DETERMINATION OF

ESSENTIAL
MACRONUTRIENTS IN
SOILS, EXTRACTED WITH
THE MEHLICH 3
EXTRACTION SOLUTION
AND MEASURED BY
SPECTROPHOTOMETRIC
METHODS.

Prepared by:
Ricardo Martinez

Chemical
Engineering
Technology
Student
Cambrian
College
 DETERMINATION OF ESSENTIAL MACRONUTRIENTS IN SOILS,
EXTRACTED WITH THE MEHLICH 3 EXTRACTION SOLUTION AND
MEASURED BY SPECTROPHOTOMETRIC METHODS.

Prepared for:

Mr. Hadi Fergani

CHLP Coordinator/Professor
Cambrian College

Prepared by:

Ricardo Martinez
Chemical Engineering Technology Student
Cambrian College

December 8th, 2022

i
 Summary

The knowledge of the concentrations of essential macronutrients in soils is a critical information

for farmers. For this reason, the Sudbury Shared Harvest organization was very interested on
measuring Nitrogen, Phosphorus and Potassium in a land that they have received as a
donation. By doing this they will know better the properties of this land and will be able make the
optimize the crop production in there as this is an organization whose mission is to support
people in gaining the knowledge, skills, and resources needed to access or grow their food.

After sampling in the land and further research, it was determined that the best approach to
measure phosphorus and potassium in soils was by extracting them first with the use of the
Mehlich 3 extraction solution and then use the UV-Vis Spectrometer to measure phosphorus by
a colorimetric method and to use the Flame Atomic Absorption Spectrometer to measure
potassium. At the beginning it was intended to use the HACH TNT 836 method to determine
Nitrogen in the soil’s samples but after further research it was determined that this method is not
suitable for that purpose. In order to guarantee the reliability of results, it was decided to
evaluate linearity, precision and trueness to determine accuracy. However, because the certified
reference material was not suitable for this purpose it was not possible to determine trueness.

Some recommendations are made in this report if the investigation is going to be continued on
how to solve the problem with the reference material and one possible method to determine
Nitrogen in soils.

ii
 Acknowledgments

I want to thank God, my family, my friends, and my teachers for helping me to complete
this report that at the same time means the end of my studies as a Chemical
Engineering Technologist. Also want to give a special acknowledgement to the
Research and Development department at Cambrian College for letting me use their
facilities and equipment to carry out the analysis necessary to complete this report.

iii
 TABLE OF CONTENTS

1. INTRODUCTION ................................................................................................................ 1
1.1. PURPOSE ................................................................................................................... 1
1.2. BACKGROUND ........................................................................................................... 1
1.3. LIMITATIONS .............................................................................................................. 1
1.4. SCOPE ........................................................................................................................ 1
2. SOILS AND THEIR MACRONUTRIENTS ........................................................................... 2
2.1. SOIL ............................................................................................................................ 2
2.2. MACRONUTRIENTS AND MICRONUTRIENTS IN SOILS .......................................... 2
2.3. NITROGEN, PHOSPHORUS, AND POTASSIUM IN SOILS ........................................ 3
2.3.1. NITROGEN (N) IN SOILS ..................................................................................... 3
2.3.2. PHOSPHORUS (P) IN SOILS ............................................................................... 4
2.3.3. POTASSIUM IN SOILS ......................................................................................... 5
3. CHEMICAL ANALYSIS OF SOILS ...................................................................................... 6
3.1. SOLUTION EXTRACTION FOR N, P AND K............................................................... 7
3.2. METHODS FOR DETERMINATION OF N, P, AND K IN SOILS .................................. 7
3.2.1. PHOSPHORUS ANALYSIS .................................................................................. 7
3.2.2. POTASSIUM ANALYSIS ...................................................................................... 8
3.2.3. NITROGEN ANALYSIS ........................................................................................ 8
4. SPECTROPHOTOMETRIC METHODS USED IN SOIL ANALYSIS ................................... 9
4.1. UV-Vis SPECTROSCOPY ........................................................................................... 9
4.2. FLAME ATOMIC ABSORPTION SPECTROSCOPY ..................................................11
4.2.1. AA SPECTROMETER SAMPLE INTRODUCTION SYSTEM ..............................13
4.2.2. THE FLAME AA SPECTROMETER BURNER.....................................................13
4.2.3. THE LIGHT SOURCE (HOLLOW CATHODE LAMP) ..........................................14
4.2.4. THE MONOCHROMATOR ..................................................................................15
4.2.5. THE DETECTOR .................................................................................................15
4.2.6. COMPUTERIZED INSTRUMENT CONTROL ......................................................16
5. SAMPLING OF SOIL .........................................................................................................16
5.1. PROCEDURE FOR SAMPLING .................................................................................16
5.2. DESCRIPTION OF LOCATION WHERE SAMPLE WAS TAKEN ...............................17
6. PERFORMANCE EVALUATION OF A METHOD OF ANALYSIS ......................................17
6.1. LINEARITY .................................................................................................................18

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 6.2. EVALUATION OF THE ACCURACY OF THE METHOD ............................................19
6.3. OUTLIERS ..................................................................................................................19
6.4. EVALUATION OF PRECISION ...................................................................................19
6.5. EVALUATION OF TRUENESS ...................................................................................20
7. METHODOLOGY...............................................................................................................21
7.1. SAMPLE PREPARATION ...........................................................................................21
7.2. PREPARATION OF MEHLICH 3 SOLUTION .............................................................22
7.3. EXTRACTION.............................................................................................................23
7.4. PHOSPHORUS ANALYSIS ........................................................................................24
7.5. POTASSIUM ANALYSIS ............................................................................................26
7.6. NITROGEN ANALYSIS ..............................................................................................26
8. DATA AND CALCULATIONS.............................................................................................27
8.1. PHOSPHORUS ANALYSIS ........................................................................................27
8.2. POTASSIUM ANALYSIS ............................................................................................36
9. DISCUSSION ....................................................................................................................44
10. CONCLUSION ...............................................................................................................47
11. RECOMMENDATIONS ..................................................................................................48
12. REFERENCES ...............................................................................................................49

v
 TABLE OF FIGURES

Figure 1:The soil P cycle (from Pierzynski et al., 2000) ....................................................................... 5

Figure 2:Generalized soil cycle of K in the soil. (Potassium for Crop Production, n.d.) .................. 6
Figure 3:The electromagnetic spectrum, with the visible light section expanded ............................ 9
Figure 4:A schematic diagram of a simple spectrophotometer. (Zumdahl, Zumdahl and
DeCoste, 2017, p. A18) ........................................................................................................................... 10
Figure 5:An electron is in an excited state when it temporarily occupies an energy state higher
than its ground state. If an electron receives additional energy, such as when it absorbs a
photon, or packet of light, it might become excited. (Background: Atoms and Light Energy, n.d.)
.................................................................................................................................................................... 12
Figure 6:Components inside an AA Spectrometer (Atomic Absorption Spectroscopy, How Does
AAS Work, AAS FAQs | Agilent, n.d.-b) ............................................................................................... 12
Figure 7:Flame AA sample introduction system. ................................................................................. 13
Figure 8: Light from the hollow cathode lamp light source is passed along the burner and
through the flame created by the burner slit. ....................................................................................... 14
Figure 9: Schematic of a single element hollow cathode lamp used for AA spectrometer analysis
.................................................................................................................................................................... 15
Figure 10: Diagram of the photomultiplier tube ................................................................................... 15
Figure 11: Core sampler (A&L Canada Laboratories Inc., n.d.) ....................................................... 16
Figure 12: Location of the site where samples were taken (Image retrieved from Google maps
on November 16th 2022) ........................................................................................................................ 17
Figure 13:Wanup sample being air-dried in the lab ............................................................................ 21
After what has been described, it was necessary to sieve the samples using a 2mm sieve
(Figure 14). This is the size recommended for all samples when the Mehlich 3 extraction
solution will be used. ................................................................................................................................ 22
Figure 14: 2 mm sieve used for soil sample preparation. .................................................................. 22
Figure 15: Soil samples in the reciprocating shaker ........................................................................... 23
Figure 16: Samples being filtered after the extraction with Mehlich 3 solution ............................... 24
Figure 17: Standard calibration curve solutions ready to read in the UV-Vis spectrometer. ........ 25
Figure 18: Calibration curve of phosphorus. ........................................................................................ 27
Figure 19: Probability plot of the absorbances obtained from measuring ten times the
phosphorus concentration in the soil sample from Wanup. ............................................................... 29

vi
Figure 20: Outlier plot obtained from measuring ten times the phosphorus content in the soil
sample from Wanup. ................................................................................................................................ 30
Figure 21: Probability plot of the readings obtained from repetition 1 of phosphorus test in the
reference material .................................................................................................................................... 33
Figure 22: Probability plot of the readings obtained from repetition 2 of phosphorus test in the
reference material .................................................................................................................................... 33
Figure 23: Probability plot of the readings obtained from repetition 3 of phosphorus test in the
reference material .................................................................................................................................... 34
Figure 24: Outlier plot obtained from the readings of repetition 1 of phosphorus test in the
reference material. ................................................................................................................................... 34
Figure 25: Outlier plot obtained from the readings of repetition 2 of phosphorus test in the
reference material. ................................................................................................................................... 35
Figure 26: Outlier plot obtained from the readings of repetition 3 of phosphorus test in the
reference material. ................................................................................................................................... 35
Figure 27: Calibration curve of potassium ............................................................................................ 37
Figure 28: Probability plot of the absorbances obtained from measuring ten times the potassium
concentration in the soil sample from Wanup. ..................................................................................... 39
Figure 29: Outlier plot obtained from measuring ten times the potassium content in the soil
sample from Wanup. ................................................................................................................................ 40

TABLE OF TABLES

Table 1: pH of each portion that was air dried from the Wanup sample ......................................... 22
Table 2: Amount of reagents used to prepare 2.0 liters of Mehlich 3 extraction solution ............. 23
Table 3:Modified volumes used to prepare standard and samples to read phosphorus by the
UV-Vis spectrometry method. ................................................................................................................ 25
Table 4: Absorbances read from standard solution of phosphorus. ................................................. 27
Table 5: Absorbance readings from the ten repetitions of the phosphorus measuring of the
Wanup sample. ......................................................................................................................................... 28
Table 6: Absolute differences between repetitions of the ten measurements phosphorus content
in the soil sample from Wanup. .............................................................................................................. 31
Table 7: Concentrations of phosphorus in the ten repetitions of phosphorus test using the
Wanup Sample. ........................................................................................................................................ 31
Table 8: Absorbances read in triplicate from three repetitions of the three measurements of
phosphorus of the reference material. .................................................................................................. 32
Table 9: Concertation of phosphorus in every repetition carried out with the certified
measurement material. ............................................................................................................................ 36

vii
Table 10: Absorbances obtained in the Flame AA with different standard concentrations .......... 37
Table 11: Absorbance readings from the ten repetitions of the potassium measuring of the
Wanup sample. ......................................................................................................................................... 38
Table 12: Absolute differences between repetitions of the ten measurements potassium
concentrations in the soil sample from Wanup. ................................................................................... 41
Table 13: Concentrations of potassium in the ten repetitions using the Wanup sample. ............. 41
Table 14: Absorbances from the potassium test read in duplicate in the three different samples
taken from the certified reference material........................................................................................... 42
Table 15: Evaluation of precision for measurements of absorbances in the potassium test for the
certified reference material. .................................................................................................................... 43
Table 16: Calculation of potassium concentration in Wanup sample .............................................. 43
Table 17: Functions of different reagents of Mehlich 3 extraction solution. .................................... 44
Table 18: Function of every reagent used in the determination of available phosphorus by the
colorimetric method.................................................................................................................................. 45

TABLE OF APPENDIXES

Appendix 1:Mehlich 3-Extractable Elements ............................................................................ A1

Appendix 2: Hach Company TNTplus 835/836 Nitrate Method 10206...................................... A2
Appendix 3: Certificate of analysis of the reference material .................................................... A3
Appendix 4: Results from Flame AA potassium test (calibration curve, sample and reference
material) ................................................................................................................................... A4

viii
 1. INTRODUCTION

1.1. PURPOSE
This report describes how to sample soils and measure Nitrogen, Phosphorus and Potassium
by methods of UV-Vis Spectrophotometry, Flame Atomic Absorption Spectrophotometry and a
commercial HACH method. Moreover it explains how to evaluate some important performance
parameters for each parameter.

1.2. BACKGROUND
This project was proposed by the Sudbury Shared Harvest organization, which supports people
in gaining the knowledge, skills, and resources needed to access or grow their food. They have
recently received the donation of a piece of land in the township of Wanup, Ontario, and they
intend to use it to cultivate different crops. For this reason, they want to know as many
properties as possible from this land. The project presented in this report is not the only one
related to Sudbury Shared Harvest and the Wanup piece of land as they want to measure as
many parameters as possible at that place to have the best idea possible on how to make the
most of that land.

1.3. LIMITATIONS
The original idea for this report was to measure nitrogen by a commercial HACH
Spectrophotometric method, phosphorus by a colorimetric method (using an UV-Vis
spectrophotometer) and Potassium by Flame Atomic Absorption Spectrophotometry. However,
at the end it was only possible to measure phosphorus and potassium because after further
research it was possible to conclude that the HACH Spectrophotometric method was not
suitable for the measurement of nitrogen. Moreover, another limitation was that even though to
evaluate the appropriate performance of a new method it is necessary to validate the method,
because of time the only performance parameters analyzed in this report were linearity,
precision and trueness.

1.4. SCOPE
This report covers theory about soils and the main macronutrients present in soils (Nitrogen,
Phosphorus and Potassium) and how to extract them from the soil. Moreover, after these
important elements have been extracted, this report explains how to measure them by different
Spectrophotometric methods and evaluate the linearity, precision and trueness of these
methods.

1
 2. SOILS AND THEIR MACRONUTRIENTS

2.1. SOIL
Soil is defined as the loose surface layer that covers the majority of the land. It consists of
inorganic matter and organic particles. In addition to being a water source and nutrients, soil
gives agricultural plants the structural support they need to grow. (Department of Jobs,
Precincts and Regions, 2022)

Soils vary greatly in their chemical and physical properties. The interaction of many processes,
including leaching, weathering, and microbiological activity, creates diverse soil types. Each
variety has unique advantages and disadvantages in terms of agricultural production.
(Department of Jobs, Precincts and Regions, 2022)

According to the United Nations “Soils are essential for achieving food security and nutrition and
have the potential to help mitigate the negative impacts of climate change.” (UN Official, 2018)

2.2. MACRONUTRIENTS AND MICRONUTRIENTS IN SOILS

In addition to carbon dioxide (CO2) and water (H2O), plants cannot grow without some essential
nutrients found in soil, without them plants are not able to produce biomass. Fourteen essential
plant nutrients are known and they are classified in two categories (Macronutrients and
Micronutrients) depending on the amounts of nutrients that plants require. The amount of
macronutrients taken up by plants per hectare ranges from 10 to 100 kg, whereas the amount of
micronutrients taken up by plants per hectare is often only a few grams. Macronutrients are
nitrogen (N), phosphorus (P), Calcium (Ca), potassium (K), magnesium (Mg) and sulfur (S).
Micronutrients are Manganese (Mn), Boron (B), Iron (Fe), Copper (Cu), Zinc (Zn), Nickel (Ni),
Molybdenum (Mo), and Chlorine (Cl). (NPK: What Is It and Why Is It so Important?, 2020)

Based on the amount required by crops, it is considered that the most essential three
macronutrients are Nitrogen (N), Phosphorus (P), and Potassium (K), often referred as NPK. In
the case of N and P, they primarily serve as components of proteins and nucleic acids, which
are significant parts of plant tissue. Potassium is considered a key element because it is
important in regulating processes in the plant, such as osmosis and enzyme activities. (NPK:
What Is It and Why Is It so Important?, 2020)

2
 2.3. NITROGEN, PHOSPHORUS, AND POTASSIUM IN SOILS
2.3.1. NITROGEN (N) IN SOILS

Nitrogen is a crucial element in plant growth. It is found in all plant cells, proteins, hormones, and
chlorophyll.
There are two primary sources of nitrogen reserves for plants, the first is atmospheric air, which
has 78% nitrogen in molecular state (N2). The other significant reserve of nitrogen is the soil's
organic matter since it is known more than 90% of soil nitrogen is forming organic compounds.
However, these two forms are unavailable for plants as molecular nitrogen is inert and Plants do
not absorb organic nitrogen. When soil organisms break down organic materials and convert it to
inorganic nitrogen, organic nitrogen becomes available. (Admin, 2020).

All types of nitrogen undergo chemical alterations in the soil before becoming plant-available
nitrogen. The three primary methods of nutrient transformation in soil are

 Mineralization: Soil microbes transform organic nitrogen forms in plant residues or

organic soil amendments into a plant-available form of nitrogen – ammonium.
 Immobilization: This is the opposite of mineralization. As organisms break down plant
matter and other organic materials, they use the nitrogen that is already present in the soil
and turn it into organic nitrogen molecules. Immobilization momentarily reduces plant-
available nitrogen, but it returns to an available form after organisms further break down
these organic molecules.
 Nitrification: Ammonium is quickly converted to nitrate by soil bacteria in warm, moist
soils so that plant roots can absorb it more easily. (Admin, 2020).

Once transformed into the inorganic nitrogen, it is predominantly in the form of nitrate (NO3) and
ammonium (NH4). While laboratories investigating tree nursery and forest soils frequently find both
NO3 and NH4, soil testing laboratories typically measure NO3 to estimate available N in
agricultural soils. (Carter & Gregorich, 2007)

3
 2.3.2. PHOSPHORUS (P) IN SOILS

Phosphorus is an essential nutrient for plants since it is used as a catalyst in many important
biochemical reactions as well as a component of several important plant structural compounds.
Phosphorus is also very well-known particularly for its function in collecting and transforming solar
energy into beneficial plant chemicals. (Plant Nutrients in the Soil, n.d.)

Many factors influence the content of soil phosphorus:

 Type of parent material from which the soil is derived

 Degree of weathering and erosion
 Climatic conditions
 Crop removal and fertilization

There are two types of phosphorus in soil: organic and inorganic. The total phosphorus in the soil is
composed of these two types. The amount of organic phosphorus varies between 30 and 65
percent, unavailable to plants, and 35 to 70 percent is in inorganic forms. Organic phosphorus can
be found in soil microorganisms and remains from dead plants and animals. These organic forms
of phosphorus are processed and converted by soil microbes into forms that are useful to plants.
(Alabama Cooperative Extension System, 2021)

The ionic inorganic forms of phosphorus that are made available in the soil solution are
absorbed by plants. This comprises HPO42- and H2PO4-, which are two easily available forms.
These Phosphorus ion compounds are usually called orthophosphates and are the primary
forms of phosphorus taken up by plants, denominated available phosphorus. (Soil Management,
n.d.)
Generally, the majority of the orthophosphate is present as either HPO42-. (pH >7.2) or H2PO4–
(pH 4.0–7.2) within the normal pH range (4.0–9.0) of most soils. (Pierzynski et al., 2015)

Phosphorus cycling in soils is a complex phenomenon strongly influenced by the nature of the
inorganic and organic solid phases. Other factors are also the forms and extent of biological
activity occurring, chemistry of the soil solution (e.g., pH, ionic strength, redox potential), and
many environmental factors such as soil moisture content and temperature (Figure 1)
(Pierzynski et al., 2015).

4
 Figure 1:The soil P cycle (from Pierzynski et al., 2000)

2.3.3. POTASSIUM IN SOILS

An important component for plant growth and a component of food crops is potassium (K).
Potassium, improves fruits and vegetables and helps plants use water and resist droughts. If
soluble potassium is deficient in soil it can stunt growth and cause other symptomatic issues.
(How Does Potassium Help Plants Grow?, 2019)

Frequently, soils have a total K content of more than 20,000 ppm. (parts per million). Although
the overall K supply in soils is rather large, only relatively small amounts are available for plant
growth. That’s because nearly all of this K is in the structural component of soil minerals and
isn’t available for plant growth. (Potassium for Crop Production, n.d.)

In the soil, there is an equilibrium between the three types of K (unavailable, slowly available or
fixed, and easily available or exchangeable). Figure 2 illustrates the general relationship among
these forms. (Potassium for Crop Production, n.d.)

5
 Figure 2:Generalized soil cycle of K in the soil. (Potassium for Crop Production, n.d.)

Potassium that is considered readily available for plant growth is described as:
 Dissolved in soil water (water soluble).
 Held on clay particles’ exchange sites, which are found on the surface of clay particles.
This form of K is called exchangeable K, measured by the routine soil testing procedure.
(Potassium for Crop Production, n.d.)

3. CHEMICAL ANALYSIS OF SOILS

Chemical analyses of soils are generally carried out to know or estimate available or extractable
forms of nutrients contained in the soil. It is important to mention that the amount of nutrient that
is extracted of the soil, indicates a proportion of the total nutrient present in the soil. Moreover,
the amount of nutrient that is extracted is not equal to the amount of nutrients that the soil
absorbs; however, it is closely related to it. The term “available” refers to the amount of nutrient
that is in rapid equilibrium with the soil solution (Correndo & García, 2017).

The chemical extracting solutions simulate the action of the root; that is, they extract in solution,
an amount of nutrients similar to what the plant would normally take in its growth cycle
(Correndo & García, 2017). It is important to mention that the extracting solutions, give values of

6
availability of nutrients. Furthermore, there is no a universal extractant solution; but each
extractant is suitable depending on factors like the elements or compounds to extract and the
technique used to analyze the sample after extraction is complete.

3.1. SOLUTION EXTRACTION FOR N, P AND K.

Over the past few years, numerous approaches and procedures have been developed to
measure soil nutrient availability. Among these methods, Mehlich 3 (M3), is considered an
appropriate and affordable chemical procedure because it works with various soil types and can
operate as a "universal" soil test extractant. (Sims 1989; Zbiral 2000a; Bolland et al. 2003).
Mehlich 3 solution was developed by Mehlich (1984) as multielement soil extraction and is
widely used, especially in agronomic studies. It is used to evaluate soil nutrient status and
establish fertilizer recommendations mainly for P and K in humid regions. The following
elements can be analyzed using M3 extracting solution: P, K, Ca, Mg, Na, Cu, Zn, Mn, B, Al,
and Fe. The extracting solution is composed of 0:2 M CH3COOH, 0:25 M NH4NO3, 0:015 M
NH4F, 0:013 M HNO3, and 0.001 M ethylene diamine tetraacetic acid (EDTA).

It is important to mention that nitrate is water soluble and that several solutions, including water,
have been employed as extractants. Exchangeable NH4 is NH4 that may be extracted using a
neutral K salt solution at room temperature. Various molarities have been used, such as
0:05 M K2SO4, 0:1 M KCl, 1:0 M KCl, and 2.0 M KCl (Keeney and Nelson 1982). The
most common extractant for NO3 and NH4 is 2.0M KCl (e.g., Magill and Aber
2000; Shahandeh et al. 2005).

3.2. METHODS FOR DETERMINATION OF N, P, AND K IN SOILS

3.2.1. PHOSPHORUS ANALYSIS

As mentioned before, Mehlich 3 is widely used to evaluate available phosphorus. Once the
available phosphorus has been extracted with this solution, a colorimetric method can be used
to measure the phosphorus concentration in the sample. Even though this is not the only
method to evaluate phosphorus, once the extraction has been performed with Mehlich 3
solution, some laboratories still hesitate to use more advanced techniques like ICP. This is
mainly because colorimetric phosphorus determination methods were initially used to calibrate
phosphorus fertilizers recommendations (Zbíral & Němec, 2000).

One official methodology for a colorimetric technique to measure phosphorus after using the
Mehlich 3 extraction solution can be found at the book Soil Sampling And Methods of Analysis

7
pages 83-85. This is included in Appendix 1 as part of this report and for future consultations.
The method explained is based on the intensity of a blue color that develops after adding
appropriate chemicals to the soil extract.

3.2.2. POTASSIUM ANALYSIS

Potassium is among the possible elements that can be extracted from soil by using the Mehlich
3 solution. Once in solution, it is possible to use techniques like ICP, Flame emission and Flame
atomic absorption spectroscopy. For the purposes of this investigation and the availability of
equipment in the Applied Research Lab, at Cambrian College, the technique used will be the
Flame atomic absorption spectroscopy. The preparation of the sample to be analyzed by this
technique is described at the book Soil Sampling And Methods of Analysis pages 83-85. This is
included in Appendix 1 as part of this report and for future consultations.

3.2.3. NITROGEN ANALYSIS

The nitrogen analysis is performed by the use of the kit for analysis “Hach Company TNT plus
Nitrate Reagent, Cat. No. TNT836” to run this kit it is necessary to have one of the following
options of equipment available: Hach Company DR 5000, DR 3800, or DR 2800
spectrophotometer.

According to the technical information of this method (Appendix 2), it is equivalent or better in
performance to SM 4500-NO3- E (Cadmium Reduction Method) for the purposes of regulatory
reporting of nitrate and nitrate-nitrite. This is why it has been decided to use this Hach kit to test
nitrates in soils because the Cadmium Reduction Method is the method recommended by the
Canadian Society of Soil Sciences in their book Soil Sampling and Methods of Analysis (2nd
ed.) page 73 numeral 6.3.

However, this method's principle is different from the Cadmium Reduction Method. The Hach
TNTplus Nitrate is the typical example of electrophillic aromatic substitution in which nitrate in
the presence of sulfuric acid yields a nitronium ion (+NO2) and HSO4-. To create intermediate
nitro-carbonium ions, nitronium ions, which are electrophiles, attack the aromatic ring of the
dimethylphenol reagent. The nitro-carbonium intermediate is then converted by the basic HSO4-
ion into a stable substitution product (o, or p-nitro-dimethylphenol). The visual absorption

8
spectra of the nitrodimethylphenol product can be used to measure its intensity due to its
intense color, which is directly related to the nitro functional group. Test results are measured at
345 nm.

Something important to mention here is that nitrate is not a compound that can be extracted
with the Mehlich 3 extraction. For this reason, nitrate is extracted from soil by using a 2.0 M KCl
solution. This method is also mentioned in section 6.2 of Soil Sampling and Methods of
Analysis (2nd ed.).

4. SPECTROPHOTOMETRIC METHODS USED IN SOIL ANALYSIS

4.1. UV-Vis SPECTROSCOPY

Ultraviolet (UV) and visible radiation are a short part of the electromagnetic spectrum. The UV
range normally extends from 100 to 400 nm, with the visible range from approximately 400 to
800 nm.

Figure 3:The electromagnetic spectrum, with the visible light section expanded

An ultraviolet-visible (UV-Vis) spectrophotometer uses a light source to illuminate a sample at a

wavelength within the Ultraviolet range or the visible range. The sample absorbs, transmits, or
reflects some of the light.

The quantity of light absorbed by a substance can be measured by the spectrophotometer as

shown schematically in Figure 4.

9
 Figure 4:A schematic diagram of a simple spectrophotometer. (Zumdahl, Zumdahl and
DeCoste, 2017, p. A18)

This instrument compares the intensity of incident light I 0 to the intensity of light after it has
passed through the sample 𝐼. The ratio 𝐼 𝐼 , called the transmittance is a measure of the

fraction of light that passes through the sample. (Zumdahl, Zumdahl and DeCoste, 2017, p.
A18)

The absorption of photons causes the radiant power, I, to decline exponentially as it passes
through the absorbing sample:

𝑇= = 10 (1)

In Equation (1), the constant 𝜀 is called the molar absorptivity (it has units of liters per mole per
centimeter), b is the cell path length in centimeters, and c is the concentration of the absorbing
sample in units of moles per liter. Taking the logarithm (to the base 10) of both sides, we obtain
a quantity, called the absorbance, A, that is proportional to the concentration of the sample:

𝐴 = 𝑙𝑜𝑔 = 𝜀𝑏𝑐 (2)

Equation (2) is referred to as the Beer-Lambert law. As long as the path length, b, remains
constant, a plot of absorbance versus concentration should be linear. (Postma, J.M., Roberts, J.
L., & Roberts, A. (2016), p 20-4).

Knowing what is mentioned above, different dilutions of known concentration of an analyte of

interest (called standards) are prepared, and each standard's absorbance measured at a
particular wavelength. With this data and using a spreadsheet program such as Microsoft Excel,
a graph of absorbance (y-axis) versus standard concentration (x-axis) can be generated.

10
Ideally, the graph will show a straight line that goes through the origin (0,0). The slope (𝜀𝑏) of
the resulting line can be determined by linear regression analysis, which gives the equation for
the line that best fits the data points. Using the equation for the best-fit line from the standard
curve, the concentration of the analyte of interest in a sample of unknown concentration can
then be determined by using the following equation:

𝑐= (3)

taking into account the y-intercept. The measured concentration will only be valid as long as the
measured absorbance falls within the standards' range. If this does not happen, it would be
necessary to dilute the unknown sample by a known factor so that the absorbance falls within
the range shown on the graph. This dilution factor must be accounted for when determining the
final concentration. (Postma, J.M., Roberts, J. L., & Roberts, A. (2016), p 20-4).

4.2. FLAME ATOMIC ABSORPTION SPECTROSCOPY

Atomic Absorption Spectroscopy is an instrumental analytical technique useful to determine the
concentrations of certain elements (analytes) in a sample. The principle behind this technique is
that atoms (and ions) can absorb light at a specific and unique wavelength. At this wavelength,
light is absorbed by the atom which causes electrons in the atom to move from the ground state
to an excited state (Figure 4). The amount of light that is absorbed is measured, and with this
information, it is possible to calculate the concentrations of the elements being analyzed.
(Atomic Absorption Spectroscopy, How Does AAS Work, AAS FAQs | Agilent, n.d.

11
Figure 5:An electron is in an excited state when it temporarily occupies an energy state higher
than its ground state. If an electron receives additional energy, such as when it absorbs a
photon, or packet of light, it might become excited. (Background: Atoms and Light Energy, n.d.)

Atomic absorption spectroscopy is studied using an atomic absorption spectrometer. This is a

device that uses the principle that atoms (and ions) can absorb light at a specific, unique
wavelength. A simplified diagram of the components of an atomic absorption spectrometer is
shown below (Figure 5):

Figure 6:Components inside an AA Spectrometer (Atomic Absorption Spectroscopy, How Does

AAS Work, AAS FAQs | Agilent, n.d.-b)

Each of its important parts is explained in greater detail below:

12
 4.2.1. AA SPECTROMETER SAMPLE INTRODUCTION SYSTEM

A capillary tube transports the sample into the nebulizer. The pneumatic nebulizer accelerates
the solution stream by using the Venturi effect. The accelerated fluid impacts a glass bead
creating a fine spray usually known as an aerosol which is passed up into the spray chamber.
The function of the mixing paddles is to remove large droplets (to maintain a homogenous flow),
minimize burner blockage and ensure a better mixing of the oxidant and the acetylene gas with
the sample droplets. The spray chamber bung is part designed mostly for safety reasons as it
will release upon any abnormal build-up of gases in the sample introduction system. (Atomic
Absorption Spectroscopy, How Does AAS Work, AAS FAQs | Agilent, n.d.-b)

Figure 6:
Figure 7:Flame
FlameAA
AAsample
sampleintroduction
introductionsystem.
system

4.2.2. THE FLAME AA SPECTROMETER BURNER

The aerosol mixture produced by the spray chamber; the burner converts it into free ground
state atoms. The most common mixtures of gases to fuel the flame are air-acetylene and nitrous
oxide-acetylene. Each one of these mixtures is used with different purposes. In the case of air-
acetylene, it produces a flame of around 2300°C which is suitable for most elements. In the
other hand nitrous oxide-acetylene creates a flame of around 2700°C which suitable for
elements prone to form oxides.

13
The aerosolized solution goes through four main stages in the burner and they are:

1. Desolvation or drying. Evaporation of the solvent happens here, resulting in dry nano
particles of the sample remaining.
2. Vaporization. The particles from the last stage are converted to the gaseous phase.
3. Atomization. This is the most important stage, where the freely dissociated atoms in the
ground state, which are the target for the AAS analysis, are created.
4. Ionization. Some of the free atoms are converted to ions. This depends on the gas mixture
and the ionization gas potential of the analytes in the solution.

The slit of the burner is specially designed, and it is 5-10 cm long and depends on the type of
burner used. It is along the slit where the free-ground atoms' population exists. The light path
passing through the atoms is determined by the length of the flame (Figure 7) (Atomic
Absorption Spectroscopy, How Does AAS Work, AAS FAQs | Agilent, n.d.-b).

Figure 8: Light from the hollow cathode lamp light source is passed along the burner and
through the flame created by the burner slit.

4.2.3. THE LIGHT SOURCE (HOLLOW CATHODE LAMP)

The most common source of light used with an Atomic Absorption Spectrometer is the hollow
cathode lamp (HCL). This is a critical component because what is measured with this technique
is the absorbance of the light from the lamp as it passes through the atomized sample.

These lamps are usually filled with argon or neon at low pressure. A metal cathode coated in
the desired element is placed in opposition to an anode (Figure 8). The two electrodes are
subjected to a high voltage, which ionizes the filler gas and accelerates ions toward the
cathode. An atom plume is produced when the cathode is hit by these ions with a high enough

14
energy to cause the metal atoms in the cathode material to be ejected or "sputtered." Additional
collisions between metal atoms inside the atom plume cause them to reach an excited state.
Radiation is emitted to an specific wavelengths when the atoms return to their respective ground
state for that particular element.

Figure 9: Schematic of a single element hollow cathode lamp used for AA spectrometer analysis

4.2.4. THE MONOCHROMATOR

A monochromator is an optical device used only to collect light with various wavelengths and
isolate (select) a narrow wavelength band.

4.2.5. THE DETECTOR

The photomultiplier tube (PMT) is the atomic absorption spectrometer's primary detector. No
other device provides the same sensitivity for the wavelength range needed for atomic
absorption analysis. The PMT measures the light's intensity by converting incident light into an
electrical signal. A sequence of dynodes amplifies the signal before being collected (measured)
on an anode and used to estimate the absorption. The series of dynodes can amplify the signal
by 100 million times.

Figure 10: Diagram of the photomultiplier tube

15
 4.2.6. COMPUTERIZED INSTRUMENT CONTROL

The device feeds the controlling computer with the data it has gathered. Specialized instrument
control software determines the concentration of each element in the sample by using the
calibration curve that was read prior to the sample analysis.

5. SAMPLING OF SOIL
Generally speaking, sampling means to select a group of individuals from a total population to
carry out measurements in this group. The measurement performed on this reduced group are
used to estimate properties of the total population. Sampling is necessary when analyzing soils
because measurement of the total population is impossible (Soil Sampling and Methods of
Analysis (2nd ed.))

Theoretically, sampling soil can be carried out at any time during the year. However this Is not
particularly true in Canada because of the extreme changes in weather that occur from the
summer and fall seasons to the winter.

5.1. PROCEDURE FOR SAMPLING

The most common sample design is the systematic sampling using transects or grids. This
consists to take a sample from the points on an imaginary grid on the terrain to sample. This
method is particularly useful when the terrain is regular and with minimum or no slope.

To take the sample a core sampler for soils is used as the one shown in Figure 10. This tool is
used to collect virtually undisturbed soil samples. (Soil Sampling and Methods of Analysis (2nd
ed.)

Figure 11: Core sampler (A&L Canada Laboratories Inc., n.d.)

16
When sampling is suggested to take the samples to a depth of six inches. However when
sampling for nitrate-nitrogen, it is recommended to sample at 1 foot increments down to 3 feet. It
is suggested that the depths be kept separate so that a more accurate assessment can be
made regarding soil fertility and soil physical conditions.

5.2. DESCRIPTION OF LOCATION WHERE SAMPLE WAS TAKEN

The location where samples were taken was 4217 Hwy 537, Wanup (46.383, -80.8212). The
procedure described previously was followed in this place.

Figure 12: Location of the site where samples were taken (Image retrieved from Google maps
on November 16th 2022)

6. PERFORMANCE EVALUATION OF A METHOD OF ANALYSIS

If the result of an analysis cannot be trusted then it has little value and the analysis might as well
have not been carried out. it is necessary to demonstrate that its performance characteristics
are adequate for use for a particular purpose. In the case of this technical report there will be
three performance parameters that will be evaluated: Linearity, precision and trueness. It is
important to mention that once precision and trueness have been evaluated it is possible to

17
evaluate the accuracy of the method because those are the two components of accuracy.
(Eurachem guide, 2014)

6.1. LINEARITY
When using an instrumental method, it is usually needed to carry out a calibration procedure.
This is done by using a sequence of samples (standards) that each have a known concentration
of the analyte to be measured. A calibration curve is created by measuring the instrumental
signal for each standard and graphing this response against concentration. The concentration of
the test sample (unknown) can be extrapolated graphically from the calibration curve if the same
experimental conditions are used to measure the standards and the test sample. (Jeffery, 1989)

To establish if there is a linear relationship between two variables x1 and y1, one of the most
used parameters is the square of the Pearson’s correlation coefficient, which is defined as:

∑ ∑ ∑
𝑟 = (4)
∑ (∑ ) ∑ (∑ )

Where n is the number of data points.

The value of r2 lies between 0 and 1. The closest to 1 is the value of r2 then the greater the
probability that a definite linear relationship exists between the variables x and y.

Once it has been determined that the data obtained has a great probability of being linear, then
it is possible to evaluate the best straight line by linear regression (the method of least squares).
To obtain the regression line it is necessary to consider that any straight line has an equation in
the form of 𝑦 = 𝑚𝑥 + 𝑏, where m is the slope and b is the intercept to the y-axis. Each of these
parameters can be obtained in the following way:

∑ ∑ ∑
𝑚= ∑ (∑ )
(5)

𝑏 = 𝑦 − 𝑚𝑥̅ (6)
where 𝑥̅ is the mean of all values 𝑥 and 𝑦 is the mean of all values of 𝑦 .

18
 6.2. EVALUATION OF THE ACCURACY OF THE METHOD

As mentioned before, the two components of accuracy are trueness and precision. However, it
is important to mention that before evaluating these two parameters, it is important to consider
the concept and evaluation of outliers.

6.3. OUTLIERS
An outlier is defined as a result or more than one result from an experiment that appears to
differ unreasonably from the other values obtained. Then, it is necessary to determine
statistically what to do with these values if they should be kept or rejected. Among the many
ways to evaluate if a given value is an outlier, ISO recommended the denominated Grubbs’ test.
This test compares the deviation of the suspect value from the sample mean with the standard
deviation of the sample. The suspect value is the value that is furthest away from the mean.

To use Grubbs’ test for an outlier the null hypothesis (H0) is set as “there are no outliers in the
population”, therefore, the alternative hypothesis (Ha) would be set as “there is exactly one
outlier in the data set. The Statistic G is then calculated:

| ̅|
𝐺= (7)

Where 𝑥̅ (mean of values) and s (standard deviation of values) are calculated with the suspect
value included. Something important to mention here is that the Grubbs’ test assumes that the
population is normal. This means that they come from a population with a normal distribution.
(Miller & Miller, 2005)

For the purposes of this technical report, the evaluation of normality and outliers is performed by
using the statistical software Minitab 3.0, which already provides the options to evaluate these
two important parameters.

6.4. EVALUATION OF PRECISION

Precision or precision measurement is defined as how close results are to one another. Usually,
statistical parameters like the standard deviation help or relative standard deviation help to
express precision when calculated from replicate measurements under specified conditions.

19
For the purpose of this technical report, it will be considered a parameter called “precision limit”.
This allows to determine if there is a significance difference between replicates obtained under
specified conditions. Something important to mention is that in this report only is considered the
precision limit under repeatability conditions which means that the different measurements have
been performed by a single analyst using the same equipment over a short timescale.
(Eurachem guide, 2014)

Considering different factors like the degrees of freedom and the level of confidence as well as
the two tailed Student t-value, it is determined that:

𝑟 = 2.8𝑆 (8)
Being r the precision limit under repeatability conditions, and Sr the standard deviation
calculated from the results obtained under repeatability conditions. The value of r is the
maximum absolute difference between any two replicates to consider that the method is
precise. (Eurachem guide, 2014).

6.5. EVALUATION OF TRUENESS

The measurement of trueness expresses how close the average of an infinite number of results
is to a reference value. However, it is impossible to take an infinite number of measurements, so
trueness cannot be measured. Nonetheless, it is possible to make a practical assessment of
trueness which is called “bias”. (Eurachem guide 2014)

A practical way to determine the bias is the comparison between the average of the results of
the candidate method and a reference value. One of the most common approaches is that this
reference value comes from the analysis of a reference material (true value) which is usually
certified. (Eurachem guide 2014).

“A reference material (RM) is defined as a material characterized by a metrologically valid

procedure for one or more specified properties, accompanied by an RM certificate that provides
the value of the specified property, its associated uncertainty, and a statement of metrological
traceability “.

20
 7. METHODOLOGY

7.1. SAMPLE PREPARATION

After samples were taken from the field as described in section 2.8.1, it was necessary to dry
them. This is not done in any furnace but by air drying. This is because air-drying allows soil
samples to be stored for analyses even after a long time, since dried soil samples are perceived
to undergo minimal changes caused by microbial and chemical reactions compared to field-
moist samples. (OBALUM, 2017)

Six portions of the sample then are air-dried during two days as shown in figure 13.

Figure 13:Wanup sample being air-dried in the lab

After the sample is air-dried for two days, it was necessary to measure the pH of each portion
that was dried to get an average of the six results. The results were as follows:

21
 Table 1: pH of each portion that was air dried from the Wanup sample

Soil sub Mass Temperature

pH
sample (g) °C
1 1.61 6.409 23.8
2 1.64 6.418 23.8
3 1.58 6.433 23.9
4 1.55 6.078 23.1
5 1.66 6.201 23.1
6 1.64 6.068 23.7
Average 6.268 23.6

Then all dried portions were mixed and stored at 4°C in Nalgene bottles. This allows preserving
the sample for a long time.

After what has been described, it was necessary to sieve the samples using a 2mm sieve
(Figure 14). This is the size recommended for all samples when the Mehlich 3 extraction
solution will be used.

Figure 15: 2 mm sieve used for soil sample preparation.

7.2. PREPARATION OF MEHLICH 3 SOLUTION

The extraction solution Mehlich 3 (M3) was prepared according to the procedure described in
the section 7.2. (Carter, 2022) (Appendix 1). However, in the proceure it is described how to

22
prepare 10 liters of this solution, and the amount of reagents necessary to prepare this amount
are given in table 2. It is important to mention that for the stock solution M3, the volumes
described in the procedure were prepared as described.

Table 2: Amount of reagents used to prepare 2.0 liters of Mehlich 3 extraction solution

Mehlich 3 Stock Solution

Ammonium Fluoride (NH4F) 55.56 g
EDTA 29.23 g
Deionized water 1000.00 ml
Mehlich 3 Solution (Ready to use)
Ammonium nitrate (NH4NO3) 40.02 g
Concentrated acetic acid (NH4NO3) 23.00 ml
10% v/v nitric acid 16.40 ml
M3 Stock solution 20.00 ml
Deionized water 2000.00 ml

7.3. EXTRACTION
The sample was extracted using Mehlich 3 solution as described in section 7.3.1 of (Carter,
2022) (Appendix 1). In this case nothing was changed from the procedure. (Figures 15 and 16).

Figure 16: Soil samples in the reciprocating shaker

23
 Figure 17: Samples being filtered after the extraction with Mehlich 3 solution

Once the filtration is complete, these samples can be stored at 4°C, but they have to be
analyzed as soon as possible because their expiration date is the same as the batch of Mehlich
3 solution used in the extraction (10 days after preparation).

7.4. PHOSPHORUS ANALYSIS

The preparation of solutions necessary for phosphorus analysis was carried out as described in
section 7.2 numeral 8 of (Carter, 2022) (Appendix 1).

The determination of phosphorus for the calibration curve and samples was carried out by the
manual colorimetric method whose procedure is in section 7.3.2 of (Carter, 2022) (Appendix 1).
However, there was an important change made, because the volumes specified in the
procedure were reduced by ten times to reduce the amount of reagents used. This meant that
the following volumes were used to prepare a sample for phosphorus testing:

24
 Table 3:Modified volumes used to prepare standard and samples to read phosphorus by the
UV-Vis spectrometry method.

Reagent Volume
Clear filtrate obtained from soil
sample (or standard when preparing
the calibration curve) 0.2 mL (200μL)
Distilled water 1.5 mL (1500 μL)
Solution B 0.4 mL (400 μL)
Second addition of distilled water 0.4 mL (400 μL)
TOTAL VOLUME 2.5 mL

This volume fits in a UV cuvette and allows to prepare the standards for the calibration curve
and samples directly in the cuvettes where they will be read in the UV-Vis Spectrometer. The
model used was Cary 60 UV-Vis Spectrophotometer, which is the one available at the Research
and Development lab at Cambrian College.

The calibration curve for this test was designed by using a standard solution prepared by
dissolving 0.4393 g of KH2PO4 in 1 L of deionized water and later preparing standard solutions
of 0, 0.5, 1, 2, 5 and 10 ppm of phosphorus by diluting with Mehlich 3 solution. This procedure is
described in section 7.2 numeral 8c of (Carter, 2022) (Appendix 1). In Figure 16, it can be seen
how each standard was prepared in the cuvette.

Figure 18: Standard calibration curve solutions ready to read in the UV-Vis spectrometer.

25
 7.5. POTASSIUM ANALYSIS

The preparation of solutions necessary for potassium analysis was carried out as described in
section 7.2 numeral 9 of (Carter, 2022) (Appendix 1).

The determination of potassium for the calibration curve and samples was carried out by atomic
absorption. The procedure is in section 7.3.3 of (Carter, 2022) (Appendix 1). However, there
was an important change made. In the procedure is specified that the determination by this
technique has to be performed by using flame emission. Still, the analysis was carried out by
using the Flame Absorption technique. The model of device used for this was the Agilent 200
Series AA

The calibration curve for this test was designed using a standard solution prepared from a
certified atomic absorption standard and prepared solutions of 0.5 ,1.0, 1.5, 2.0 ppm as
described in section 7.2 numeral 9c of (Carter, 2022) (Appendix 1).

7.6. NITROGEN ANALYSIS

Even though it was specified in Section 3.2.3 that the Nitrogen Analysis was going to be
performed by the use of the kit for analysis “Hach Company TNT plus Nitrate Reagent, Cat. No.
TNT836” further research showed that this commercial test is not suitable for this purpose. The
reasons why on how this was determined are discussed in the Discussion section (Section 9) of
this report.

26
 8. DATA AND CALCULATIONS
8.1. PHOSPHORUS ANALYSIS
After preparing standard solutions at different concentrations for phosphorus analysis as
described in section 7.2 numeral 8c of (Carter, 2022) (Appendix 1), the results obtained were as
follows:

Table 4: Absorbances read from standard solution of phosphorus.

Standard
Concentration Absorbance readings
(ppm)
Repetition 1 Repetition 2 Average
0.0 0 0 0
0.5 0.0191 0.0178 0.0185
1.0 0.0458 0.0466 0.0462
2.0 0.0995 0.0979 0.0987
5.0 0.2370 0.2381 0.2376
10.0 0.4884 0.4944 0.4914

With these results is possible to obtain the following graph:

Absorbance vs Phosphate concentration

0.6

0.5 y = 0.0489x - 0.0025

R² = 0.9997

0.4
Apsorbance

0.3

0.2

0.1

0
0.0 2.0 4.0 6.0 8.0 10.0 12.0
Phosphorus concentration (ppm)

Figure 19: Calibration curve of phosphorus.

27
With a Pearson’s correlation factor of 0.9997, this calibration curve is accepted and the following
equation is defined as the calibration curve to evaluate phosphorus:

𝐴𝑏𝑠 = 0.0489(𝐶) − 0.0025 (9)

Or re-arranged it would be:

.
C= (10)
.

Where C is the concentration of phosphorus and Abs is the absorbance read by the UV-Vis
Spectrometer.

Once the calibration curve was determined, it was possible to read the sample. As explained
before (Section 6.4), the measurement of phosphorus in the sample from Wanup was measured
ten times because this number allows to have enough data to evaluate normality of results, look
for any outliers and at the same time to determine how precise the method is. It was also
decided to include a process blank. The result for all the measurements were the following:

Table 5: Absorbance readings from the ten repetitions of the phosphorus measuring of the
Wanup sample.

Repetition Absorbance

Process
blank 0.0054
1 0.2491
2 0.2425
3 0.2510
4 0.2557
5 0.2509
6 0.2359
7 0.2292
8 0.2390
9 0.2436
10 0.2290

28
After applying the Anderson Darling test to this data to determine if they belong to a normal
distribution, it was possible to obtain the following probability plot:

Figure 20: Probability plot of the absorbances obtained from measuring ten times the
phosphorus concentration in the soil sample from Wanup.

For the Anderson Darling test, the null hypothesis is that the data are normally distributed; the
alternative hypothesis is that the data are non-normal. The test was performed using the
Software Minitab 3.0 and it was set for a significance level of 5% (Confidence level of 95%).
From the probability plot obtained it is possible to see that the P-value is 0.613 and as this is
greater than 0.05, so the null hypothesis is be accepted.

Now that it is known that the result obtained belong to a normal distribution it is necessary to
evaluate for any outliers, this is done by running a Grubbs test using the Software Minitab 3.0.
obtaining the following outlier plot:

29
 Figure 21: Outlier plot obtained from measuring ten times the phosphorus content in the soil
sample from Wanup.

For the Grubbs test, the null hypothesis is that there are no outliers in the dataset; the
alternative hypothesis is that either the smallest or largest data value is an outlier. The test was
performed using the Software Minitab 3.0 and it was set for a significance level of 5%
(Confidence level of 95%). From the outlier plot obtained it is possible to see that the P-value is
1.000 and as this is greater than 0.05, so the null hypothesis is be accepted.

Due to the fact that all ten values obtained belong to a normal distribution and that there are no
outliers in the dataset, it was possible to determine if the method is precise, this is done as
described in (Section 6.4), for which it is necessary to calculate the standard deviation of the
dataset (Sr), then the precision limit (2.8 Sr),

The staandard deviation of the ten repetitions is calculated to be 0.0093 when using excel, so
the precision limit can be calculated as:

𝑟 = 2.8𝑆 = 2.8 ∗ (0.0093) = 0.02601

This value allowed to prepare the prepare the following matrix:

30
 Table 6: Absolute differences between repetitions of the ten measurements phosphorus content
in the soil sample from Wanup.

Absorbances 0.2491 0.2425 0.2510 0.2557 0.2509 0.2359 0.2292 0.2390 0.2436
0.2491
0.2425 0.0066
0.2510 0.0019 0.0085
0.2557 0.0066 0.0132 0.0047
0.2509 0.0018 0.0084 1E-04 0.0048
0.2359 0.0132 0.0066 0.0151 0.0198 0.015
0.2292 0.0199 0.0133 0.0218 0.0265 0.0217 0.0067
0.2390 0.0101 0.0035 0.012 0.0167 0.0119 0.0031 0.0098
0.2436 0.0055 0.0011 0.0074 0.0121 0.0073 0.0077 0.0144 0.0046
0.2290 0.0201 0.0135 0.022 0.0267 0.0219 0.0069 0.0002 0.01 0.0146

As it can be seen only two values in the absolute values of the differences between repetitions
are greater than the precision limit, so these values would not be precise between them, but as
all the values have been checked to be from a normal distribution and that there are not outliers
present at a confidence level of 95%, then the test can be considered precise.

After this analysis and by using Equation 10, it is possible to determine the average
concentration present in the soil sample:

Table 7: Concentrations of phosphorus in the ten repetitions of phosphorus test using the
Wanup Sample.

Concentration of
Phosphorus
Repetition Absorbance (Considering process
blank correction)
(ppm)
1 0.2491 5.01
2 0.2425 4.88
3 0.2510 5.05
4 0.2557 5.15
5 0.2509 5.05
6 0.2359 4.74
7 0.2292 4.61
8 0.2390 4.81
9 0.2436 4.90
10 0.2290 4.60
Average 4.88

31
This value of 4.88 ppm (mg/L) is the concentration in solution, so the concentration in soil would
be calculated as:

4.88 𝑚𝑔 𝑃 0.03𝐿
× = 48.8 𝑚𝑔/𝑘𝑔
𝐿 0.003𝑘𝑔

Once defined that the method is precise, it is necessary to evaluate the trueness of the method.
This is be done by the comparison between the average of the results of the candidate method
and a reference value. For the purposes of this investigation, it was possible to acquire a
certified reference material whose certificate of analysis is included in the Appendix 3. This
reference material was treated exactly like another sample, with the exception that it was only
measured three times (triplicate) because for economic reasons, it was decided to us as less as
possible of the 30.0g of reference material available because it will be also useful for future
investigations. The results obtained after measuring absorbance three times were the following:

Table 8: Absorbances read in triplicate from three repetitions of the three measurements of
phosphorus of the reference material.

Amount of Absorbances Average

reference material
absorbance
used (g)
Reading 1 Reading 2 Reading 3
3.0101 0.2401 0.2303 0.2371 0.2358
3.0124 0.2461 0.2345 0.2301 0.2369
3.0138 0.2420 0.2312 0.2435 0.2389

Normality and outliers are evaluated again by the Anderson Darling test and Grubbs test
respectively, both of them at a 95% confidence level are evaluated for each repetition, giving the
following results:

32
 Figure 22: Probability plot of the readings obtained from repetition 1 of
phosphorus test in the reference material

Figure 23: Probability plot of the readings obtained from repetition 2 of phosphorus test in the
reference material

33
Figure 24: Probability plot of the readings obtained from repetition 3 of phosphorus test in the
reference material

Figure 25: Outlier plot obtained from the readings of repetition 1 of phosphorus test in the
reference material.

34
Figure 26: Outlier plot obtained from the readings of repetition 2 of phosphorus test in the
reference material.

Figure 27: Outlier plot obtained from the readings of repetition 3 of phosphorus test in the
reference material.

35
As it can be verified from Figures 20-25, none of the P-values are under 0.05, which means that
all data come from normal distributions and there are no outliers.

Then by using Equation 10 it is possible to determine the concentration of three repetitions and
obtain a general average which will correspond to the phosphorus concentration of the
reference material obtained by the method in which the element to measure is extracted by the
Mehlich 3 solution extraction and the concentration of phosphorus is measured by a colorimetric
method using the UV-Vis spectrometer. The results obtained are as follows:

Table 9: Concertation of phosphorus in every repetition carried out with the certified
measurement material.

Concentration
Average of
Absorbance phosphorus
(ppm)
0.2358 4.85
0.2369 4.87
0.2389 4.92
Average
Concentration
4.88
of
phosphorus

This value of 4.88 ppm (mg/L) is the concentration in solution, so the concentration in soil would
be calculated as:

4.88 𝑚𝑔 𝑃 0.03𝐿
× = 48.8 𝑚𝑔/𝑘𝑔
𝐿 0.003𝑘𝑔

According to the certificate of analysis of the reference material Appendix 3, the phosphorus
certified value should be 294±26 ppm. However, as it can be seen, the value obtained by the
method perform is far from this value. The possible reasons for this are explained in the
discussion section.

8.2. POTASSIUM ANALYSIS

After preparing standard solutions at different concentrations for potassium analysis as
described in section 7.2 numeral 9c of (Carter, 2022) (Appendix 1) the results obtained were as
follows:

36
 Table 10: Absorbances obtained in the Flame AA with different standard concentrations

Standard Absorbance
concentration
0.0 0.0000
0.3 0.0754
0.5 0.1483
1.0 0.2852
1.5 0.4250
2.0 0.5573

From here obtaining the following graph:

Absorbance vs Potassium concentration

0.6000
y = 0.2781x + 0.0052
0.5000 R² = 0.9997

0.4000
Absorbance

0.3000

0.2000

0.1000

0.0000
0.0 0.5 1.0 1.5 2.0 2.5
Potassium Standard Concentration (ppm)

Figure 28: Calibration curve of potassium

With a Pearson’s correlation factor of 0.9997, this calibration curve is accepted and the following
equation is defined as the calibration curve to evaluate phosphorus:

𝐴𝑏𝑠 = 0.2781(𝐶) − 0.0052 (11)

Or re-arranged it would be:

.
C= (12)
.

37
Where C is the concentration of potassium and Abs is the absorbance read by the UV-Vis
Spectrometer.

Once the calibration curve was determined, it was possible to read the sample. As explained
before (Section 6.4), the measurement of potassium in the sample from Wanup was measured
ten times because this number allows to have enough data to evaluate normality of results, look
for any outliers and at the same time to determine how precise the method is. It was also
decided to include a process blank. The result for all the measurements were the following:

Table 11: Absorbance readings from the ten repetitions of the potassium measuring of the
Wanup sample.

Repetition Absorbance

Process
blank 0.0042
1 0.0920
2 0.0934
3 0.0919
4 0.0874
5 0.0931
6 0.0838
7 0.0816
8 0.0927
9 0.0883
10 0.0850

After applying the Anderson Darling test to this data to determine if they belong to a normal
distribution, it was possible to obtain the following probability plot:

38
Figure 29: Probability plot of the absorbances obtained from measuring ten times the potassium
concentration in the soil sample from Wanup.

For the Anderson Darling test, the null hypothesis is that the data are normally distributed; the
alternative hypothesis is that the data are non-normal. The test was performed using the
Software Minitab 3.0 and it was set for a significance level of 5% (Confidence level of 95%).
From the probability plot obtained it is possible to see that the P-value is 0.156 and as this is
greater than 0.05, so the null hypothesis is accepted.

Now that it is known that the result obtained belong to a normal distribution it is necessary to
evaluate for any outliers, this is done by running a Grubbs test using the Software Minitab 3.0.
obtaining the following outlier plot:

39
 Figure 30: Outlier plot obtained from measuring ten times the potassium content in the soil
sample from Wanup.

For the Grubbs test, the null hypothesis is that there are no outliers in the dataset; the
alternative hypothesis is that either the smallest or largest data value is an outlier. The test was
performed using the Software Minitab 3.0 and it was set for a significance level of 5%
(Confidence level of 95%). From the outlier plot obtained it is possible to see that the P-value is
1.000 and as this is greater than 0.05, so the null hypothesis is be accepted.

Due to the fact that all ten values obtained belong to a normal distribution and that there are no
outliers in the dataset, it was possible to determine if the method is precise, this is done as
described in (Section 6.4), for which it is necessary to calculate the standard deviation of the
dataset (Sr), then the precision limit (2.8 Sr),

The staandard deviation of the ten repetitions is calculated to be 0.0043 when using excel, so
the precision limit can be calculated as:

𝑟 = 2.8𝑆 = 2.8 ∗ (0.0043) = 0.01210

This value allowed to prepare the prepare the following matrix:

40
 Table 12: Absolute differences between repetitions of the ten measurements potassium
concentrations in the soil sample from Wanup.

Absorbances 0.0920 0.0934 0.0919 0.0874 0.0931 0.0838 0.0816 0.0927 0.0883
0.0920
0.0934 0.0014
0.0919 0.0001 0.0015
0.0874 0.0046 0.006 0.0045
0.0931 0.0011 0.0003 0.0012 0.0057
0.0838 0.0082 0.0096 0.0081 0.0036 0.0093
0.0816 0.0104 0.0118 0.0103 0.0058 0.0115 0.0022
0.0927 0.0007 0.0007 0.0008 0.0053 0.0004 0.0089 0.0111
0.0883 0.0037 0.0051 0.0036 0.0009 0.0048 0.0045 0.0067 0.0044
0.0850 0.007 0.0084 0.0069 0.0024 0.0081 0.0012 0.0034 0.0077 0.0033

As it can be seen, there are no values in the absolute values of the differences between
repetitions are greater than the precision limit, then the test can be considered precise.

After this analysis and by using Equation 12, it is possible to determine the average
concentration of potassium present in the soil sample:

Table 13: Concentrations of potassium in the ten repetitions using the Wanup sample.

Concentration of
Phosphorus
Repetition Absorbance (Considering process
blank correction)
(ppm)
1 0.0920 0.30
2 0.0934 0.30
3 0.0919 0.30
4 0.0874 0.28
5 0.0931 0.30
6 0.0838 0.27
7 0.0816 0.26
8 0.0927 0.30
9 0.0883 0.28
10 0.0850 0.27
Average 0.29

41
This value of 0.29 ppm (mg/L) is the concentration in solution and it is necessary to consider
that it was necessary to dilute the sample with a factor of 1/100, so the concentration in soil
would be calculated as:

0.29 𝑚𝑔 𝐾 0.03𝐿
× × 100 = 290 𝑚𝑔/𝑘𝑔
𝐿 0.003𝑘𝑔

Once defined that the method is precise, it is necessary to evaluate the trueness of the method.
This is done by the comparison between the average of the results of the candidate method and
a reference value. The same reference material used for the Phosphorus test is used here
because potassium is among the certified elements whose concentration is known in the
acquired reference material. Moreover, potassium is among the elements that can be extracted
by the use of the Mehlich 3.

This reference material was treated exactly like another sample, with the exception that it was
only measured three times (triplicate) because for economic reasons, it was decided to us as
less as possible of the 30.0g of reference material available because it will be also useful for
future investigations. It is important to mention that as Phosphorus and Potassium are both
extractable with Mehlich 3, then the same extracted solution used for the Phosphorus test was
used for the Potassium test. The results obtained after measuring absorbance three times were
the following:

Table 14: Absorbances from the potassium test read in duplicate in the three different samples
taken from the certified reference material.

Amount of Absorbances
reference material Average
used (g) Repetition Repetition
1 2
3.0101 0.2194 0.2093 0.2144
3.0124 0.2157 0.1992 0.2075
3.0138 0.2272 0.2207 0.2240

In this case for being only two repetitions, three was not neither normality test nor outlier’s test.
The only thing verified between repetitions was that the difference between the two values
would not exceed the precision limit.

42
Table 15: Evaluation of precision for measurements of absorbances in the potassium test for the
certified reference material.

Absorbances Difference Standard Precision

Repetition Repetition between deviation limit
1 2 repetitions
0.2194 0.2093 0.0101 0.0071 0.0200
0.2157 0.1992 0.0165 0.0117 0.0327
0.2272 0.2207 0.0065 0.0046 0.0129

As it can be seen from table 14, the values can be accepted and the concentrations of
potassium then calculated for the certified reference material by using Equation 12.

Table 16: Calculation of potassium concentration in Wanup sample

Amount of Absorbances Potassium

reference material Average concentration
used (g) Repetition Repetition (ppm)
1 2
3.0101 0.2194 0.2093 0.2144 0.79
3.0124 0.2157 0.1992 0.2075 0.76
3.0138 0.2272 0.2207 0.2240 0.82
Average 0.79

This value of 0.79 ppm (mg/L) is the concentration in solution and it is necessary to consider
that it was necessary to dilute the sample with a factor of 1/100, so the concentration in soil
would be calculated as:

0.79 𝑚𝑔 𝐾 0.03𝐿
× = 790 𝑚𝑔/𝑘𝑔
𝐿 0.003𝑘𝑔

However, the potassium concentration specified in the Reference Material Certificate is

6087±141 ppm. The possible reasons for this are explained in the discussion section.

43
 9. DISCUSSION

The reason why it is necessary to dry the soil sample is to stop soil microbial activity and to stop
ion exchange reactions. This needs to be by air drying and not in a furnace because

Mehlich 3 solution is used as the extraction solution because according to the different
bibliography consulted, it is appropriate for the extraction of macronutrients such as Phosphorus
and Potassium. The reason for this is because phosphorus is extracted by a reaction with acetic
acid and fluoride compounds while exchangeable base cations (calcium, magnesium and
potassium) are extracted by ammonium nitrate and nitric acid. (Ústřední kontrolní a zkušební
ústav zemědělský [Central Institute for Supervising and Testing in Agriculture] & Horová, 2014)

Now, talking about the composition of Mehlich 3 solution the different reagents to prepare it
have the following functions:

Table 17: Functions of different reagents of Mehlich 3 extraction solution.

Reagent Function in extraction solution Mehlich 3

Buffers the extracting solution to pH 2.5 when all
Acetic acid (CH3COOH) reagents are added and mixed, thus preventing calcium
from being precipitated as calcium fluoride.
Facilitates extraction of basic cations such as calcium,
Ammonium nitrate (NH4NO3) magnesium, sodium and potassium and
reacts with acetic acid to form ammonium acetate.
Extracts a portion of calcium phosphates and
Nitric acid (HNO3) its acid component [H+] aids in extraction of basic and
micronutrient cations.
The fluoride extracts iron and aluminum phosphates &
Ammonium Fluoride the NH4+ ion complements ammonium nitrate in
extracting basic cations.
Ethylenediaminetetraacetic acid Chelates micronutrients (particularly copper) &
(EDTA) prevents precipitation of calcium fluoride.
(North Carolina Department of Agriculture and Consumer Services Agronomic Division, n.d.)

Here it is important to mention that Mehlich 3 solution is used to measure available phosphorus
and potassium in soils, which means those forms of phosphorus of potassium in soils that can
be absorbed by plants as macronutrients (inorganic phosphorus in the form of orthophosphate).

44
For the colorimetric method used to determine available phosphorus it is necessary to prepare
two solutions (named A and B in the procedure), the reagents used and the reasons why every
reagent is necessary are explained below:

Table 18: Function of every reagent used in the determination of available phosphorus by the colorimetric
method.

SOLUTION A
Reagent Function in the analysis of available phosphorus
Ammonium molybdate Reducing agent.
((NH4)6Mo7O24 ·4H2O)
Antimony potassium tartrate Ammonium molybdate and antimony potassium tartrate
react in an acid medium with dilute solutions of
phosphorus to form an antimony-phospho-molybdate
complex
Sulfuric acid Acidify the solution which is useful also when analyzing
the sample.

SOLUTION B
Ascorbic acid To reduce antimony-phospho-molybdate complex. This
complex to an intensely blue-colored complex
(Phosphorous, All Forms (Colorimetric, Ascorbic Acid, Two Reagent), n.d.)

The phosphate standard solutions are prepared by dissolving 0.4393 g of KH2PO4 (Potassium
dihydrogen phosphate) in 1 L of deionized water, and this allows to prepare a stock solution of
100 ppm of phosphorus, which can be used to prepare the different solutions at different
concentrations necessary to prepare a calibration curve. However, the reason to use KH2PO4
is because it is considered a primary standard which means that it is a reagent that we can use
to dispense an accurately known amount of analyte. (Libretexts, 2021)

The wavelength at which phosphorus is read is set in the UV-Vis spectrophotometer as 845nm.
This is determined by the method as the Lambda max (λmax) (The wavelength at which a
substance has its strongest photon absorption).

After performing the measurement of phosphorus ten times in the Wanup sample, it was
determined that the method was precise enough to be accepted as described in section 8.1 of
this report. However, after measuring by the same method the phosphorus in the reference
material, the result was 48.8 mg/Kg, if we compare this result with the concentration of
phosphorus reported in the certificate of analysis which is 294±26, the result is about 15.25% to
18.2%. The reason for this is that the reported value in the reference material’s certificate is

45
Total Phosphorus, while the phosphorus measured by using the Mehlich 3 extraction solution is
available phosphorus (Orthophosphate). According to what is mentioned in section 2.3.2,
available phosphorus is ideally between 30-65% of total phosphorus.

For the potassium analysis in soil sample, the preparation of the sample involves to add a
Cesium Chloride Lanthanum Chloride Buffer Solution, this is done because this solution is an
auxiliary spectroscopic buffer reagent for flame AAS (atomic absorption spectrum) applications.

Precipitation problems can result from the mixture of the CsCl---LaCl2 solution with the M3
extract, this is why it is recommended by the method that the extracts have to be diluted (at
least 1:10 final dilution). However, it was necessary to carry out a 1:100 dilution because with a
1:10 dilution the read values were out of the calibration curve.

In the method followed for the determination of potassium, it is specified that potassium has to
be carried out by using the Flame emission technique, but the Flame atomic Absorption
technique was used instead. The key difference between flame emission spectroscopy and
atomic absorption spectroscopy is that during flame emission spectroscopy,
certain wavelengths are emitted from atoms, whereas during atomic absorption spectroscopy,
certain wavelengths are absorbed by atoms. It was decided that the Flame AA technique was
going to be used because the research lab already counts with a potassium lamp.

After performing the measurement of potassium ten times in the Wanup sample, it was
determined that the method was precise enough to be accepted as described in section 8.2 of
this report. However, after measuring by the same method the potassium in the reference
material, the result was 790.0 mg/Kg, if we compare this result with the concentration of
phosphorus reported in the certificate of analysis which is 6087±141 ppm, the result is about
12.68% to 13.28%. The reason for this is that the reported value in the reference material’s
certificate is specified as only potassium, while the potassium measured by using the Mehlich 3
extraction solution is available or inorganic potassium. Readily available forms of potassium
make up only 0.1 to 2 percent of the total potassium in the soil, and consist of potassium
dissolved in the soil solution and held on the exchange positions of the clay and organic
matter. (Mosaic Crop Nutrition, n.d.)

Finally, as it was mentioned in section 3.2.3, the original intention was to carry out the
measurement of nitrogen by using the commercial Spectrometric HACH test TNT836. However,
after further research there were some factors that were considered as a limitation to use this
kit, the reasons are mentioned below:

46
  Originally this method was designed for the determination of nitrate in drinking water,
surface water, domestic and industrial wastes.
 According to the book Soil Sampling and Methods of Analysis (2nd ed.), page 72,
extraction of nitrate (NO3) and ammonium (NH4) to measure total nitrogen is done by
extracting them from the soil sample with a 2.0 M KCl solution. However, this is specified
like that because the extraction is carried out like that with the purpose of perform the
determination of nitrate by the Cadmium reduction method. The problem to use the
TNT836 HACH method with this kind of samples is that according to the official
specifications of this method (Appendix 4), potassium ion in a concentration greater than
500 ppm and chloride ion concentrations greater than 500 ppm cause interferences with
the method.
 It was also seen that nitrogen is not one of the certified parameters in the reference
material, so it was not going to be possible to evaluate the trueness of the method.

10. CONCLUSION

Phosphorus and potassium were analyzed in a soil sample by UV-Vis’s spectroscopy and
Flame Atomic Absorption Spectroscopy respectively. Initially, these two elements were
extracted by the use of the extraction solution Mehlich 3 and then preparing every sample
according to the method that was going to be used. It was possible to come to the conclusion
that the determination of phosphorus and potassium is precise when determined by the
methods previously mentioned. However, when it comes to the determination of trueness of the
method, the measurement of phosphorus and potassium was performed in a certified reference
material obtaining for phosphorus between 15.25% to 18.2% of the certified value and for
potassium between 12.68% to 13.28%. Even though it is considered that Mehlich 3 only
extracts available sodium and potassium in soils, the percentages reported does not coincide
with the values expected according to the bibliography consulted. Therefore, neither trueness
nor accuracy can be determined for these two methods at least with the available reference
material.

The determination of nitrogen in the sample was not performed as it was determined that the
HACH method TNT836 was not suitable for this kind of analysis in soils. The main reasons for
this conclusion were related to the type of matrix and interferences of the method with the

47
extraction solution used for nitrates in soils. Moreover, the reference material chosen does not
have nitrogen as one of its certified values, so it would not be possible to evaluate trueness
either.

11. RECOMMENDATIONS

It is necessary to corroborate the trueness of the spectrophotometric methods for the

determination of phosphorus and potassium. As the Mehlich 3 solution only extracts the
available forms of phosphorus and potassium in soil samples, instead of a reference material to
evaluate trueness, it would be recommended to use the values measured of those two elements
by a certified lab and then consider their value as the true value. One of the main reasons to
suggest this is also because after researching the options available in the market, there is no
reference material that contains only available forms of phosphorus and potassium in its matrix.

The cadmium reduction method is the more recommended to carry out when measuring nitrates
in a soil sample. How to perform this test is described in the book “Soil Sampling and Methods
of Analysis (2nd ed.)” pages 73-77. So it is recommended for the Applied research lab at
Cambrian College to acquire the necessary reagents and the laboratory glassware specified in
the mentioned source.

In order to formally validate a method, the Eurachem Guide: The Fitness for Purpose of
Analytical Methods page 8, suggest the following performance parameters: selectivity, limit of
detection (LOD), Limit of quantification (LOQ), working range, Analytical sensitivity, trueness,
precision, uncertainty and ruggedness. It is recommended that all this parameters are evaluated
for the potassium, phosphorus and nitrogen tests. The purpose of this is to be sure that reliable
results are being produced in the lab.

48
12. REFERENCES
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How does Potassium Help Plants Grow? (2019, February 5). Phoslab Environmental

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52
Appendix 1:Mehlich 3-Extractable
Elements

A-1
Appendix 2: Hach Company TNTplus
835/836 Nitrate Method 10206

A-2
Appendix 3: Certificate of analysis of the
reference material

A-3
Appendix 4: Results from Flame AA
potassium test (calibration curve,
sample and reference material)

A-4