Food Analysis - Complete Lecture

Content of this lecture
FOOD ANALYSIS Introduction

BTFT302IU Important of the course
Aim
Assoc. Prof. PHAM VAN HUNG Content of the course
Department of Food Technology Lab work and Assignment
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
Information of food products Important of food analysis

Is an important part of quality assurance
program in food processing.
In formulating and developing new
products.
In evaluating new process for making new
products.
In identifying the source of problem with
unacceptable products.
5
WHY DO WE NEED TO ANALYZE FOOD?

Food analysis is critical to ensure the food
Government regulations require it for certain
products with standards of identity (e.g.% fat and
moisture in meat products). is safe, free from harmful contaminants
Nutritional Labeling regulations require it.
is compositionally correct
Quality Control- monitor product quality for
consistency. contains only permitted additives
Research and Development- for the development is correctly described and labeled
of new products and improving existing products.
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
Steps in analysis What must a food scientist be able to do?

Select and prepare SAMPLE
Obtain a representative sample and convert the Know chemical properties of foods
sample to a form that can be analyzed.
Understand food composition and quality
Perform the ASSAY
The assay is unique for each component or C
Controll compositional
i i l iinteractions
i
characteristic to be analyzed and may be unique Know current analysis procedures
for a specific type of food product.
Select appropriate procedure for a food
Calculate and interpret the RESULTS
To make decision and take action based on the Be aware of developments and changes
results obtained.
Criteria for Selecting an

A Good Analytical Technique
Analytical Technique
There are many techniques to analyze foods Precision Sensitivity
but not all methods are convenient to use.
Accuracy Specificity
You must select the technique that is R
Reproducibility
d ibilit S f t
Safety
required or fits into your system. Simplicity Official Approval
Must be familiar with the principle and the Cost
critical steps of the method.
Speed
Depend on the objective of the measurement
Instrumental systems are available
13
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)
- American Association of Cereal Chemists (AACC)
15
Spectrometry Chromatography
The study how the chemical compound

interacts with different wavelenghts in a given Chromatography basically involves the separation of
region of electromagnetic radiation is called mixtures due to differences in the distribution
spectroscopy
t or spectrochemical
t h i l analysis.
l i coefficient of sample components between 2 different
phases.
The collection of measurements signals - a mobile phase

(absorbance) of the compound as a function of - a stationary phase.
electromagnetic radiation is called a spectrum.
Lecture 1 (chapter 6):

Determination of moisture content
Moisture or water is by far the most
common component in foods ranging in
Course syllabus content
t t ffrom 60 95%.
95%
The two most common moisture
considerations in foods is that of total
moisture content and water activity.
19
Lecture 1 (chapter 7): Lecture 2 (chapter 9):

Determination of ash content
Total protein analysis
Ash: total mineral content; inorganic residue
remaining after ignition or complete oxidation of Kjeldahl measures the amount of nitrogen
organic matter
in a sample.
Dry ashing
high temperature
Protein fractions
Wet ashing
oxidizing agent and/or acid
Low-temperature plasma ashing
dry ashing in partial vacuum at low temperature
21
Lecture 2 (chapter 8): Lecture 3 (Chapter 21):

Total lipid analysis Basic principles of spectroscopy
Extraction Methods determine total lipid Learn about the production, measurement,
Continuous Goldfinch and interpretation of spectra which come from
S i C ti
Semi-Continuous- S
Soxhlet
hl t the
h iinteraction
i off electromagnetic
l i radiation
di i
Discontinuous- Mojonnier with matter.
Learn about the principles of spectroscopic
Oil quality analysis
methods.
23
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

Lecture 6 (Chapter 24):
Total phenolic compound analysis
Atomic absorption spectroscopy
a large class of plant secondary metabolites:
Simple structures, e.g. phenolic acids Learn about principle of atomic absorption
polyphenols
Polymeric compounds: polyphenols. spectroscopy
p py
Learn about structure and operation of AAS
Chemical reaction
Develop color with chemical solution
Measure absorbance using UV/Vis Spectroscopy
27

Basic principles of Chromatography HPLC
Learn about history of chromatography Learn structure and operation of HPLC

Learn about physicochemical principles of Learn about application in HPLC
chromatographic separation
Learn about analysis of chromatographic
peaks

GC Amino acid composition analysis
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
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

Vitamin analysis Mineral analysis
Fat-soluble vitamins
A: Retinol or -carotene 1. Preparation: standard & sample
D: Cholecalciferol or Ergocalciferol 2. Ashing
K: Phylloquinone
E: Tocopherol 3. Forming solution
Water-soluble vitamins 4. Running sample by AAS or ICP-AES or ICP-MS
B complex: Thiamin (B1), Riboflavin (B2), Pyridoxine
5. Calculation
(B6), Folic acid (B9), Cyanocobalamin (B12)
C: ascorbic acid
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
Presentation 1: Vietnamese standards Assignment: 30%

Midterm: 30%
Presentation 2: Article reading Final
Fi l exam: 40%
39 40
Introduction
MOISTURE AND TOTAL SOLID ANALYSIS Moisture content in foods

Importance of moisture analysis
Assoc. Prof. Pham Van Hung Methods
Department of Food Technology Oven drying methods
Other methods
Introduction What is moisture content?
Moisture or water is by far the most common

component in foods ranging in content from 0 Moisture content express the amount of water
95%.
present in a moist sample.
Moisture
M i t assays can b
be one off the
th mostt iimportant
t t
analyses performed on a food product. Moisture content can be expressed on wet basis or
dry basis.
The two most common moisture considerations in
foods is that of total moisture content and water
activity.
What is water activity? Importance of Moisture Analysis
1- Moisture is a quality factor in the preservation of

The ratio of the vapor pressures of pure water some products and affects stability in
and a solution. a. dehydrated vegetables and fruits
b. dried milks
p c. powdered eggs
p gg
aW = d. dehydrated potatoes
p0 e. spices and herbs
2- Moisture is used as a quality factor for
Where
a. jams and jellies, to prevent sugar crystallization
p = the vapor pressure of water in the food at T
b. sugar syrups
p0 = the vapor pressure of pure water at the same T
c. prepared cereals conventional,4-8%; puffed, 7-8%
Importance of Moisture Analysis Importance of Moisture Analysis

3- Reduced moisture is used for convenience in
packaging or shipping of 5- Computations of the nutritional value of
a. concentrated milks
foods require that you know the moisture
b. liquid cane sugar (67% solids) and liquid corn sweetener
(80% solids) content.
c. dehydrated products
d
d. concentrated fruit juices
4- Moisture (or solids) content is often specified in 6- Moisture data are used to express results
compositional standards (i.e., Standards of of other analytical determinations on a
Identity)
a. Cheddar cheese must be 39% moisture. uniform basis (i.e., dry weight basis).
b. Enriched flour must be 5% moisture.
c. Pineapple juice must have soluble solids of 10.5 Brix
(conditions specified).
d. Glucose syrup must have 70% total solids.
e. The percentage of add water in processed meats.
Forms of water in foods Moisture contents in foods
Food Item % of Moisture
Free water:
White bread, enriched 13.4
This water retains its physical properties and thus acts as Corn flakes cereal 3.0
the dispersing agent for colloids and the solvent for salts. Milk, whole, fluid, 3.3% fat 88.0
Ice cream, vanilla 61.0
Adsorbed water: Margarine, regular, hard, corn 1 6.7
Oil 0
This water is held tightly or is occluded in cell walls or
Oranges, raw 86.8
protoplasm and is held tightly to proteins. Apples, raw, with skin 83.9
Water of hydration: Beef, ground, extra lean, raw 6 3 .2

Egg, whole, raw, fresh 75.3
This water is bound chemically, for example, lactose Peanuts, all types, dry roasted with salt 1.6
monohydrate; also some salts such as Na2SO4 10H2O. Sugar, granulated 0
Sugar, brown 1 .6
Cucumbers, with peel, raw 96 .0
Total moisture Content assay Total moisture Content assay
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
Determination of Moisture: Methods Drying Methods
Testing sample Weight (Ws)

1. Drying Methods
Oven drying methods
Container Weight (W1)
2. Distillation method
Dean and Stark Method
Heating machine
3. Chemical Methods
Karl Fisher
Cooling
Gas production
4. Physical Methods
Container + dried sample Weight (W2)
How to calculate moisture content How to calculate moisture content
Moisture Content on Wet Basis Moisture Content on Dry Basis

Moisture content on wet basis is the amount Moisture content on dry basis (db) is the
of water per unit mass of moist- or wet- amount of water per unit mass of dry solids
sample. in the sample.
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
Convection or forced draft ovens (AOAC) Objective

- Very simple; Most common Determine the moisture content of cereal flour
Vacuum Oven using a forced draft oven method.
- Sample is placed in oven under reduced pressure thereby reducing the
gp
boiling point of water. Principle of Method
Microwave Oven The sample is heated under specified conditions
- Uses microwave as a heat source; Very fast method and the loss of weight is used to calculate the
Infrared Drying moisture content of the sample.
- Uses infrared lamp as a heat source; Very fast
Equipment
Rapid moisture analyzer
- cover a wide range of applications within the food industry
Forced draft oven
- offers quick and accurate results within minutes. Analytical balance, 0.1 mg sensitivity
Forced draft ovens VACUUM OVEN
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
VACUUM OVEN MICROWAVE DRYING OVEN
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).
MICROWAVE DRYING OVEN NEAR INFRARED ANALYZER
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
Procedure Using a digital balance, the

test sample is placed on an
Follow instructions from manufacturer for use of the near aluminum pan and the
infrared analyzer, regarding the following: constant temperature is
Turning on instrument and warming up applied to the test sample.
Calibrating instrument Instrument automatically
Testing sample weighs and calculates the %
of moisture or solids
Obtaining results
Rapid Moisture Analyzer WATER ACTIVITY MACHINE
Follow instruction manual from manufacture

Turning on instrument and warming up
Specifying settings
Tarring instrument
Placing the sample pan in the tester
Selecting test material
Testing sample
Obtaining results
Introduction
Ash: total mineral content; inorganic residue remaining
after ignition or complete oxidation of organic matter
Total ash analysis Minerals:
Macro minerals (>100 mg/day)
C P
Ca, P, N
Na ,K,
K MMg, Cl,
Cl S
Assoc. Prof. PHAM VAN HUNG, PhD
Trace minerals (mg/day)
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
Ash Contents in Foods Importance of ash analysis

Nutritional labeling. The concentration and type of minerals
Wheat flour, whole grain 1.6% present must often be stipulated on the label of a food.
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
Methods for Determining Ash Dry Ashing
Dry ashing Principles

high temperature Separation of minerals from organic compounds of
food products using a muffle furnace capable of
Wet ashing maintaining high temperature (>525
(>525C).
C).
oxidizing agent and/or acid Water and volatiles are vaporized.
Microwave ashing Organic substances are burned in the presence of oxygen in

air to CO2 and oxides of N2.
Available for both dry and wet ashing The remaining is ash. Most minerals are converted to oxides,
sulfates, phosphates, chlorides, and silicates.
5 6
Dry Ashing General Procedure for Dry Ashing
Weigh a 510 g sample into a tared crucible. Predry if the

Instrumentation sample is very moist.
Muffle furnace Place crucibles in a cool muffle furnace. Use tongs, gloves,
and protective eyewear if the muffle furnace is warm.
Crucible
Ignite 12
1218
18 h (or overnight) at about 550 C.
550C
porcelain
Turn off muffle furnace and wait to open it until the
temperature has dropped to at least 250C, preferably
lower. Open door carefully to avoid losing ash that may be
fluffy.
Using safety tongs, quickly transfer crucibles to a desiccator
with a porcelain plate and desiccant. Cover crucibles, close
desiccator, and allow crucibles to cool prior to weighing.
7 8
General Procedure for Dry Ashing Dry Ashing
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
wt after ashing - crucible wt Disadvantages

% ash (wb) = 100 loss of volatiles
Sample wt
interaction
long time and expensive equipment
9 10
Wet Ashing Wet Ashing
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
6. Dry on a hot plate or steam bath and then return to a Advantages

525C furnace for 12 h. - Minerals will usually stay in solution.
- Little or no loss from volatilization because of the
7. Repeat steps 5 and 6 if carbon persists. lower temperature.
8 Dissolve the ash in 5 ml of 1M HNO3 by warming on a
8. - The oxidation time is short and requires a hood,
hood hot
hot plate for 23 min to aid solution. Transfer to an plate, and long tongs, plus safety equipment.
appropriate size volumetric flask (i.e., 50 ml), then
repeat with two additional portions of 1M HNO3. Disadvantages
- It takes virtually constant operator attention,
9. The specific type of mineral being analyzed using AAS corrosive reagents are necessary.
or ICP-MS. - Only small numbers of samples can be handled at
any one time.
13
Review lecture 2
1. What is ash content? 1. What is moisture content?
2. What is principle of ash 2. What is principle of
PROTEIN ANALYSIS
content analysis? moisture content analysis?
3 Methods
3. M h d ffor ddetermination
i i 3. Methods
3 M h d ffor ddetermination
i i Assoc. Prof. PHAM VAN HUNG
of ash content? of moisture content?
Department of Food Technology
4. How to calculate the ash 4. How to calculate the
content? moisture content?
In this lecture Introduction
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.
Procedure of total protein analysis

Different proteins have different chemical
Protein fraction analysis properties and structures.
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?
Introduction Importance of protein analyses

Nutrition labeling
Protein fractions in foods:
Pricing: The cost of certain commodities is based on the protein
Four classes content as measured by nitrogen content (e.g., cereal grains; milk for
making certain dairy products, e.g., cheese).
Albumins
Globulins Functional property investigation: Proteins in various types of
food have unique food functional properties: for example,
example gliadin and
Gliadins (Prolamins) glutenins in wheat flour for breadmaking, casein in milk for
coagulation into cheese products, and egg albumen for foaming.
Glutelins
Biological activity determination: Some proteins, including
enzymes or enzyme inhibitors, have actions on food quality: for
instance, the proteolytic enzymes in the tenderization of meats,
pectinases in the ripening of fruits, and trypsin inhibitors in legume
seeds are proteins.
Importance of protein analyses Methods for protein analysis
Protein analysis is required when you want to
know: Total protein
Total protein of food
1. Total protein content
Total protein of each protein fractions
2. Protein content during isolation and purification of
a protein
Amino acid composition
3. Profile of a particular protein in a mixture
4. Amino acid composition
5. Nonprotein nitrogen
6. Nutritive value of a protein
Kjeldahl Method - Principle

Total Protein Determination Methods
is a method for the quantitative determination of nitrogen in
foods developed by Johan Kjeldahl in 1883.
1. Kjeldahl Method.
The method consists of heating a sample with sulphuric
2 Dye Binding Method. acid, which decomposes the organic substance by oxidation
to
o liberate
be a e thee reduced
educed nitrogen
oge as aammonium
o u su sulphate.
p a e.
3. Biuret Method. Then the solution is then distilled with a small quantity of
sodium hydroxide, which converts the ammonium salt to
4. Lowry Method. ammonia. The amount of ammonia present, and thus the
5. Ultraviolet Method. amount of nitrogen present in the sample, is determined by
back titration.
Procedure Conversion Factors from Nitrogen to Protein for Foods

1. Step 1: Sample preparation
2. Step 2: Digestion The protein content in foodstuffs is estimated by multiplying
the determined nitrogen content by a nitrogen-to-protein
3. Step 3: Neutralization and Distillation
conversion factor.
[2]
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).
Conversion Factors from Nitrogen to Protein for Foods Apparatus
6.25 6.38 5.83 5.70 5.30
Corns Milk Whole wheat Wheat flour Nuts
Eggs Barley
Peas Oats
Meat Rye
Beans Millet
Apparatus Apparatus
Application Analysis of protein fractions

Advantages:
1. Applicable to all types of foods
2. Inexpensive (if not using an automated system) Separation of protein fractions
3. Accurate; an official method for crude protein Using different solvents: water, alcohol, Alkaline
content
4. Has been modified (micro Kjeldahl method) to
measure microgram quantities of proteins Determine total protein
Disadvantages:
1. Measures total organic nitrogen, not just protein Determine molecular weight of protein
nitrogen
2. Time consuming (at least 2 h to complete)
3. Poorer precision than the biuret method
4. Corrosive reagent
Analysis of protein fractions Analysis of protein fractions
Sample Residue
Add distilled water Add NaCl
Stirring SDS-PAGE Kjeldah

Centrifuge Supernatant (Albumin)
Centrifuge Supernatant (Globulin)

SDS-PAGE Kjeldah
Analysis of protein fractions Analysis of protein fractions

Residue 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
Shaking SDS-PAGE Kjeldah Shaking SDS-PAGE Kjeldah
Centrifuge Supernatant (Glutenin) Centrifuge Supernatant (Gliadin)

Analysis of protein fractions
Alb i
Albumin Gl b li
Globulin
Gliadin Glutenin
Review lecture 3 Review lecture 3
1. What is protein? Protein fraction? 1. What is lipid?
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?
5. Why is this analysis important? 5. Why is this analysis important?

1. Introduction of spectroscopy
2. Electromagnetic radiation
SPECTROSCOPY
3. Energy state of matter
4. Energy level transitions
Assoc. Prof. PHAM VAN HUNG
Department of Food Technology 5. Spectroscopic techniques
6. Quantitative absorption spectroscopy
7. Beers Law
3 4
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
1. Introduction 2. Electromagnetic radiation

Atoms and molecules may absorb and/or emit Electromagnetic radiation is a type of energy
electromagnetic radiation (EMR). that is transmitted through space as a transverse
wave at enormous velocity.
The patterns of absorption (wavelengths It takes numerous forms known as
absorbed and to what extent) and/or emission
electromagnetic spectrum. The electromagnetic
(wavelengths emitted and their respective
intensities) are called spectra. spectrum include gamma ray, X-ray, ultraviolet
(UV), visible, infrared (IR), microwave and radio-
wave radiation.
7 8
Properties of electromagnetic radiation (EMR) Properties of electromagnetic radiation (EMR)
1- Wave Properties
The wave properties of electromagnetic radiation are described in
2- particle properties
terms of
EMR can be viewed as a stream of discrete wave packets of distinct
-The wavelength (): the distance between successive maxima or particles called photons.
minima of a wave (nm)),
The energy E of photon depends upon the frequency of the
- The frequency
q y (():
) the number of oscillation of the field p
per second. radiation
The velocity of light, c, is given by the equation: E = h
c= Where:
Another unit commonly used h = Plancks constant (6.626 x 10-34 J s)
is the wavenumber, which is
linear with energy: = frequency of the radiation (most common units = cm-1)
Energy is inversely proportional to wavelength

9 10
Principal regions of electromagnetic radiation UV-Vis regions of electromagnetic radiation
The ultraviolet region extends from about 10 to 380 nm

The most analytically useful region is from 200 to 380
nm, called the near-ultraviolet region or quartz UV
region.
g
The visible (VIS) region extends from about 380 to 780

nm.
Violet Indigo Blue Green Yellow Orange Red 11 12
Infrared regions of electromagnetic radiation 3. Energy states of matter

Atoms and molecules, under normal conditions,
The infrared (IR) region extends from about 0.78 m to
300 m.
exist predominantly in the ground state (the
lowest energy).
The near-infrared (IR) region extends from about 0.78
Atoms and molecules at the ground state can
m to 2.5
m.
gain
i energy, in
i which
hi h case they
th will
ill be
b elevated
l t d
The far-infrared (IR) region extends from about 2.5 m to one of their higher energy states, referred to
to 16 m. as excited states.
The quantum nature of atoms and molecules
puts limitations on the energy levels that are
available to these species.
13 14
Absorption vs. Emission

4. Energy level transitions
Energy is emitted by
electrons returning to
lower energy levels The absorption of light energy by organic compounds
3rd in the visible and ultraviolet region involves the
Excited 2nd
States
promotion of electrons in , and n-orbitals from the
,
1st ground state to higher energy states. This is also called
energy transition. These higher energy states are
Energy is absorbed as molecular orbitals called antibonding.
electrons jump to
higher energy levels
Ground State
15 16
Sigma, pi and n-orbitals
Sigma bonds ( bonds) are the strongest type of
Energy level transitions
covalent chemical bond. They are formed by head-on
overlapping between atomic orbitals. Antibonding
*
Pi bonds ( bonds) are covalent chemical bonds where
* Antibonding
two lobes of one involved atomic orbital overlap two
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

5. Spectroscopy Types of spectroscopy
Utilises the Absorption and Emission of electromagnetic

radiation by atoms
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
Spectroscopic Techniques and Common Uses Spectrum

Type of spectroscopy EMR Application
Quantitative analysis Is a plot of photon
UV-vis UV-vis region /Beers Law energy vs. the
Quantitative analysis relative absorbance
Atomic Absorption UV-vis region /Beers Law of radiation.
Fourier transform
infrared (FT-IR) IR/Microwave Functional Group Analysis
The shape of
spectrum is
Raman IR/UV Functional Group Analysis determined by the
Nuclear magnetic relative absorptivity
resonance (NMR) Radio waves Structure determination of photons of
X-Ray Spectroscopy X-rays Elemental Analysis different energy.
X-ray Crystallography X-rays 3-D structure Analysis
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
Transmittance and Absorbance 7. Beers Law

There is a logarithmic dependence between the transmission, T, of light
through a substance and the absorption coefficient of the substance, , and
Light the distance the light travels through the material, .
I0 I
The absorption coefficient can, in turn, be written as a product of either a
molar absorptivity (extinction coefficient) of the absorber, , and the molar
Glass cell filled with concentration c of absorbing species in the material.
concentration of solution (C)
Transmittance is defined as the ratio of the electromagnetic radiations power

exiting the sample, I, to that incident on the sample from the source, I0,
I not directly proportional to the

T= concentration of the absorbing
I0 analyte in the sample solution.
An alternative method for expressing the attenuation of electromagnetic The molar absorptivity give, in effect, the probability that the analyte will absorb
radiation is absorbance, A, which is defined as a photon of given energy. As a result, value for depend on the wavelength of
electromagnetic radiation. Compound x has a unique e at different wavelengths.
directly proportional to the
A = - Log T = - Log
I = Log
I0 concentration of the absorbing Unit of : L*cm-1*M-1
I 0 I species in the solution. 27 28
Beers Law Steps in Developing a Spectrometric Analytical Method

slit cuvette
source
detector
1. Run the sample for spectrum
2. Obtain a monochromatic
g for the maximum
wavelength 2.0
ance
Absorba
absorption wavelength.
3. Calculate the concentration of 0.0
your sample using Beer Lambert 200 250 300 350 400 450
A = -logT = log(Io/I) = bc Equation: A = lc Wavelength (nm)
30
UV/Vis Spectrophotometer
Spectrophotometer
An instrument which can measure the absorbance of a

sample at any wavelength.
Light Lens Slit Monochromator Slits
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
- Polychromatic radiation from the source Tungsten slit
enters the manochromator and is dispersed Filament (vis)
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)
Detector Readout device

- The light transmitted through the reference or
sample cell is quantified by means of a detector. - The signal from the detector is generally
amplified and then displayed in a usable form to
- The detector is designed to produce an electric the analyst.
signal when it is struck by photons.
photons
- Digital readout device express the signal as
Phototube detector numbers on the face of a meter.
Photomultiplier tube detector
Photodiode detector
Cuvette UV-vis Spectrophotometer Application
UV Spectrophotometer Protein
Quartz ((crystalline
Q y silica))
Amino Acids (aromatic)
Visible Spectrophotometer Glucose Determination
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?
Quantitative measurements Sample preparation
Sample preparation
Determination of appropriate wavelength
Standard curve formation Grinding
Absorbance measurement
Calculation
Determination of appropriate wavelength Calibration curves

Prepare standards of known concentration
Measure absorbance at max of solution
at different concentration
Plot A vs
vs. concentration
Obtain slope
Use slope (and intercept) to determine the
concentration of the analyte in the
unknown
Prepare standards of known concentration Measure absorbance at max of solution at

Prepare stock standard solution (100 g/ml)
different concentration
Prepare series of standards with different concentration
(0, 20, 40, 60, 80, 100 g/ml) Standard sample Concentration Absorbance
(mg/ml)
Standard Concentration Volume of stock Volume of
sample (g/ml) standard water 1 0 0.000
1 0 0 10 2 20 0 048
0.048
2 20 2 8
3 40 0.110
3 40 4 6
4 60 0.169
4 60 6 4
5 80 0.226
5 80 8 2
6 100 10 0 6 100 0.284

Determine the concentration of the
Typical Beers Law Plot analyte in the unknown
Total phenolic cotent (Ferulic acid standard) Total phenolic cotent (Ferulic acid standard)
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)
Characteristics of Beers Law Plots
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
Dye Binding Method

Total protein analysis using a Principle:
spectrophotometer - At low pH, basic groups of protein are (+) charged. These will quantitatively
bind a (-) charged dye.
- Proteins bind the dye to form an insoluble complex. The unbound soluble dye
Dye binding method is measured after equilibration of the reaction and the removal of insoluble
complex by centrifugation or filtration.
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
Dye Binding Method Dye Binding Method

Acid Orange 12: Absorbance of dye bound by protein = A initial (free) die concentration -
A. filtrate die concentration
-
SO3
HO
2
N=N
2
x
x
Skim milk
Procedure: 11 x
1. Mix protein, dye, buffer pH = 2. x

6 8 10 12 14 16
2. Filter or centrifuge. % Protein (Kjeldahl)
3. Measure absorbance of filtrate.

Biuret Method
Dye Binding Method
Principles: Cu++ in alkaline solution form complexity with peptide bonds -
give pinkish-purple color.
Measure the intensity of color at 540 nm. Standard is bovine serum

Factors Influencing Dye Binding determination: albumin (BSA).
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
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
Determination of total carbohydrates Determination of total carbohydrates

in foods in foods
Mono-, di- and Polysaccharide
oligosaccharide
Objectives Food product
Food product
Determine a concentration of total carbohydrates in Sample preparation

foods including all classes of sugars, including sugar Sample preparation
derivatives and oligo- and polysaccharides using an Hydrolysis

anthron
th th d which
method hi h is
i a simple
i l colorimetric
l i t i method.
th d Sugar extraction
Sugar extraction
Total carbohydrate Total carbohydrate

analysis analysis
Principle of total carbohydrate analysis

Sample preparation (Phenol-Sulfuric acid method)
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
These products condense with themselves and other

product to produce brown and black substances
They also condense with phenol to produce colored
compounds.
Procedure of Phenol-Sulfuric acid method Calculation
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
Advantages Determination of Total Reducing Sugar
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.
Principle of total Reducing Sugar analysis

Procedure of Somogyi-Nelson Method
(Somogyi-Nelson Method)
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.
Glucose Oxidase System Total starch

Glucose Oxidasse
D-Glucose + O2 Gluconic Acid + H2O2
Principle:
- Complete conversion of starch into D-glucose
H2O2+ 0 - Dianisidine Peroxidase 2 H2O + Oxidized 0-Dianisidine
(Colorless) (Brown)
- Amylase
- Amyloglucosidase
- Determination of the D-glucose released

H 3 CO OCH3 H 3 CO OCH3
H2N NH 2 HN NH
Total starch
Dietary fiber analysis
Dietary fiber is essentially the sum of the nondigestible

components of a foodstuff or food product.
Most, but not all, dietary fiber is plant cell-wall material
(cellulose, hemicelluloses, lignin) and thus is composed
primarily of polysaccharide molecules.
Insoluble dietary fiber: cellulose, lignin, hemicelluloses
entrapped in a lignocellulosic matrix, and resistant
starch.
starch
Soluble dietary fiber: Hemicelluloses not entrapped in
a lignocellulosic matrix, much of the native pectin, and
the majority of hydrocolloids/food gums.

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
Vitamin analysis D: Cholecalciferol or Ergocalciferol

K: Phylloquinone
E: Tocopherol
Assoc. Prof. Pham Van Hung Water--soluble vitamins
Water
Department of Food Technologyy B complex: Thiamin (B1)
(B1),, Riboflavin (B2), Pyridoxine (B6),
Folic acid (B9), Cyanocobalamin (B12)
C: ascorbic acid
Extraction Methods Extraction Methods

Vitamin B1 and B2:
Vitamins A, E, K or D: Boiling or autoclaving in acid plus enzyme treatment
treatment..
Organic solvent extraction, saponification, and rere-- Folate::
Folate
extraction with organic solvents
solvents.. For unstable Enzyme extraction with -amylase, protease and -glutamyl
vitamins, antioxidants are routinely added to inhibit hydrolase(conjugase).
hydrolase(conjugase).
oxidation..
oxidation Niacin::
Niacin
Autoclaving in acid (noncereal products) or alkali
products).
products).
Ascorbic acid:
acid:
Cold extraction with metaphosphoric acid/acetic acid..
acid..
Vitamin analysis Vitamin C analysis
Bioassay Methods:
involving humans and animals
vitamins
it i B12 andd D D. Vitamin C:
Microbiological Assays: Vitamin C or L-ascorbic acid, acid, or simply
making use of protozan organisms, bacteria, and yeast. ascorbate (the anion of ascorbic acid), is an
water-soluble vitamins. essential nutrient for humans and certain other
The growth of a certain microorganism in an extract of a animal species
species..
vitamin-containingg sample
p is compared
p against
g the g Ascorbate may also act as an antioxidant against
this microorganism in the presence of known quantities of that
oxidative stress
stress..
vitamin.
Physicochemical assays:
include spectrophotometer, fluorometric, chromatographic,
enzymatic, immunological, and radiometric methods.
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
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
Introduction Major minerals

Accurately measuring trace amounts of Macrominerals (7)
mineral elements. Needed in > 100 mg/day
Application in food analysis, nutrition, Calcium
biochemistry and toxicology.
biochemistry, toxicology Phosphorus
Magnesium
Application of plasma as excited sources
Sodium
for Inductively coupled plasma - atomic
Chloride
emission spectroscopy: ICP-AES Potassium
Recently, ICP-MS are used. Sulfur
6
Trace minerals Toxic mineral

Inorganic atoms or molecules
Microminerals or trace elements Aluminum
< 100 mg/day needed
Iron Arsenic
Zinc Cadmium
Iodine
selenium
Lead
copper Mecury
manganese
fluoride
chromium
molybdenum
7 8
Atomic Absorption Spectroscopy:
Energy transitions in atoms
An Aussie Invention
Atomic absorption spectra are produced when
Developed by Alan Walsh (below) of the CSIRO in
ground state atoms absorb energy from a
early 1950s. radiation source.
Atomic emission spectra are produced when
excited neutral atoms emit energy on returning
t the
to th ground d state
t t or the
th lower
l energy state.
t t
The absorption of photon of radiation causes an
outer shell electron to jump to a higher energy
level.
Atoms absorb or emit radiation of discrete
wavelength because the allowed levels of
electrons in atoms are fixed.
9
Absorption and Emission Atomization

Atomic spectroscopy requires that atoms
Excited States
of element of interest in the atomic state
Not combined with other elements in a
p
compound
Ground State They are well separated in space
Absorption Emission In foods, all elements are present as
When atoms absorb light, the incoming energy excites compounds or complexes.
an electron to a higher energy level.
Electronic transitions are usually observed in the Must be converted to neutral atoms
visible or ultraviolet regions of the electromagnetic
11
spectrum.
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
Principles of AAS Atomic Absorption Spectroscopy

The metal vapor absorbs energy from an A solution of a metal compound is sprayed into a
external light source, and electrons jump from flame and vaporises
the ground to the excited states The metal atoms absorb light of a specific
The ratio of the transmitted to incident light q y, and the amount of light
frequency, g absorbed is a
energy is directly proportional to the direct measure of the number of atoms of the
concentration of metal atoms present metal in the solution
A calibration curve can thus be constructed

Metal Zn Fe Cu Ca Na
[Concentration (ppm) vs. Absorbance]
(nm) 214 248 325 423 589
15 16
Atomic Absorption/Emission/
Fluorescence Spectroscopy Methods for atomization
17
Atomic Absorption Spectroscopy Atomic Absorption Spectroscopy

The analyte concentration is determined from the It is possible to measure the concentration of an
amount of absorption. absorbing species in a sample by applying the
Beer law:
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
Radiation Sources Hollow-Cathode Lamps
Hollow-Cathode Lamps (most common).

can be used to detect one or several atomic species
simultaneously
Lasers (more specialised).

more sensitive
can detect only one element at a time (disadvantage) - A hollow tube filled with argon or neon.
- An anode made of tungsten
- a cathode made of the metallic form of the element being
23 measured 24
Hollow-Cathode Lamps Atomisation
Atomic Absorption Spectroscopy (AAS) requires
that the analyte atoms be in the gas phase.
Vapourisation is usually performed by:

Flames
When voltage is applied across the electrode, the Furnaces
lamp emits radiation characteristic of the metal in the Plasmas
cathode.
HCLs for about 60 metallic elements are available.
25 26
Flame Atomisation Flame Atomization

Flame AAS can only analyze
solutions.
Three types of flames
Stoichiometric flame is produced from
A slot-type burner is used to stoichiometric amounts of oxidant and fuel so
increase the absorption path the fuel is completely burned and the oxidant
l
length
h (recall
( ll Beer
B l )
law). i completely
is l t l consumed.d
Oxidizing flame is produced from a fuel-lean
Solutions are aspirated with mixture. The hottest flame and a clear blue
the gas flow into a appearance.
nebulising/mixing chamber
to form small droplets prior to Reducing flame is produced from fuel-rich
entering the flame. mixture. A cool flame and a yellow color.
27
Flame Atomisation Furnaces

Degree of atomisation is temperature dependent. Improved sensitivity over flame sources.
Vary flame temperature by fuel/oxidant mixture. Less sample is required.
Generally, the same temp range as flames.

Fuel Oxidant Temperature (K)
Acetylene Air 2,400 - 2,700
Acetylene Nitrous Oxide 2,900 - 3,100 More difficult to use, but with operator skill at the
Acetylene Oxygen 3,300 - 3,400 atomisation step, more precise measurements can
Hydrogen Air 2,300 - 2,400 be obtained.
Hydrogen Oxygen 2,800 - 3,000
Cyanogen Oxygen 4,800
29 30
Furnaces Inductively Coupled Plasmas
Enables much higher temperatures to be achieved.

Uses Argon gas to generate the plasma.
Temps ~ 6,000-10,000 K.
Used for emission spectroscopy rather than absorption
spectroscopy due to the higher sensitivity and elevated
temperatures.
Atoms are generated in excited states and
spontaneously emit light.
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
AAS - Calibration Curve Calibration Curve for Sodium

The instrument is calibrated before use by testing the
absorbance with solutions of known concentration. A
b 1.0
Consider that you wanted to test the sodium content of s
bottled water (A = 0.650?). o 0.8
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
Use of Calibration curve to determine sodium

concentration {sample absorbance = 0.65} Sample Problem 1
The nickel content in river water Determination of Nickel
was determined by AA analysis Content by AA
A after 5.00 L was trapped by ion 120
b 1.0
exchange. Rinsing the column
with 25.0 mL of a salt solution y = 5.6x + 20
s released all of the nickel and
e Units
o 0.8 the wash volume was adjusted 80

to 75.00
75 00 mL; 1010.00
00 mL aliquots
Absorbance
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)
2 4 6 8 Answer: 0.375 g/L

Concentration (ppm) 39 40
Review
Metals in Foods
Chromatography Radiation regions of AAS
Classification of AAS
Energy transition in atoms
Atomization
Principle of AAS
Structure of AAS
Determination of metal concentration
In this lecture Definition

Introduction
Chromatography is a technique exploiting
Definition
the interaction of the components of a
History of chromatography
mixture with a stationary phase and a mobile
Principle of chromatography
phase
p (solvent)
( ) in order to separate
p the
Classification of chromatograph
components.
Chromatographic properties
Components are separated by different levels
TLC
of adsorption to the stationary phase and
Column chromatography solubility in the mobile phase.
The separation techniques before Chromatography First invented chromatography

Extraction
- is based on the difference in solubility. The Russian botanist Mikhail Tswett
- Material is grounded, placed with a solvent which dissolves soluble coined the term chromatography in
compounds. The mixture is separated by filtration.
1906 to describe his experiments.
Crystallization Tswett used a glass column filled
- also based on the difference of solubility. The solubility is solved in
with finely divided chalk (calcium
a fixed volume of solvent.
- The purified compound crystallizes as solution cools, evaporates or carbonate)
b t ) tto separate
t plant
l t
diffuses pigments. He observed the
Distillation separation of colored zones or
- separates components based on their volatility typically via bands along the column, hence
vaporization-condensation method name chromatography,
Filtration In Greak, chroma means color and
- separate components of a mixture based on their particle size. graphein means write.
Used most often to separate a liquid from a solid
How to separate individual compound in a mixture?
Brief History of Chromatography History of the Main techniques

1938 Thin Layer chromatography by Russian
1906 Tswett, a Russian botanist scientist N.A Izamailov and M.S Shraiber
coined the term chromatography.
1930s Affinity Chromatography was
1915, German Chemist developed for the study of enzymes and
other proteins
win Nobel Prize for similar experiment.
1941 Liquid-Liquid
q q partition
p chromatography
g p y
1922,
1922 L.SSPPalmer,
l American
A i scientist
i ti t developed by Archer John, Porter Martin and
used Tswetts techniques on various Richard Laurence Millington Synge
natural products.
1944 Paper Chromatography one of the
1931 Richard Kuhn used most important methods in the development British chemist Archer John Porter Martin, co-
chromatography to separate isomers of of biotechnology recipient, with Richard L. M. Synge, of the 1952
Nobel Prize in chemistry, "for their invention of
polyene pigments; this is the first known partition chromatography."
1945 Gas Chromatography 1st analytical
acceptance of chromatographic
gas-solid (adsorption) chromatography
methods.
developed by Fritz Prior
History of the Main Techniques Principles of Chromatography
1950,Gas Liquid Chromatography by Martin and Physical method of separation that distributes
Anthony James; Martin won the Nobel Prize in components to separate between two phases
1952 moves in a definite direction.
1966, HPLC named by Csaba Horvath, but didnt
become a popular
b l method
th d until 1970s
til 1970
Two types of phases
1950s, Ion-Exchange chromatography
1) Stationary phase
declassified this technique
2) mobile phases
1970s, Ion Chromatography was developed by
Hamish Small and co-workers at the Dow
Chemical company
Purpose of Chromatography Classification of Methods
Analytical - determine chemical composition There are two classification schemes:

of a sample
mobile phase
Preparative - purify and collect one or more
components of a sample attractive forces
Classification based on Mobile Phase

gas (GC)
Gas Chromatography Pyrolysis GC -
heat solid materials
water (LC) to 500 1000 oC
Mobile Phase so they decompose
into gaseous products
Organic solvent (LC) Gas - solid Gas - liquid
Stationary Phase
Supercritical fluid (SCFC)
Sample MUST be volatile at temperatures BELOW 3500C
Classification based on Mobile Phase Classification based on Attractive Forces

Adsorption - for polar non-ionic compounds
Liquid chromatography (LC)
Ion Exchange - for ionic compounds
Anion - analyte is anion; bonded phase has positive charge
Cation analyte is cation; bonded phase has negative charge
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:
Thin-layer chromatography A mobile phase (developing solvent) and

A stationary phase (a plate or strip coated with a form of
(TLC) silica gel)
Analysis is performed on a flat surface under atmospheric
pressure and room temperature
TLC is one of the simplest, fastest, easiest and least
expensive of several chromatographic techniques
used in qualitative and quantitative analysis to
separate organic compounds
Advantages of TLC Application of TLC

High sample throughput TLC is applied in many fields
Low cost Environmental
The possibility to analyze several samples Clinical
and standards simultaneously Forensic
Pharmaceutical
Minimal sample preparation
Food
A plate may be stored for later identification
Flavors
and quantification
Cosmetics
Stationary phase for TLC Stationary phase for TLC
1. Thin layer of sorbent (stationary phase)

2. An inert support
Glass plate (20 x 20 cm)
Plastic sheets
Aluminum foil
Mobile phase for TLC Classifications of TLC
Perfluoroalkane (weakest),
Hexane,
Pentane,
The two most common classes of TLC are:
Carbon tetrachloride,
Benzene/Toluene, Normal phase
Dichloromethane,
Diethyl ether, Reversed phase
p
Ethylacetate,
Acetonitrile,
Acetone,
2-Propanol/n-Butanol,
Water,
Methanol,
Triethylamine,
Acetic acid,
Formic acid (strongest)
Normal Phase Reversed Phase

Normal phase is the terminology used when Reversed phase is the terminology used
when
the stationary phase is polar; for example
silica gel, the stationary phase is a silica bonded with an
organic substrate such as a long chain aliphatic
the mobile phase is an organic solvent or a acid like C-18.
mixture of organic solvents which is less polar
than the stationary phase. the mobile phase is a mixture of water and
organic solvent which is more polar than the
stationary phase.
Steps in TLC Analysis Running of TLC
The following are the important components of The sorbent is first activated by
drying for a specific time and
a typical TLC system: temperature
1. Prepare apparatus (developing chamber) Apply a concentrated drop of sample ()

with a capillary or dropping tube to
2. Prepare stationary phase layer and mobile phase bottom of plate (origin pencil line)
Stand plate in a sealed vessel.

3. Prepare sample and Apply it on the sorbent
Carefully add solvent (keep
4. Develop the plate (running) solvent level below sample).

5. Detect analyte Allow solvent to adsorb up the plate.
Calculation of the results Qualitative analysis by TLC

The ratio of distance travelled by the component A solution of a mixture is applied as a spot/band
(from origin) compared with the distance at the bottom of the plate and allowed to travel
travelled by the solvent front (from origin) is with the solvent up the plate.
called the Rf value (the relative mobility value).
Mixed Unknown +
x standards standards standards
Solvent front a Rf of = a/x
b
c Rf of = b/x
Rf of = c/x

A B C A+B+C A+B+C ?
Quantitative analysis by TLC
Carefully cut a square around a

spot of analyte.
Immerse these cut sorbent

Column chromatography
contaning sample into solvent to (CC)
release it.
A+B+C ?
Determine concentration using
a spectrophotometer based on
Beers law.
Introduction Column Chromatography

Column chromatography is one of the most Similar to thin layer
useful methods for the separation and chromatography
purification of both solids and liquids. Stationary phase =
silica gel on support
This is a solid liquid technique in which the Mobile phase = liquid solvent
stationary
stat o a y phase
p ase is solid
s a so daand
d mobile phase
ob e p ase Mixture of components
is a liquid. dissolved in the mobile
In column chromatography, this stationary phase is introduced into the
phase is packed into a vertical glass column. column.
Mobile phase moves down the column as a Components moves
depending upon their
result of gravity. relative affinities
Column Chromatography Adsorbents of Column Chromatography
Example of column chromatography separation: The usual adsorbents employed in column

Blue compound = more polar chromatography are silica, alumina, calcium
Adsorb more to the carbonate, calcium phosphate, magnesia,
silica gel
starch, etc
Elutes slower
Yellow compound = Alumina is generally suitable for chromatography
less polar of less polar compounds.
Spends much of its
time in the mobile Silica gel gives good results with compounds
phase containing polar functional groups.
Elutes faster
Mobile phase of Column chromatography Column characteristics

The choice of the solvent is depend on the solubility The main function of all the columns is to support
characteristics of the mixture. The solvents should the stationary phase.
also have sufficiently low boiling points to permit The material of the column is mostly good quality
ready recovery of eluted material. neutral glass since it shouldnt be affected by
Polarity is the most important factor in adsorption solvents.
chromatography. A ordinary
An di burette
b tt can also
l beb used
d as column
l for
f
separation.
Increasing polarity order: Petroleum ether, carbon
Column dimensions Length:diameter ratio = 10:1,
tetrachloride, cyclohexane, ether, aceton, benzene,
30:1 or 100:1
toluene, esters, water, etc.
Various accessories are attached to the top and
It can be used in either pure form or as mixture of bottom of the column for maintenace of the elution
solvents process.
Column packing Dry packing technique
Adsorbent is packed in the column in dry
There are two types of preparing the column:
form.
Dry packing / dry filling
After filling tapping can be done to remove
void spaces
Wet packing / wet filling Fill the solvent, till equilibrium is reached.
Wet packing technique Injection of the sample

Ideal and common technique
The sample which is usually a mixture of
The material is slurried with solvent and components is dissolved in minimum quantity
generally added to the column in portions. of the mobile phase.
Stationary phase settles uniformly and no The entire sample is injected into the column
crack in the column of adsorbent. at once and get adsorbed on the top portion
Solid settle down while the solvent remain of the column.
upward.
From this zone, individual sample can be
This solvent is removed then again cotton separated by a process of elution.
plug is placed.
Procedure of CC Detection of the sample

If the compounds separated in a column
chromatography procedure are colored, the
progress of the separation can simply be
monitored visually.
h compounds to be
If the b isolated
i l d fromf l
column
chromatography are colorless. In this case,
small fractions of the eluent are collected
sequentially in labelled tubes and the
composition of each fractions is analyzed by
TLC
Column chromatography Factor affecting column efficiency

1. Dimension ofthe column: column eciency has been
improved by increasing length/width ratio ofthe column.
2. Particle size of column packing: separation to be improved
by decreasing the particle size ofthe adsorbent.
3. Activityy of the adsorbent
4. Temperature ofthe column: The speed ofthe elution
increases at higher temperatures.
5. Packing ofthe column
6. Quality of solvents: solvents having low viscosities is giving
better results.
Application COLUMN CHROMATOGRAPHY
Separation of mixture of compounds Advantages of C.C

Any type of mixture can be separated
Purication process
Any quantity of mixture can be separated
Isolation of active constituents Wider choice of Mobile Phase
Estimation of drugs in formulation AAutomation
i iis possible
ibl
Isolation of active constituents Disadvantages of C.C
Determination of primary and secondary glycosides Time consuming
in digitalis leaf. more amount of Mobile Phase are required
Automation makes the techniques more complicated &
expensive
Introduction
Classification
High Performance Liquid
Components of HPLC
Chromatography (HPLC) Pump
Injector
Assoc. Prof. Pham Van Hung Column
Detector
Data system
Qualitative and Quantitative analysis
Introduction Chromatography Theory
Even though each method utilizes different

Chromatography techniques to separate compounds, the
principles are the same.
Common to all:
Stationary phase
TLC CC HPLC GC a solid or a liquid supported on a solid
Mobile phase
A liquid or gas
Chromatography Theory Introduction

As the mobile phase passes through the
HPLC: improved form of column
stationary phase, it carries the components of
chromatography
the sample mixture with it.
The components of the sample will be attracted Instead of the mobile phase moving through
to the stationary phase,
phase but there will also be a the column as a result of gravity, it is forced
competing attraction for the mobile phase. through the column under high pressure.
Each component will have its own characteristic Typical operating pressures: 500-6000psi
balance of attraction to the mobile/stationary
A smaller sized packing material (<10m) is
phase.
required to get improved separation.
So the components will not move at the same
speed and are separated.
HPLC History Diagram of HPLC Apparatus

1966: Horvth Mobile phase pumped
Built the first HPLC instrument and gave it its name through column at high
pressure.
HPLC = High Pressure Liquid Chromatography.
Sample is injected into the
1970s: HPLC became popular with an increase in system.
technology
gy
S
Separation
ti occurs as the
th
Improved columns and detectors mobile phase and sample
Production of small silica packing material are pumped through the
By 1972 particle sizes less than 10m were introduced column. The response of the
This allowed for more precise and rapid separations. Each component will elute detector to each component
from the column, one at a eluted will be displayed on a
As new technology continued to develop, HPLC
time, and will be detected by chart or computer screen.
became more efficient.
one of several possible
HPLC = High Performance Liquid Chromatography Known as a chromatogram.
detector types.
Running HPLC Data analysis
Each compound eluted will show up as a peak on the
A mixture is injected into a steel-jacketed chromatography chromatogram.
column and eluted with solvent at high pressure (4000psi or
approx 130 atm).
Inject sample A solid component can be dissolved in solvent but a solvent
peak will also be seen.
Elute with solvent UV detector
Retention Time- tR An HPLC system

Solvent reservoir
The elapsed time between the time of analyte Solvent manager
injection and the time which the maximum peak Sample manager
height for that compound is detected. Column manager
Different compounds will have different retention Detector
times.
times Read-out
Read out device
Each compound will have its own characteristic

balance of attraction to the mobile/stationary phase.
So they will not move at the same speed.
Running conditions can also effect tR:
Pressure used, nature of the stationary phase,
mobile phase composition, temperature of the
column
Diagram of HPLC system Design & Operation of an HPLC Instrument
1) Mobile phase degassing:

Dissolved gases in the mobile phase can come out of
solution and form bubbles as the pressure changes
from the column entrance to the exit.
May block flow through the system
Sparging is used to remove any dissolved gas from
the mobile phase.
An inert and virtually insoluble gas, such as helium, is forced
into the mobile phase solution and drives out any dissolved
gas.
Degassing may also be achieved by filtering the
mobile phase under a vacuum or using sonicator.
Design & Operation of an HPLC Design & Operation of an HPLC

Instrument Instrument
2) Solvent reservoirs: 3) Mobile phase mixing:
Individual reservoirs store the mobile phase Solvent proportioning valve can be programmed
components until to mix specific
they are mixed amounts of solvent
and used. 2.
from the various
May also manually reservoirs to
prepare the mobile produce the
phase mixture and
desired mobile 3.
store in a single
reservoir. phase composition.
3) Mobile phase mixing: 4) HPLC pump:
Isocratic elution: Fill stroke: mobile phase is pulled from the
operate at a single, constant mobile phase composition solvent side
Gradient elution: Exhaust stroke:
Vary the mobile phase composition with time the mobile phase 4.
If there is a wide polarity range of components to be eluted. is pushed from the
Allows for faster runs. injector to the
Ex: mobile phase composition can be programmed to vary column head.
25% water: 75% acetonitrile at the end of the run. This is where the
More polar components will tend to elute first.
high pressure is
More non-polar components will elute later in the gradient. generated

5) Injector: 6) Column:
Introduces the sample Usually constructed of stainless steel
into the mobile phase glass or Tygon may be
stream to be carried used for lower pressure
into the a pplications (<600 psi)
applications
column. Length: 5-100cm
Syringe = impractical 10 to 20cm common
for use in highly Diameter:
pressurized systems.
Typical: 2.1, 3.2, or
Rotary injection 6.
5. 4.5mm
valve is used. Up to 30mm for preparative applications

Column packing: 6) Column:
Usually spherical silica particles of
uniform diameter (2-10m) Guard column: Protects the analytical column
The smaller p
particles yyield higher
g Particles
separation efficiencies. http://hplc.chem.shu.edu/NEW/HP
The silica particles are very porous

LC_Book/Adsorbents/ads_part.html
Interferences
Allows for greater surface area for Prolongs the life of the analytical column
interactions between the stationary
phase and the analytes. Analytical column: Performs the separation
Other packing materials may also be
used:
Zirconia (ZrO2)
http://www.lcresources.com/resources/getstart/3a01.htm
Design & Operation of an HPLC Detection in HPLC

Instrument
*There are six major HPLC detectors:
7) Detector:
Refractive Index (RI) Detector
The component that
emits a response due Evaporative Light Scattering Detector (ELSD)
to the eluting sample UV/VIS Absorption Detectors
compound and The Fluorescence Detector
subsequently signals
Electrochemical Detectors (ECDs)
a peak on the
chromatogram. Conductivity Detector
A wide variety of 7. * The type of detector
detectors exist.
utilized depends on the
Must have high sensitivity- characteristics of of
small sample sizes are used the analyte interest.
with most HPLC columns
http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4
Advantages of HPLC Application in Food System
Speed many analyses can be Carbohydrates
accomplished in 30 min or less. Amino acids, proteins
A wide variety of stationary phases Vitamins, A, D, E, K
Nucleosides (purines and pyrimidines)
Improved resolution
Fatty acids, fats
Greater sensitivity various detectors Aflatoxins
can be employed. Antioxidants
Easy sample recovery less eluent Contaminants of packaging materials
volumn to remove. Carotenoids, chlorophylls
Saccharines
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
Normal phase HPLC Reversed phase HPLC

Analysis The stationary phase is non-polar adsorbant
Fat-soluble vitamins Octadecylsilyl (ODS) bonded silica matrix: An
octadecyl (C18) chain; Octyl (C8) or Butyl (C4).
Biologically active polyphenols from natural Phenyl groups
plant sources such as grape and cocoa
cocoa.
Polar vitamins: A, D, E and K.
The mobile phase is polar solvent.
Natural carotenoid pigments Water mixed with methanol, acetonitrile or
Highly hydrophilic components such as tetrahydrofuran.
carbohydrates.
Reversed phase HPLC Ion exchange HPLC

Analysis The stationary phase is functional organic
Plant proteins resins.
Cereal proteins Macroporous resins are most effective due to
Water- and fat-soluble vitamins their rigidity and permament pore structure.
Antioxidants: bytylated hydroxyanisole (BHA) Pellicular packings also are used.
and butylated hydroxytoluene (BHT).
Phenolic flavor compounds: vanillin The mobile phase is an aqueous buffer.
Pigments: chlorophylls, carotenoids,
Gradient elution is frequently employed.
anthocyanins
Ion exchange HPLC Size exclusion HPLC
Analysis Size-exclusion chromatograpy (SEC)
Simple inorganic ions fractionates solutes solely on the basis of
Carbohydrates size, with larger molecules eluting first.
Amino acids Prepacked columns of microparticulate
Preparative purification of protein media are available in a wide range of
oligosaccharides pore sizes
Aqueous buffers are used for biopolymers
such as proteins and polysaccharides
Size exclusion HPLC Sample analysis

Determination of average molecular In most cases,
sample peaks
weight and molecular weight range of on the
polysacchrides: amylose, amylopectin, chromatogram
other soluble gums and water-soluble can be used to
estimate the
cellulose
ll l d
derivatives.
i ti amount of a
compound
present.
The more
concentrated,
the stronger
the signal, the
larger the
peak. http://www.waters.com/WatersDivision/Contentd.asp?watersit=JDRS-6UXGZ4
Selectivity Resolution Equation

Ratio of Net Retention Time of 2 components (Distribution
Coefficient). Resolution is the
Selectivity represents the separation power of particular V2 - V1
parameter R=
adsorbent to the mixture of this particular components. 1/2(W1 + W2)
describing the
R esp on se
separation power
X 2
of the complete
- 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
Column selectivity refers to the distance, or relative Volumes
separation, between two peaks
Resolution General Factors Increasing Resolution

Increase column length
Decrease column diameter
Decrease flow-rate
Pack column uniformly
U uniform
Use if stationary
t ti phase
h ((packing
ki material)
t i l)
Decrease sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution
What is Ultra Performance Liquid
Chromatography?
2004: Further advances in column technology
and chromatography instrumentation
Utilized even smaller packing particle sizes
(1 7m)
(1.7m)
Higher pressures (15000psi)
Allowed for significant increases in LC speed,
reproducibility, and sensitivity.
New research utilizing particle sizes as small
as 1m and pressures up to 100,000psi!
WHO KNOWS WHAT THE FUTURE MAY BRING!

Review
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
Gas chromatography (GC) Gas chromatography (GC)
Definition The mobile phase is a carrier gas, usually an

A gas chromatography is a chemical analysis
inert gas such as helium or an unreactive
instrument for separatin
separating
p g chemicals in a gas such as nitrogen.
complex sample using a carrier gas as a mobile The stationary phase is a microscopic layer of
phase and a microscopic layer of liquid or liquid or polymer on an inert solid support,
polymer on an inert solid support as a inside a piece of glass or metal tubing
stationary phase.
phase. called a column
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
Filters/Traps Data system

H
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.
Injector Split injection

The sample must be vaporized in
the injection port in order to pass
through the column for
separation..
separation
Instant vaporization off the
sample, 280 C
Carrier gas transports the sample
into the head of the column
The injection port may serve the additional function of splitting
Purge valve controls the fraction the injection so that only a portion of the analyte goes on the
of sample that enters the column column
Split injection Splitless injection
The sample may be diluted
with carrier gas to accomplish
a split (1:50 to 1:100
preferred). In splitless injection, the split
Only a small portion (1 part) of vent valve is closed and all
th l t
the analyte goes on th the of the analyte goes on the
column, and the majority (44 column.
99 parts) of the analytes are
vented to the split vent. Used for limited sample
This is common method
High split ratio typically gives a
sharp, narrow peak.
Column Configurations Columns

Two general types of columns are encountered
in gas chromatography, packed and open Packed
tubular,, or capillary
tubular capillary..
Chromatographic
g p columns varyy in lengthlengg from
less than 2 m to 50 m or more. They are
constructed of stainless steel, glass, fused silica,
or Teflon. In order to fit into an oven for Capillary
thermostating, they are usually formed as coils
having diameters of 10 to 30 cm.
Open Tubular Capillary Column Polar vs. nonpolar

Separation is based on the vapor pressure and
0.32 mm ID polarity of the components.
Mobile phase
(Helium) Within a homologous series (alkanes, alcohol,
g at 1
flowing Liquid
q
Stationary
olefins
olefins,, fatty acids) retention time increases with
mL/min
phase
0.1-5 m
chain length (or molecular weight)
Polar columns retain polar compounds to a
greater extent than non-
non-polar
C18 saturated vs. C18 saturated methyl ester
15-60 m in length
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
Characteristics of the Ideal Detector

Detector:: The
ideal detector for gas chromatography has the
following characteristics:
characteristics:
160C
q
1. Adequate sensitivityy
2. Good stability and reproducibility
3. A linear response to solutes that extends over
several orders of magnitude.
magnitude.
50C 4. A temperature range from room temperature
to at least 400oC.
0 60
Time (min)
Characteristics of the Ideal Detector Detectors

5. A short response time that is independent of
Flame Ionization Detectors (FID)
flow rate.
rate.
Electron Capture Detectors (ECD)
6. High reliability and ease of use.
use. Electron impact/chemical
p / ionization ((EI/CI)
/ )
7. Similarity in response toward all solutes or a Mass spectrometry
highly selective response toward one or more
classes of solutes.
solutes.
8. Nondestructive of sample.
sample.
Flame Ionization Detectors Flame Ionization Detectors

Effluent exits column and enters an A general detector for organic compounds
air/hydrogen flame Very sensitive (10-13 g/s)
The gas-
gas-phase solute is pyrolized to form
electrons and ions Linear response (107)
All carbon species are reduced to CH2+ ions Rugged
These ions collected at an electrode held above Disadvantage: specificity
the flame
The current reaching the electrode is amplified
to give the signal
Electron Capture Detectors SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
Ultra
Ultra--sensitive detection of halogen
halogen--containing Detector Response
species C18 Peak Area (cm2 )
10
Pesticide analysis C16 8
O h ddetectors b
Other
ther id MS
besides 6
C14 4
IR 2
AE 0.5 1.0 1.5 2.0 2.5 3.0

Sample Concentration (mg/ml)
Retention Time
C1 4
T h e c o n te n t % o f C1 4 fa tty a c id s = 100
C + C 1 6+ C 18
14
= 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
Mixture of known compounds

RT= 4.0 min on SE-30
Hexane
Octane
Decane
1.6 min = RT
Hexane
GC Retention Time on SE-30
GC Retention Time on Carbowax-20 (min)
Response
Response
RT= 4 min on SE-30

Unknown compound may be Hexane
Unknown compound
1.6 min = RT
GC Retention Time on SE-30

Retention Time on Carbowax-20 (min)
GC ADVANTAGES DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250C without decomposition.
1. Very good separation

Fatty Acids Methylester
2. Time (analysis is short)

O O
3. Small sample is needed - l R C OH +CH 3 OH + H 2 SO 4 R C O CH 3
Reflux Volatile in Gas
4. Good detection system O
Chromatography
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
Gas Chromatogram of Methyl Esters of Fatty Acids

In this lecture
Introduction to Amino acids

AMINO ACID COMPOSITION Importance of amino acid analysis
ANALYSIS Methods of amino acid analysis
Assoc. Prof. PHAM VAN HUNG
Introduction Amino Acids

an amino group (hence "amino"
Amino acids acid).
The monomer unit of proteins
a carboxyl group (-COOH). This
Important constituents of foods both in nutrition gives up a proton and is thus an acid
and quality of foods (hence amino "acid").
one of 20 different "R" groups. It is
Classification the structure of the R group that
20 amino acids determines which of the 20 it is and
Different side chain (R groups) its special properties.
R groups vary in size, shape and polarity
Amino acids can be classified by R groups
Amine group
pKa = 10.5 i id
imidazo
pKa = 3.9 pKa = 4.1
guanidino
pKa = 12.5
Aspartic acid Glutamic acid

Basic amino acids Acidic amino acids
Amino acids can act as acids and bases
Nonionic and zwitterionic forms
Titration of glycine
of amino acids
pH 7
indole
ring
pH 1
Titration curves predict the

electric charge of amino acids
pH 12
Isoelectric point (or isoelectric pH)
Amphoteric
(ampholytes - amphoteric electrolytes) pI = (pk1 + pk2) = (2.34 + 9.60) = 5.97
Amino acids differ in their acid-base properties
Amino acids with

pka of the COOH group: 1.8 2.4 Important of analysis
R groups that do not ionize pka of the NH3+ group: 8.8 11.0
Amino acids with ionizable R groups

Amino acid analysis can be used to:
e.g. to quantify protein and peptides
to determine the identity of proteins or
peptides
id b d on their
based h i amino
i acid
id
composition
and to detect atypical amino acids that might
be presentin a protein or peptide
three stages (three ionization steps three pka values)
Principle of analysis Determination for Amino Acid Compositions of Proteins
A protein/peptide is hydrolyzed into its A. Hydrolysis

individual amino acid constituents and then 1. Overnight in 6 M HCl at 100 oC.
amino acid compositions are analyzed based
2. Enzymes.
on a chromatographic method.
method
Ion-exchange chromatography, reversed-
phase liquid chromatography, and gasliquid B. Separation by HPLC.
chromatography are three separation
techniques used.
Protein Hydrolysis Amino acid composition hydrolysis
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.
Method for analysis Method for analysis

When the amino acid reacts with ninhydrin, the
reactant has characteristic purple or yellow color. A detector is usually present as an ultraviolet-
Amino acids, exceptimino acid, give a purple color, visible or uorescence detector .
and show the maximum absorption at 570 nm.
The imino acids such as p proline ggive a yyellow color,,
and show the maximum absorption at 440 nm. A recording
di device
d i (e.g.,
( integrator)
i ) is
i used
d for
f
The postcolumn reaction between ninhydrin and transforming the analog signal from the
amino acid eluted from column is monitored at 440 detector and for quantication.
and 570 nm.
and the chromatogram obtained is used for the
determination of amino acid composition.
1. Amino acids are pre- or

Mechanism of Ion-Exchange Chromatography of
postcolumn derivatized with Amino Acids
ninhydrin, or o-phtalaldehyde + pH 2
Na
(OPA) form fluorescent H3N
+
adducts.
-
SO 3
COOH
2. The amino acids are identified Na
+
OH
according to their retention
- +
So 3 H3N
times on HPLC Exchange Resin

COOH
3. Amounts of aas present are

determined fluorescent -
SO 3 H 3N+
pH3.5
intensities. COOH
4. Sensitive: can detect less than 1 So 3

-
OH
H3N+
pmol of each amino acid. Na
+
COO
-
H
+ -
OH = H 2 O
+
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
Chromatogram of Amino Acids
Moles/Liter VAL
ALA
HIS LEU
ASP
LYS GLU
pH 2.25 pH 3.25 pH4.25

Structure of Lipids
Fatty acid composition analysis
Assoc Prof
Fatty acids Saturated Fatty Acids

a fatty acid is a carboxylic acid with a long
aliphatic tail (chain).
- either saturated or unsaturated. O
8 7 6 5 4 3 2 1
- Most naturally occurring fatty acids have a CH3 CH2 CH2 CH2 CH2 CH2 CH2 C OH
chain of an even number of carbon atoms, Octanoic Acid
from 12 to 28.
Unsaturated Fatty Acids Cis and Trans Fatty Acids
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
Short hand: 8:1 (3) 8:2 (3,6)

H
Trans 9 - Octadecenoic Acid (elaidic acid)
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)
Procedure for Fatty Acids Analysis

Procedure for Fatty Acids Analysis
1. Extract fat.
2. Saponify (hydrolysis under basic condition).
3. Prepare methyl ester (CH3ONa). The esterification is
carried out with a reagent
g such as boron trifluoride in
methanol (BF3 in methanol).
4. Chromatography methyl ester.
5. Determine peak areas of fatty acids.
Fatty acids are identified by retention time.
6. Compare with response curve of standard.
Fatty Acids composition

Carbohydrates
Definition
Sugar composition analysis any of a large group of organic compounds occurring in
foods and living tissues containing hydrogen and oxygen
in the same ratio as water (2:1) and typically can be
Assoc. Prof. Pham Van Hung broken down to release energy in the animal body.
Department of Food Technology Classification

Monosaccharides
International University
Disaccharides
Olygosaccharides
Polysaccharides
Simple carbohydrates Sugar composition analysis

Monosaccharides
Glucose Principle
p
Total carbohydrate
Fructose determination Sugars are extracted from foods, cleaned up and
Galactose analyze by liquid or gas chromatography.
Reducing sugar
analysis Techniques
Disaccharides
sacc a es 1. Thin Layer chromatography
1
Maltose Sugar composition 2. HPLC
Sucrose analysis 3. GC
Lactose
TLC technique TLC system

TLC is a simple and rapid technique commonly
used to resolve mixtures of components into
their individual constituent parts.
Different sugars such as glucose, fructose,
sucrose, raffinose, and others have been
separated
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)
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
Principle of mineral analysis Principles of Flame Atomic Absorption Spectroscopy
The sample solution is nebulized (dispersed into

Solid/liquid
Solution
Nebulisation Molecules in tiny droplets), mixed with fuel and an oxidant,
sample Desolvation gas phase
Sample M+ X-
MX(g)
and burned in a flame produced by oxidation of
preparation Vaporisation
the fuel by the oxidant.
(g) + X(g)
M(g) (g) Atomisation
Atomisation=
Atoms in gas Dissociation Atoms and ions are formed within the flame as
phase
analyte compounds are decomposed by the high
M+ Ionisation
Excitation temperatures.
Ions
Excited Atoms and ions of the same element produce
ICP-MS and other
MS methods
Atoms
different spectra so they absorb radiation of
different wavelengths.
Principles of Inductively Coupled Plasma-Atomic

Procedure of mineral composition analysis
Emission Spectroscopy
In ICP-AES, a plasma is used as the atomization and

excitation source. 1. Preparation: standard & sample
A plasma is defined as gaseous mixture containing 2. Ashing
significant concentrations of cations and electrons.
3 Forming
3. F i solution
l ti
Temperatures in plasmas are very high (in the
neighborhood of 500010000K) resulting in very 4. Running sample by AAS or ICP-AES or ICP-MS
effective atomization.
5. Calculation
Excessive ionization of sample atoms is not a
problem, probably because of the high concentration
of electrons contributed by the ionization of the argon.
Analysis of Natural Waters Analysis of Natural Waters

Scope Sample Preparation and Analysis
This method describes the determination of calcium, copper, lithium,
magnesium, manganese, potassium, sodium, strontium and zinc in Filter each sample through a 0.45 micron micropore
natural waters and may be applicable to other elements.
membrane filter.
Reagents
Aspirate each sample directly, except for calcium and
Lanthanum
L th solution,
l ti 5% ((w/v).
/ ) PPrepare as d
described
ib d under
d ththe St
Standard
d d magnesium. For calcium and magnesium, dilute with
Conditions for La.
Hydrochloric acid, HCl, concentrated. 5% (w/v) La solution and HCl to give a final solution
concentration of 0.25% (w/v) La and 5% (v/v) HCl.
Standard Solutions
Prepare all standard solutions except calcium and magnesium by Determinethe concentration of the element of interest
suitable dilutions of the stock solutions described under the Standard by using AAS or ICP-AES.
Conditions for each element. For calcium and magnesium, dilute the
stock solutions with the 5% (w/v) La solution and HCl to give dilute
standards which contain 0.25% (w/v) La and 5% (v/v) HCl.
Analysis of Meat and Meat Products Analysis of Meat and Meat Products
Scope Sample Preparation

o Accurately weigh a 4-6 g sample into a Vycor crucible, add 2.5 mL of
This method describes the determination of lead and 50% (w/v) magnesium nitrate hexahydrate solution, and pre-ash for 1
copper in meats and meat products and may be to 2 hours until the sample is completely charred, under an infrared
applicable to other elements. lamp on a hot plate.
o Place the ppre-ashed samplep in a muffle furnace and ash at 500 C.
For meatt samples,
F l a dry
d ashinghi procedure
d i used.
is d It After one hour remove the sample from the furnace, carefully wet the
requires minimum operator attention and there is no loss ash with HNO3 and replace in the furnace.
due to spattering, volatilization or retention on crucibles. o Repeat the wetting procedure hourly until the ash is white (usually
about two hours).
After ashing, samples are dissolved in acid and diluted. o After ashing, quantitatively transfer the sample to a 10 mL volumetric
flask by carefully washing the crucible with 1 mL HNO3, then two 1-mL
portions of dilute HNO3. Transfer all washings to the volumetric flask,
repeating the washing procedure twice. Dilute the solution to volume
with deionized water.
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.

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Food Analysis - Complete Lecture

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FOOD ANALYSIS Introduction

Information of food products Important of food analysis

WHY DO WE NEED TO ANALYZE FOOD?

Steps in analysis What must a food scientist be able to do?

Criteria for Selecting an

- American Association of Cereal Chemists (AACC)

The study how the chemical compound

The collection of measurements signals - a mobile phase

Lecture 1 (chapter 6):

Lecture 1 (chapter 7): Lecture 2 (chapter 9):

Lecture 2 (chapter 8): Lecture 3 (Chapter 21):

Lecture 5 (chapter 10):

Lecture 7 (chapter 27): Lecture 8 (chapter 28):

Learn about history of chromatography Learn structure and operation of HPLC

Lecture 9 (chapter 29):

Lecture 13 (chapter 11): Lecture 14 (chapter 24):

Presentation 1: Vietnamese standards Assignment: 30%

MOISTURE AND TOTAL SOLID ANALYSIS Moisture content in foods

Introduction What is moisture content?

Moisture or water is by far the most common

What is water activity? Importance of Moisture Analysis

1- Moisture is a quality factor in the preservation of

Importance of Moisture Analysis Importance of Moisture Analysis

Water of hydration: Beef, ground, extra lean, raw 6 3 .2

Total moisture Content assay Total moisture Content assay

Determination of Moisture: Methods Drying Methods

Testing sample Weight (Ws)

How to calculate moisture content How to calculate moisture content

Moisture Content on Wet Basis Moisture Content on Dry Basis

Convection or forced draft ovens (AOAC) Objective

Forced draft ovens VACUUM OVEN

VACUUM OVEN MICROWAVE DRYING OVEN

MICROWAVE DRYING OVEN NEAR INFRARED ANALYZER

Procedure Using a digital balance, the

Rapid Moisture Analyzer WATER ACTIVITY MACHINE

Follow instruction manual from manufacture

Ash Contents in Foods Importance of ash analysis

Methods for Determining Ash Dry Ashing

Dry ashing Principles

Microwave ashing Organic substances are burned in the presence of oxygen in

Dry Ashing General Procedure for Dry Ashing

Weigh a 510 g sample into a tared crucible. Predry if the

wt after ashing - crucible wt Disadvantages

Wet Ashing Wet Ashing

Wet Ashing Wet Ashing

6. Dry on a hot plate or steam bath and then return to a Advantages

In this lecture Introduction

Procedure of total protein analysis

Introduction Importance of protein analyses

Kjeldahl Method - Principle

Procedure Conversion Factors from Nitrogen to Protein for Foods

Conversion Factors from Nitrogen to Protein for Foods Apparatus

6.25 6.38 5.83 5.70 5.30

Corns Milk Whole wheat Wheat flour Nuts

Application Analysis of protein fractions

Analysis of protein fractions Analysis of protein fractions

Add distilled water Add NaCl

Stirring SDS-PAGE Kjeldah

Centrifuge Supernatant (Globulin)

Analysis of protein fractions Analysis of protein fractions

Shaking SDS-PAGE Kjeldah Shaking SDS-PAGE Kjeldah

Centrifuge Supernatant (Glutenin) Centrifuge Supernatant (Gliadin)

1. What is protein? Protein fraction? 1. What is lipid?