivatization
Usually compounds analyzed are low molecular weight

Compounds for HPLC can be polar and non-volatile, also does not require derivatization
Compounds must be soluble in liquid solvents, can be smaller or larger or ionic

Mass spectrum + MS vs MS/MS

• Mass spectrum: shows the mass of the molecule and the masses of pieces from it

  • Commonly interfaced with GC, but also increasingly interfaced with HPLC

    • HPLC-MS for larger molecules?

  • Tandem MS aka MS/MS: additional stage of ion fragmentation done before detection, to give structural information → more specific and sensitive, good for more complex samples

Ionization

• Ionization: placing a charge on a molecule and convert it to an ion

  • Occurs in ion source

  • 2 kinds electron and chemical ionization

electron ionization

• electron ionization EI → for interpretation of fragments (structure identification), harsher method

  • Compound in ion source exposed to beam of e- emitted from glowing filament 

  • E- move across ion chamber toward positive electrode

  • As e- pass through source region, they strike the sample and knock off an e- to form a + ionized molecule

  • Ionized molecule contains high internal E → further fragments into smaller fragments

  • + charged molecules and fragments get repelled toward quadrupole mass analyzer

  • Interpret fragment data

Chemical ionization

• chemical ionization 

  • CI, soft ionization, knocks e- off gently, produces less fragments → for molecular weight, to determine molecular ion

  • Uses reagent gas (like methane) and ionizes it with EI to form ions that then ionize sample molecule through charge exchange

Mass spectrometry instrumentation

Sample inlet → ion source (ionization of sample) → mass analyzer → detector → data system

Typically ion source is an electron beam from a filament

Mass analyzer in mass spectrometer

Mass analyzer: generated ions are then filtered according to their mass-to-charge ratio (m/z) by subjecting them to electrostatic fields, then detected

• Via quadrupoles (Q, separates major fragments) 

  • quadrupole MS - 4 rods divided into 2 groups, each group has 2 equal, but out-of-phase electronic fill/potential → separates fragments

• or ion traps (IT, traps larger, more stable fragments and then put that into second MS → MSMS… very good for protein analysis because you cannot get detailed fragments from the first MS)

• Charged molecular ions and its fragments are separated according to their mass-to-charge ratio (m/z)

Detector in mass spectrometer

• Detection of separated and charged fragments is done by a detector

Mass spectrometry peak/data analysis

• Base peak: maximum response → highest relative abundance or intensity (100%)

  • Usually 15 lower than parent ion bc methane groups are very easy to remove

• Parent peak: from OG molecule (molecular ion peak)

  • Parent molecular weight, peak with highest mass number → from positively charged intact molecule with m/Z = molecular mass

• Key fragment ions/daughter ions: smaller fragments, due to stepwise cleavage of large fragments

  • The important ones are the ones in same ratio as base peak

  • Daughter ions are from like next generation (MS-MS)

• Use % of each fragment to interpret results → display pattern of ions used to identify unknown compounds

• Follow mclafferty law → common fragmentation processes for organic molecules are McLafferty rearrangement and alpha cleavage

Mass spectrometry sample introduction

• Static method (direct injection) - like GC, with a syringe

• Direct insertion probe method → used with solids that are at least somewhat volatile

  • Need pure compound… he doesn’t like this one

  • 2mg to get a good signal

• Dynamic method/separation

  • Sample separated into individual compounds, then analyzed by MS → like separated by GC or HPLC first → interfaced directly into MS 

  • Required for complex mixtures of several compounds

  • Done by an interface in cases of GC or HPLC connected to MS

GC-MS

• Gas Chromatography - Mass spectrometry (GC-MS)

  • Identify and confirm peaks

  • Identify unknown compounds, using computer assisted search of library with MS spectra → for smaller or easier compounds

  • Determine purity of peaks eluted

  • Connects capillary GC column to MS source with heated capillary transfer line

LC-MS

• Liquid chromatography - Mass spectrometry (LC-MS)

  • Converts liquid from LC into gas phase ions (sampled by MS) by desolvation in presence of highly charged electrical field at atmospheric pressure

  • Types of LC-MS ionization interfaces:

    • Electrospray: ESIP

    • Atmospheric pressure chemical ionization: APCI

Applications of MS

• Structure ID

  • MS patterns = fingerprint for specific compounds and structure elucidation

• Quantitative:

  • Nutrient analysis: Se, I, vitamins, proteins, CHO, omega-3 FA, cholesterol, others

  • Bioactive Compound analysis: phenolics, flavonoids, carotenoids and other phytochemicals

  • Toxin analysis: Pesticides, herbicides, and other toxic compounds

• Stable isotope (no radiation) ratios useful to identify source of samples

  • Can enrich foods w/ compounds containing specific isotopes and track movement of the metabolites through the body

  • Help determine location and purity of compound

Major ions/fragments in EI mass spectrum of methanol

Base beak at 31
Parent peak at 32

Key fragments:
CH3+ (15)
OH+ (17)
CHO+ (29)

CH3+ + OH+ → 15 +17 = 32 = MW of CH3OH

GMO, GMO food

• GMO = genetically modified organism

  • GMO Food = any food made of/from or containing GMO ingredients

GMO foods in the US

• Soy → its in a lot of things! → >94% are probably GM, since 1996 → for herbicide tolerance

• Cottonseed → since 1996 >90% → for herbicide tolerance

• Corn since 1996 >89%

  • Its in everything

• Canola oil (Canadian oil; low acid)

• Papaya >90% produced in Hawaii is GM → to withstand ringspot virus

Types of techniques for GMO detection

• If you find foreign DNA or proteins, then you KNOWW its gmo

  • Ex: like a fish gene in a plant..

  • If we can detect the known border sequences then you know

  • If you can detect the mRNA that are not known in the guy they you know

• DNA-based techniques: most widely used method → PCR (polymerase chain reaction)

  • To amplify foreign DNAs

• mRNA-based techniques: reverse transcription PCR (RT-PCR), quantitative real time PCR (qPCR)

  • We basically turn it into DNA and analyze it like DNA so.. Same thing

• Protein-based techniques; western blot, ELISA (enzyme-linked immunoabsorbent assay)

PCR for GMO detection

• Amplify a particular piece of DNA (make so many copies of it) → can make billions of copies of target sequence of DNA in a few hours

• you must know the sequence you want to amplify, so basically you need to know what foreign DNA may possibly be in there

• 3 main steps

  • Basis: temperature changes that affect DNA

  • Repeat these steps many times

  • 1. Denature DNA (95C), separate 2 strands

    • Mimics helicase activity

  • 2. Anneal primers (40-65C) to their complementary sequences on the single strands of DNA

    • If T too low → low stringency → false positives, primer matches elsewhere

    • If T too high → high stringency → false negatives, primer fails to match

    • Stringency = specificity of amplified DNA product, affected by annealing temperature

    • Other factors:

      •  GC% → more stringent than AT pairs

      • Salt and buffer

  • 3. DNA polymerase extends the DNA chain (72C) by adding nucleotides to 3’ ends of primers

    • If its too high you will cook the proteins

• Uses heat stable DNA pol bc PCR occurs at high T → taq (from Thermas AQuaticus, who likes to live in hot springs) DNA pol

PCR copy accumulation

• Number of copies is x2 after each cycle, size of DNA fragment produced is dependent on the primers

  • # of copies you start out with → if more, increases accumulation rate (reaches faster saturation), if less, decreases accumulation rate (reaches saturation slower)

  • In theory, PCR product accumulation should be exponential, but there may be exponential and non-exponential phases

    • Slows down because reaction: A + B → C, reaction rate = ([A][B])/[C], so as concentration of C increase, A and B decreases, reaction rate decreases over time

So we can detect the foreign DNA but how do we quantify how much is in there

•  qPCR

  • Can make a standard curve of things with known amount foreign DNA → you can know by doing the same # cycles and graph how many products is made for each sample vs. % GMO→ makes curve

  • Can also make have each make same amount of product and graph how many cycles that required vs % GMO → makes linear inverse relationship, preferred for quantitative analysis

Protein based gmo detection techniques

Immunoassays

• Enzyme-Linked immunosorbant Assay (ELISA)

  • Premise: must know foreign proteins (gene identity) that may be in there, and you must have the antibodies in there, the gene must be expressed to produce the protein, need training and equipment

  • plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones

  • the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then complexed with an antibody that is linked to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product

  • can be used quantitatively (use standard curve) or qualitatively

  • False negatives common

• Lateral flow test

What separation techniques you have learned from this class?

Extraction, distillation, precipitation, crystallization, drying, digestion, chromatography

Extraction and distillation techniques

Extraction:

Extraction - mejonnier, vit A analysis 

• Continuous - soxhlet

• Column (solid) - chromatography

Distillation - good for low water content foods

Direct: for alcohol
Steam: like Kjeldahl (after digesting with sulfuric acid, steaming to trap nitrogen by converting it into ammonium sulfate (NH4)2SO4)
Vacuum: adding vacuum to speed up distillation

Precipitation and crystallization techniques

Precipitation

• Fiber: Insoluble fiber is collected by filtration. Soluble fiber is precipitated in 78% ethanol and collected by filtration (can also do with protein and DNA??)

• Protein: Zinc sulfate to remove interfering proteins for CHO analysis

• DNA: 75% ethanol

• Also oversaturation can cause precipitation
  
  Crystallization
  NaCl 

• For purification of compounds → if you grow a crystal it pure :D

Supercritical extraction, GCFID, and HPLC analyze…

GCFID
Very good for determining fatty acid lipid profiles → measures sat and unsat FA, cholesterol, omega 3, trans fatty acids, conjugated linoleic acid
(soxhlet and mojonnier aint got nothing on tis one)

HPLC-UV
Can measure fat soluble vitamins (ADEK), cholesterol, carotenoids (beta carotene, beta cryptoxanthin, lutein, zeaxanthin, lycopene), and conjugated linoleic acid isomers

SFE
Uses a CO2 fluid under specific pressure and temperature → when CO2 behaves differently as a solvent… like changes in polarity??
Removes cholesterol from milk and leaves fatty acid and vitamins alone → but also we need some dietary cholesterol
Very expensive, not possible for commercial use

Removing water from a food sample

Drying methods: Oven drying - to remove water

• Desiccator - to remove water and moisture

  • Calcium sulfate

• Drying agents 

  • Sodium sulfate to remove water from organic solvents

  • Calcium sulfate in desiccators to keep things dry because they do not remove water from organic solvents

• Freeze-drying

• Infrared drying

• Microwave drying

Digestion methods

• Acid - like in Kjeldahl and Babcock or for sugar analysis

• Enzymatic - like to remove CHO in fiber analysis (alpha amylase and amylglucosidase) and protease to remove proteins

• Base - whole grain analysis, to digest polyphenols in the grains → before we used to underestimate whole grain by like 85%

Common analytical methods we learned

• Gravimetric methods: for moisture, ash, total solids, total fat, fiber

  • Body weight

• Titration: for acid, base, sodium, protein (Kjeldahl), vitamin C

• Spectroscopy: absorption (spectrophotometer with UV and visible light, atomic absorption spectroscopy), emission (fluorescence  and atomic emission)

• HPLC, GC, MS

  • Common solvent properties: Miscibility, polarity, viscosity, UV cutoff

Volumetric glassware and balances

• Pipettes

  • Serological pipettes for solvent transfer

  • Volumetric pipettes for making standard solutions

• Volumetric flasks also for making standards because they are calibrated

• Balances**** 

  • Analytical balance sensitivity: 0.1mg

    • Ex: if you have very little of a substance so whats the minimum you can weigh while not wasting money 

      • If you use 1mg (10x sensitivity) → already have 10% variation (no good)

      • 5% is the variation that is acceptable → so you should use 10mg so you get only 1% variation*******