Method Liquid chromatography

History 1890s: Mikhail Tswett used chromatography in chlorophyll research 1952: Archer Martin & Richard Synge won Nobel Prize for Chemistry for partition chromatography

Principles Physical method of separation in which components to be separated are between 2 phases: 1 stationary, 1 mobile Components separated based on affinity Both qualitative and quantitative o Qualitative: retention time o Quantitative: concentration Data output: chromatogram Types: o Adsorption: solid stationary phase o Partition: liquid stationary phase

Instrumentation Paper chromatography o Stationary phase is liquid o Dissolved sample applied as spot from edge of strip of paper, then allowed to dry o Dry strip is suspended in closed container in which atmosphere is saturated with solvent o Techniques: ascending, descending, or horizontal Thin layer chromatography o Thin layer of stationary phase formed on flat surface coated with absorbent o Mobile phase ascends (capillary action) o Chromatogram dried prior to detection (physical,

Applications In general: o Materials engineering o Analytical chemistry o Proteomics o Quality control Paper: o Highly polar compounds Thin layer: o Lipids o Carbs o Vitamins o Amino acids o Natural pigments Column: o Compound separation after organic synthesis o Purification of compounds o Detection of trace compounds o Construction of component profiles

Advantages and Disadvantages Paper: o Advantages: simple, low-cost, unattended, does not need expensive instruments o Disadvantages: timeconsuming Thin Layer: o Advantages: extremely rapid, more sensitive than paper chromatography o Disadvantages: uses corrosive agents and high temperatures, sample cannot be recovered once loaded onto plate Column: o Advantages: easily adjusted, no chemical reaction, officially accepted o Disadvantages: time-consuming, constant monitoring, can be expensive

Supercritical fluid (SCF) chromatography

1879: Hanny & Hogarth 1960s: major developments 1962: Cowin & Turin 1981: HewlettPackard 1982: Navotry

SCF: both liquid and vapor phases Obtained by heating above Tc and compress above Pc Properties: o density o diffusion o viscosity o Can dissolve large non-volatile solutes SCF acts as mobile phase; samples separate into bands Mobile phase blocks reactive sites on stationary phase

chemical, biological) Column liquid chromatography o Stationary phase is either solid or liquid o Sample is passed through column packed with solid particles o Types: flash, quick column, size exclusion, ionexchange o Methods: dry, wet Modifier (usually CO2, good for non-polar matrices) Pump: flow control o Syringe pump for capillary column (constant pressure) o Reciprocating pump for packed column (allows mixing) Injector: inject sample into column Oven: precise temperature control (ambient to 300C) Restrictor: maintains pressure in column, prevents clogging Detector: specific

Non-polar compounds Usually for lipids Used in forensics, pharmaceutical studies, food analysis, toxicology, life sciences

Advantages: o diffusibility o viscosity o density o solvating power o Use of non-toxic reagents o Cost-effective o Fast, convenient o Easy recovery of analytes after extraction o Same results quality as HPLC Disadvantages: o Poor capability in polar compound separation o Modifier limits 2

Method Gas chromatography

History 1901: Michael Tsvett discovered chromatography color writing 1941: Arthur Martin & Richard Synge 1951: Arthur Martin & Anthony James GC principles 1954: Griffin & George 1st commercial GC system

Principles Method of separation of a mixture of compounds that can be vaporized Both qualitative and quantitative o Quantitative: retention time o Qualitative: area under peak Factors affecting analyte migration: o Sample volatility o Analyte polarity

Instrumentation Carrier gas: pressurized container o Pushes sample into column o Usually inert gas o Also contains gauge and flow meter o May have molecular sieve or trap for particles Injector port: where sample is injected o Temperature is 150-250 C to vaporize sample o Uses rubber stopper o Must be fast (slow injection causes band broadening, resolution loss) Columns o Packed or capillary o Depends on needed resolution o column length effectivity (to a limit only)

Applications Separation, identification, quantitation of unknown compounds Separation of flavour and aroma components for sensory evaluation

Advantages & Disadvantages Advantages: o Good separation o Simple, rapid o Sensitive o Cheap Disadvantages: o Works only with volatile sample

Electrophoresis

Theodere Svedberg: invented moving boundary electrophoresis in his study of colloids 1950s: zone and gel

Migration of charged particles through a matrix due to electric field Matrix: o Gel (temperaturedependent) o Capillary

o Pressure differentials must be avoided Detector o TC cell: thermal conductivity cell (measures resistance of hot wire; signal concentration) o FID: flame ionization detector (ionic fragments from burning are collected and produce electric current; for oxidizable compounds only) Oven: regulates temperature (maintain state of vapor and components) Capillary: o Set pH o Diameter aids separation o Buffer pulls components to detector o Columns are selected based on

Detection of microorganisms Protein determination

Advantages: o Good for macromolecules o Fast o band distortion o resolution Disadvantages: o Uses toxic reagents o Complex 4

electrophoresis 1960s: isoelectric, capillary, SDSPAGE

Fluorescence spectroscopy 1865: Nicolas Monandes opalescence in Mexican wood 1950: de Saliagun isolated contli Acua coatlein B resembled fluorescence 1913: Heimstaedt & Lehmann microscope Jablonski father of spectroscopy

(withstand high voltage, small samples only) Depends on size, shape, charge , friction, chemical composition size net charge speed + cathode - anode Identification based on velocity, mobility Band visibility via dyes or UV rays Both qualitative and quantitative Fluorophores: capable of fluorescence Absorb and re-emit energy at specific wavelengths Both qualitative and quantitative

resistance, diameter, transparency to UV rays, ability to dissipate heat Gel: o Molecules are buffered o Polyacrylamide for proteins, agar for nucleic acids o Gel pore size is adjustable

o Detection time varies with matrix o Expensive

Light source excitation monochromator SAMPLE emission monochromator photomultiplier fluorescent signal detector Emission monochromator is positioned at 90 Slits in monochromator are adjustable to control amount of light passing through

Detection of adulterants Quality control Characterization and differentiation of oils and wines Evaluation of nonenzymatic browning and photoxidation

Advantages: o 2 wavelengths used o Simple, easy, rapid o Can be used online o selectivity, sensitivity o Non-invasive o signal noise Disadvantages: o Not for turbid systems (A > 0.1) o Quenching lowers intensity (collisional, static, and resonance)

Method Infrared spectroscopy

History 1800s: Herschel discovered infrared region 1903: Coblentz 1st modern analysis; foundation of infrared spectroscopy WWII: air force, penicillin structure

Principles Infrared: between visible & microwave Wave number frequency Absorbed and converted into vibrations (stretch and bend); specific to functional group For molecule to be IR-active, there must be change in dipole moment (change intensity) Frequencyvibration = FrequencyIR radiation: molecules absorb Vibrational motion is quantized; follows selection rule

Instrumentation IR source SAMPLE monochromator dispersion medium wavelength selection detector Detectors are either thermal or photosensitive IR source must have wide wavelength range; not have fluctuations (e.g. Nernst glower, Globar, nichrome wire) Thermoscope: combination of 2 metals (energy focused on junction; change in temperature ensures voltage) Bolometer: measures change in resistance; more sensitive Golay cells: measures change in pressure Sample cells: must be carried downward; treated with dry solvents, not exposed to large changes in T

Applications Detection of adulterants, contaminants Estimation of component content

Advantages & Disadvantages Advantages: o Rapid o Non-destructive o Environmentally friendly o Online o Can detect specific functional groups Disadvantages: o Transparent matrix needed o False radiation due to heat

Method Atomic absorption spectroscopy (AAS)

History

Principles Allows measurement of amount of specific metals by absorption of light Lamp emits same wavelength as target metal (via excitation of metal inside it) Remaining light after absorption of metal is measure of substance present Both qualitative and quantitative

Instrumentation Source cropper furnace atomizer monochromator detector Light source: depends on element to be analysed o Hollow cathode lamp (sputtering) o Electrode-less discharge lamp (radio frequency) Chopper: removes emission interference from furnace and/or flare (peaks: lamp; plateaus: flame) Atomizer: produces ground state atoms (specificity) o Flame atomizer (nebulizer + Bunsen burner) o Electrothermal atomize (graphite furnace AAS) Techniques: o Cold vapor (mercury) o Hydride generation (NaBH4)

Applications Quality assurance Safety testing Detection of heavy metals (mercury, lead) Assessment of food packaging

Advantages & Disadvantages Advantages: o Highly applicable o Simple, fast o Accurate o Small samples Disadvantages: o Samples must be dissolved o Interference from other light sources

Method Inductively coupled plasma atomic emission spectroscopy (ICP-AES)

History 1960s: FAAS & FAES Albright & Wilson, Stan Greenfield; plasma-based instruments 1975: Fassel & Winge determine trace pollutants in water 1979: establishment of ICP-AES for waste water treatment 1980s: 2 designs (simultaneous, sequential) 1990s: axial viewed torches

Principles Elemental concentration and identification Plasma: heat source, gaseous mix of cations and electrons Absorption of heat causes excitation and energy in the form of light Emission of energy at specific wavelengths Movement of electrons: o Absorb E: from ground to excited state o Release E: from excited to ground state Both qualitative and quantitative Atomic nuclei spin on axis o Creates magnetic field o Distinct quantum # o Parallel or antiparallel

Instrumentation Nebulization excitation detection calibration Uses ICP torch for excitation Sample: liquid (2-5% HNO3, 50 ppm) Similar to xerography in terms of reactions (electron attraction) Calibrate to minimize instrumental drift

Applications Food analysis o Confirm purity o Detect adulteration Environmental Biological Geological

Advantages & Disadvantages Advantages: o Reliable, simple o Precise, accurate o Rapid and simultaneous o analytical working range o detection limits o Applies to elements not under AAS Disadvantages: o Costly, regular maintenance o Dissolved samples only (no solids) o Cannot distinguish isotopes o Uses silicon tetrafluoride (toxic) o Not for ultra-trace results o Cannot detect CHON, inert gases Advantages: o Rapid o Wide application (atom-specific) o Non-invasive o Can be used with other identification methods 8

Nuclear magnetic resonance (NMR) spectroscopy

1944: Rabi wins Nobel prize for discovery of NMR 1946: Purell & Block wins Nobel prize for NMR

Atoms oriented in strong magnetic field RF beamed onto sample, exciting atoms RF released by atoms Released RF detected Sample held in

Chemical information (shift, rate constant, conductivity) Structure determination of molecules

experiments 1966: Ernst & Anderson discovered Fourier transformation 1985: Wthrich wins Nobel prize for use of NMR in 3D structures

Electron spin resonance (ESR) spectroscopy

1944: Zavoisky discovered ESR during study of Cu2+ 1965: McConmel spin labels 1989: Hutbel SOSI strategy

o Precession in spin causes Larmor frequency RF pulse: excites large range of frequencies; right angle to EM field Data measured: free induction decay (FID) Processed by Fourier transformation into NMR spectrum Qualitative: structure and behavior Electron spin resonance Similar to NMR Interaction of unpaired electrons Constant magnetic field while microwave frequency varies (and vice versa) Conditions: o Frequency = spectrum between energy levels o Formation of

borosilicate straight glass tube of uniform thickness and length Solvent: COCl3 Blank: Si(CH3)4

Disadvantages: o Low temperature o Impurities affect result o Expensive o sensitivity

Microwave source attenuator circulator sample cavity + magnet detector Microwave source: klystron Attenuator: controls microwave power Circulator: 2 ports for direction/ transmission ESR spectra is first derivative of absorption spectra

Detection of foods with paramagnetic species (free radicals) Detection and analysis of irradiated foods

Advantages: o Irradiation-specific o Time-efficient o Non-destructive o Reproducible Disadvantages: o Low temperature o Impurities affect results

Mass spectroscopy 1800s: Dalton atomic theory 1887: Thompson discovery of electron, cathode rays 1898: Wien study of positive rays 1907: Thompson use of particle deflection as means of determining mass and charge 1919: Aston discovery of isotopes

dipole o Presence of paramagnetic substance Electron + external magnetic field = spin o Nuclear hyperfine intersection o Dipole-dipole interaction Both qualitative and quantitative Deflection and separation of ion beams by electromagnetic field Classified according to mass or charge Amount of deflection = mass of sample Both quantitative and qualitative

Ionization acceleration of ions deflection detection Ionization is based off clastogram; ions are fragmented Accelerated to carry electromagnetic field Parent peak: last to be detected, least fragmented Base peak: highest peak, many ways of fragmenting

Determination of unknown compounds

Advantages: o Highly sensitive and selective o Can be coupled with other separation methods Disadvantages: o Sample must be purified before analysis o Costly o Cannot distinguish isomers o Not reliable for mixtures o Hard to clean

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Method Voltammetry

History Galvan Galvanism Volta invented voltaic battery 1800: Nicholson & Carlisle 1800: Cruikshanks

Principles Current-voltage relationship Applied potential generates current due to redox reaction at surface of electrode Current concentration Based off electrochemical principles

Instrumentation Wave form generator potentiostat cell current to voltage converter Electrodes: working, reference, counter Peak of voltammogram: limiting current Dropping mercury electrode (DME) determines redox substances Cyclic o Applies potential to working electrode o Charges with time (forward, reverse) o Records current as function of time o Triangle graph (peak indicates change in concentration) Anodic stripping o Mercury drop hung at capillary o Allows for oxidation o Straight line on graph shows deposition, slant shows stripping

Applications Adsorption processes Measurement of kinetics Detection and determination of adulterants Determination of vitamin content

Advantages & Disadvantages Advantages: o Selective o Wide range o Rapid o Simultaneous determination Disadvantages: o Costly o Training required o Regular service and maintenance o Mercury can dissolve at high (+) potentials

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Method Electron microscopy

History 1591: Janssen first microscope 1886: Abbe & Zoss compound light microscope 1931: Knoll & Ruska 2-stage TEM 1933: Knoll & Ruska TEM with 3 magnetic lenses 1938: von Ardenne SEM

Principles Makes use of electron beams as illumination source Vacuum environment (beams must be unfiltered by gas) Both qualitative and quantitative

Instrumentation Parts: electron gun, electromagnetic lenses, fluorescent screen (for viewing and analysis) TEM o Beam passed through ultra-thin specimen o Sample must be live, trimmed, concentrated; fixed and dehydrated, then infiltrated with transitional solvent o Produces 2D image SEM o Uses secondary electrons o Focused scanned collected cathode ray tube o Produces 3D image Spectroscopic method Types: o Direct (antigen is adsorbed onto plate then enzymelabelled antibodies are added) o Indirect (primary

Applications Structure analysis Microbiology assays

Advantages & Disadvantages TEM o Advantages: magnification, resolution o Disadvantages: potentially carcinogenic reagents, costly, labor-intensive SEM o Advantages: depth of field, sample size, less tedious o Disadvantages: magnification, resolution, costly

Enzyme-linked immunosorbent assay (ELISA)

1798: Jenner discovered antigen-antibody relation using cowpox 1897: Krauss

Uses antigenantibody relation (epitope identification) Passive adsorption: adsorption of antigen to solid phase (polystyrene),

Determination of antigens Fields o Medicine o Agriculture o Food science Uses o Detection o Quantification

Advantages: o Simple o Time-efficient o Sensitive o Minimum sample o Low cost Disadvantages: o Low resolution

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Toxicity bioassay Dioscorides Matthieu Orfila 1st formal discussion on toxicology Paracelcus dosage makes things not poisons Trevan pioneered LD-50 Draize Draize test

allows for separation Enzyme-substrate reaction: antibody conjugates with enzymes to react with substrate, produces colored solution Both qualitative and quantitative Use of organisms Toxic: dosage beyond limit (measures potency) Organisms are dosed through inhalation, ingestion, or absorption Classification basis: o Biological organization used o Response (quantal, graded) o Intent (absolute, comparative) o Exposure (cutaneous, ingestion, intramuscular, inhalation) Both qualitative and quantitative

antibody added as intermediate before addition of enzyme-labelled antibodies) o Sandwich o Competitive/ inhibitive

o Identification o Assessment

Set-up protocol experimentation parameter analysis results Considerations in extrapolation of data o Reproducibility o Relative approximation of dosage o Design, evaluation of bioassay o Consensus on interpretation o Availability of data for decision-making

Regulatory Effect screening Research & teaching Biomonitoring Hazard/ risk assessment

Advantages: o Wide range o Can test for multiple parameters simultaneously o Real-time results Disadvantages: o Time-consuming o Labor-intensive o Can be expensive o Not necessarily applicable to humans

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Method Protein quality bioassay

History

Principles Protein quality: usefulness of protein to body (ideal ratios) More qualitative than quantitative Types o Digestibility (true, apparent) o Body weight gain (protein efficiency ratio, net protein ratio, multilevel/ slope ratio) o Nutrient balance (biological value, net protein utilization) Glycemic index (increase/ decrease) Sugar levels in blood are regulated by insulin Fasting (8-12 hours) releases glucose in plasma due to glycogen hormone Normal levels: 7099 mg/dL Area under peak

Instrumentation Selection of method Selection of test animals Preparation of animals Management and experimentation Determination of protein content (usually Kjeldahl method)

Application Analysis of feeds Processing procedures Bioavailability testing

Advantages & Disadvantages

Blood sugar level bioassay

Photometric assay Orthotoluidine or otolidine (colorimetric, aldohexose reaction) Ferricyanide (glucose reduces, yellow to colorless at 420 nm) Hexokinase (glucose reduced in ATP, forms NADH at 340 nm) Glucose oxidase (O2 reaction with phenol aminophenazone)

Diet planning Diabetes maintenance Determination of glycemic index of foods (GI: complex carbs; GI: simple carbs)

Advantages: o Widely accepted o More accurate o Easy, fast Disadvantages: o Large biological variability o Pre-analytical variations

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Method Antioxidant assay

History

Principles Antioxidants neutralize effects of free radicals (can be primary or secondary) Measure antioxidant content/ capacity Capacity: extent of radical scavenging; duration of lag phase of probe during peroxidation reaction (TRAP) or how much oxidant is reduced (DPPH) Quantitative

Instrumentation Fluorescence or UV spectrophotometer Oxidation initiator Suitable substrate Appropriate endpoint Types: o Hydrogen atom transfer (HAT): competitive, adds targets; based on reaction kinetics o Single electron transfer (SET): noncompetitive; based on color changes

Applications Food analysis

Advantages & Disadvantages Advantages: o Independent of solvent o Not timeconsuming o Sensitive o Wide range of applications Disadvantages: o Can be costly o Affected by presence of other compounds

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