FA All ASSAYS

Dry Ashing

used for proximate analysis and sample prep of ash/minerals

Principle: sample incinerated and organic materials volatilize

Procedure:

• weigh empty crucible then weigh crucible and sample

• incinerate sample at high volume

• cool and weigh

Wet ashing

used for sample prep

procedure:

• sample oxidized with strong acid

• organic acid volatilized and inorganic mineral remains

• mineral soluble in acid

EDTA

• used to determine 1-2 individual minerals in foods

for Ca and Mg

principle: EDTA forms a chelated complex around mineral of interest

procedure:

• add sample and indicator → turns pink

• titrate EDTA → turns purple, as EDTA has better chelation ability than indicator

• when there’s excess EDTA, indicator is no longer bound and turns blue

Mohr titration

Forward precipitation

• used to measure Cl-

Procedure:

• titrate ag+ into sample

• excess Ag reacts which reacts with CrO4 indicator to form red/brown color

Volhard titration

• measures the chlorine in a sample

• backwards titration

procedure:

• add excess Ag+ to sample

• add KSCN and excess reactions with Fe+ to form a red colored complex

ion selective electrodes

principle:

• measures voltage between two electrodes

• voltage is proportional to ion activity

Procedure

• measure sample with glass electrode specific to specific ion

• same concept as pH meter

Atomic absorption spectroscopy

used to measure multiple minerals in foods

procedure

• heat atoms to excite electrons and they absorb specific wavelengths of light

atomic emission spectroscopy

• used to measure multiple minerals in foods

procedure

• heat atoms and as they relax to ground state energy is emitted as a specific wavelength

oven drying

method to measure moisture

procedure

• weigh empty pan then weigh pan and sample

• dry in oven and find difference

distillation

physical moisture analysis method

measure water loss directly

ideal for low moisture sample

procedure:

• mix sample with solvent that is immiscible with water

• boil to evaporate solvent

• condense in reflux condensor

• water on bottom layer- measure amt collected

hydrometer

physical method of measuring moisture content

for liquid products only

solution density measured and converted to moisture content

temperature dependent and only works on simple solutions

refractometer

RI is proportional to dissolved solids

cheap easy and portable

only works on simple solutions

cryoscope

physical method of measuring moisture

principle:

• more solutes dissolved, lower freezing point

Mostly used to detect dilution of raw milk

Karl Fischer titration

chemical method of analyzing moisture content

procedure

• Karl Fischer reagent added to sample until it is all oxidized by water

• excess KFR measured by electrodes, as I2 forms current

ideal for low moisture high carb samples

Titratable acidity

measures the free and bound H+ ions in solution

-measured using titration with base until all acid is neutralized

reported in main acid equivalents of solution

pH

measures free acid equivalents

measured with pH meter

Goldfisch

crude lipid analysis continuous solvent extraction

fat measured as: amount of fat removed or sample weight loss

principle: sample is continuously exposed to boiling solvent which extracts lipid

procedure

• weigh sample into thimble

• reflux solvent for four hours, fat extracted continuously

• remove solvent and weigh fat

Soxhlet

semi continuous solvent method of analyzing crude lipids

principle: sample soaks in condensed solvent repeatedly to extract lipids

fat measured: loss of sample

procedure

• add sample to thimble

• refluxed solvent accumulates in sample chamber and fat is siphoned into boiling flask

• the solvent evaporates and leaves just the fat in the flask

• process repeats every 5-10 minutes

• dry defatted sample

• cool and weigh

Mojonnier

Discontinuous solvent extraction for crude lipid analysis

Principle: lipids are extracted discretely from hydrolyzed sample

fat measured as weight of fat removed

procedure:

• add sample to mojonnier flask

• add ammonium hydroxide, ethanol, and ether

• centrifuge and decant organic layer

• repeat then remove solvent and weigh

Folch

discontinuous solvent extraction for crude lipid analysis

principle: lipids extracted discreetly with folch solvent (methanol-chloroform)

procedure

• add sample to test tube and homogenize

• add folch solvent

• add KCl to separate layers

• remove organic layer

• evaporate solvent

best as prep for future analysis

extracts fat from foods of many different matrices

Babcock method for milk fat

non solvent method to analyze milk fat

principle: sulfuric acid digests proteins in milk and releases lipids that are measure directly in Babcock flask

fat measured as percent fat

procedure

• add milk into flask and add sulfuric acid to digest

• centrifuge and add hot water so lipids float to the top

• measure lipids in the neck of the flask

GC for crude lipid analysis

analyze total lipids with GC

• most common method used for nutrition labeling

GC for fatty acids

to prep: derivative FA into FAMEs which decreases polarity and increases volatility for GC

procedure

• mix IS and sample

• extract lipids using folch method

• hydrolyze and saponify acylglycerols which separates cholesterol and FFA

• derivative lipid component s

  • FFA → FAMES

  • Cholesterol → TMS cholesterol

• Analyze

Cholesterol with GC

used to measure specific lipid copmpoent: cholesterol

procedure:

• extract lipids

• saponify (break ester bonds), turning the lipid into cholesterol (nonpolar, stays in tact) and FFA+glycerol (polar)

• extract cholesterol with benzene

• derivative to make more volatile

• analyze with GC

refractometer

physical method of analyzing fats

principle: the higher the RI, the more DB (unsaturation)

heating, FFA, and oxidation impacts RI

melting point (lipids)

physical analysis to characterize lipids

principle: lipids have distinct melting points, the more double bonds, the lower the melting point

procedure

• use thermometer to record when lipid in water bath melts

cloud point test

physical method of lipid characterization

principle: lipids cool and solidify at specific temps

procedure:

• heat sample to 130

• place thermometer in sample and measure when it first solidifies and gets cloudy

iodine value (lipids)

chemical method to characterize lipids

principle: iodine reacts with C=C

the more double bonds, the higher the IV

helps track hydrogenation and oxidation

procedure

• dissolve lipid in organic solvent

• add excess ICl to react with C=C

• add KI to make free I2

• titrate free I2

Saponification value

chemical method of lipid characterization

amount of KOH needed to saponify (turn into FFA and glycerol) a lipid

the higher the MW of FFA, the lower the saponification value (need less KOH to saponify the lipids)

procedure

• refluxing a sample with alcoholic KOH,

• followed by back-titrating the unreacted KOH with standard hydrochloric acid (HCl) using a phenolphthalein indicator

FFA and AV

chemical method of lipid analysis

principle: measured unesesterified fatty acids by titration with strong base

purpose: monitor lipid quality

procedure:

• titrate sample with strong base to endpoint 8.2

peroxide value

principle: peroxides form as primary lipid oxidation products

procedure

• add excess KI to sample

• titrate with sodium thiosulfate and blue starch indicator

• turns clear when reacted

measure conjugated dienes and trienes

primary oxidation products

dienes: measured at 232nm

trienes: measure absorbance at 270nm

measure TBARS

early secondary oxidation

principle: lipid oxidation generates unsatriuattd carbonyls MDA which react with TBA to form purple chromagen

measure p anisidine

late secondary oxidation products

principle: unsaturated aldehydes react with p anisidine to form colored product which can be measured on spec

measure VOCs

late secondary oxidation: VOCS

measured using GC

oven storage test

used to measure oxidative stability

principle: elevated heat expedites lipid oxidation

procedure

• measure baseline primary oxidation products

• forced draft over at 40-60°C and remove aliquots over a month

kjeldahl

measures crude organic protein

principle: measures organic nitrogen titrated as borate ions can multiply by 6.25 to get total protein value

procedure:

• digest sample with Na2SO4 and heat

• neutralize with base

• distill ammonia with boric acid to form borate

• titrate borate with HCl

dumas

measures total organic and inorganic nitrogen in sample

official AOAC method

principle: sample is combusted and converted into N2 gas which is measured using GC

procedure:

• combust sample in pure O2

• reduce sample using copper catalyst

• analyze using GC

TCA precipitation

separates NPN
principle: protein is separated from NPON by precipitation

procedure

• TCA mixed with sample which precipitates protein

  • NPN stays dissolved in solution

• analyze NPN in filtrate via kjeldahl

• convert to protein with conversion factor

anionic dye binding

dye method to analyze crude protein

principle: protein+ excess dye → precipitate and remaining dye

procedure

• mix protein and excess dye

• centrifuge to remove precipitated protein

• measure excess dye with spec

remaining dye is inversely proportional to protein content

anionic dye binding best for:

milk, wheat flour, soy, meats

Bradford assay

dye method for analyzing crude protein

principle: coomassie dye interacts with peptide bond in proteins and produces blue color

procedure

• mix Bradford reagent (coomassie dye, acid, ethanol)

• measure absorbance at 595

• measures change in absorbance from bound dye to unbound dye

• use BSA curve

best applications of Bradford assay

beer and potatoes

biuret method

copper method for analyzing crude protein

principle: copper ions complex with peptide bonds in alkaline solution

procedure

• mix biuret reagent (NaOH + Cu2SO4 + NaK tartrate) with sample

• measure absorbance (use BSA std)

test measures peptide bonds

applications of biuret method

cereal meat soybean animal feed

lowry method

copper method of analyzing crude protein

principle: biuret reagent + folin reagent cause color change when reacting with aromatic AA

procedure

• add sample, biuret reagent and folin reagent, Na2CO3 and heat

• measure absorbance (blue) and use BSA std

measures reaction with aromatic amino acids tryptophan and tyrosine

Abs 700: low protein

abs 500: high protein

100x more sensitive than biuret

BCA method

Copper ion method used to analyze crude protein

principle: peptide bonds reduce Cu2+ to Cu under alkaline conditions

measures peptide bonds

procedure

• add protein, copper, BCA, and NaOH

• BCA is green and turns purple when Copper is reduced

• measure absorbance

100x more sensitive than biuret

UV spec @280

principle: proteins absorb at 280nm bc of the aromatic AA

procedure

• solubilize sample in base

• read abs @ 280

• measures tryp and tyro

applications of UV spec @ 280

milk and meat products

UV spec @ 190-220

principle: peptides have max abs at 190-220

procedure:

• solubilize proteins in base and read abs at 190-220

use in low trp and tyr foods

salting out

precipitation method for separating proteins

principle: proteins precipitate as ionic strength of solution increased

procedure:

• add ammonium sulfate slowly to solution with protein

• centrifuge just below ppt

• centrifuge just above ppt

solvent fractionation

principle: organic solvent reacts with charged protein surface causing precipitation

procedures: add ethanol or acetone to decrease protein solubility

• 5-60% ethanol or acetone

• at 0°C prevents denaturation

isoelectric precipiattion

principle: proteins precipitate at their pI because no electrostatic repulsion

procedure:

• adjust pH until pH=pI

• at pI protein precipitates

• centrifuge

denaturation

precipitation method of separating proteins

principle: heat or extreme pH denatures proteins

procedure

• heat or alter pH to denature

dialysis

size method of protein separation

• semi permeable membrane separates proteins of different sizes over time until equilibrium reached

membrane process filtration

size method of separating proteins

filtration separates smaller proteins than dialysis

principle: filter solution through membrane with specific pore size to separate proteins of different sizes

size exclusion chromatography

methods to separate proteins

separate proteins by size, larger first

PAGE electrophoresis

principle: proteins separated based on charge and size

procedure

• load sample

• add dye

• apply voltage

• separate proteins

smaller proteins move faster and further

Native PAGE s

separate based on charge size and shape

SDS Page

seperate based on size alone

Isoelectric focusing IEF

principle: proteins migrate to where pH=pI

procedure:

• A- load sample and apply voltage

  • proteins migrate to isoelectric point

• B- proteins form distinct bonds

  • use for further experiments

two dimensional gel electrophoresis

combine two electrophoresis techniques

Amino acid analysis

principle: quantify individual AA via LC

procedure

• hydrolyze protein to AA with strong acid

• separate amino acids with chromatography

• quantify with spec

Amino acid score

principle: composition compared to requeriemn of preschool age children

calculated based on FLAA

Protein digestibility corrected amino acid score PDCAAS

principle: estimate protein nutritional quality from AAS and in vivo digestibility

• rats digest proteins similar to humans

procedure

• calculate AAS based on FLAA

• conduct rat feeding study

  • 10% vs 0% protein diet for 28 days

  • measure food consumption and fecal unabsorbed nitrogen

• calculate digestibility

  • use kjeldahl

• calculate PDCAAS

PER

principle: measure rat growth with test protein compared to casein reference protein

application: detrmine % DV for infant foods

procedure

• determine nitrogen content and calculate protein

• formulate test protein diet and control casein diet

• feed male weanling rats for 28 days and monitor weight gain

• calculate protein efficiency rate (total weigh gain/total protein consumed)

anthrone test

quantify total carbs

principle

• sulfuric acid degrades carbs into furans which react with anthrone to make chromophore

purpose

• quick hexose analysis

procedure

• mix solution with H2SO4

  • hydrolyze polysaccharides into monosaccharides

  • monosaccharides dehydrated into furan derivatives

• add anthrone

• quantify w spectrophotometer at 620nm

phenol sulfuric acid method

quantify total carbs

principle: sulfuric acid and heat degrade carbs into furan which reacts with phenol to form chromophore

purpose: estimate all carbs in solution

procedure

• mix unknown solution with H2SO4

  • forms furan derivatives

• add phenol which creates orange solution

quantify w spec @ 490

somongyi nelson

quantifies reducing sugars

principle: reducing sugars reduce Cu2+ → Cu+ in alkaline solution

procedure

• reducing sugars reduce Cu2+. to Cu

• Cu+ reduces AsMO complex to blue Mo

• measure absorbance @ 520

most common method for reducing sugars

Benedicts test

semi quantitative method of analyzing reducing sugars

procedure

• mix sample with Benedicts reagent (copper II citrate)

• heat + reducing product + benedicts reagent → colored product

goes from blue to red

Fehlings test

procedure

• mix fehlings A and B

• heat and reducing sugar and Cu2+ → forms red precipitate

lane eyons

procedure

• boil fehlings A and B

• add unknown solution via burette and titrate

• endpoint reached when solution changes from blue to red

anion exchange LC for carbs

instrumental method of analyzing carbs

stationary phase: cation with anion counter ion

mobile: acidic → basic

carbs have OH group that makes them negative at high pH

normal phase LC for carbs

stationary: polar

mobile: nonpolar

carbs are highly polar

GC analysis of mono and oligosaccharides

1. reduce aldehyde to hydroxyl group

2. convert all hydroxyls into volatile derivatives

3. detect with flame ionization detector

GOPOD

enzymatic method of mono and oligosaccharides

principle: glucoxidase reduces glucose forming peroxide which reacts with dye and peroxidase to form colored compound

procedure:

• mix sample with glucooxidase and oxygen

• mix H2O2 with dye and peroxidase

• measure color on spec

total starch assay

principle: hydrolyze starch to glucose using gelatinization and enzymes

procedure

• gelatinize starch

  • exposes resistance starch

• digest with alpha amylase to make starch fragments

• digest with glucoamylase

  • break glucose-glucose bonds

• analyze using GOPOD

measure: peroxides via GOPOD

degree of gelatinization

principle: deb ranching followed by depolymerization of non gelatinized starch

procedure

• suspend water in sample

• debranch

• convert amylose to maltose with beta amylase

• measure reducing sugars

total dietary fiber

principle: remove moisture, lipids, sugars, starches, and protein to analyze fiber

procedure

• dry and defat

• remove sugars with hot ethanol

• remove starch with amylase and glucoamylase

• deproteinate with protease

• precipitate all dietary fiber with ethanol → and weigh

insoluble and soluble dietary fiber

principle: remove moisture, lipids, proteins, sugars, starches and separate soluble and insoluble fiber

procedure

• dry and defat

• remove sugars with hot ethanol

• remove starch with amylase and glucoamylase

• deproteinate with protease

• add water to dissolve soluble fiber

• filter to separate soluble and insoluble water

• insoluble portion can be weighed

• precipitate soluble fiber from remaining solution → remove → weigh

direct elisa

immunoassay to mesure specific molecules in solution

procedure:

• stationary antigen

• antibody-enzyme binds to form complex

• chromagen + antibody + antigen complex → colored product

indirect elisa

1. stationary antigen

2. 1° antibody binds to stationary antigen

3. 2° antibody binds to primary antibody

4. chromagen and secondary antibody form colored product

sandwich Elisa

1. stationary antibody

2. 1° antibody binds antigen

3. 2° antibody binds primary antibody

4. chromagen and secondary antibody form colored product

competitive elisa

principle: sample has the same antigen/antibody as ELISA which creates competition for binding

results are inversely proportional to amount of antigen/antibody in sample

methods to analyze pesticides

single residue method, multiple residue method

Quenchers

multiple residue method to analyze pesticides

rapidly detects residues (6 samples in 30 mins)

monier williams

measures sulfites in sample

principle: sulfites acidified to sulfur dioxide gas which oxidize hydrogen peroxide to sulfuric acid

procedure

• heat samples with HCL, converting sulfite to SO2 gas

• SO2 bubbles through solution, oxidizing H2O2 to H2SO4

• H2SO4 measured by gravitimetric or turbidimetric method

FDA method of analyzing sulfate containing foods

Ripper method

principle: sulfites acidified with sulfur dioxide which is then oxidized via titration with iodine

procedure:

• sample mixed with sulfuric acid, starch, and heat to produce SO2

• SO2 oxidized with iodine

• excess iodine reacts with starch

free and total SO2 quantified

used by wine industry