OBJECTIVE

To determine the amount of mercury in dried anchovy using Hg-analyzer.

INTRODUCTION

Mercury is a heavy, silver-white liquid metal and a fair conductor of electricity.

It is the only common metal which sometime called quicksilver is liquid at ordinary
temperatures. Mercury alloys easily with may metals, such as gold, silver and tin.
These alloys are called amalgams. Mercury can present in several ways, either
organic (CH3Hg) or organic (HgI). The most harmful form is organic because it can
absorb onto skin, lung as well as blood. Mercury compounds have many uses.
Generally, Mercuric chloride (corrosive sublimate, HgCl 2) is used as an insecticide, in
rat poison, and as a disinfectant. Mercuric oxide is used in skin ointments. Mercuric
sulphate is used as a catalyst in organic chemistry. Vermilion, a red pigment, is
mercuric sulphide; another crystalline form of the sulphide (also used as a pigment)
is black. Mercury fulminate, Hg(CNO)2, is used as a detonator.

Mercury is compound that can be found naturally in the environment. It can be

found in metal form, as mercury salts or as organic mercury compound. Some forms
of human activity release mercury directly into soil or water. For example, the
application of agricultural fertilizers and industrial wastewater disposal. All mercury
that is released in the environment will eventually end up in soil or surface waters.
Mercury concentration in fish usually greatly exceed the concentrations in water they
live. It is become more serious when contamination of mercury is dominant in fish.
Fish accumulates substantial concentration of mercury in their tissue thus can
represent as the major source of this element to human. Due to this, the analysis of
mercury in marine organism such as fish has gained many scientists. In this
expeiment, anchovy was used as an animal tissue sample for the determination of
the content of mercury.

APPARATUS AND INSTRUMENT

100 mL volumetric flasks, 50 mL volumetric flasks, beakers, measuring cylinder, hot

plate, conical flask, Whatman’s filter paper.
CHEMICALS

100 ppm of stock solution of mercury, 70% Hydrogen peroxide (H2O2), Concentrated
Nitric acid (HNO4), Sodium borohydride (NaBH4), Hydrochloric acid (HCl), Sodium
hydroxide, deionized water

PROCEDURE

A. Preparation of sample ( Wet digestion method)

The sample was dried in the oven at 60 – 70 C for 24 hours. 0.5 – 1.0 g of
animal tissue was weighed accurately and placed into a conical flask. 7 – 10 mL
of concentrated nitric acid was added. The conical flask was placed in the fume
hood and the sample allowed to digest overnight. The digested sample was
heated using a hot plate until the red fume evolving from the conical flask. The
conical flask was cooled down at room temperature. 1 – 2 mL of 70% H 2O2 was
added and heated again until clear solution was obtained. The solution was
filtered using a Whatman’s filter paper, into a 100 mL volumetric flask. The
solution was diluted with deionized water to the mark. The solution was
transferred into a plastic bottle. The sample was prepared in triplicate and was
kept for further analysis.

B. Preparation of 100 ppb standard solution of mercury.

Stock solution of Mercury = 100 ppm = 100000 ppb
Stock solution of mercury is 100000 ppb. 0.5 mL of stock solution used to
prepare 1000 ppb in 50 mL volumetric flask. Then, 25 mL of 1000 ppb of stock
solution used to prepare 500 ppb in 50 mL volumetric flask. Lastly,10 ml of 500
ppb of stock solution used to prepare 100 ppb of standard solution.

C. Preparation of standard solutions of mercury.

The volume of 100 ppb standard solution of mercury that was needed to prepare
2.0, 4.0, 6.0, 8.0 and 10.0 ppb in 100 mL volumetric flask was calculated. The
calculated volume was transferred into five separate 100 mL volumetric flask.
3% of Hydrochloric acid was added into each flask and up to the mark.

All dilutions done using 3% HCL for procedures B and C

D. Preparation of the reagents.
D1. Preparation of 0.2% NaBH4 in 0.05% NaOH
0.25 g of sodium hydroxide was weighed. Sodium hydroxide was added and
stirred into a beaker which contains 300 mL of distilled water. 1 g of NaBH 4
was weighed and added into the sodium hydroxide solution. The solution
was transferred into a 500 mL volumetric flask. Distilled was added up to the
mark.

D2. Preparation of 3% Hydrochloric Acid

30 mL of concentrated hydrochloric acid was measured using measuring
cylinder. The acid was poured and stirred into the beaker containing 200 mL
of distilled water. The diluted hydrochloric acid was transferred into 1000 mL
volumetric flask. Distilled was added up to the mark.

RESULTS

Concentration of
Absorbance Volume (mL)
mercury standard (ppb)

0 0.0000 0

2.0 0.0079 2

4.0 0.0274 4

6.0 0.0329 6

8.0 0.0444 8

10.0 0.0547 10
Table 1. Table of absorbance and volume of the standards.
 Sample Concentration (ppb) Percent weight (w/w%)

A 1.5536 1.5536 x 10-7 %w/w

B 0.4643 4.643 x 10-8 %w/w

C 0.875 8.75 x 10-8 %w/w

Averag 0.9643 9.643 x 10-8 %w/w
e
Table 2. Table of concentration and percent weight of the tea samples.

Mean concentration 9.643 x 10-8 %w/w

Standard deviation 0.550113

Relative Standard deviation 57.05%

Table 3. Statistical table for mercury in tea samples.

GRAPH

Calibration curve of Mercury

0.06
f(x) = 0.01 x + 0
0.05 R² = 0.98

0.04
Absorbance

0.03

0.02

0.01

0
0 2 4 6 8 10 12

Concentration (μ/L)

Graph 1. Calibration curve of Mercury.

DISCUSSION

Direct Mercury Analyzer (DMA) was used in this experiment for the
determination of the amount of mercury in anchovy. It is used in this experiment to
analyze the mercury because it has wider dynamic range that can be achieved as
compared to AAS. DMA can analyze solid, liquid and gas matrices with equal
precision. It uses the principle of thermal decomposition, amalgamation and atomic
absorption. All mercury is released from the sample through thermal decomposition.
There are six steps to use the Hg-analyzer. First, warming up the mercury analyzer.
After switch on the instrument, wait until it complete warm up. After approximate 15
minutes, it is ready to work. At this point it would be possible to install the
amalgamator. Next, installation of the catalyst tube. After that, calibration of the unit
perform. If any calibration is not selected the analysis cannot start. Then proceed
with conditioning the catalyst tube. If the condition are not verified, it is necessary to
run further cleaning. Next, stabilized the aqueous solution. The solution must be
fresh and stabilized with 1-2 % HCL. Lastly, verification of stability test. All the
sample must be selected to verify the correct value of the relative standard deviation.
If both conditions are verified, then the test is passed.

Then, by using the formula y=mX+c, we have obtained the value of the
concentration. Absorbance versus concentration graph was plotted and the linear
equation was calculated in Microsoft excel. The linear equation of standard addition
curve for mercury is y =.0.0056X + 0.0001 while the value of R 2 is 0.9827. The value
of R2 is 0.9827 which is approximately to 1 is the additional information of a best fit
for all points are on the straight line. The actual concentration of the sample in
average after taking the dilution into account is equal to 0.9643 ppb .With that value,
weight percent of the amount of mercury in the triplicate sample is 9.643 x 10 -8
%w/w. This value falls within the safe amount of mercury to be consumed by human.
By referring to Environmental Protection Agency (EPA), safe levels of mercury to be
consumed are between 0.5 mg/L and 1 mg/L of mercury in the fish. The amount of
mercury in fish depend on the species and the levels of population in its
environment. Larger and longer-lived fish usually contain higher levels of mercury.
. The standard deviation and relative standard deviation were calculated in
Microsoft excel in order to ensure the accuracy between the triplicate samples. The
 standard deviation and relative standard deviation are 0.550113 and 57.05%
respectively. The smaller the value of the relative standard deviation indicates the
more precise data of the samples. There might be some error during performing this
experiment that affect the accuracy of the result. There still need to be improved in
the sample preparation in pipetting and diluting the samples.

CONCLUSION
In conclusion, the concentration of mercury in anchovies sample is 0.9643
ppb and the %w/w is 9.643 x 10 -8 . The anchovies are safe to be consumed since the
value is in the range of safe amount.

PRE-LABORATORY QUESTIONS

1. Explain the principle of Hg-analyzer spectrophotometer

Hg-analyzer spectrophotometer uses the principle of thermal decomposition,

amalgamation and atomic absorption. It can analyze both solid and liquid matrices
with equal precision. Analysis takes only 5 minutes per sample and does not require
any sample preparation. All mercury is released from the sample through thermal
decomposition. This eliminates the need for any sample preparation and,
subsequently, purchasing, handling and disposing of hazardous chemicals.
No sample preparation is required, the typical bottleneck in the analytical
laboratory is eliminated. Therefore, analysis is reduced to only 5 minutes per sample,
and at a fraction of the cost typically associated with traditional mercury techniques,
such as ICP-AES or ICP-MS. Typical applications that the Hg-analyzer
spectrophotometer is used for include environmental, geochemical, petrochemical,
food and feed, clinical and polymer samples.

2. Briefly explain the component of Hg-analyzer spectrophotometer.

The major component of Hg-analyzer spectrophotometer are sample dosing
system, thermal process furnaces, atomic absorption spectrophotometer and system
controller. Sample dosing system built-in 40 position auto-sampler for high
throughput unattended operation, for solid and liquid sample. The maximum sample
weight and sample volume are 500 mg and 500 μl. There are four steps in thermal
process systems which are drying, decomposition, catalysis and amalgam. Oxygen
used as the carrier and decomposition gas. Next, atomic absorption
spectrophotometer. Single bean spectrophotometer with sequential flow through of
measurement cells was used. Light source used is low pressure mercury lamp at
wavelength 253.65 nm. The last component is system controller. System controller is
a standard bench-top computer to export the data and act as data storage.

REFERENCES

DMA-80 Direct Mercury Analyzer. (2017). Retrieved April 29, 2020, from
https://docplayer.net/20874836-Dma-80-direct-mercury-analyzer.html
Hamid, G. A. (2017, February 22). DMA Direct Mercury Analyzer. Retrieved April 29,
2020, from https://www.slideshare.net/GamalAbdulHamid/dma-direct-
mercury-analyzer
Mercury contamination - Water Treatment Solutions. (n.d.). Retrieved April 29, 2020,
from https://www.lenntech.com/periodic/elements/hg.htm
Mercury: how much is safe? (2016, September 5). Retrieved May 2, 2020, from
https://www.greenleft.org.au/content/mercury-how-much-safe

APPENDIX

Calculation of serial dilution from stock solution of Mercury.

Concentration of stock solution of Mercury = 100 ppm = 100000 ppb

To prepare 100 ppb stock solution of mercury in 50 mL volumetric flask,

M1 V 1 = M 2 V 2

Concentration of stock Volume (mL)

solution of Mercury (ppb)

1000 (100000)(V1) = (1000)(50)

V1 = 0.5

500 (1000)(V1) = (500)(50)

V1 = 25

100 (500)(V1) = (100)(50)

V1 = 10
Calculation for the preparation of the standard solution of Mercury.

Fomula: M1V1 = M2V2

Concentration of Absorbance Volume (mL)

standard solution (ppb)

0 0.0000 0

2.0 0.0079 (100)(V1) = (2.0)(100)

V1 = 2

4.0 0.0274 (100)(V1) = (4.0)(100)

V1 = 4

6.0 0.0329 (100)(V1) = (6.0)(100)

V1 = 6

8.0 0.0444 (100)(V1) = (8.0)(100)

V1 = 8
10.0 0.0547 (100)(V1) = (10.0)(100)
V1 = 10
Table 4. Calculation for the preparation of standard solution

Absorbance Concentrarion (ppb)

0.0088 0.0088 = 0.0056 (X) + 0.0001
X = 1.5536
0.0027 0.0027 = 0.0056 (X) + 0.0001
X = 0.4643
0.0050 0.0050 = 0.0056 (X) + 0.0001
X = 0.875
Table 5. Calculation for concentration of Mercury in the sample.
 Sampl Absorbance Concentration Percent weight (w/w%)
e (ppb)
A 0.0088 1.5536 (1.5536 x 10-4 mg/100000mg) X100%
= 1.5536 x 10-7 %w/w
B 0.0027 0.4643 (4.643 x 10-5 mg/100000mg) X100%
= 4.643 x 10-8 %w/w
C 0.0050 0.875 (8.75 x 10-5 mg/100000mg) X100%
= 8.75 x 10-8 %w/w
Table 6. Calculation for percent weight of mercury in the sample.

Calculation for the concentration and mass of mercury in the sample (% w/w).
Average concentration of mercury in the sample = 0.9643 ppb

= 0.9643 μ/L

= 0.0009643 mg/L

Weight of sample solution = 100 mL = 100000 mg

Weight of mercury = 0.0009643 mg/L X 0.1 L

= 9.643 x 10-5 mg

weight
Mercury concentration as % w/w, X 100%
100000mg

(9.643 x 10-5 mg/100000mg) X100% = 9.643 x 10-8 %w/w

Calculation for the % relative standard deviation of replicated samples.

Relative standard deviation
= (standard deviation/mean concentration) X 100%
= (0.550113/0.9643) X 100%
= 57.05%