Food Analysis 3 Review Topics Part 1

Front 1

Basic components of a MS

Back 1

Sample Introduction >

Ion Source >

Mass Analyzer >

Detector >

Data System

Front 2

Principle involved in MS

Back 2

The MS performs three basic functions. There mustbe a way to ionize the molecules, which occurs inthe ion source by a variety of techniques such aselectron impact, matrix-assisted-laser-desorption, oratmospheric pressure ionization. The charged molecularion and its fragments must be separated accordingto their m /z , and this occurs in the mass analyzer section(e.g., quadrupoles, ion traps, time-of-flight (TOF),Fourier transform). Finally the separated, chargedfragments must be monitored by a detector

Front 3

Information obtained in MS analysis (compared to other types of detectors for HPLC orGC)

Back 3

Detects and identifies unknown compound. Useful for identifying unknown.

Front 4

Types of mass analyzers

Back 4

There areseven basic types of mass analyzers: quadrupoles (Q), ion traps (IT), time-of-flight (TOF), magneticsectors , isotope ratio MS , Fourier-transform-basedion cyclotrons (FT-ICR) and Orbitraps (OT), andaccelerator mass spectrometers (AMS).

Front 5

Advantages of MS/MS vs. single MS

Back 5

MS/MS is better because

- Greater selectivity

- Better quality spectra

- Additional structural info

- Less sample cleanup

- Improved detection

Front 6

Adsorption vs. partition chromatography, regarding nature of stationary andmobile phases, and how solute interacts with phases

Back 6

Adsorption - Stationary is solid, Mobile is liquid

Partition - Stationary is liquid, Mobile is liquid

Adsorption - Solute adsorbs to stationary phase (By van der waals, electrostatic, H-Bonds, Hydrophobic)

Partition - Solute partitions between mobile and stationary phases according to partition coefficient.

Front 7

Normal vs. reversed phase chromatography, regarding nature of stationary andmobile phases, and order of elution of polar vs. nonpolar compounds

Back 7

Normal - Stationary is polar, Mobile is non

Reversed - Stationary is nonpolar, Mobile is polar

Polar stationary phase = Polar elutes last

Nonpolar stationary phase = Nonpolar elutes last

Front 8

How does changing polarity of mobile phase affect elution of solutes?

Back 8

Changing the polarity of the mobile phase changes the order of the solutes

Front 9

Cation vs. anion exchange chromatography, regarding charge on column andcharge of compounds that bind;

Back 9

Anion column has a positively charged stationary phase, while the charge of the compounds that bind is negative

Cation column has a negatively charged stationary phase, while the charge of the compound that binds is positive.

Front 10

How are bound compounds unbound? Why doesthis work?

Back 10

– Change mobile-phase pH (changes charge ofsolute, so it no longer binds) (However, forproteins, changing charge can mean passingthrough isoelectric point, so it can precipitate out ofsolution)

OR– Increase ionic strength (e.g., add NaCl) to m

Front 11

What is charge of an amino acid/protein above vs. below itsisoelectric point?

Back 11

The charge of an amino acid/protein above the isoelectric point is negative

The charge of an amino acid/protein below the isoelectric point is positive

Front 12

Ion exchange vs. affinity chromatography: Similarities? Differences?

Back 12

Ion exchange is less specific than affinity chromatography. Because Ion exchange is based on charges like positive or negative and polarity of the molecule being purified. You can purify proteins in ion exchange based on their overall net charge.

Affinity chromatography on the other hand uses antigen antibody, or ligand and receptor or enzyme substrate which is VERY specific. This way you only purify what you are looking for.

Front 13

Internal vs. external standards, regarding the nature of the standards, when/whyeach is used, how they are handled related to the sample, and what is plotted onstd. curve? How is internal standard selected?

Back 13

 External Standards

– Standard solutions arechromatographed, and peak area(or height or mass) are plotted vs.concentration on standard curve

Internal Standards

– Amount of each component insample is determined bycomparing area (or height ormass) of component peak to thatof the internal standard peak

– Need to run set of standards atvarying concentrations, with eachsolution containing constantamount of internal standard

Front 14

TLC vs. column liquid chromatography, regarding nature and location ofstationary phase and mobile phase, how sample is applied, and how separatedsolutes are identified

Back 14

TLC: Thin layer (250 μm thick) of sorbent orstationary phase bound to inert support Different techniques can be applied to detector visualize the components:– Colorimetrically (ninhydrin, sulfuric acid, iodinevapor)– Measure of absorbed or emitted radiation(fluorescence)– Measure of radioactivity of radioactive labeledcompounds Quantitative evaluation can be carried out bymeans of:– Densitometer– Scraping off the zone, eluting the compound andthen analyze the resulting solution

Column liquid chromatography

Mobile phase is liquid; Stationary phase is a solid, ora liquid supported by an inert solid Stationary phase is packed in column andequilibrated with mobile phase Mobile phase moves through column by gravity flowor peristaltic pump

Elution may be isocratic (constant mobile phasecomposition) or a gradient (changing nature ofmobile phase) Solutes are separated based on str

Front 15

HETP vs. N. vs. L, regarding what they stand for and their relationship; HowHETP, flow rate, L, N and column efficiency are related?

Back 15

HETP = height equivalentto a theoretical plate (i.e.,plate height, H)

L = column length

N = number of theoreticalplates

Lower HETP = More Accuracy

Front 16

How does stationary phase particle size affect column efficiency?

Back 16

Efficient column gives narrowpeaks and keep bands fromspreading

Front 17

Bonded support vs. coated support for partition chromatography; Advantage ofbonded support?

Back 17

Coated Supports:– Liquid coating on a solid matrix» Silica gel (hydrated silica)» Cellulose» Glass beads– Disadvantage: liquid stationary phase is oftenstripped off (overcome with Bonded Support)

Bonded Supports:– Liquid stationary phase covalently bonded to asupport via a chemical reaction– Very common in reversed-phase HPLC (Silicabonded to C8 or C18)

Front 18

Polystyrene vs. polysaccharide-based stationary phase, regarding nature ofstationary phase and appropriate applications

Back 18

Stationary phase is cross-linked polyelectrolytes

– Polysaccharide ion exchangers:Useful for separation and purification of large molecules, e.g.,protein and nucleic acid

Polystyrene ion exchangers

Extent of crosslinking controls rigidity and porosity, todetermine optimal us

Front 19

Void volume, total volume, and elution volume in size exclusion chromatography:How determined? How used to calculate Kav? How Kav is used to estimate M.W.of proteins?

Back 19

Void volume (Vo) = volume of mobile phase in column

Vi = volume of liquid insidethe sorbent pores Elution volume

Ve, is the volume required to elute acertain solute

Kav = (Ve-Vo)/(Vt-Vo)

Front 20

Gradient vs. isocratic elution: How used in HPLC? Advantage of gradientelution in HPLC?

Back 20

Gradient elution system usedto vary mobile phase conc.during the run (by mixingfrom two or more reservoirs)

Gradient HPLC isextremely important for the effective elution of allcomponents of a sample and for optimal resolution.It is routinely applied to all modes of HPLC exceptsize-exclusion chromatography.

Front 21

Guard column: What is it and why is it used?

Back 21

Guard Columns (or cartridge):– Installed between injector and analytical column– Filled with either pellicular media or microparticulatepacking material, identical to that of analytical column

Often are used to protect the analyticalcolumn from strongly adsorbed sample components

Front 22

HPLC column packing materials: Requirements and types?

Back 22

Must have:» Good chemical stability» Sufficient mechanical strength to withstandpressure used» Well-defined particle size (narrow distribution)

Nature of stationary phase determinestype of separation mode: normal-phase,reversed-phase, ion-exchange, etc.

Front 23

HPLC detectors: Types?

Back 23

UV-Vis, Fluorescence, Refractive

Front 24

Ion chromatography: Description and applications?

Back 24

high-performance ion-exchange chromatographyusing a relatively low-capacity stationaryphase (either anion- or cation-exchange) and, usually,a conductivity detector .

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Times} determination of organic and inorganic ions in milk;organic acids in coffee extract and wine

Front 25

Basis of separation using HPLC columns, and example applications?

Back 25

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Front 26

Methods to isolate solutes from foods for GC analysis: headspace, distillation, solventextraction, solid-phase extraction; Food applications for each method? For solid-phaseextraction, what are its applications, and its advantages compared to liquid-liquidextraction?

Back 26

Headspace Methods Direct injection of headspace vapors above a foodproduct is a simple method to isolate volatilecompounds

Distillation MethodsProduct moisture or outside steam isused to heat and codistill volatiles fromfood product

Solvent Extraction Often preferred method for recovery ofvolatiles from food

Solid-Phase Extraction Original Procedure:– Liquid sample is passed throughcartridge or filter withchromatographic material (e.g.,ion-exchange resins; reversedornormal-phase packings

-Advantages ofSolid-PhaseExtraction overLiquid-LiquidExtractions:– Less solvent required– Speed– Less glassware needed– Better precision andaccuracy– Minimal solventevaporation for furtheranalysis– Readily automated

Front 27

Derivatization of solutes for GC analysis: What does this mean? Why needed? Exampleapplications?

Back 27

Some compounds must be derivatized(chemically modified; e.g., use silyl reagents,esterifying reagents) before GC analysis,because of the following:– Are thermally unstable– Are tool low in volatility (e.g., sugars, amino acids)– Yield poor chromatography separation due topolarity (e.g., pehnols, acids)

Front 28

Capillary column: What is a split injection? Why is it needed? Advantages?

Back 28

Split Injection– Only a portion of the sample injected goes ontothe column (because capillary columns havelimited capacity)– Sample gets diluted with carrier gas, for 1:50 to1:100 split (e.g., 1 part goes to column, 99 partsare vented to the split vent)– High split ratio usually gives sharp, narrow peak

Front 29

Packed column: What is meant by the “percent load”, and what does this affect?What is column bleeding, and what typically causes it?

Back 29

– Liquid loading applied to solid support at 1-10% byweight of solid support– Loading influences analysis time, resolution, andcolumn bleeding

column bleeding (i.e., liquid coating volatilized andlost at high temp.)

Front 30

Nature of packed vs. capillary columns? - - differences; characteristics of each

Back 30

Packed columns

0.5 – 5 m long– Solid support commonly made of diatomaceousearth, of definite mesh size– Liquid loading applied to solid support at 1-10% byweight of solid support

Capillary

– 5-100 m long; very thin walls– Inner diameters are 0.1 mm (microbore), 0.23 –0.32 mm (normal capillary), or 0.53 mm(megabore)– Stationary phase: Liquid coating chemicallybonded to glass walls of column, and internallycrosslinked to thickness of 0.1 – 5 um– Get more column bleeding with thicker stationaryphase film

Front 31

GC detectors: Types?

Back 31

Thermal Conductivity (TCD)Flame Ionization (FID)Electron Capture (ECD)Flame Photometric (FPD)Pulsed Flame Photometric (PFPD)Photoionization (PID)Electrolytic Conductivity (ELCD)Thermionic (i.e., Nitrogen PhosphorusDetector, NPD)

Front 32

Basis of separation using GC columns?

Back 32

Separation depends on several types ofchromatography: Size-exclusion chromatography– e.g., Separate gases such as nitrogen, oxygen,and hydrogen; Separate chiral compounds Adsorption chromatography– E.g., Separate very volatile polar compounds onporous polymer columns Partition chromatography– Workhorse for GC separation, used for manyseparations

Front 33

GC vs. HPLC – characteristics and applications?

Back 33

GC

- Mobile phase is inert gas

- Stationary phase is typically liquid

- Long column length

- Temp gradient

- App: Triglyerides, pesticides drugs

HPLC

- Mobile phase is liquid

- Stationary phase is typically solid

- Short Column length

- Gradient is either mobile phase or isoelectric

- App: Carbs, Proteins, pigments

Front 34

Electron Input ionization

Back 34

-Fragmentation mention

- Once in ion source, compound is exposed to beam of electrons emitted from a filament composed of rhenium or tungsten metal

- When direct current is applied, it heats and emits electrons that move across ion chamber towards positive electrode to other side.

- As electrons pass through source region, they come in close proximity to sample molecule and extract an electron, forming ionized molecule

- Once ionized, molecules are unstable and breaks into smaller molecular fragments

Front 35

Chemical Ionization

Back 35

Fragmentation method

- Gas is ionized, which directly ionies modlecule

- Soft ionization

- Only few fragments produced

- Most important use to determine molecular ion

Front 36

What is the base peak on a mass spectrum?

Back 36

(BASE ION) Fragment that has highest abundance or intensity

Front 37

What is the molecular ion peak?

Back 37

(MOLECULAR ION) Peak that has the highest mass number and represents positively charged intact molecule with an m/z equal to molecular mass.

Front 38

Electrospray

Back 38

Charges are formed on the liquid droplets as the solvent evaporates. Then the ions are emitted from the cone

Front 39

APCI

Back 39

Similar set up to electrospray but there is a charged needle at the point of exit of the roplets from the vaporized tube. A high voltage discharge forms ions from both LC mobile phase and nitrogen nebulizing gas. These charged ions then can further react with the sample, producing charged species.

Front 40

Major differences between magnetic, quadrupole, ion trap and fourier transform mass analyzer?

Back 40

Magnetic - Uses a magnet to sepearte ions, it is slower and takes up more lab space, but it can have a higher resolution

Quadrupole- uses four rods with varying electrical potentials that selectively filter very rapidly to scan a range of masses. Fast and detector can be made very small which explains its popularity in bench top MS, however resolution is not very good.

Ion Trap - Has been called a 3D quadrupole and is somewhat similar except ALL ions are trapped then released over time to produce MS spectra. It is also small in size and fast. Resolution is not as good as the magnetic sector and is about the same as quadrupole. With good resolution ion trap can perform tandem MS experiments.

Fourier transform mass analyzer - iUnique from other mass analyzers because they are nver resolved in space or time, nor are they detected upon a detector. Instead, the frequency is measured as a function of applied electronalysis or magnetic field. This results in sub part per million mass accuracy.