AS245: Bachelor in Science Applied Chemistry Title: Analysis of Animal Tissue Course: Spectrochemical Methods of Analysis (CHM580) Name: Mohamad Nor Amirul Azhar b. Kamis Matrix no:
2014647344 (AS245 3S)
Date of experiment: 1st April 2015 Date of submission: 29th May 2015 Group members: 1. Nurnailah bt. Noorazlan 2. Nor Amirah bt. Ahmad Azuan 3. Noramira bt. Saad It is my responsibility as a student of UiTM to adhere to truthfulness and avoid dishonesty, fraud or deceit of any type in connection with write up and conduct of this experiment. Signature:__________ Date:
TITLE:
Determination of mercury (Hg) in animal tissue (anchovies) by using mercury analyser.
ABSTRACT: The sample used is the anchovies that had been treated with wet digestion by using strong acid to extract the analyte (mercury) from the sample. The experiment was done by using the Mercury analyser to know that amount of mercury in the sample. The sample has been done triplicate to ensure the accuracy and getting more accurate data. In order to make sure the amount of mercury in samples is in the range of standard solutions, the samples had been diluted with dilution factor of 20. The average amount of mercury in the diluted triplicate samples is 4.432ppb with relative standard deviation of 1.213. The actual average concentration of samples is 88.64ppb. Smaller relative standard deviation value indicates the more precise data between the triplicates.
INTRODUCTION: Fishes are important for a healthy diet. Fish such as anchovies is lean, high-quality protein, essential nutrients, low saturated fat, contain omega-3 fatty acids and lowcalorie source of protein for a good diet. However, some of the fishes may contain high amount of harmful chemical such as mercury that is not safe to be consumed in high amount for human. Mercury enters atmosphere from natural sources estimated 1600 to 4000 tonnes each year. Mercury is generated by industries, burning fossil fuels and as by-product of some bacteria. High amount of mercury consumed may harm an unborn baby or young child’s developing nervous system. According to Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA), fishes that contain high level of mercury that is not safe to be consumed are shark, swordfish, king mackerel and tilefish. For this experiment, the sample used was anchovies that is known been contaminated with mercury. On average, by calculation made by the FDA, the average amount of mercury in anchovies is
0.04mg/kg. 0.5ppm to 1.0ppm is considered safe amount of mercury to be consumed by human according to EPA. OBJECTIVES: To perform wet digestion method to extract mercury out from sample anchovies and determine the amount of mercury in anchovies.
EXPERIMENTAL: A. Preparation of reagent: 1. Preparation of 3% HCl solution: i. 30mL of concentrated HCl solution was pipetted into 1000mL volumetric flask and distilled water was used to mark the solution until the calibration mark. 2. Preparation of 0.2% NaBH4 in 0.05% NaOH i. 0.125g NaOH powder and 0.5g NaBH4 was poured in a beaker and was dissolved with distilled water. The mixture then was transferred into a 250mL volumetric flask and distilled water was used to mark up the mixture until the calibration mark. B. Preparation of Hg standards: 1. 5mL of 100ppm Hg stock solution was pipetted into a 50mL volumetric flask and 3% acetic acid solution was used to mark up the solution until the calibration mark to make a 10ppm Hg solution. C1V1= C2V2 100V1 = 10(50) V1 = 5mL 2. 5mL of 10ppm Hg solution was pipetted into a 50mL volumetric flask and 3% acetic acid solution was used to mark up the solution until the calibration mark to make a 1ppm (1000ppb) Hg solution. 3. 5mL of 1000ppb Hg solution was pipetted into a 50mL volumetric flask and 3% acetic acid solution was used to mark up the solution until the calibration mark to make a 100ppb Hg solution. 4. 100ppb Hg solution was pipetted into 6 different 50mL volumetric flasks to make the following concentration:
TABLE 1:Preparation of standard solutions Standard
Volume of Hg
concentration, ppb 100ppb, (mL) 0 0 2 1 4 2 6 3 8 4 10 5 Sample calculation: standard 1
Blank Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
C1V1= C2V2 100V1 = 2(50) V1 = 1.0mL 5. Each of the volumetric flasks was mark up with 3% acetic acid solution until the 50mL calibration mark.
C. Preparation of sample: 1. Day 1: a. The anchovies were cut to smaller size and were dried in oven at 110˚C overnight to remove water moisture in the tissue. 2. Day 2: a. 1.017g (A), 1.0885g (B) and 1.1291g (C) of the dried anchovies was weighed and was mixed with 7mL nitric acid, then was let stand overnight with cover. 3. Day 3: a. The mixture was heated until the red fume was observed. b. The mixture was let to cool at room temperature. c. 1mL of 70% H2O2 was added into the mixture and was reheating again until the mixture become concentrated. d. 5mL of the concentrated sample mixture then was diluted into 100mL volumetric flask and was mark up with 3% acetic acid solution until the calibration mark. Dilution factor calculation: 100 DF = 5 = 20
D. Operational instrument: 1. Select method, new method, element (Hg), NaBH 4, fill title. 2. Select setting, time (12s), replicate (3). 3. Select sampler, speed pump 1 (100), equation (linear through zero). 4. Select standard concentration, fill id (std 1-5 and blank), fill concentration 5. 6. 7. 8.
for standards and blank, unit (ppb or μg/L). Select method Ed, save as, name of method. Select samp info, fill sample id (1-3), file, sample info file, save as (name). Select manual, FIAS, calibration, result (on ribbon), on pump. Analyse blank, standards and finally samples.
RESULT: TABLE 2: ID
Result from instrumental analysis
Entered
Calculated
Mean signal
concentration (μg/L)
concentration
(Abs)
Blank Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
0.0 2.0 4.0 6.0 8.0 10.0
(μg/L) 0.000 2.257 4.443 6.346 7.974 9.523
0.0000 0.0198 0.0390 0.0557 0.0700 0.0836
Sample 1 Sample 2
-
4.404 4.494
0.0386 0.0394
Sample 3
-
4.398
0.0386
Graph of entered concentration vs calculated concentration 10 9 f(x) = 0.95x + 0.33 R² = 0.99 8 7
calculated concentration (μg/L)
6 5 4 3 2 1 0 0
2
4
6
8
10
12
entered concentration (μg/L)
FIGURE 1:
Graph of entered concentration versus calculated concentration
Graph of mean signal vs calculated concentration 0.09 0.08
f(x) = 0.01x - 0 R² = 1
0.07 0.06 0.05 mean signal (μg/L) 0.04 0.03 0.02 0.01 0 0
1
2
3
4
5
6
7
8
9
10
calculated concentration (μg/L)
FIGURE 2:
Graph of mean signal versus calculated concentration
Calculation for samples:
Mean =
Mean =
∑N N 4.404+ 4.494+ 4.398 3
= 4.432 Actual concentration of sample
= mean calculated concentration × dilution factor = 4.432 × 20 = 88.64ppb = 0.08864ppm
Standard deviation =
√
1 ∑ ( x i−mean)2 N −1 i
Standard deviation =
√
1 2 2 2 [( 4.404−4.432 ) + ( 4.494−4.432 ) + ( 4.398−4.432 ) ] 3−1
N
= 0.054
Relative standard devation, %RSD
=
standard deviation mean
=
0.054 4.432
× 100%
× 100%
= 1.213 Mean weight of sample (instrumental)
= 0.08864mg/L × 0.1L = 0.008864mg
Mean sample weight (actual weight)
=
1.017+ 1.0885+ 1.1291 3
= 1.0782g = 1078.2mg w/w percentage
=
0.008864 mg 1078.2mg × 100
= 8.221 × 10-4 DISCUSSION: Figure 1 shows the graph of entered concentration into the instrument versus the calculated concentration from the instrument. From this graph, it can show how the standard solutions were prepared. The value of calculated concentration of wellprepared standard solutions will be only minor error with the entered concentration. It still needs improvement in the preparation of the standard solutions. The laboratory skills like pipetting and diluting need improvement. In figure 2, graph of mean absorbance signal versus calculated concentration of the standard solution. The graph shows a straight line passing all the points showing the accurate reading. The R2 value of 1 is the additional information of a best fit for all points are on the straight line. The triplicates samples were ensure the precision by calculating the relative standard deviation. The smaller value of the relative standard deviation indicates the more precise data between the triplicates. The mean concentration of the triplicates samples is 88.64ppb. This value falls within the safe amount of mercury to be consumed by human. By referring to EPA, safe levels of mercury to be consumed are between 0.5ppm to 1.0ppm of mercury in the fish. The mercury analyser instrument is used in this experiment to analyse the mercury because it has wider dynamic range can be achieved as compared to AAS. The mercury analyser has two configurations which are employing simple atomic fluorescence and employing gold amalgamation to pre-concentrate mercury prior to measurement by atomic fluorescence. It can be concluded that, mercury can analyse mercury analyte better compared to AAS.
CONCLUSION:
The concentration of mercury in anchovies sample is 88.64ppb that is equal to 0.08864ppm and the %w/w is 8.221 × 10 -4. Since the value is in the range of safe amount, the anchovies are safe to be consumed.
REFERENCES: 1. Fish
Consumption
Advice,
Retrieved
April
14,
from
http://www.epa.gov/mercury/advisories.htm 2. What You Need to Know about Mercury in Fish and Shellfish, United States Environmental
Protection
Agency,
Retrieved
April
14,
from
http://water.epa.gov/scitech/swguidance/fishshellfish/outreach/advice_index.c m 3. Should I be concerned about mercury in fish and what fish are safe to eat?, Retrieved
April
14,
tname=george&dbid=103 4. Mercury: how much
from is
http://www.whfoods.com/genpage.php? safe?,
Retrieved
April
15,
from
https://www.greenleft.org.au/node/17431 5. K. Scoffin (2013), Mercury Analyzer in the Laboratory, Retrieved April 16, from http://ww.labcompare.com/10-Featured-Articles/133134-Mercury-Analyzer-inth-Laboratory/ 6. D. Pfeil (2012), Mercury Analysis, Which Techmique is right for you?, Retrieved
April
17,
from
http://info.teledyneleemanlabs.com/blog/bid/123607/Mercury-Analysis-WhichTechnique-is-right-for-you
