NATURAL WELLNESS INDUSTRIES SDN BHD

ANALYTICAL PROTOCOL
ACTIVATED CHARCOAL

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No. Test Release Specification Shelf Life Specification Reference

01 Appearance Black, light powder free Black, light powder free BP 2018
from grittiness from grittiness

02 Solubility Practically insoluble in all Practically insoluble in all BP 2018

usual solvents usual solvents

03 Identification:

When heated Burns slowly without a Burns slowly without a BP 2018

to redness flame flame

04 Acidity and Not more than 0.75ml of Not more than 0.75ml of
alkalinity 0.02M hydrochloric acid is 0.02M hydrochloric acid is
required to change the required to change the BP 2018
colour of the indicator to colour of the indicator to
yellow. yellow.

05 Assay: The fumes released do not The fumes released do not BP 2018
Sulfides turn lead acetate paper turn lead acetate paper
brown brown

06 Microbial
limit test:

Total aerobic NMT 1 x 10³ cfu/g NMT 1 x 10³ cfu/g

microbial BP 2018
count

Total yeast NMT 1 x 102 cfu/g NMT 1 x 102 cfu/g

and mould
count

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ANALYTICAL PROTOCOL
ACTIVATED CHARCOAL

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07 Heavy Metal
Test:

Lead NMT 10ppm NMT 10ppm

Note:
BP 2018 : British Pharmacopeia 2018
NMT : Not more than
# Microbial Limit Test for product under stability to be done yearly and at the end of shelf
life (ESL).

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ANALYTICAL PROTOCOL
ACTIVATED CHARCOAL

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No. Tests Methods

01 Appearance Visual Inspection
02 Solubility Test Procedure
1. Weight 10g of the activated charcoal.
2. Add 10g of the activated charcoal into 100ml water.
3. Observe the solubility of the material.
4. Repeat step 1 to 4 by using alcohol 99%.
5. Observe and record the solubility of the material.

03 Identification Test Procedure

1. Weight 10g of the activated charcoal and place it in crucible.
2. Put the crucible in the oven and heat for 30 mins at 200℃
3. Observe and record the results.

04 Acidity and Test Procedure

Alkalinity 1. Add 2.0g of activated charcoal into 40ml of distilled water and
boil for 5 min.
2. Cool the solution and restore to the original mass with distilled
water and filter.
3. The first 20ml of the filtrate is rejected.
4. To 10ml of the filtrate, add 0.25ml of bromothymol blue solution
R1 and 0.25mL of 0.02M sodium hydroxide.
5. The solution is blue. The solution is titrated with 0.02M
hydrochloric acid.
6. Record the volume of hydrochloric use to change the color of
indicator to yellow. Not more than 0.75ml of 0.02M
hydrochloric acid is required to change the color of the indicator
to yellow.

04 Assay: Test Procedure

Sulfides
Lead Acetate Paper

1. Immerse filter paper weighing about 80 g/m2 in a mixture of 1

volume of dilute acetic acid and 10 volumes of lead acetate
solution.
2. After drying, cut the paper strips 15 mm by 40 mm.

Test

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ANALYTICAL PROTOCOL
ACTIVATED CHARCOAL

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1. Add 1.0 g of sample, 5 mL of Hydrochloric Acid (HCl) and 20

mL in conical flask
2. Heat to boiling
3. The fumes released do not turn lead acetate paper to brown.

05 Microbial Limit Test: Procedure Analysis

i. Total Aerobic Negative Control

Microbial Count
(TAMC) 1. Aseptically pipette 10mL of sterile purified water into sterile
90mL Buffered Peptone Water (BPW). Shake vigorously to
ii. Total Yeasts and homogenize the sample.
Moulds 2. Aseptically pipette out 10mL of the prepared sample into
Count(TYMC) sterile universal bottle.
3. Mix and homogenize the sample.
4. Aseptically pipette out 1mL of pretreated specimen in
duplicate sterile petri dishes.
5. Pour approximately 15-20 mL of sterile TSA and SDA
cooled to about 45°C in each plate. Mix the plates by gently
rotating clockwise and anti-clockwise direction.
6. Allow to solidify to room temperature.
7. Invert the TSA plates, incubate at 30-35°C for 72-120 hours
(3-5 days) for TAMC.
8. Invert the SDA plates, incubate at 20-25°C for 120-168
hours (5-7 days) for TYMC.
9. Observe the plates for presence of any colonies.

Negative Product Control

1. Aseptically prepare sample by dissolve 10g of product under

test into sterile 90mL BPW. Shake vigorously to homogenize
the sample.
2. If necessary, warm the sample in water bath which the
temperature is not more than 450C as to de-solidify the
sample.
3. Aseptically pipette out 10mL of the prepared sample into
sterile universal bottle.
4. Mix and homogenize the sample.
5. Aseptically pipette out 1mL of the pretreated specimen in
duplicated sterile petri dishes.
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ACTIVATED CHARCOAL

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6. Pour approximately 15-20mL of sterile TSA and SDA

cooled to about 45°C in each plate. Mix the plates by gently
rotating clockwise and anti-clockwise direction.
7. Allow to solidify to room temperature.
8. Invert the TSA plates, incubate at 30-35°C for 72-120 hours
(3-5 days) for TAMC.
9. Invert the SDA plates, incubate at 20-25°C for 120-168
hours (5-7 days) for TYMC.
10. Observe and count the number of colony forming units
(CFU) developed in each plate.
11. Report the highest number of CFU obtained in the plates.
Take mean count.

Interpretation results of TAMC and TYMC

1. Count for each plate and take average count. Calculate

the number of CFU/g of product.

Number of colonies (CFU/g or CFU/ml) = C

avd

C = total colonies on plates counted

a = average number of plates counted (duplicate)
v = volume applied to each plate
d = dilution from which the count obtained

2. For TAMC, count all colonies including fungi growth on

TSA. Use those counts of duplicate that showing the highest
number of colonies less than 250 CFU.
3. If the plates with more than 250 CFU, report as Too
Numerous To Count (TNTC).
4. For TYMC, count all colonies including bacteria growth on
SDA. Use those counts of duplicate that showing the
5. Highest number of colonies less than 50 CFU.
6. If the plates with more than 50 CFU, report as Too
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ANALYTICAL PROTOCOL
ACTIVATED CHARCOAL

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Numerous To Count (TNTC).

7. For negative control, there should be no growth of
microorganisms on TSA and SDA plates. If there is growth,
all test results are invalid. Report Out-Of-Specification
(OOS).

06 Heavy Metal Test: Lamp – EDL

Lead Correlation Coefficient: > 0.99

Chemical & Reagent

1. Nitric Acid concentrated
2. Hydrogen Peroxide
3. Lead Standard Solution 1000ppm
4. Hydrochloric Acid

Standard Preparation
1. Prepare 100ppm stock solution by pipette 10.0mL of
1000ppm Lead standard solution into 100.0mL volumetric
flask.
2. Top up to final volume with distilled water.
3. Continue the blank and standard preparation as mentioned in
the Table 2 below:

100ppm of Pb 37%
Standard Final
intermediate stock concentrated
Solution Volume
solution (mL) HCl (mL)
Blank 0.0 2.0 100.0
1.00ppm 1.0 2.0 100.0
2.00ppm 2.0 2.0 100.0
5.00ppm 5.0 2.0 100.0
10.00ppm 10.0 2.0 100.0
6.00ppm
6.0 2.0 100.0
(QC sample)

Sample Preparation
1. Weigh 0.5g of sample in the 50.0mL beaker.
2. Add 10.0mL concentrated Nitric Acid, HNO3.
3. Heat the sample until the color of its fume change from
brown fume to white fume.
4. Let the sample to cool down for few minutes.
5. Add 2.0mL of 30% H2O2 drop by drop. And then shake.
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ANALYTICAL PROTOCOL
ACTIVATED CHARCOAL

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6. Filter the sample solution into 50.0mL volumetric flask and

top up with DI water to the mark.
7. Analyze by AAS.

Apparatus & Parameters

Method – Flame Atomic Absorption Spectrometer
Wavelength – 283.31 nm
Lamp - HCL
Correlation Coefficient: > 0.99

HISTORY:

Date Effective Reason for Revision Revision No.

30/01/2020 First protocol written 00
22/08/2023 Add identification test 01
Add solubility test procedure
Add acidicty and alkalinity test

Shahnas Oli
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Date: Date: Date:
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