method of analysis of food additives

1. METHOD OF ANALYSIS OF
ARTIFICIAL SWEETENERS , FLAVORS,
FLAVOR ENHANCERS, STABILIZERS,
THICKENING AND JELLING AGENTS
MADE BY:- RAJAT RANA
1ST SEM
M.PHARMACY (ANALYSIS)

2. Sweeteners
• Sweeteners: is a substance used to sweeten food or drink, especially one
other than sugar.
• Types of sweetener
• Natural sweeteners : exist or are produced by nature, without added
chemicals or fancy machinery. The only sugars that are optimal to eat are
wild, non-hybridized, seeded fruits, and the natural sugars and starches in
living vegetables, trees, seeds, nuts, and roots.
• Artificial sweeteners : which are also called sugar substitutes, alternative
sweeteners, or nonsugar sweeteners, are substances used to replace sugar in
foods and beverages. They can be divided into two large groups: nutritive
sweeteners, which add some energy value (calories) to food; and non-
nutritive sweeteners, which are also called high-intensity sweeteners because
they are used in very small quantities, adding no energy value to food.

3. Examples of natural sweeteners:- Honey, Maple Syrup, Coconut Palm Sugar and
molasses
Examples of artificial sweeteners:- Aspartame, Sucralose, Saccharin, Neotame,
Sodium Cyclamate
• Determination of saccharin in food, the following analytical techniques
are:
1. Biosensors
2. Spectophotometry
3. Electroanalysis
4. Chromatography
Chemicals and reagents
• • Reference standards.
• • Water
• • Acetonitrile was HPLC purity. All other reagents were analytical purity.
Sample preparation
• Yogurt: 5 mL was diluted with 5 mL methanol, and then the mixture was
stirred and centrifuged. The sample was filtered with a 0.45 µm filter prior to
injection.

4. • • Diet cola: The sample was treated with an ultrasonic for 10 minutes, and
then was filtered with a 0.45 µm filter prior to injection.
• Chromatographic conditions
Column: Agilent TC-C18(2), 4.6 x 250 mm, 5 µm
Mobile phase: A = 20 mM KH2PO4 buffer, pH 3.0; B = acetonitrile
Gradient:0 min 15 %B 5 min 35 %B 10 min 80 %B
Flow rate: 1 mL/min
Wavelength: 214 nm
Injection volume: 5 µL
Temperature: 30 °C
• A mixture of acetonitrile-water (1:1) was used to extract these additives from
solid food matrices and acetonitrile was used to extract them from liquid food
matrices. Saccharin was identified and determined in the negative ionization
mode, using single reaction monitoring (SRM).

5. • Thickeners
Thickeners are substances which, when added to the mixtur
e, increase its viscosity without substantially modifying its ot
her properties. Ex:- alginin, guar gum, locust bean gum,
and xanthan gum.
Analysis
• Sample Preparation :-
• 1. Your sample needs to be highly purified. No corrosive
chemicals, such as strong acids or bases, are allowed. Otherwise,
it will permanently damage the instrument.
• 2. Dissolve 1-2 mg of your sample in HPLC grade chloroform at
least one hour prior to measurement. There should be no
precipitates in the chloroform solution; otherwise you need to
filter the solution to remove the precipitates.

6. Procedure
1. Turn on the binary pump.
a. To set the flow rate and whether it will be single or binary pump system, use the
Breeze software on the computer next to the instrument.
2. Turn on the Autosampler.
a. To load the samples, open the door on the bottom of the sampler and put the
corresponding slot numbers in the Breeze software.
3. Turn on the UV detector and the RI detector.
a. Increase the flow rate gradually. Don’t jump the flow rate from low flow to higher
flow rate at one time. If you do this, the column and the cells 3 may be damaged.
You have to keep the flow rate at 0.1 ml/min when the instrument is not in use.
b. Each time you make an adjustment to the flow rate (especially when increasing
the flow rate), make the adjustments in gradual steps and make sure the pressure has
been stabilized before making the next adjustment.
4. Wait until both the UV and RI detectors are stabilized.
5. Wait at least 1hour until the flow rate stabilizes.
6. Run the Breeze software to analyze your samples.

7. • Jelling agents:- agents which help in forming a gel include
• alginate,
• pectin,
• carrageenan,
• gellan,
• gelatin,
• agar,
• modified starch,
• methyl cellulose
• hydroxypropylmethyl cellulose.
• Stabilizers are substances that increase stability and thickness
by helping foods remain in an emulsion and retain physical
characteristics.
• Ex:- Lecithin, agar-agar, carrageenan and pectin

8. Stabilizers and Jelling agents:-Pectin
• BUFFERS AND REAGENTS:
1. 50 mM Tris/HCl buffer plus 1 mM CaCl2.
2. 0.5 M NaOH.
3. 0.5 M HCl.
4. 1 M HCl.
5. 2-Propanol (100%).
SAMPLE PREPARATION:
1. Moisten 50 mg (0.05 g) of the sample with 2 drops of 2-propanol.
2. Add 50 mL of deionised water and stir gently on a magnetic stirrer for 20-
30 min (until the pectin dissolves).
3. Adjust the pH to 12 by careful addition of 0.5 M NaOH, and leave the
solution for exactly 15 min at room temperature.
4. Lower the pH to 8.0 by dropwise addition of 0.5 M HCl.
5. Adjust the volume to 100 mL with deionised water.
Add the following to quartz cuvettes, mix the contents well and measure the
absorbance values at 235 nm after 30 min.

9. • RESULTS:
• The increase in absorbance for a given sample on incubation with pectate lyase is measured as follows:
Blank Absorbance = Enzyme Blank + Sample Blank (measured after 30 min).
ΔAbsorbance = Reaction Absorbance – Blank Absorbance.
From the increase in absorbance (ΔA) the amount of unsaturated product produced can be calculated as:
Unsaturated product = ΔA/L x ε
where: ΔA = Reaction Absorbance (after 30 min) – Blank Absorbance.
L = path-length of the reaction cuvette (= 1 cm).
ε = the molar extinction coefficient of the reaction product (4600 M-1 cm-1).

10. • Flavor enhancers
Flavor enhancers enhance a food's existing flavors. They may be extrac
ted from natural sources (through distillation, solvent extraction, macerat
ion, among other methods) or created artificially. Ex. MSG
• Some flavor enhancers are as follows:
a) Dioctyl sodium-sulfosuccinate - used in processed foods.
b) Disodium guanylate - used in canned meats, meat based foods.
c) Hydrolyzed vegetable - used in mixes, stock, processed meats.
d) Monosodium glutamate
(MSG) used in Chinese food, dry mixes, stock cubes, and canned, processed, a
nd frozen meats.

11. Monosodium Glutamate:-
Standard and sample solutions
• Monosodium l-glutamate (100 mg) was accurately weighed into a
100-mL volumetric flask, dissolved in water, and the solution was
diluted to volume with the same solvent to furnish a working
standard.
• Accurately weighed sample equivalent to 1 g and transferred to a
100-mL volumetric flask, dissolved in water (50 mL), sonicated for
15 min in an ultrasonicator, and made up to the volume with the
same solvent.
• The solution was then filtered through Whatmann’s No. 42 filter
paper.
• One milliliter of the filtrate was taken in a 10-mL volumetric flask
and diluted to the volume with methanol and used for analysis.
.

12. Chromatography Procedure
• Chromatography was performed on aluminum-backed silica gel 60 GF254TLC
plates prewashed with methanol.
• Standard solutions of MSG were transferred to different 10 mL volumetric flasks
and diluted to volume with the methanol such that the final concentrations were
0.4–1 μg/μL.
• Standards and three different sample solutions were applied to the TLC plates as 8
mM bands with 9 mM space between two bands using a Camag Linomat IV sample
applicator.
• Plates were developed with a mobile phase of methanol–chloroform–formic acid 5
+ 5 + 1 (v/v) in a TLC twin trough chamber.
• After development, the plates were derivatized with 1% ninhydrin solution in
acetone and dried at 60°C for 5 min. The quantification of the standards and
samples were performed by means of a Camag TLC scanner III controlled by
WinCATS 4.06 version software.
• The amount of MSG in the sample solutions were computed from the calibration
plot.

13. Flavors
Flavors are additives that give food a particular taste or smell, and may be derived
from natural ingredients or created artificially.
Type Description
Natural flavoring substances
These flavoring substances are obtained from
plant or animal raw materials, by physical,
microbiological, or enzymatic processes. They can
be either used in their natural state or processed
for human consumption, but cannot contain any
nature-identical or artificial flavoring substances.
Nature-identical flavoring substances
These are obtained by synthesis or isolated
through chemical processes, which are chemically
and organoleptically identical to flavoring
substances naturally present in products
intended for human consumption. They cannot
contain any artificial flavoring substances.
Artificial flavoring substances
These are not identified in a natural product
intended for human consumption, whether or
not the product is processed. These are typically
produced by fractional distillation and additional
chemical manipulation of naturally sourced
chemicals, crude oil, or coal tar. Although they
are chemically different, in sensory characteristics
they are the same as natural ones.