BIOANALYTICAL CHEMISTRY LABORATORY BSB2442

EXPERIMENT 2:
ELEMENT ANALYSIS

Group Morning session (Section B) : 9am – 10am

Group member 1. Nurul Syafiqah Binti Nazari
(SB20039)
2. Chen Yoke Ching
(SB20041)
Course BSB2442 Bioanalytical Chemistry
Laboratory
Section B1
Date of assignment 8 November 2021
Date of assignment submission 22 November 2021

Report Outline Person-In-Charge

Introduction Chen Yoke Ching

Methodology Chen Yoke Ching

Results Nurul Syafiqah Binti Nazari

Discussion Nurul Syafiqah Binti Nazari

Conclusion Nurul Syafiqah Binti Nazari

References Chen Yoke Ching

Appendices Nurul Syafiqah Binti Nazari

INTRODUCTION

Elemental analysis is a technique to analyse compounds, molecules, isotopes or

material in a sample. Elemental analysis can be divided into two categories: quantitative and
qualitative. Quantitative elemental analysis is to determine the amount of each of the
compounds present in the sample. Qualitative is to identify what compounds are present in
the sample. The tools for analysis of elements must be extremely sensitive to detect the small
amount of compounds present in the sample. The examples of the tools are Absorption
Atomic Spectroscopy (AAS), X-Ray Fluorescence and Inductively Coupled Plasma (ICP)
techniques such as ICP-Mass Spectrometry (ICP-MS), and ICP-Optical Emission
Spectroscopy (ICP-OES).

The tools used in this experiment is Absorption Atomic Spectroscopy and Inductively
Coupled Plasma-Mass Spectrometry. A light source emits a particular wavelength of light.
The sample was sucked up to the mixing chamber to be mixed with a volatile and flammable
gas. The sample compound then pass through a flame at slit burner and atoms or ions of the
sample will be produced. Then, the metal atoms of sample absorb the light energy from the
light emitted. When the metal atoms absorb a certain wavelength of light energy, the electron
will be promoted to excited state, the electrons move to an outer shell of electron orbital.
There are lights which absorbed by metal atoms to promote to excited state and lights which
passed through the metal atoms and are not absorbed by metal atoms. The atoms then pass
through a slit, monochromator and then to detector. The function of monochromator is to
separate the spectral lines with different wavelengths emitted by the hollow-cathode lamp.
The amount of light absorbed by the metal atoms is measured and the concentration of the
element in the sample can be calculated.
 Inductively Coupled Plasma Mass Spectrometry is a technique to detect elements with
low concentration. ICPMS is the combination of Inductively Coupled Plasma (ICP) and Mass
Spectrometer (MS). The ICP converts atoms in the sample into ions. These ions are separated
and detected by the MS. The word “plasma” is one of the physical states of matter which
consists of free electrons, positive ions and neutral atoms. The Argon gas is electrically
heated to a high temperature plasma. Then, the plasma is ionized by inductively heating the
gas with an electromagnetic coil. The sample passed through the high temperature of Argon
plasma to become ionised. Then, the sample ions enter into an electric field and are separated
according to their mass to charge ratio. A graph of mass spectrum was construct to interpret
the elements in the sample.

There are some differences between Atomic Absorption Spectrometry (AAS) and
Inductively Coupled Plasma Mass Spectrometry (ICPMS). AAS can only measure metal
atoms while ICPMS can measure almost all of the elements in Periodic Table. However,
some of the gaseous elements such as helium and hydrogen cannot be detected by ICPMS.
AAS only can measure one element in one time but ICPMS can measure multielement in one
time. To measure the concentration of certain element in sample, AAS need to compare the
concentration of sample element with a standard curve while ICPMS does not.

A standard curve of absorbance against concentration of sodium chloride solution was

plotted. The standard curve obeys Beer-Lambert law. Beer-Lambert law states that the
concentration of sample and path length is directly proportional to the absorbance of the light.
The absorbance of different concentration of sodium chloride solution measured was plotted.
The linear equation and the R2 value of the graph was obtained. The R2 value indicates how
well a linear regression model fits the data. The closer the R2 value to 1, the lesser the
variations in the absorbance obtained.
OBJECTIVES

1. To measure and analyse the amount of sodium in chicken essence, mineral water and
unknown solution.
2. To learn the techniques to use Atomic Absorption Spectroscopy (AAS) and
Inductively Coupled Plasma Mass Spectrometry (ICPMS).
3. To plot a sodium standard curve and compare the sodium amount in chicken essence,
mineral water and unknown solution.
METHODOLOGY

Apparatus:

1. Mortar and pestle

2. Filter paper and funnel
3. Volumetric flask (1L, 100mL)
4. Atomic Absorption Spectrophotometer with air-acetylene flame
5. Sodium hollow cathode lamp

Materials:

1. Distilled water
2. Mineral water
3. Chicken stock
4. Multivitamin supplement

Methods:

Part 1: AAS Analysis

1. A series of standards for sodium chloride was prepared.

2. The sodium chloride was used to prepare a concentrated stock solution. The stock
solution was diluted to prepare the analytical standards.

3. Sodium chloride stock solution, 1g/L:

100ml of sodium chloride stock solution was prepared by dissolving 0.05g of sodium
chloride in distilled water. The five following standard concentrations (25mg/L,
10mg/L, 15mg/L, 20mg/L, and 25mg/L) was prepared from the stock solution. Three
replications of each concentration were prepared to get an average reading using 100-
mL volumetric flasks.
 4. Preparation of the samples (mineral water and chicken stock) for sodium
analysis:
10 ml of mineral water was prepared.
100μL of chicken stock was diluted with distilled water until 10mL to obtain the ratio
of 1:100.

5. Setting up the atomic absorption spectrophotometer:

The operation manual was consulted for the specific instrument being used to make
sure all adjustments are properly made. It is important to ensure that the instrument is
properly adjusted to provide the optimum conditions for analysis of each element. The
instrument was ensured to equip with a sodium hollow cathode lamp. The instrument
was turned on.

6. Analysis of sodium concentration in mineral water and chicken stock solution.

Both samples were run on the AAS to measure their sodium concentration.

Part 2: ICPMS ANALYSIS

1. Preparation of the multi-vitamin supplement for ICPMS:

A third multivitamin tablet (using the same brand as in the zinc analysis) was crushed
thoroughly in a mortar and pestle. 20mL 7.9 M HNO3 was added and mixed
thoroughly, and allowed to stand for 5 minutes. The solution was quantitative
transferred to a funnel fitted with Whatman #1 filter paper and filtered into a 100-mL
volumetric flask. The extraction was repeated two or more times with 20mL dilute
HNO3 each time. The filter was rinsed with 0.16M HNO3 and brought the volume to
100mL with 0.16 M HNO3.

2. ICPMS analysis
The sample was sent to Central Laboratory for multiple element analysis (Ag, Al, As,
Ba, Be, Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Se, U, V, Zn, K)
 RESULTS

I. PREPARATION OF SODIUM STANDARD SOLUTION

Sample Concentration of sodium Volume of sodium Final concentration of Final volume of

stock solution (mg/L) stock solution (mL) sodium solution (mg/L) solution after (mL)
Blank 1000 0.00 0.00 100
1 1000 0.50 5.00 100
2 1000 1.00 10.00 100
3 1000 1.50 15.00 100
4 1000 2.00 20.00 100
5 1000 2.50 25.00 100
Table 1: Dilution of the sodium standard solution

By using 𝑀1 𝑉1=𝑀2 𝑉2 formula,

Where:

𝑀1 = Concentration of sodium stock solution (mg/L)

𝑉1 = Volume of sodium stock solution (mL)

𝑀2 = Final concentration of sodium solution (mg/L)

𝑉2= Final volume of solution after (mL),

The volume of sodium stock solution needed to prepare calculated.

Preparation of sample 1

𝑀1 𝑉1=𝑀2 𝑉2

1000 x 𝑉1= 5.00 x 100

𝑉1 = 0.50 mL

Volume of sodium stock solution needed is 0.50 mL

Preparation of sample 2

𝑀1 𝑉1=𝑀2 𝑉2

1000 x 𝑉1= 10.00 x 100

𝑉1 = 1.00 mL

Volume of sodium stock solution needed is 1.00 mL

Preparation of sample 3

𝑀1 𝑉1=𝑀2 𝑉2

1000 x 𝑉1= 15.00 x 100

𝑉1 = 1.50 mL

Volume of sodium stock solution needed is 1.50 mL

Preparation of sample 4

𝑀1 𝑉1=𝑀2 𝑉2

1000 x 𝑉1= 20.00 x 100

𝑉1 = 2.00 mL

Volume of sodium stock solution needed is 2.00 mL

Preparation of sample 5

𝑀1 𝑉1=𝑀2 𝑉2

1000 x 𝑉1= 25.00 x 100

𝑉1 = 2.50 mL

Volume of sodium stock solution needed is 2.50 mL

II. AAS analysis of sodium solution standard concentration

No. of Standard concentration (mg/L) Mean of absorbance (A)

calibration
standard
Blank 0.00 0.660
1 5.00 0.808
2 10.00 1.229
3 15.00 1.469
4 20.00 1.641
5 25.00 0.749
Table 2: Absorbance of sodium standard solution by AAS analysis
Only the absorbance reading against the concentration of sodium solution from calibration
standard 0 to 4 is shown in the graph below. The data of calibration standard 5 is not included
in the calibration curve due to an error.

Absorbance against Standard Concentration of Sodium Solution

1.8
1.641
1.6 1.469

1.4 y = 0.0525x + 0.6368

1.229 R² = 0.9752
1.2
Absorbance (A)

1
0.808
0.8 0.66

0.6

0.4

0.2

0
0 5 10 15 20 25
Standard Concentration of Sodium Solution (mg/L)

Graph 1: Absorbance against standard concentration of sodium solution

III. The analysis of sodium concentration in mineral water and chicken stock
solution.

Sample Mean of absorbance (A)

Mineral water 0.977
Chicken stock 1.085
Table 3: Absorbance of mineral water and chicken stock obtained by using AAS
analysis.
The sodium concentration of mineral water and chicken stock can be estimated using a linear
equation derived from the standard curve shown in graph 2.

𝑦 = 0.0525𝑥 + 0.6368

Concentration of mineral water

𝑦 = 0.0525𝑥 + 0.6368

0.977 = 0.0525𝑥 + 0.6368

0.977 − 0.6368
𝑥=
0.0525

𝑥 = 6.48 mg/L

Concentration of chicken stock

𝑦 = 0.0525𝑥 + 0.6368

1.085 = 0.0525𝑥 + 0.6368

1.085 − 0.6368
𝑥=
0.0525

𝑥 = 8.537 mg/L
IV. ICPMS Analysis

No. Parameter Concentration Unit

1 Beryllium (Be) 0 ppb
2 Vanadium (V) 0 ppb
3 Chromium (Cr) 0 ppb
4 Cobalt (Co) 0 ppb
5 Nickel (Ni) 0 ppb
6 Copper (Cu) 0 ppb
7 Arsenic (As) 0 ppb
8 Selenium (Se) 0 ppb
9 Silver (Ag) 0 ppb
10 Cadmium (Cd) 0 ppb
11 Antimony (Sb) 0 ppb
12 Barium (Ba) 0 ppb
13 Lead (Pb) 0 ppb
14 Molybdenum (Mo) 32.41 ppm
15 Magnesium (Mg) 371.90 ppm
16 Manganese (Mn) 1416.31 ppm
17 Iron (Fe) 1657.20 ppm
18 Zinc (Zn) 2571.10 ppm
19 Aluminium (Al) 2944.76 ppm
20 Potassium (K) 3597.20 ppm
21 Sodium (Na) 5819.90 ppm
22 Calcium (Ca) 6872.60 ppm
Table 4: ICP-MS Analysis of multivitamin arranged from low concentration to high
concentration
DISCUSSION

To determine the mean of absorbance of sodium in different concentrations, they were

measured by using AAS analysis. Atomic absorption spectrometry (AAS) uses certain
wavelengths of electromagnetic radiation from a light source to detect components in liquid
or solid samples. Individual elements absorb wavelengths in different ways, and their
absorbances are compared to standard values. We can determine the concentration of many
separate elements in a sample using an atomic absorption spectrometer. Sodium is usually
measured at one of two wavelengths: 589.0 nm or 589.6 nm, which are the most sensitive.
The sodium standard solutions in the unit of mg/L were prepared start with 0, 5, 10, 15, 20,
and 25. All subsequent sample readings are corrected using the “blank” mean absorbance
value to reflect the actual analyte absorbance. The “blank” would have 0 absorbance if there
was no analyte contamination. Contamination can occur in practice, needing the modification
of absorbance values in tests.

According to table 2, the concentration for "blank" is 0 mg/L, and the mean absorbance
is meant to be 0 A, however the AAS analysis revealed a reading of 0.660 A. It proved there
was contamination that occurred. Furthermore, as the standard sodium concentrations
increases, the mean absorbance increases. However, when standard concentration was 25
mg/L the reading of mean absorbance was 0.749 A. It should be increased, or more precisely,
the value must exceed the earlier value of 1.641 A, rather than decreasing below it. The error
may have occurred because the capillary tube was not thoroughly cleaned between each
reading. That is a reasonably common mistake that occurs frequently.

In Graph 1, the absorbance reading is plotted against the concentration of sodium

solution from calibration standard 0 to 4. Due to an error that occurred, the mean of
absorbance for sodium solution concentration from calibration standard 5 is not included. It
has been observed that the absorption rate will increase correspondingly as the sodium
solution increase intensity. A straight line was drawn between the points obtained by plotting
the observed absorption at each concentration. Small measurement errors are one reason why
data may not fit squarely on a line. The calibration curve's line equation was y = 0.0525x +
0.6368, where y shows the average absorbance readings, 0.0525 indicates the graph's
gradient, x measures the concentration of sodium standard solution, and 0.6368 indicates the
graph's y-intercept. The calibration curve has an R² value of 0.9752. R-squared is a statistical
measure of how closest the data are to the regression line that has been fitted. The greater the
R-squared, the better the model matches the data. The R-squared value practically reaches 1
as a result of the analysis. This demonstrates a positive linear relationship between the mean
of absorbance and the concentration of the sodium standard solution, because as the
concentrations increase, so does the absorbance.

The value of the mean of absorbance of chicken stock is higher than mineral water,
according to Table 3, which presents the absorbance of mineral water and chicken stock
generated by AAS analysis. The line regression y = 0.0525x + 0.6368 was used to determine
the concentration of sodium in both mineral water and chicken stock. The mean absorbance
values, 0.977 for mineral water and 1.085 for chicken stock, were substituted into y. As the
result the sodium concentration in chicken stock is higher than in mineral water which is
8.537 mg/L and 6.48 mg/L sodium concentration was found in the mineral water.

ICP-MS (inductively coupled plasma mass spectrometry) is a form of mass

spectrometry in which the sample is ionized using an inductively coupled plasma. It atomizes
the material, resulting in atomic and tiny polyatomic ions that are identified. It is well-known
for its ability to identify metals and non-metals in liquid samples at very low concentrations.
It can distinguish between different isotopes of the same element, making it a useful isotopic
labelling technique. Beryllium (Be), Vanadium (V), Chromium (Cr), Cobalt (Co), Nickel
(Ni), Copper (Cu), Arsenic (As), Selenium (Se), Silver (Ag), Cadmium (Cd), Antimony (Sb),
Barium (Ba), and Lead (Pb) in ppb are among the elements that cannot be detected because
the value obtained is less than 0.5, according to Table 4. The table's elements were organized
in order of lowest to highest concentration.
 There are some precautions to be taken during this experiment to ensure the accurate
results was obtained. Cleaning the capillary tube with distilled water is suggested when using
AAS analysis, as is cleaning the capillary tube with a thin wire. If there are solids in the flask,
the capillary tube should not be placed on the bottom of the flask during the readings.
Furthermore, we must change the pipette tips every time we want to use it for a different
element or solution in order to avoid any stuck-up elements in the tips ruining the readings.

CONCLUSION

AAS analysis was used to determine the amounts of sodium standard solution at
various concentrations. The standard solution's concentrations were 5, 10, 15, 20, and 25
mg/L, respectively. Except for reference solution 5, which had an error, when the
concentration increased, the mean of absorbance increased as well. However, the AAS
analysis experiment's overall results are as expected. In comparison to mineral water, chicken
stock contains greater sodium. Chicken stock has an 8.537 mg/L concentration, while mineral
water has a concentration of 6.48 mg/L. Substituting the mean of absorbance for both mineral
water and chicken stock into y in the line equation y = 0.0525x+0.6368 yielded the
concentrations. The R² value of the calibration curve is 0.9752. R-squared is a statistic that
indicates how near the data are to the fitted regression line. In addition, 22 elements were
observed for ICP-MS analysis to assess their concentration. Table 4 shows that 13 elements
were unable to identify their concentrations since the value was less than 0.5, whereas 9
others were successful. The elements were ordered in order of concentration, starting with the
lowest and working up to the highest. Atomic Absorption Spectroscopy (AAS) and
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) techniques were studied. The skill
and method of the user are crucial to the accuracy of AAS and ICP-MS. To avoid inaccurate
calibration curves and contamination, extreme caution must be exercised when preparing
standards and samples.
REFERENCES

1. Raja, P. M. V., & Barron, A. R. (2021, March 22). Introduction to Elemental

Analysis. Rice University. https://chem.libretexts.org/@go/page/55810
2. García, R., & Báez, A. P. (2012). Atomic absorption spectrometry (AAS). Atomic
absorption spectroscopy, 1, 1-13.
3. Thomas, R. (2008). Practical guide to ICP-MS: a tutorial for beginners. CRC press.
4. Vanhaecke, F., & Degryse, P. (Eds.). (2012). Isotopic analysis: fundamentals and
applications using ICP-MS. John Wiley & Sons.

5. Shah, K. (2020, July 16). Atomic Absorption spectroscopy Principle, Instrumentation,

Application & MCQ for GPAT, GATE, NET JRF. Gpatindia: Pharmacy Jobs,
Admissions, Scholarships, Conference,Grants, Exam Alerts.
https://gpatindia.com/atomic-absorption-spectroscopy/
6. Wilschefski, S. C., & Baxter, M. R. (2019). Inductively Coupled Plasma Mass
Spectrometry: Introduction to Analytical Aspects. The Clinical biochemist.
Reviews, 40(3), 115–133. https://doi.org/10.33176/AACB-19-00024
7. Peter Boer, Branko Braam, René Fransen, Walther H. Boer, Hein A. Koomans,
Reliable atomic absorption analysis of sodium and potassium in rat renal tubular fluid,
Kidney International, Volume 45, Issue 4, 1994, Pages 1211-1214, ISSN 0085-2538,
https://doi.org/10.1038/ki.1994.160.
(https://www.sciencedirect.com/science/article/pii/S008525381558441X)
8. Guerrero, Jose. (2020). Re: In AAS (atomic absorption), I'm not getting the consistent
value of the concentration of metal ion for the same sample. Can anybody tell why it
is so?. Retrieved from: https://www.researchgate.net/post/In-AAS-atomic-absorption-
Im-not-getting-the-consistent-value-of-the-concentration-of-metal-ion-for-the-same-
sample-Can-anybody-tell-why-it-is-
so/5e20ccde2ba3a10324638100/citation/download.

9. Choueiry, G. (n.d.). Relationship Between r and R-squared in Linear Regression.

Retrieved from QuantifyingHealth: https://quantifyinghealth.com/relationship-
between-r-and-r-squared/

10. Wikipedia. (2021, October 6). Inductively coupled plasma mass spectrometry.
Retrieved from Wikipedia:
https://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry
 APPENDICES

The picture above shows the sample of sodium standard solution from blank to 25 mg/L
and the multi-vitamin sample.

The picture above shows samples for both mineral water that labelled as G and chicken stock as F.