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Food Analysis - Complete Lecture
Food Analysis - Complete Lecture
FOOD COMPONENTS
COMPOSITION OF FOODS
Water (moisture)
Carbohydrate COMPONENT % Water %Carbohydrates %Protein % Fat % Min/Vit
Protein Milk
Beef
87.3
60.0
5.0
0
3.5
17..5
3.5
22.0
0.7
0.9
Chicken
Fat or oil Fish
66.0
81.8
0
0
20.2
16.4
12.6
0.5
1.0
1..3
Cheese 37.0 2.0 25.0 31.0 5.0
Ash (minerals) Cereal grains
Potatoes
10-14 58-72 8-13 2-5 0.5-3.0
78.0 18.9 2.0 0.1 1.0
Vitamins Carrots
Lettuce
88.6
94.8
9.1
2.8
1.1
1.3
0.2
0.2
1.0
0.9
Apple 84.0 15.0 0.3 0.4 0.3
Melon 92.8 6.0 0.6 0.2 0.4
it is important that we
understand how and how much
these components are measured 3
7
OBJECTIVES Types of samples need to analyze
This module is a very brief overview of Raw materials
common methods and equipments of food
analysis used in food processing Process control samples
organizations. Finished product
You will know: Competitors sample
1. Common methods in food analysis Complaint sample
2. Instruments used in food analysis
References on Analytical
Techniques Instruments in Food Analysis
Official Methods;
- Vietnamese Standards (TCVN)
Spectroscopy
p py
- Association of the Official Analytical Chemists (AOAC)
Chromatography
- American Oil Chemists Society (AOCS)
15
Spectrometry Chromatography
19
23
Lecture 5 (chapter 10):
Lecture 4 (Chapter 22):
Total carbohydrate and phenolic
Ultraviolet - visible spectroscopy compound analysis
Learn about the principle of quantitative Next to water, carbohydrates are the most
absorption spectroscopy.
spectroscopy abundant food component
%carbohydrate=100% - (H2O + ash + fat + protein)
Learn about structure and operation of
UV-Vis spectroscopy
Chemical reaction
Develop color with chemical solution
Measure absorbance using UV/Vis Spectroscopy
26
27
Learn structure and operation of GC Amino acids are the building blocks of protein.
Learn about application in GC Method and equipments to analyze amino acid
composition
composition.
32
Lecture 11 (chapter 14): Lecture 12 (chapter 10):
Fatty acid composition analysis Sugar composition analysis
Types of carbohydrates include;
Fatty acid: a carboxylic acid consisting of a
hydrocarbon chain and a terminal carboxyl group. monosaccharide: glucose, fructose, galactose
Method and equipments to analyze fatty acid disaccharide: sucrose,, lactose,, maltose
composition. oligosaccharids: raffinose
polysaccharide: starch, cellulose
HPLC
Determine individual sugar
33 34
Lab work
SUMMARY
Lab 1: Lab safety, glasses, balance, using pippette and
balance.
This lecture has presented the topic of Food Lab 2: Moisture, total dried basis and ash content..
Analysis by discussing Lab 3: Crude lipid (Soxhlet method).
why we analyze food, Lab 4: Protein content in food (Kjeldahl
( j methods).)
the components of food generally analyzed for Lab 5: Carbohydrate analysis: standard calibration; beer
and other food samples.
(water, protein, fat, carbohydrates)
Lab 6: Dietary fiber digestion: food sample.
The instruments used.
Lab 7: Vitamin C analysis: Titration method.
Lab 8: Total phenolic content using Colorimetric method:
Tea and coffee.
37
Lab 9: HPLC for Caffeine in soft drink. 38
Assignment Assessment
39 40
Content of this lecture
Introduction
The total moisture content of foods is generally Sometimes, moisture content also reported as total
determined by some forms of drying method solid
whereby all the moisture is removed by heat and
Total solid is a measure of the amount of material
moisture is determined as the weight lost. remaining after all the water has been evaporated
measure the mass of a food before and after the water Advantages
is removed by drying.
Cheap, easy to use, many samples can be analyzed
Basic Principle simultaneously
Water has a lower boiling point than the other major
Disadvantages
components within foods such as lipids, protein and
carbohydrate. Destructive, time consuming
Where: Where:
%W wb: Moisture content on wet basis %W db: Moisture content on dry basis
mH20: mass of water (kg) mH20: mass of water (kg)
msample = total mass of moist - or wet - sample (kg) mts = total mass of the dry solids in the sample (kg)
Drying Methods Forced draft ovens
Procedure Objective
Determine the moisture content of sample by the vacuum oven
Weigh accurately dried pan with lid (Note identifier
method, with and without the addition of sand to the sample.
number on pan and lid.)
Principle
Place 23 g of sample in the pan and weigh accurately.
The sample
Th l is
i heated
h t d underd conditions
diti off reduced
d d pressure to
t
Place in a forced draft oven at 130C for 1 h. Be sure remove water and the loss of weight is used to calculate the
metal covers are ajar, to allow water loss. moisture content of the sample.
Equipment
Remove from oven, realign covers to close, cool, and
store in desiccator until samples are weighed. Vacuum oven (capable of pulling vacuum
to <100 mm of mercury)
Calculate percentage moisture (wt/wt) as described
below. Analytical balance, 0.1 mg sensitivity
Procedure Objective
Determine the moisture content of sample (corn syrup and
Label weighing pans (i.e., etch identifier into milk) using a microwave drying oven.
tab of disposable aluminum pan) and weigh
accurately. Principle
The
h sample l is h
heatedd using microwave energy, andd the
h lloss
Place 5 g of sample in the weighing pan and of weight is used to calculate the moisture content of the
weigh accurately. sample.
Dry at 70C and a vacuum for 24 h. Equipment
Store in a desiccator until samples are cooled Microwave drying oven
to ambient temperature. Weigh. (e.g., from CEM Corporation,
Matthew, NC).
Objective
Procedure Determine the moisture content of cereal flour using a
Follow instructions from manufacturer for use of the near infrared analyzer.
microwave drying oven, regarding the following:
Principle
Turning on instrument and warming up
Specific frequencies of infrared radiation are absorbed
Loading method for specific application (i.e., sets time, by the functional groups characteristic of water. The
power, etc.) concentration of moisture in the sample is determined
Tarring instrument by measuring the energy that is reflected or
Testing sample transmitted by the sample, which is inversely
proportional to the energy absorbed.
Obtaining results
Equipment
Near infrared analyzer
NEAR INFRARED ANALYZER Rapid Moisture Analyzer
Macaroni, dry, enriched 0.7% Quality. The quality of many foods depends on the concentration and
type of minerals they contain, including their taste, appearance, texture
Milk, whole, fluid 0.7% and stability.
Butter, with salt 2.1% Microbiological stability. High mineral contents are sometimes
used to retard the growth of certain microorganisms.
Apple, raw with skin 0.3%
Nutrition. Some minerals are essential to a healthy diet (e.g., calcium,
Banana, raw 0.8% phosphorous, potassium and sodium) whereas others can be toxic (e.g.,
lead, mercury, cadmium and aluminum).
Egg, whole, raw 0.9%
Processing. It is often important to know the mineral content of foods
Hamburger, regular, plain 1.7% during processing because this affects the physicochemical properties of
foods.
3 4
5 6
Advantages
The ash content is calculated as follows
safe and easy
wt after ashing - crucible wt no chemical
% ash (db) = 100 manyy samples
p handled at one time
S
Sample
l wtt solid%/100
lid%/100
resultant ash for further mineral analysis
9 10
a procedure for oxidizing organic substances by 1. Evaporate moist samples (2550 ml) in an appropriate
using acids and oxidizing agents or their dish at 100C overnight or in a microwave drying oven
combinations. until dry.
Minerals are solubilized without volatilization. 2. Heat on a hot plate until smoking ceases.
Its primary use is preparation for specific mineral 3. Ash in a 525C furnace for 38 h.
analysis and metallic poisons. 4. Remove dish from furnace and allow to cool. Ash
Analytical testing laboratories use only wet ashing in should be grayish white to white and free from carbon.
preparing samples for certain mineral analyses (e.g., 5. Cool and wet with deionized distilled water plus 0.5
Fe, Cu, Zn, P), because losses would occur by 3.0 ml of HNO3.
volatilization during dry ashing.
11 12
Introduction to Protein
Definition: Proteins are a sequence
Importance of protein analysis (polymers) of amino acids joined together by
Principle
p of total p
protein analysis
y peptide bonds.
Introduction Introduction
Protein is one of nutritional component of Three main group of proteins
food. Simple Proteins
Proteins
Conjugated Proteins
Lipids
Derived Proteins
Carbohydrates
Minerals
Vitamins Classification of proteins (protein fractions)
Based mostly on the solubility of proteins in
What is difference between proteins and different solvents
other nutritional components?
C d protein
Crude i (%) = Nitrogen
i content (%) x C
CF
4. Step 4: Titration
Why do we need conversion factor?
5. Step 5: Calculations A factor is used to convert percent N to percent crude protein.
Most proteins contain 16% N, so the conversion factor is 6.25
[5]
(100/16 = 6.25).
Eggs Barley
Peas Oats
Meat Rye
Beans Millet
Apparatus Apparatus
Sample Residue
Extracted 3 times with Extracted 2 times with Tris- SDS: Sodium dodecyl sulfate
70% (v/v) ethanol HCl (pH 8.8) + SDS + DTT DTT: Dithiothreitol
Alb i
Albumin Gl b li
Globulin
Gliadin Glutenin
Review lecture 3 Review lecture 3
2. What is principle of protein content analysis? 2. What is principle of lipid content analysis?
3. Methods for
f determination off protein content?
? 3 Methods for determination of lipid content?
3.
4. How to calculate the protein content? 4. What are oil quality indicator?
1. Introduction 1. Introduction
Spectroscopy is the Daily observations of color can be related
study of the interaction to spectroscopy.
between matter and
radiated energy. Neon and other noble gases have
characteristic emission frequencies (colors)
It deals with the
production, measurement, Inks, dyes and paints include chemical
and interpretation of compounds selected for their spectral
spectra arising from the characteristics in order to generate specific
interaction.
An example of spectroscopy.
colors and hues.
6
5
n *
n *
*
*
*
lobes of the other involved atomic orbital.
Energy
n Nonbonding
Non-bonding orbitals (n-orbitals) are the equivalent in
Bonding
molecular orbital theory of the lone pairs in Lewis
structures. Bonding
Antibonding orbital is a form of molecular orbital,
that are normally higher in energy than bonding
orbitals. 17 18
Spectrum
Observed electronic transitions C C > 135 nm
From the molecular orbital diagram, there are several possible electronic
transitions that can occur, each of a different relative energy: C C > 165 nm
H
alkanes C O n > 183 nm weak
carbonyls
C O > 150 nm
unsaturated cmpds. n > 188 nm
Energy n > 279 nm weak
n
n O, N, S, halogens 180 nm
n carbonyls
C O
A
279 nm
19 20
Absorption:
bso pt o :
Low energy electrons absorb energy to move to higher energy level
Emission:
Excited electrons return to lower energy states
21 22
23 24
6. Quantitative Absorption Quantitative Absorption
Spectroscopy Spectroscopy
The objective of quantitative absorption In actual practice, the solution to be analyzed is
spectroscopy is to determine the concentration contained in an absorption cell and placed in the path of
radiation of a selected wavelength(s).
of analyte in a given sample solution.
The amount of radiation passing through the sample is
The determination is based on the measurement of
then measured relative to a reference sample.
sample
the amount of light absorbed from a reference beam
as it passes through the sample solution. The decrease in radiant power as the beam passes
through the solution is due to the capture (absorption) of
The presence of analyte in the solution will affect the
photons by the absorbing species.
amount of radiation transmitted through the solution
and, hence, the relative transmittance or absorbance The relationship between the power of the incident and
of the solution may be used as an index of analyte exiting beams typically is expressed in terms of either
concentration. the transmittance or the absorbance of the solution.
25 26
absorption wavelength.
3. Calculate the concentration of 0.0
your sample using Beer Lambert 200 250 300 350 400 450
30
UV/Vis Spectrophotometer
Spectrophotometer
The optics of the light source in UV-visible spectroscopy Sample Detector Quantitative Analysis
allow either visible [approx. 400nm (blue end) to 750nm
(red end) ] or ultraviolet (below 400nm) to be directed at
the sample under analysis (common range: 200 800 nm).
Light Sources
UV Spectrophotometer
1. Deuterium (200-400 nm)
Visible Spectrophotometer
1. Tungsten Lamp (350-2500 nm)
* Function: providing an energy
Monochromator
Monochromator Scanning Instrument
monochromator
according
di tot wavelength,
l th andd monochromatic
h ti
radiation of a selected wavelength exits the
monochromator. Photomultiplier
tube
slit
cuvette
Deuterium lamp
Filament (UV)
UV Spectrophotometer Protein
Quartz ((crystalline
Q y silica))
Amino Acids (aromatic)
Glass, Plastic
Vitamins determination
Fat-quality determination
Enzyme Activity (Hexokinase)
Review lecture 4
Lecture 5. Quantitative analysis - What is an electromagnetic radiation?
- Properties of electromagnetic radiation?
Assoc. Prof. Dr. Pham Van Hung
g - Principle regions of electromagnetic radiation?
- Energy states of matter: what is ground state? What is
excited state?
- Absorption and Emission: What? How?
C0 C1 C2 C3 C4 C5
- How do the electrons in organic compounds transfer their
energy?
- What is spectrum?
- Beers law?
- Components of UV-Vis spectrophotometer?
Sample preparation
Determination of appropriate wavelength
Standard curve formation Grinding
Absorbance measurement
Calculation
0.300 0.300
y = 0.0028x y = 0.0028x
0.250 R2 = 0.9986 0.250 R2 = 0.9986
0.200 0.200
Ab
Ab
0.150 0.150
0.100 0.100
0.050 0.050
0.000 0.000
0 20 40 60 80 100 120 0 20 40 60 80 100 120
FA (microgram/ml) FA (microgram/ml)
One wavelength
Lecture 5. Total protein analysis
Good plots have a range of absorbances from
0.010 to 1.000
Assoc. Prof. Dr. Pham Van Hung
Absorbances
Ab b over 1
1.000
000 are nott th
thatt valid
lid and
d
should be avoided
Biuret method NH 3
+
CH 2 H
Lowry method CH 2
Lysine
CH 2 N C NH 2
+
CH 2 CH 2 NH 2
Ultra-violet absorpton CH 2
CH
H
N
O
C
CH 2
CH
Arginine
N C C N
H H
O CH 2
+
C NH
HC CH Histidine
N
H
1. Temperature
2. Non-proteins.
3. Buffers systems.
4. Protein quality.
% Protein (Kjeldalh)
Lowry Method
The Lowry method combines the biuret reaction with the reduction of the Folin
Ciocalteau phenol reagent (phosphomolybdic-phosphotungstic acid) by
Procedure
tyrosine and tryptophan residues in the proteins. 1. Proteins to be analyzed are diluted to an appropriate
Cu++ in alkaline solution to form complexity with protein. range (20100 g).
Cu++ catalyses oxidation of phenol group of tyrosine with phosphomolybdic- 2. K Na Tartrate-Na2CO3 solution is added after cooling
phosphotungstic acid. and incubated at room temperature for 10 min.
3. CuSO4-K Na Tartrate-NaOH solution is added after
cooling and incubated at room temperature for 10min
10min.
4. Freshly prepared Folin reagent is added and then the
reaction mixture is mixed and incubated at 50C for 10
min.
5. Absorbance is read at 650 nm.
6. A standard curve of BSA is carefully constructed for
estimating protein concentration of the unknown.
Application
Advantages:
Ultra-violet Absorption (UV) at 280 nm
1. Very sensitive
(a) 50100 times more sensitive than biuret method 1. Proteins show strong absorption in the region at ultraviolet (UV) 280nm,
(b) 1020 times more sensitive than 280-nm UV absorption method primarily due to tryptophan and tyrosine residues in the proteins.
2. Less affected by turbidity of the sample.
3. More specific than most other methods. Chromophoric side chains of aromatic amino
4. Relatively simple; can be done in 11.5 h. acids (Tyrosine, Tryptophan).
Disadvantages: 2. Because the content of tryptophan and tyrosine in proteins from each
1. Color varies with different proteins to a greater extent than the food source is fairly constant, the absorbance at 280nm could be used to
biuret method. estimate the concentration of proteins, using Beers law.
2. Color is not strictly proportional to protein concentration.
3. The reaction is interfered with to varying degrees by sucrose,
lipids, phosphate buffers, monosaccharides, and hexoamines.
4. High concentrations of reducing sugars, ammonium sulfate,
and sulfhydryl compounds interfere with the reaction.
Problem 1
Procedure A 20 ml protein fraction recovered from a column
chromatography was analyzed for protein using the BCA
1. Proteins are solubilized in buffer or alkali. method. The following data were the means of a duplicate
2. Absorbance of protein solution is read at 280nm analysis using BSA as a standard:
against a reagent blank.
BSA Mean Absorbance
3. Protein concentration is calculated according to the
equation (mg/ml) at 562nm
A = lc
where: 0.2 0.25
A = absorbance 0.4 0.53
= absorptivity 0.6 0.74
l = cell or cuvette path length 0.8 0.95
c = concentration 1.0 1.15
The average absorbance of a 1-ml sample was 0.44. Calculate
protein concentration (mg/ml) and total protein quantity of this
column fraction
Carbohydrates
Monosaccharides
Carbohydrate analysis
Disaccharides
Olygosaccharides
Assoc. Prof. Pham Van Hung
Polysaccharides
Department of Food Technology
Carbohydrates Carbohydrates
Monosaccharides Polysaccharides
Total carbohydrate Total
T t l carbohydrate
b h d t
Glucose
Fructose determination Starch determination
Galactose Pectin
Disaccharides Reducing sugar Cellulose Total starch
Maltose analysis determination
Dietary fiber (DF)
Sucrose
Lactose Soluble DF Total dietary fiber
Individual sugar
Olygosaccharides
analysis
Insoluble DF analysis
y
Carbohydrates y by
are destroyed y strong
g acids and/or
high temperatures.
Continued heating in the presence of acid produces
various furan derivatives.
Sugar extraction
1. A clear, aqueous solution of carbohydrate(s) is transferred using a A standard curve must be used.
pipette into a small tube. A blank of water also is prepared. Ideally, the standard curve will be prepared using mixtures of the
same sugars present in the same ratio as they are found in the
2. An aqueous solution of phenol is added, and the contents are unknown.
mixed.
If this is not possible or if more than one sugar is present either as
3. Concentrated sulfuric acid is added rapidly to the tube so that the free sugars in unknown => D-glucose is used to prepare the
stream produces good mixing. The tube is agitated. (Adding the standard curve.
sulfuric acid to the water produces considerable heat.) A yellow-
orange color results.
results % TS = (TG * V * DF)/Ws
4. Absorbance is measured at 490 nm. - %TS: Total carbohydrate of sample (mg/g sample)
- TG: total glucose of the solution (mg/g sample)
5. The average absorbance of the blanks is subtracted, and the
- V; volume of the solution (ml)
amount of sugar is determined by reference to a standard curve. - DF: dilution factor
- Ws: mass of sample used for sugar extraction
Objective
j
This method
Thi i l rapid,
th d iis simple, id sensitive,
iti t
accurate,
specific for carbohydrates, and widely applied. Determination of reducing sugars that have an
aldehydo group (aldehyde group or ketone group) in
The reagents are inexpensive, readily available, and the sugar solution.
stable. All monosaccharides are reducing sugars, along with
some disaccharides, oligosaccharides, and
A stable color is produced
produced, and results are polysaccharides.
l h id
reproducible.
Reducing sugar have an aldehyde group 1. A solution of copper(II) sulfate and an alkaline buffer are
added
dd d by i tt to
b pipettes t a solution
l ti off reducing
d i sugars(s) ( ) andd
This group reacts with an oxidizing agent to produce a
a water blank.
carboxylic group.
2. The resulting solution is heated in a boiling water bath.
3. A reagent prepared by mixing solutions of acidic
The SomogyiNelson method is based on the reduction ammonium molybdate and sodium arsenate is added.
of Cu(II) ions to Cu(I) ions by reducing sugars. 4 After mixing,
4. mixing dilution,
dilution and remixing,
remixing absorbance is
Cu(I) reduces an arsenomolybdate complex (ammonium measured at 520 nm.
molybdate [(NH4)6Mo7O24] + sodium arsenate 5. After subtracting the absorbance of the reagent blank,
(Na2HAsO7)+Sulfuric acid) to produce an intense, stable the A250 is converted into glucose equivalents using a
blue color. standard plot of micrograms of glucose vs. absorbance.
H2N NH 2 HN NH
Total starch
Dietary fiber analysis
Sta c es a
Starches anddpproteins
ote s a are
e removed
e o ed by
enzymes
Lipids are removed easily from the
sample with organic solvents.
Protein and minerals that are not Total fiber
emoved from the sample during the
solubilization steps should be corrected
for by Kjeldahl nitrogen analysis and by
ashing portions of the fiber residue.
Vitamin
Fat--soluble vitamins
Fat
A: Retinol or -carotene
Principle of vitamin
vitamin C analysis Procedure of vitamin
vitamin C analysis
L-ascorbic acid is oxidized to L-dehydroascorbic
Sample Preparation
acid by the oxidation
oxidationreduction indicator dye, 2,6-
Weigh and extract by homogenizing test sample in metaphosphoric acid-acetic
ddichloroindophenol
d p OR iodine.
iodine
d . At the endpoint,
dp , acid
id solution
l ti (i.e., 15 g off HPO 3 and
(i d 40 mll off HOAc
HOA in
i 500 mll off deionized
d i i d
excess unreduced dye appears rosepink in acid H2O). Filter (and/or centrifuge) sample extract, and dilute appropriately to a
final concentration of 10100 mg of ascorbic acid/100 ml.
solution..
solution
Standard Preparation
Weigh 50 mg of USP L-ascorbic acid reference standard and dilute to 50 ml
with HPO3-HOAc extracting solution.
Titration
Titrate three replicates each of the standard (i.e., to determine the
concentration of the indophenol solution as mg ascorbic acid equivalents to 1.0
ml of reagent), test sample, and blank with the indophenol reagent (i.e.,
prepared by dissolving 50 mg of 2,6-dichloroindophenol sodium salt and 42 mg
of NaHCO3 to 200 ml with deionized H2O) to a light but distinctive rose pink
endpoint lasting 5 sec.
Calculation
mg of ascorbic acid/g or ml of sample
= (X B) (F/E) (V/Y)
where:
X = average ml for test solution titration
B = average ml for test blank titration
F = mg ascorbic acid equivalents to 1.0-ml
indophenol
i d h l standard
d d solution
l i
E = sample weight (g) or volume (ml)
V = volume of initial test solution
Y = volume of test solution titrated
Review
Beers law
Atomic Absorption What is spectra?
Standard calibration formation
Spectroscopyy (AAS)
Spectroscop Quantitative analysis of unknown compounds
Characteristics of absorbance measurement
Assoc. Prof. PHAM VAN HUNG Spectrophotometer: Light sources,
Monochromator, Detector, readout device, cuvette
Department of Food Technology
Practise problem
1 2
In this lecture
Introduction
Atomic absorption spectroscopy (AAS)
AAS is an analytical method based on the
History
absorption of ultraviolet or visible radiation
General principle of AAS by free atoms in the gaseous state.
state
Instrumentation of AAS
Practice problem AAS is commonly used for analysis mineral
nutrients and toxicants in foods
3 4
Atomization Atomisation
Atomization involves separating particles
into individual molecules (vaporization)
and breaking molecules into atoms.
Expose the analyte to high temperatures in a MX M M+
flame or plasma.
Solution Solid Gas Atom Ion
Vaporize and decompose the analyte to
atoms that may absorb radiation or become Excitation
excited and subsequently emit radiation.
M* M+* 14
17
Abs = cb
I
Abs = log
Io
19 = extinction coefficient 20
Overview of AA Sample
spectrometer. Compartment
Atomic Absorption Spectroscopy
Instrumentation
Radiation Sources
Atomizer
A i
Light Source Detector
Monochromator
Detector
21 22
27
31 32
Analytical procedure Wet Ashing
a procedure for oxidizing organic substances by
Standard preparation using acids and oxidizing agents or their
Sample preparation combinations.
Absorbance measurement Minerals are solubilized without volatilization.
It primary
Its i use isi preparation
ti f specific
for ifi mineral
i l
Calculation analysis and metallic poisons.
Analytical testing laboratories use only wet ashing in
preparing samples for certain mineral analyses (e.g.,
Fe, Cu, Zn, P), because losses would occur by
volatilization during dry ashing.
33 34
1. Evaporate moist samples (2550 ml) in an appropriate 6. Dry on a hot plate or steam bath and then return to a
dish at 100C overnight or in a microwave drying oven 525C furnace for 12 h.
until dry.
7. Repeat steps 5 and 6 if carbon persists.
2. Heat on a hot plate until smoking ceases.
8 Dissolve the ash in 5 ml of 1M HNO3 by warming on a
8.
3. Ash in a 525C furnace for 38 h.
hot plate for 23 min to aid solution. Transfer to an
4. Remove dish from furnace and allow to cool. Ash appropriate size volumetric flask (i.e., 50 ml), then
should be grayish white to white and free from carbon. repeat with two additional portions of 1M HNO3.
5. Cool and wet with deionized distilled water plus 0.5 9. The specific type of mineral being analyzed using AAS
3.0 ml of HNO3. or ICP-MS.
35 36
r 0.6
The following data was collected using solutions of b
sodium chloride of known concentration a 0.4
n
c 0.2
Concentration (ppm) 2 4 6 8 e
Absorbance 0.18 0.38 0.52 0.76 2 4 6 8
37 Concentration (ppm) 38
r 0.6
of this solution were analyzed
b by AA after adding a volume of 40
0.0700 g Ni/mL to each. A
a 0.4 plot of the results are shown
n below. Determine the
Concentration concentration of the Ni in the 0
c 0.2
Na+ = 7.3ppm river water. 0 5 10 15
e Volum e of Nickel Added(m L)
mobile phase
Preparative - purify and collect one or more
components of a sample attractive forces
Stationary Phase
Supercritical fluid (SCFC)
Sample MUST be volatile at temperatures BELOW 3500C
Thin layer
y Column Partition - based on the relative solubility of analyte in
(adsorption) (gravity flow) mobile and stationary phases
Normal analyte is nonpolar organic; stationary phase MORE
polar than the mobile phase
Reverse analyte is polar organic; stationary phase LESS
polar than the mobile phase
Glass column High performance Ultra performance Size Exclusion - stationary phase is a porous matrix;
(gravity flow) (pressure flow) (high pressure) sieving
Types of Chromatography Chromatographic Properties
Thin Layer Chromatography (TLC) 1) immiscible stationary and mobile phases
2) an arrangement where a mixture is deposited at
Column Chromatography (CC)
one end of the stationary phase
3) flow of the mobile phase towards the other end of
Gas Chromatography (GC) the stationary phase
4) different rates of partitioning for each component
High Performance Liquid Chromatography (HPLC)
5) means for visualizing the separation of each
component
Ultra Performance Liquid Chromatography (UPLC)
Principles of TLC
TLC is a form of liquid chromatography consisting
of:
The following are the important components of The sorbent is first activated by
drying for a specific time and
a typical TLC system: temperature
A B C A+B+C A+B+C ?
Quantitative analysis by TLC
injection and the time which the maximum peak Sample manager
height for that compound is detected. Column manager
times.
times Read-out
Read out device
Classifications of HPLC
Normal phase HPLC
There are numerous types of HPLC which The stationary phase is a polar adsorbant.
vary in their separation chemistry. Silica attached with polar nonionic functional
All chromatographic modes are possible: groups: hydroxyl, nitro, cyano, or amino.
Ion-exchange
Ion exchange
Size exclusion The mobile phase is a nonpolar solvent.
Also can vary the stationary & mobile Hexane added with a more polar modifier such
phases: as methylene chloride to control solvent
strength and selectivity.
Normal phase HPLC
Reverse phase HPLC
- chromatographic
Response
X2 X0
system relative to
-
= V2
X1 X0 X1
the particular
X0 components of the V1
mixture.
1 3 6 W
1 W2
R e te n t io n T im e W1 W2
What is a HPLC?
GAS CHROMATOGRAPHY Advantages of HPLC?
Components of HPLC system?
A
Assoc. P
Prof.
f PHAM VAN HUNG Retention time?
Qualitative and quantitative analysis of
chemicals using HPLC
GC Principle Functions of GC
Separation of volatile organic compounds Separation and analysis of organic compounds
An inert carrier gas carries the gaseous compounds
through the column Testing purity of compounds
The gaseous compounds being analyzed interact Determine relative amounts of components
p in
with the stationary phase.
phase. mixture
Separation occurs as a result of unique equilibria Compound identification
established between the solutes and the stationary
phase (the GC column)
column).. Isolation of pure compounds (microscale work)
Each compound will be eluted at a different time,
known as the retention time of the compound
compound..
Characteristics of GC Application
Similar to column chromatography, but differs in 3 In general, substances that vaporize below 300 C (and
ways: therefore are stable up to that temperature) can be
measured quantitatively
quantitatively..
Partitioning process carried out between Moving
Gas Phase and Stationary y Liquid
q Phase Examples:: fatty acids, triglycerides, cholesterol and
Examples
Temperature of gas can be controlled other sterols, gases, solvent analysis, water, alcohols,
and simple sugars, as well as oligosaccharides, amino
Concentration of compound in gas phase is a
acids and peptides, vitamins, pesticides, herbicides,
function of the vapor pressure only.
food additives, antioxidants, nitrosamines,
GC also known as Vapor-Phase Chromatography polychlorinated biphenyls (PCBs), drugs, flavor
(VPC) and Gas-Liquid Partition Chromatography more..
compounds, and many more.
(GLPC).
Components Schematic Diagram of Gas Chromatography
RESET
Regulators Syringe/Sampler
Injector
Oven
gas system
Detectors
Injector
Gas Carrier
Hydrogen
Air
Column column
Oven
detector
data system
Carrier Gas
Gas--Supply Carrier Gas
Gas--Supply
Carrier gases, which must be High-quality pressure regulators must
High-
chemically inert, include helium, be used to provide a stable and
nitrogen, and hydrogen.
hydrogen. continuous gas supply
supply..
Must be at a constant flow
flo rate so All ggas lines must be clean and contain
that retention times & retention no residual drawing oil
oil..
volumes may be equated.
equated.
The carrier gas line should have traps
The gases used must be of high (moisture trap, oxygen trap, and
purity and all regulators, gas lines, hydrocarbon trap) in line to remove
and fittings must be of good quality
quality.. any moisture and contaminants from
the incoming gas
gas..
Injector Injector
The injection port serves the The injection port contains a
purpose of providing a place soft septum that provides a gas-
gas-
for sample introduction, its tight seal but can be penetrated
vaporization, and possibly by a syringe needle for sample
some dilution and splitting.
splitting. introduction.
A GC syringe penetrates a
septum to inject sample into
the vaporization chamber.
C16:0 C18:1
C18:2 Oven
C18:0
C16:1
Programmable
Polar column RT (min) Isothermal-- run at one constant temperature
Isothermal
Temperature programming - Start at low
temperature and gradually ramp to higher
C18:2 temperature
C18:1 More constant peak width
C16:0
Better sensitivity for components that are retained
C18:0
C16:1 longer
Much better chromatographic resolution
RT (min)
Non-polar column Peak refocusing at head of column
Typical Temperature Program Detection Systems
220C
Ultra
Ultra--sensitive detection of halogen
halogen--containing Detector Response
species C18 Peak Area (cm2 )
10
O h ddetectors b
Other
ther id MS
besides 6
C14 4
IR 2
= th e c o n te n t % o f C1 4 fa tty a c id s
TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS Retention Times
Response
Response
Hexane
Response
Response
1.6 min = RT
CH 2 O C R
5. Quantitatively analyzed
O CH 3 ONa O
CH O C R + CH 3 OH 3 R C O CH 3
Volatile in Gas
O Chromatography
CH 2 O C R
Amine group
pKa = 10.5 i id
imidazo
pKa = 3.9 pKa = 4.1
guanidino
pKa = 12.5
pH 7
indole
ring
pH 1
Acid hydrolysis is the most common method for Free amino acids can be obtained from proteins by strong
acid hydrolysis:
hydrolyzing a protein sample before amino acid
analysis.
6 N HCl
However, some of the amino acids can be Protein Amino acids
destroyed 100 C,, 24 h,,
in vacuo
A time-course study (i.e., amino acid analysis at
acid hydrolysis times of 24, 48, and 72 hours) is 3 of the standard aas are lost during acid hydrolysis treatment:
often employed to analyze the starting
concentration of amino acids that are partially Asparagine Aspartic acid Amides go to
destroyed or slow to cleave Glutamine Glutamic acid acids
Tryptophan Decomposed
Method for hydrolysis Method for analysis
Hydrolysis Solution:
6 N hydrochloric acid containing 0.1% to 1.0% of phenol. (phenol is
Ion-exchange chromatography with postcolumn ninhydrin
to prevent halogenation oftyrosine) detection is one ofthe most common methods employed
for quantitative amino acid analysis.
Liquid Phase Hydrolysis
a Li-based cation-exchange system is employed for the
a. Place the protein sample in a hydrolysis tube, and dry.
analysis
y of the more complex p physiological
p y g samples,
p , and
b. Add 200 L of Hydrolysis Solution per 500 g of protein. the faster Na-based cation-exchange system is used for
c. Freeze the sample tube in a dry ice-acetone bath, and ame seal the more simplistic amino acid mixtures.
in vacuum.
Separation ofthe amino acids on an ion -exchange column
d. Samples are typically hydrolyzed at 110C for 24 hours in is accomplished through a combination of changes in pH
vacuum or inert atmosphere to prevent oxidation. Longer
and cation strength.
hydrolysis times (e.g., 48 and 72 hours) are investigated ifthere
is a concern thatthe protein is not completely hydrolyzed. A temperature gradientis often employed to enhance
separation.
adducts.
-
SO 3
COOH
2. The amino acids are identified Na
+
OH
according to their retention
- +
So 3 H3N
+
Na
-
SO 3 H3N
+
- + -
COO H OH = H 2 O
OPA-amino acid analysis using reverse-phase HPLC - + pH4.5
*note OPA does not react with proline so another reagent must be used (FMOC) So 3 Na
Moles/Liter VAL
ALA
HIS LEU
ASP
LYS GLU
Assoc Prof
Assoc. Prof. Pham Van Hung
Department of Food Technology
O H H O
8 7 6 5 4 3 2 1
CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C OH CH3 (CH2 )7 C C (CH 2 )7 C OH
3 - Octenoic Acid 10 9
O Ci 9 - Octadecenoic
Cis O d i Acid
A id ((oleic)
l i )
8 7 6 5 4 3 2 1
CH 3 CH2 CH 2 CH2 CH 2 CH2 CH2 C OH H O
3, 6 - Octadienoic Acid CH3 (CH2 )7 C C (CH2 )7 C OH
Importance of fatty acid composition analysis Principle of fatty acid composition analysis
1. Quantitative analysis of fat and oil 1. Fat and fatty acids are extracted from food by
hydrolytic method.
2. Determination of fat and oil quality
2. Fat is hydrolyzed into fatty acids and fatty
3 Determination of fatty acid composition
3.
acids are methylated to fatty acid methyl esters
4. Determination of essential fatty acids (FAMEs) to increase volatility, improve peak
5. Determination of trans fats symmetry, and decrease sample activity.
3. FAMEs are quantitatively measured by GC.
CHEMICAL EQUATION CHEMICAL EQUATION
(Hydrolysis of a fat)
(Transesterification of fat)
O O
CH2O C R CH2O C R
O O O
CH2OH R C O R C OH
O R C O CH3
-
OH O O O CH2OH
CHO C R' CHOH + H3O O
R' C O R' C OH KOH
CHO C R' CHOH +
CH2OH O O CH3OH R' C O CH3
O
Glycerol
R" C O R" C OH CH2OH O
CH2O C R" O
Fatty Acid salts Fatty Acids
Glycerol
fat
forms CH3O-K+ R" C O CH3
CH2O C R"
and H2O Fatty acid methyl esters
Vegetable oil (FAMES)
Solvent
For Spot
p A C
A B
Rf = S
S A
A B
Procedure for sugar analysis by TLC Procedure for sugar analysis by TLC
1. Preparation 2. TLC running
a. Sample preparation Fill chromatography tray with eluent.
Sample is extracted and purified from other components Take care that the liquid is below the
b Standard
b. St d d preparationti application line (eluent level about 1
Dissolve the standards in 20 mL water including 10 mg D(+)- cm high).
glucose-monohydrate, 10 mg Sucrose, 10 mg Fructose, 10 mg
Maltose, 10 mg Maltotriose. Put up to TLC-plates with samples in
the tray. Use small notched rods to
c. Preparation of stationary phase
keep plates upright and evenly spaced.
Glass plates precoated with 0.25 mm dry silica gel
d. Preparation of solvents Put lid on the tray using silicon fat to
Solvent system which consists of a mix ture of chloroform, acetic make
k an airtight
i ti ht chamber.
h b
acid, and water (3:3.5:0.5) by volume, respectively.
Take plates from the tray when the
c. Preparation of dye eluent is at or near the top of the plates
Spraying agent made from 1 gram diphenylamine and 1 ml of aniline (5 8 h) and dry in fumehood or stove.
in 100 ml acetone. This mixture is further mixed with 85% If required dried plates can be eluted
orthophosphoric acid prior to use (10 : 1 v/v, respectively) . again (to obtain better separation).
Procedure for sugar analysis by TLC
HPLC technique
3. Detection
HPLC systems are commonly used to separate and analyze
Visualisation can be effected through chemical, thermal or optical
sugars because of its availability.
means.
However traditional reversed phase columns cannot be used
for underivatized sugars, as the stationary phase will not
Lane 1 Glucose standard provide the required retention and specialized columns are
Lane 2 Sucrose standard necessary.
Lane 3 Fructose standard
Lane 4 Maltose standard Detection of sugars faces troubles as their structure contains
Lane 5 Maltotriose standard no chromophores. Detection by UV-VIS, as commonly used in
Lane 6 Mix of all standards HPLC, is not p
possible in sugar
g analysis.
y
Lane 7 Blank lane
Lane 8 Fermentation Day 0 Another type of liquid chromatography that can be used for
Lane 9 Fermentation Day 3 sugar analysis is high-performance anion exchange
Lane 10 Fermentation day 6
chromatography with pulsed amperometric detection (HPAEC
PAD). Anionic separation and PAD detection improves the
sensitivity and specificity compared to other LC methods.
HPAEC-PAD system
Anion exchange chromatography (AEC)
Procedure for sugar analysis by HPLC
Columns specifically for carbohydrate anion-exchange chromatography.
These columns permit the separation and analysis of mono-, oligo-, and 1. Preparation of sample
polysaccharides.
polysaccharides
The Dionex CarboPac PA1 and Dionex CarboPac PA100 are packed It is recommended that all samples other than pure
with a unique polymeric, nonporous, Thermo Scientific Dionex standards be passed through a 0.45-m nylon filter prior to
MicroBead pellicular resin. injection to remove particulates.
Dionex MicroBead resins exhibit rapid mass transfer, high pH stability
(pH 014), and excellent mechanical stability that permits back
pressures of more than 4000 psi (28 MPa).
2. Preparation
p of standards
Pulsed amperometric detection (PAD)
Pulsed amperometry permits detection of carbohydrates with excellent
signal-to-noise ratios down to approximately 10 picomoles without
requiring derivatization. 3. Running HPAEC-PAD
Carbohydrates are detected by measuring the electrical current
generated by their oxidation at the surface of a gold electrode.
Chromatogram of sugars
Running program of HPAEC-PAD
- Pump: Thermo Scientific Dionex ISO-3100 SD
- Autosampler: Thermo Scientific Dionex WPS-
3000TSL Analytical Autosampler
- Flow: Isocratic at 0.50 mL/min. with constant He
purge
- Column: Thermo Scientific CarboPak: PA20, 3 x
150 mm, 6.5 m
- Temperature: 32 C
- Injection volume: 50 L partial loop
- Mobile
M bil Phase:
Ph 50 mM
M sodium
di h d id (NaOH),
hydroxide (N OH)
prepared from pellets, 99.99%, semiconductor
grade
- EC detector: Coulochem III, Thermo Scientific
Dionex model 5040 cell with Au Target: 25 m Mylar
Introduction
Ash: total mineral content; inorganic residue remaining
after ignition or complete oxidation of organic matter
Mineral composition analysis Minerals:
Macro minerals (>100 mg/day)
Assoc. P
A Prof.
f PHAM VAN HUNG C P
Ca, P, N
Na ,K,
K MMg, Cl,
Cl S
Trace minerals (mg/day)
Department of Food Technology
Fe, I, Zn, Cu, Cr, Mn, Mo, F, Se, Si
Ultra trace minerals
Va, Tn, Ni, Sn, B
Toxic mineral
lead, mercury, cadmium, aluminum
1 2
Analysis of Meat and Meat Products Analysis of Fish and Seafood: Wet Digestion
Scope
Analysis o An acid digestion procedure may be used for sample preparation of
many elements in fish and seafood tissue including K, Na, Zn, Cu, Cr,
Cd, Fe, Ni and Pb.
o A weighed sample is placed in a digestion vessel, acid is added and the
mixture is heated for several hours.
o The samples are digested with HNO3 and HClO4 or HNO3 and H2SO4
depending on the technique and heating vessel used. After the
digestion, the samples are diluted to a specific volume and analyzed
directly or chelated and extracted into an organic solvent if the element
of interest is present in low concentration .
o The main advantage of wet digestion is that it eliminates elemental loss
by volatilization because the digestion takes place at a low temperature.
o The main disadvantages of a wet digestion procedure is that it is
subject to reagent contamination and requires operator attention.
Analysis of Fish and Seafood: Wet Digestion Analysis of Fish and Seafood: Wet Digestion
Sample Preparation
o Weigh about 5 g of sample (dry weight) into a digestion tube. Analysis
Add 5 mL of HNO3 and then 5 mL of H2SO4 to the sample.
Allow the reaction to proceed.
o When the reaction slows, place the tubes in a hot-block digestion
apparatus and heat at a low temperature (60 C) for 30 min.
o Remove the tubes from the hot block, allow to cool, add 10 mL of
HNO3, return tubes to digestion rack and heat slowly to 120 C.
Increase the temperature to 150 C.
o Remove the tubes when the samples go black, allow to cool,
then add 1 mL of H2O2. A vigorous reaction may occur.
o Return the tubes to the block. Repeat the H2O2 additions until
the samples are clear. Remove the tubes and make up to 50 mL
with deionized water.