This document describes methods for quantitatively determining preservatives, emulsifiers, and colouring materials found in foods and pharmaceuticals. It discusses using thin-layer chromatography to identify common preservatives like methyl paraben and propyl paraben, and extracting and identifying emulsifiers like polysorbate 80. Gas chromatography methods are provided for determining mono- and diglycerides. Finally, it outlines extracting and identifying food dyes from candy coatings using yarn and ammonia.

1. QUANTITATIVE DETERMINATION
OF PRESERVATIVES, EMULSIFIERS,
AND COLOURING MATERIALS
BY
1

2. PRESERVATIVES
 Preservatives are substances that commonly added to
various foods and pharmaceutical products in order to
prolong their shelf life.
 The addition of preservatives to such products, especially
to those that have higher water content, is essential for
avoiding alteration and degradation by microorganisms
during storage.
2

3. IDENTIFICATION AND QUANTITATIVE
DETERMINATION OF PRESERVATIVES
 Identification of the preservatives in pharmaceuticals by
TLC:
 The most used preservatives are- Germaben II
Imidazolidinyl urea
Methyl paraben
Propyl paraben
 Materials and methods:
 Stationary phase: silica gel 60, silica gel 60 F254
pre-coated TLC plates on Al support
 Mobile phase: mixture of solvents
 Detection: UV light, specific reagents
3

4. GERMABEN II IDENTIFICATION
5 g sample (lipogel)+ 15 ml ethanol
reflux on water bath at 70°c for 1 hr
filter in a 25 ml graduated flask, cool
adjust with ethanol up to the mark
apply on starting line of TLC plate
development and detection under UV
4

5. METHYLPARABEN AND PROPYLPARABEN
IDENTIFICATION:
2g of sample + 15ml of 0.05 N KH2PO4 + 0.6g of NaCl
stirr
6ml chloroform
stirr for 5 min
layers are separated, organic phase is removed
residue dissolved in 6ml methanol
filter
TLC
5

6. IMIDAZOLIDINYL UREA EXTRACTION:
 To identify imidazolidinyl urea, it was mixed with water.
The sample was applied on the starting line of the TLC
plate, simultaneously with 20 µl of imidazolidinyl urea
reference solution, having 1% concentration in water. The
mobile phase used is water-ethanol.
6

7. DETERMINATION OF PRESERVATIVES BY TLC
 PRINCIPLE:
 The preservatives are extracted from the acidified sample
with acetone.
 After filtration, the acetone solution is mixed with water,
and in alkaline medium the fatty acids are precipitated as
their Ca salts.
 The alkaline acetone/water mixture is extracted with
diethyl ether to remove lipophilic substances.
 After acidification the preservatives are extracted with
diethyl ether.
 An aliquot of the diethyl ether extract is spotted on a silica-
gel coated thin-layer plate.
 After development of the plate, the chromatogram
obtained is observed under UV light and visualized using
Millon’s reagent.
7

8.  PROCEDURE:
 Weigh accurately about 1g of sample into a 125ml flask.
 Add 4 drops of HCl 4M, add 40ml of acetone and mix.
 Heat the mixture to about 60°c until complete extraction.
 Cool and shake for 1 minute.
 Adjust the pH of the solution at ≤3 using 4M KOH.
 Add 1g calcium chloride dihydrate and shake
 Filter the solution into a 250ml separating funnel,
containing 75ml diethyl ether, and shake for 5 min.
 Allow the phases to separate.
 Discard the upper layer (diethyl ether phase).
 Collect the aqueous phase (lower layer) in a 100ml
separating funnel.
8

9.  Adjust the pH to 2 with HCl 4M.
 Add 10ml diethyl ether, and shake for 5 minutes.
 Allow the phases to separate.
 Discard the lower layer (aqueous phase)
 Transfer 2ml of diethyl ether phase (upper layer) into a
5ml sample vial.
 DETERMINATION:
 Activate the plates at 100°c for 10 minutes
 Apply 10µL of each of the reference solutions and 100µL of
the sample solution on base line of TLC plate.
 A stream of air can be used to facilitate evaporation of the
solvent.
9

10.  Transfer an adequate volume of the development solvent
into a developing tank of suitable size.
 Place the TLC plate in the chamber and develop at room
temperature.
 Remove the plate from development tank and dry it.
 Examine the plate under UV light.
 Visualize the preservatives in the chromatogram with
millon’s reagent.
 Calculate the Rf value for each spot.
 Compare the spots obtained from the sample solution with
those of standard solutions with respect to their Rf values,
their behavior under UV radiation and the colour after
visualization.
10

11. compound Rf colour
Methyl paraben 0.12 Pink ++++
Ethyl paraben 0.17 pink+++
Propyl paraben 0.21 Pink++
Butyl paraben 0.26 Light pink
2-phenoxy ethanol 0.29 Lemon yellow
11

12. DETERMINATION OF PRESERVATIVES BY HPLC
PRINCIPLE:
 The sample is acidified by adding sulfuric acid and then
suspended in a mixture of ethanol and water.
 After gently heating the mixture to melt the lipid phase to
promote quantitative extraction, the mixture is filtered.
 The preservatives in the filtrate are determined by reversed
phase HPLC.
 PROCEDURE:
 Preparation of standard preservative solution:
 Pipette 20ml, 10ml, 5ml, 2ml and 1ml of preservative stock
solution into each a 50ml volumetric flask.
12

13.  Add 1ml of H2SO4 2M and shake to homogenize it
 Add ethanol/water mixture 9:1 (v/v) to volume and mix
 Filter through 0.45µm membrane filter into HPLC injection
vials.
 sample preparation:
 Weigh accurately about 1 g of sample into a 125 ml flask
 Add 1ml of H2SO4 2M, and add 50ml ethanol/water
mixture 9:1 (v/v) and add 1 g of boiling chips.
 Shake vigorously for 1 minute until homogenous
suspension
 Place in water bath at(60±1)°c for 5 minutes
 Cool the flask in a stream of cold water and store in
refrigerator for 1 hour.
13

14.  Filter the solution through a membrane filter (0.45µm)-
after centrifugation when necessary
 Transfer approximately 2ml filtrate into a 5ml sample vials
 Perform determination of the filtrate by HPLC within less
than 24 hours.
 Chromatographic conditions:
 Mobile phase: tetrahydrofuran/water/methanol/acetonitril
mixture(5:60:10:25)
 Flow rate: 1.5 ml/minute
 Detection wavelength: 280nm
 Oven temperature: 25°c
14

15. DETERMINATION
 Inject 20µL of the sample solution into the chromatograph and
record the chromatogram.
 inject 20µL of one of the standard preservative solution and
record the chromatogram.
 Compare the chromatograms obtained.
 Measure the peak heights(or area)
 If the peak area of the sample is too low or too high, increase or
decrease the amount of the sample so that the peak area falls
within the calibration range.
 Calculate wi, as percentage by weight(%w/w) using the formula
 % wi(w/w)= bi × 51/(1000×a)
 bi = the concentration (µg/ml) of preservative in the test solution
as read from the calibration curve
 a=the weight of the test solution
15

16. QUANTITATIVE DETERMINATION OF METHYL
PARABEN
80mg of sample + 25ml 2M NaOH
reflux for 30 minutes, cool
25ml 0.0333M KBrO3 + 5ml 12.5% w/v KBr + 40ml GAA
cool
10ml HCl, stand for 15 minutes + 15ml KI
Titrate liberated iodine with 0.1 M sod.thiosulphate using
2ml of starch solution as indicator
16

17.  Repeat the operation without substance being examined.
 The difference between the titrations represent the amount
of potassium bromate required.
 The volume of 0.0333M potassium bromate is equivalent to
half of the volume of 0.1M sodium thiosulphate required
for titration.
 Each ml of 0.0333M KBrO3 is equivalent to 0.005072 g of
C8H8O3
17

18. EMULSIFIERS
 An emulsifier is a substance which stabilizes an emulsion,
which is a mixture of two immiscible substances.
 Emulsifiers are the surface-active substances that act at the
surface between two media (e.g. oil and water).
 emulsifiers are used to keep an emulsion stable through the
shelf life of a product, and prevent it separating into its
components. Eg: polysorbate 80, sorbitan esters
18

19. DETERMINATION OF MONO AND DIGLYCERIDES
BY CAPILLARY GAS CHROMATOGRAPHY
 PRINCIPLE:
 Conversion of mono and diglycerides with
N,N-bis(tri methyl silyl)tri fluoroacetamide(BSTFA) and
trimethylchlorosilane(TMCS) in pyridine in to more volatile tri
methyl silyl ether derivatives and quantitative determination by
capillary gas chromatography using an internal standard (n-
tetradecane).
 PROCEDURE:
 Sample solution
 Accurately weigh 10 mg of homogenized sample of emulsifier
concentrates or 50 mg of oils and fats containing emulsifiers in
to a 2.5 m l screw cap vial with Teflon faced septa.
19

20.  Add 0.1 ml o f internal standard solution containing 1 mg
n-tetradecane, 0.2 ml BSTFA and 0.1 ml TMCS to the
sample.
 Humidity is strictly excluded.
 Close vial and shake vigorously.
 Heat the reaction mixture in heating device at 70°c for 20
minutes.
 Inject 1-5 µl of the reaction mixture into the gas
chromatograph showing a stable base line.
 Avoid delay of GC analysis.
 The reaction is carried out twice and duplicate injections
are made per reaction.
20

21.  Reference solution:
 Transfer 0.10 ml of reference solution to a vial and add the
silylating agents 0.2ml BSTFA and 0.1 m l TMCS and inject.
 Use a concentration range of reference standards similar to
that of the substances to be quantified in sample solution.
A plot of response factor vs. concentration of reference
standards may be useful to check linearity .
 Check response factors periodically. Response factors
should be above 0.5.
 Lower response factors indicate some loss or
decomposition. Use concentration range of 0.5 - 10mg/ml
of components in reference and sample solutions.
21

22.  Identification
 Analyze the reference solution under the same operation
conditions as the sample solution.
 Identify peaks by comparison of retention time with known
substances or apply coupled GC/MS.
22

23.  Response factor = area of spectral peak
weight of substance injected
 Calculation of sample component content
mx(%) = 1/Rx × (mis/ms) × (Ax/Ais ) × 100
 where:
 mx(%) : percent (m/m) mass of component x in sample
 Rx: response factor of component x in sample
 mi S : mass, in mg, of internal standard in sample
 ms : mass, in mg, of sample
 Ax : peak area of the component x in sample
 Ais : peak area of the internal standard in sample
23

24. High-Performance Thin-Layer Chromatographic
Method for quantitative determination of
Emulsifiers
 Standard Stock solution and Sample preparation:
 Standard stock solutions containing 0.5 mg/mL of pure
compounds were prepared in a mixture of methanol and
chloroform(1:1, v/v) and filtered through 0.45-µm filters for
calibration studies.
 Samples were prepared from dried and powdered aerial
parts (1.000 g each) of C. inerme collected from different
locations.
 The powder was extracted with ethanol (3×10 mL),
concentrated, and fractionated with n-hexane(3×10 mL).
24

25.  The vacuum-dried hexane extract was treated with
charcoal using a chloroform–methanol (1:1, v/v) mixture to
obtain chlorophyll-free hexane extracts.
 Known amounts of extracts were taken and dissolved in
chloroform–methanol (1:1 v/v) and filtered through a 0.45-
µm filter for HPTLC analysis.
 HPTLC Procedure:
 Precoated silica gel 60F254 HPTLC glass plates were
prewashed with methanol and activated at 100°C for 5 min
prior to chromatography.
 Standard and sample solutions were spotted in the form of
bands of 5-mm width at 15 mm from both the lower and
left edge, and with a space of 10 mm between two bands,
with a microlitre syringe using the Linomat IV, under a
stream of nitrogen gas.
25

26.  Linear ascending development was carried out in 20×20-
cm twin trough glass chambers saturated with the mobile
phase.
 mobile phase consisting of toluene–acetone (94:06, v/v)
was selected for chromatography.
 Detection of the spots was carried out by dipping the
chromatoplate in freshly prepared vanillin–sulphuric acid–
ethanol (1g :5mL :95 mL) reagent using the Camag
Immersion device and subsequent heating at 110°C for 15
min on TLC plate heater.
 Quantitative evaluation of the plate was carried out with a
TLC Scanner running win CATS 1.3.3 software.
26

27.  Densitometric scanning was performed in the
reflectance/absorbance mode at 620 nm.
 The radiation source utilized was a tungsten lamp. Both
deuterium and tungsten lamps were used to record the
spectra of compounds in the range 200–800 nm.
 Concentrations of the compounds chromatographed were
determined from the intensity of the reflected light.
Evaluation was done with peak areas.
27

28. COLOURING MATERIALS
 Any material used for it’s color.
 Help to create unique appearance.
28

29. EXTRACTION AND IDENTIFICATION OF FOOD
COLOURS
 Take 6 pieces of candy-coated gum.
 Make a water bath by filling a
400-mL beaker with approximately
150 mL water and placing it on a hot plate in the fume hood.
 Set the hot plate at medium and heat the water to boiling.
use this water bath throughout the first part of this
experiment. Maintain the water at a slow boil, adding
additional water to the beaker to maintain an approximate
volume of 150 mL
 Place 6 pieces of candy, all the same color and the same
brand, into a test tube. Add enough vinegar to just cover
the candy.
29

30.  Place the test tube in the water bath and heat it until the
colored coating has just dissolved.
 Remove the test tube from the water bath and carefully
pour the colored liquid into a second, clean test tube
leaving the solid candy pieces behind.
 This solution contains the food dyes, some sugar, some
emulsifier, and the vinegar.
 The solids left from the candy, in the first test tube, can be
discarded.
 Measure 15 cm of wool yarn and add it to the test tube with
the dye solution. Add an additional 3 mL of vinegar to the
test tube.
 Place the test tube into the water bath and heat the
solution for about 5 minutes, stirring occasionally.
30

31.  After heating, remove the yarn from the test tube. Rinse it
with a little de ionized water. The yarn should be colored
from the food dye.
 Place the yarn into a clean test tube and add 1 mL of
concentrated ammonia solution. Use the stirring rod to
make sure the yarn is submerged in the ammonia. If
necessary, add up to an additional 1 mL of ammonia
solution.
 Heat the test tube in the water bath for about 5 minutes,
stirring occasionally.
 Remove the test tube from the water bath and allow it to
cool. Save this for Part B of the experiment.
31

32. B. Chromatographic Separation of the
Unknown Color Mixture
 Obtain a sheet of chromatography paper. Draw a light
pencil line across the paper about 1.0 cm from the bottom
of the sheet.
 Pour the colored liquid, from the test tube in Part A of this
experiment, into a small beaker or evaporating dish.
 Using a capillary tube, apply a small spot of the unknown
color mixture (obtained in Part A of this experiment) on
the pencil line near the center of the paper.
 Spot the paper with each of the available F D & C colors
allowing about 2.0 cm minimum distance between each
different spot. Using a pencil, note the identity of each
color at the top of the paper above each spot.
32

33. Chromatography paper in the 600-ml beaker developing
chamber
33

34.  Add 1% NaCl solution to a clean 600 mL beaker to a depth
of about 0.5 cm.
 Bend the spotted chromatography paper into a cylindrical
shape, and staple them.
 Place the chromatography paper into the beaker making
sure that the spots of dye are not below the solvent level
and that the paper is not touching the sides of the beaker.
 Cover the beaker with a piece of plastic wrap or with a
watch glass.
 Allow the solvent to move up the paper to within 1 cm of
the top.
 Remove the paper from the beaker, open it flat, and, using
a pencil, mark the solvent front.
 Lay the chromatography paper on a paper towel to dry.
34

35.  Outline each spot on the chromatography paper in pencil.
Measure and record the average distance from the origin to
the solvent front.
 Measure and record the distance each spot moved from the
origin.
 Determine the Rf values for each spot in the unknown
mixture and for each of the F D & C colors that used.
 The Rf values for the components in the unknown mixture
will be the same for the same component in the F D & C
known colors.
 By matching both the color and the Rf value of each of the
spots, we can determine the identity of the colors
extracted from the food.
35

36.  Calculations:
 The Rf value for a spot is calculated using the formula:
Rf = distance spot moved in cm
average distance to solvent line (from origin) in
cm
36

37. Determination of natural colorants in plant
extracts by high-performance liquid
chromatography
 Chemicals and reagents:
 The standard of apigenin was purchased from
Extrasynthese (Genay, France) and those of lawsone (97%),
juglone (97 %) and indigotin (95 %) from Sigma–Aldrich
(Deisenhofen, Germany).
 HPLC-grade solvents and analytical-grade chemicals were
provided by Merck.
 The water was double distilled.
 Solvents were filtered through a 0.45μm filter and degassed
in an ultrasonic bath before use.
37

38.  Stock solutions (0.4 mg mL-1) of 1–3 were prepared by
dissolving 20 mg of each powder in 50 ml methanol.
 The solutions were stored in a refrigerator.
 The working standard solutions of appropriate
concentration were prepared daily by diluting the stock
standard solutions with methanol.
 The solutions of 4 were freshly prepared (0.2 mg/mL to
0.001mg/ml) in methanol–dioxane (1:1, v/v) and kept in
vials preventing light penetration to avoid decomposition
of indigotin.
38

39.  Extracts:
 The ethanolic and propylene glycolic extracts from
chamomile (Chamomilla recutita), henna (Lawsonia
inermis), walnut (Juglans regia) and natural indigo
(Indigofera sp.) were received directly from a Bulgarian
producer.
 According to the provider, the alcoholic extracts were
obtained with 70 % ethanolic–aqueous solution.
 The ethanolic extracts (10 mL) were evaporated under
vacuum at 40 °C and reconstituted with 10 mL methanol.
 All extracts were then submitted to sonication at room
temperature for 5 min using a Sonicator and centrifuged at
10000 rpm for 10 min.
 The supernatant was collected and filtered through a 0.45
μm filter prior to injection.
39

40.  HPLC analysis:
 The chromatographic analyses were performed on a
chromatographic system equipped with a tertiary pump Model
9012, a rheodyne injector with a 20 μl sample loop, a UV/Vis
detector Model 9050 set at 335nm, 340nm, 249nm and 288nm
according to the UV absorption maxima of the compounds 1–
4(apigenin, lawsone, juglone, indigotin) respectively.
 Stationary phase: A reversed phase Hypersil ODS RP18
stationary phase was used.
 Mobile phase: The chromatographic separation was realized
using a mobile phase consisting of
 A) acetonitrile,
 B) methanol and
 C) potassium dihydrogen phosphate buffer adjusted to pH 3.20
with orthophosphoric acid.
 The flow rate was 1 mL min-1.
40

41.  Quantitative analysis:
 The analysis of the assayed compounds (1–4) was
performed.
 Working solutions containing 0.2, 0.1, 0.05, 0.02, 0.01 and
0.001 mg mL-1 of 1–3 were prepared from stock solution,
0.4 mg mL-1 in methanol, respectively.
 The employed concentrations of 4 were 0.2, 0.1, 0.05, 0.02,
0.01 and 0.001 mg mL-1 and the solutions were prepared in
dioxane–methanol (1:1, v/v).
 Triplicate analyses were performed for each concentration
and the peak area was detected at 335, 340, 249 and 288 nm
for 1–4, respectively.
 Calibration curves were constructed from the peak areas vs.
analyte concentrations.
 Slope, intercept and other statistics of the calibration lines
were calculated.
41

42. REFERENCES
 http://www.cael.pub.ro/SiteulCAEL/cicic/chimanalitica/Id
entification%20of%20the%20Preservatives%20in%20Phar
maceuticals.pdf
 http://www.asean.org/MRA-Cosmetic/Doc-5.pdf
 http://www.iupac.org/publications/pac/1991/pdf/6308x1153
.pdf
 www.ncbi.nlm.nih.gov/pubmed/17654616
 http://www.chymist.com/Chromatography%20food%20col
ors.pdf
 www.shd.org.rs/JSCS/JSCS_OnLine_First/4602_OLF.pdf
 INDIAN PHARMACOPOEIA
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43. Size: 299 × 300
Type: 438KB GIF
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