Advanced Instrumental Methods for Identification of Components in Food

Method History Principles Instrumentation Applications Advantages andDisadvantages

Liquid chromatography

1890s: Mikhail Tswett – used chromatography in chlorophyll research

1952: Archer Martin & Richard Synge – won Nobel Prize for Chemistry for partition chromatography

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 quantitativeo Qualitative:

retention timeo Quantitative:

concentration Data output:

chromatogram Types:

o Adsorption: solid stationary phase

o Partition: liquid stationary phase

Paper chromatographyo Stationary phase is

liquido 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 chromatographyo 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,

In general:o Materials

engineeringo Analytical

chemistryo Proteomicso Quality control

Paper:o Highly polar

compounds Thin layer:o Lipidso CarbsoVitaminsoAmino acidsoNatural pigments Column:o Compound

separation after organic synthesis

o Purification of compounds

o Detection of trace compounds

o Construction of component profiles

Paper:o Advantages: simple,

low-cost, unattended, does not need expensive instruments

o Disadvantages: time-consuming

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:oAdvantages: easily

adjusted, no chemical reaction, officially accepted

oDisadvantages: time-consuming, constant monitoring, can be expensive

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chemical, biological)

Column liquid chromatographyo Stationary phase is

either solid or liquido Sample is passed

through column packed with solid particles

o Types: flash, quick column, size exclusion, ion-exchange

o Methods: dry, wetSupercritical fluid (SCF) chromatography

1879: Hanny & Hogarth

1960s: major developments

1962: Cowin & Turin

1981: Hewlett-Packard

1982: Navotry

SCF: both liquid and vapor phases

Obtained by heating above Tc and compress above Pc

Properties:o↑densityo↑diffusiono↓viscosityoCan dissolve large

non-volatile solutes

SCF acts as mobile phase; samples separate into bands

Mobile phase blocks reactive sites on stationary phase

Modifier (usually CO2, good for non-polar matrices)

Pump: flow controlo Syringe pump for

capillary column (constant pressure)

oReciprocating pump for packed column (allows mixing)

Injector: inject sample into column

Oven: precise temperature control (ambient to 300°C)

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↑diffusibilityo↓ viscosityo↑ densityo↑solvating poweroUse of non-toxic

reagentsoCost-effectiveo Fast, conveniento Easy recovery of

analytes after extraction

o Same results quality as HPLC

Disadvantages:oPoor capability in

polar compound separation

oModifier limits

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Method History Principles Instrumentation Applications Advantages & Disadvantages

Gas chromatography

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

Method of separation of a mixture of compounds that can be vaporized

Both qualitative and quantitativeo Quantitative:

retention timeo Qualitative: area

under peak Factors affecting

analyte migration:o Sample volatilityo Analyte polarity

Carrier gas: pressurized containero Pushes sample

into columnoUsually inert gasoAlso contains

gauge and flow meter

oMay have molecular sieve or trap for particles

Injector port: where sample is injectedo Temperature is

150-250 °C to vaporize sample

oUses rubber stopper

oMust be fast (slow injection causes band broadening, resolution loss)

Columnso Packed or

capillaryo Depends on

needed resolutiono ↑column length

↑effectivity (to a limit only)

o Pressure

Separation, identification, quantitation of unknown compounds

Separation of flavour and aroma components for sensory evaluation

Advantages:o Good separationo Simple, rapido Sensitiveo Cheap

Disadvantages:o Works only with

volatile sample

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differentials must be avoided

Detectoro TC cell: thermal

conductivity cell (measures resistance of hot wire; signal α 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)

Electrophoresis Theodere Svedberg: invented moving boundary electrophoresis in his study of colloids

1950s: zone and gel electrophoresis

Migration of charged particles through a matrix due to electric field

Matrix:o Gel

(temperature-dependent)

o Capillary (withstand high

Capillary:o Set pHo Diameter aids

separationo Buffer pulls

components to detector

o Columns are selected based on resistance,

Detection of microorganisms

Protein determination

Advantages:o Good for

macromoleculeso Fasto ↓band distortiono ↑resolution

Disadvantages:o Uses toxic reagentso Complexo Detection time

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1960s: isoelectric, capillary, SDS-PAGE

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

diameter, transparency to UV rays, ability to dissipate heat

Gel:o Molecules are

bufferedo Polyacrylamide for

proteins, agar for nucleic acids

o Gel pore size is adjustable

varies with matrixo Expensive

Fluorescence spectroscopy

1865: Nicolas Monandes –opalescence in Mexican wood

1950: de Saliagun – isolated contli

Acuña – coatlein B resembled fluorescence

1913: Heimstaedt & Lehmann - microscope

Jablonski – father of spectroscopy

Fluorophores: capable of fluorescence

Absorb and re-emit energy at specific wavelengths


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 non-enzymatic browning and photoxidation

Advantages:o 2 wavelengths usedo Simple, easy, rapido Can be used onlineo ↑selectivity,

sensitivityo Non-invasiveo ↓signal noise

Disadvantages:o Not for turbid

systems (A > 0.1)o Quenching lowers

intensity (collisional, static, and resonance)

Method History Principles Instrumentation Applications Advantages &

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DisadvantagesInfrared spectroscopy

1800s: Herschel – discovered infrared region

1903: Coblentz – 1st modern analysis; foundation of infrared spectroscopy

WWII: air force, penicillin structure

Infrared: between visible & microwave

Wave number α 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

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

Detection of adulterants, contaminants

Estimation of component content

Advantages:o Rapido Non-destructiveo Environmentally

friendlyo Onlineo Can detect specific

functional groups Disadvantages:o Transparent matrix

neededo False radiation due

to heat


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DisadvantagesAtomic absorption spectroscopy (AAS)

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


Source → cropper → furnace → atomizer → monochromator → detector

Light source: depends on element to be analysedo 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)

Quality assurance Safety testing Detection of heavy

metals (mercury, lead)

Assessment of food packaging

Advantages:o Highly applicableo Simple, fasto Accurateo Small samples

Disadvantages:o Samples must be

dissolvedo Interference from

other light sources


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DisadvantagesInductively coupled plasma – atomic emission spectroscopy (ICP-AES)

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

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


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

Food analysiso Confirm purityo Detect

adulteration Environmental Biological Geological

Advantages:o Reliable, simpleo Precise, accurateo Rapid and

simultaneouso ↑analytical

working rangeo ↓detection limitso Applies to

elements not under AAS

Disadvantages:o Costly, regular

maintenanceo Dissolved samples

only (no solids)o Cannot distinguish

isotopeso Uses silicon

tetrafluoride (toxic)o Not for ultra-trace

resultso Cannot detect

CHON, inert gasesNuclear magnetic resonance (NMR) spectroscopy

1944: Rabi – wins Nobel prize for discovery of NMR

1946: Purell & Block – wins Nobel prize for NMR experiments

Atomic nuclei spin on axiso Creates

magnetic fieldo Distinct

quantum #o Parallel or

antiparallel

Atoms oriented in strong magnetic field

RF beamed onto sample, exciting atoms

RF released by atoms Released RF detected Sample held in

borosilicate straight

Chemical information (shift, rate constant, conductivity)

Structure determination of molecules

Advantages:o Rapido Wide application

(atom-specific)o Non-invasiveo Can be used with

other identification methods

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1966: Ernst & Anderson – discovered Fourier transformation

1985: Wüthrich – wins Nobel prize for use of NMR in 3D structures

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

glass tube of uniform thickness and length

Solvent: COCl3

Blank: Si(CH3)4

Disadvantages:o Low temperatureo Impurities affect

resulto Expensiveo ↓sensitivity

Electron spin resonance (ESR) spectroscopy

1944: Zavoisky – discovered ESR during study of Cu2+

1965: McConmel – spin labels

1989: Hutbel – SOSI strategy

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

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-specifico Time-efficiento Non-destructiveo Reproducible

Disadvantages:o Low temperatureo Impurities affect

results

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dipoleo Presence of

paramagnetic substance

Electron + external magnetic field = spino Nuclear hyperfine

intersectiono Dipole-dipole

interaction Both qualitative

and quantitativeMass 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

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 selectiveo Can be coupled

with other separation methods

Disadvantages:o Sample must be

purified before analysis

o Costlyo Cannot distinguish

isomerso Not reliable for

mixtureso Hard to clean

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Voltammetry Galvan – Galvanism

Volta – invented voltaic battery

1800: Nicholson & Carlisle

1800: Cruikshanks

Current-voltage relationship

Applied potential generates current due to redox reaction at surface of electrode

Current α concentration

Based off electrochemical principles

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

Cyclico Applies potential to

working electrodeo Charges with time

(forward, reverse)o Records current as

function of timeo Triangle graph

(peak indicates change in concentration)

Anodic strippingo Mercury drop hung

at capillaryo Allows for oxidationo Straight line on

graph shows deposition, slant shows stripping

Adsorption processes

Measurement of kinetics

Detection and determination of adulterants

Determination of vitamin content

Advantages:o Selectiveo Wide rangeo Rapido Simultaneous

determination Disadvantages:o Costlyo Training requiredo Regular service and

maintenanceo Mercury can

dissolve at high (+) potentials

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Electron microscopy

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

Makes use of electron beams as illumination source

Vacuum environment (beams must be unfiltered by gas)


Parts: electron gun, electromagnetic lenses, fluorescent screen (for viewing and analysis)

TEMo 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 SEMo Uses secondary

electronso Focused → scanned

→ collected → cathode ray tube

o Produces 3D image

Structure analysis Microbiology assays

TEMo Advantages:

↑magnification, ↑resolution

o Disadvantages: potentially carcinogenic reagents, costly, labor-intensive

SEMo 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 antigen- antibody relation (epitope identification)

Passive adsorption: adsorption of antigen to solid phase (polystyrene),

Spectroscopic method

Types:o Direct (antigen is

adsorbed onto plate then enzyme-labelled antibodies are added)

o Indirect (primary

Determination of antigens

Fieldso Medicineo Agricultureo Food science

Useso Detectiono Quantification

Advantages:o Simpleo Time-efficiento Sensitiveo Minimum sampleo Low cost

Disadvantages:o Low resolution

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allows for separation

Enzyme-substrate reaction: antibody conjugates with enzymes to react with substrate, produces colored solution


antibody added as intermediate before addition of enzyme-labelled antibodies)

o Sandwicho Competitive/

inhibitive

o Identificationo Assessment

Toxicity bioassay Dioscorides Matthieu Orfila –

1st formal discussion on toxicology

Paracelcus – dosage makes things not poisons

Trevan – pioneered LD-50

Draize – Draize test

Use of organisms Toxic: dosage

beyond limit (measures potency)

Organisms are dosed through inhalation, ingestion, or absorption

Classification basis:o Biological

organization usedo Response

(quantal, graded)o Intent (absolute,

comparative)o Exposure

(cutaneous, ingestion, intramuscular, inhalation)


Set-up → protocol → experimentation → parameter analysis → results

Considerations in extrapolation of datao Reproducibilityo 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 rangeo Can test for

multiple parameters simultaneously

o Real-time results Disadvantages:o Time-consumingo Labor-intensiveo Can be expensiveo Not necessarily

applicable to humans

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Method History Principles Instrumentation Application Advantages & Disadvantages

Protein quality bioassay

Protein quality: usefulness of protein to body (ideal ratios)

More qualitative than quantitative

Typeso Digestibility (true,

apparent)o Body weight gain

(protein efficiency ratio, net protein ratio, multilevel/ slope ratio)

o Nutrient balance (biological value, net protein utilization)

Selection of method Selection of test

animals Preparation of

animals Management and

experimentation Determination of

protein content (usually Kjeldahl method)

Analysis of feeds Processing

procedures Bioavailability

testing

Blood sugar level bioassay

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: 70-99 mg/dL

Area under peak

Photometric assay Orthotoluidine or o-

tolidine (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 acceptedo More accurateo Easy, fast

Disadvantages:o Large biological

variabilityo Pre-analytical

variations

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Antioxidant assay

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

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): non-competitive; based on color changes

Food analysis Advantages:o Independent of

solvento Not time-

consumingo Sensitiveo Wide range of

applications Disadvantages:o Can be costlyo Affected by

presence of other compounds

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Advanced Instrumental Methods for Identification of Components in Food

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