2. Vitamin analysis
Generally difficult to measure vitamin content of food
chemically heterogeneous group of essential
micronutrients
present in very small amounts
often unstable during analysis
Bioassays using humans or animals
Microbiological assays
Physico-chemical assays
titrimetric / spectrophotometric
fluorimetry
chromatographic
enzymatic
2
3. HPLC analysis of vitamins- vitamin
E (Tocopherols & Tocotrienols)
Vitamin E is present in food as eight compounds
(--- and -) tocopherols and tocotrienols
In order to estimate the Vit E activity tocopherol
equivalent
quantification of each Vit E form is required
3
5. Method
saponify
mix with 6% (wt/vol) pyrogallol in ethanol, heat at
70°C & sonicate
add 60% KOH solution digest at 70°C & sonicate
extract with hexane
filter & inject into normal phase HPLC
use florescence detector at 290 and 330 nm
quantify by external standard from peak area by linear
regression
5
7. Example of a titrimetric analysis of
vitamins
2, 6-Dichloroindophenol (DCPIP or DPIP)
titrimetric method for ascorbic acid
Sample homogenised in metaphosphoric acetic
acid solution
Filtered and diluted
Standard solutions of ascorbic acid prepared
Standards and samples titrated to pink endpoint
with dichloroindophenol dye
mg ascorbic acid/ml sample = C x V x (DF / WT)
C = mg ascorbic acid/mL dye
V = ml of dye used for titration of diluted sample
DF = dilution factor
WT = sample wgt, g
7
8. Example of fluorimetric analysis of
vitamins
Fluorometric thiamin (vitamin B1) determination
sample and standards dissolved in HCl
enzymatic hydrolysis of phosphate esters of thiamin
sample ‘clean up’ using ion exchange cartridge
converte thiamin to thiachrome using potassium
ferrocyanide
immiscible isobutyl alcohol added, shaken and left to
separate into two layers
isobutyl alcohol upper layer now containing
thiochrome decanted off into fluorescence tube
fluorescence measured (at 365-435nm) compared to
standard solutions
8
9. Comparison of methods used for vitamin
analysis
Bioassays
extremely time consuming
don’t necessarily require preparation of an extract
limited to animals rather than humans
Microbiological
limited to water extractable vitamins
require vitamin extraction but less sample preparation than
physicochemical assays
not necessarily measure of bioavailability in humans
Physicochemical (require vit. Extraction)
relatively simple, accurate & precise
can involve high capital outlay (ie HPLC)
establishing the peak identity is essential 9
10. ASH ANALYSIS
“Ash = inorganic residue remaining after either ignition or
complete oxidation of organic matter in a foodstuff”
dry ashing (proximate analysis)
whole grain, cereals and dried vegetables
wet ashing (oxidation, preparation for elemental
analysis)
meat and meat products
microwave (low temperature ashing)
volatile elements
Ash content of fresh food is rarely >5%
10
11. Dry ashing
Sample (5-10 g) weighed into crucible and pre-
prepared
Ignited in muffle furnace 12-18 hr, 550oC
water & volatiles vapourised
organic substances oxidised to water vapour, carbon
dioxide, and oxides of nitrogen
minerals converted to oxides, sulphates, phosphates,
chlorides and silicates
some elements may be partially lost through
volatilisation eg. Fe, Se, Pb, Hg
Cool furnace and open door carefully as ash may
be fluffy
Cool in desiccator and calculate ash weight as
percentage of original sample
11
12. Wet ashing
Wet oxidising of organic substances
Place 1g dried sample of food in H2SO4 &
HNO3
Heated to 200C on hot plate in fume-hood’
brown-yellow fume will evolve
sample should become colourless
Cool and transfer oxidised food solution to
50 mL volumetric flask
Make to volume with ultra pure water
Follow wash down procedure for fume-
hood
12
13. Microwave ashing
Totally automated
May carry out dry or wet ashing
Wet ashing is preformed in open or closed vessels
(Teflon, quartz or Pyrex) which withstand pressures
of >1500psi
acids may be heated past their boiling point
ensures complete digestion in 30 min.
permits use of nitric acid when normally we
would require sulfuric acid
13
14. Time, temperature, pressure & microwave power
parameters are adjustable
may ramp the temperature according to
preprogramming
Dry ashing may reach up to 1200°C
uses the same protocol and crucibles as muffle
furnace ashing
generally 20 min. in a microwave oven is equal to
4hr in a muffle furnace
14
15. Analysis of specific minerals
Flame photometry and atomic absorbtion
spectroscopy
EDTA complexation titration
Redox reactions
Precipitation titration
Colorimetric methods
Ion selective electrodes
15
16. Contamination
Milling and grinding with steel grinders
Old glassware can contaminate samples for micro-
elemental analysis
glass is acid washed & triple rinsed with ultra
pure water
Solvents including water may contain high amounts
of minerals
need pure reagents high in cost
run reagent blank
16
17. EDTA complexometric titration
17
• Formation of stable complexes of metal
ions with ethylenediaminetetraacetic acid
(EDTA)
– except alkali metals (Na)
• Via the presence of donor oxygen and
nitrogen atoms EDTA is able to form six,
five member chelate rings
18. Water hardness - EDTA titration
Adjust water sample pH to 10 by adding buffer
solution (NH4OH + Na2EDTA + MgCl2) and Calmagite
indicator solution
Titrate with 0.01 EDTA to a blue endpoint
This method is suitable to assess Ca in ashed fruits and
vegetables
18
19. Add magnesium salt and enough EDTA to bind all
magnesium.
In buffer solution the Ca replaces the Mg bound to the
EDTA.
The free magnesium binds to Calmagite,
pink magnesium Calmagite complex persists until all
Ca in the sample has been titrated with the EDTA.
Excess EDTA removes Mg from Clamagite and produces a
blue endpoint
19
20. Redox reactions for mineral
measurement
oxidation
addition of oxygen OR removal of hydrogen
removal of electrons = increase in positive
charge
reduction
removal of oxygen OR addition of hydrogen
addition of electrons = reduction in positive
charge
20
21. Iron determination using redox
reaction and colorimetry
Food sample dried and ashed
Dissolve ash in HCl and filter with rinsing
Add aliquot of filtrate containing iron to
solution of hydroxylamine hydrochloride
Add acetate buffer and colour developing
reagent such as 0.1% orthophenanthroline
Dilute and read absorbance of colour at 510
nm
To calculate iron content compare absorbance
of sample to standard curve generated by
known concentrations of iron chloride
prepared by dissolving analytical grade iron wire
in HCl 21
22. Precipitation (Mohr) titration analysis of salt
in butter
‘Mohr titration’ Based on formation of an
orange coloured solid, silver chromate after
silver from silver nitrate has complexed with
all available chloride
AgNo3 + NaCl AgCl (Cl- is complexed)+ NaNo3
2AgCl + K2CrO4 Ag2CrO4 (orange all Cl- is complexed) + 2KCl
butter is melted in boiling water in a conical flask
potassium chromate solution is added
titrated with silver nitrate until an orange brown
colour persists for 30 sec
exact normality of silver nitrate solution standardised
against known amount of potassium chloride
% salt in food calculated from concentration &
titration volume of standardised silver nitrate 22
23. Colorimetric assay for phosphorous
Sample of food is ashed
Add HCl and nitric acid
Heat to dissolved ash, cool and make up to
volume in water
Add 20 ml of molybdovanadate reagent to
aliquot of ash solution
After colour development (10 min.) due to
formation of phosphomolybdovanadate
read absorbance at 400 nm
To calculate phosphorus content compare
absorbance of sample to standard curve
generated by known concentrations of
potassium dihydrogen phosphate
23
24. Ion-selective electrodes
Similar concept to pH electrodes that measure
H+ ions can be applied to other ions and even
dissolved gases
Chemical composition of glass in electrode is
one means of making electrodes sensitive to
specific ions
71% SiO2, 11% Na2O and 18% Al2O3 for K
A typical glass membrane sodium indicating
electrode operates in the range of 1 - 10-6 M
Usually develop a calibration curve of electrode
potential (millivolts) developed in standard
solutions
plot on semilog paper electrode potential vs
logarithms of the standard concentrations
24
25. Sulphur dioxide determination
Pale coloured liquid foods or foods that can be
dispersed in water
digest food with cold alkali (pH 13),
acidify to produce un-dissociated sulphurous acid
sulphur dioxide reacts with standard iodine solution
SO2 + I2 + 2H2O 2I- + 2H+ + H2SO4 + I 2
excess iodine reacts with starch to give a dark blue endpoint
Foods not easily dispersed in water or intensely
coloured
distilling of sulphur dioxide from the acidified food
titrating the colourless sulphur dioxide directly with
standard iodine solution as it distills over
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26. Nitrates and nitrites
Levels of nitrites (NO2) & nitrates (NO3) in foods are
controlled by international regulation
have the potential to form toxic nitrosamines and
to interfere with infant metabolism
Aqueous extraction of the food
to reduce nitrate loss, pH of extract is >5
De-proteinisation of the extract at the isoelectric
point of the contaminating proteins followed by
filtration
26
27. Extracted nitrates reduced to nitrites by
nicotinamide-adenine dinucleotide phosphate
(NADPH) in the presence of the enzyme nitrate
reductase
Nitrate + NADPH nitrite + NADP+ + H2O
The nitrites originally present in the sample
plus those converted from nitrates converted to
a diazo dye
Nitrite + sulphanilamide + NED diazo dye
Level of dye can then be determined
colorimetrically with reference to a standard
curve
Nitrite and nitrates in food calculated as nitrite
27
