Umerez, Charles Michael B.

Date Performed: March 28, 2018

Group 2 Date Submitted: April 4, 2018

Exercise 7
Available Micronutrient (Fe, Zn, Cu) Analysis in Soils
I. Introduction
Micronutrients are essential elements needed by the plants in small quantities. It can range
from being deficient to excessive in amount. Micronutrient elements include Fe, Mn, Zn, Cu B, Ni,
and Mo which are generally <100 mg/kg (White, 2006). A lack of any of these micronutrients in
the soil can limit the growth and development of plants.
One of the problems involving around micronutrients is micronutrient deficiencies. From the
microelements mentioned, Zn is likely the most common microelement that is short in supply and
Fe is the most difficult to make available because it is needed in relatively large amounts and soil
chemical processes sometimes quickly make it unavailable (Lohry, 2007).
Micronutrients differ in the form they are adsorbed by the plant., their functions and mobility
in the plant, and their characteristic deficiency or toxicity symptoms. Zn is a component of many
enzymes, essential for plant hormone balance and auxin activity. Fe is a component of enzymes,
essential for chlorophyll synthesis and photosynthesis. Cu is a component of enzymes that is
involved in photosynthesis (Butzen, n.a).
Methods commonly used for extracting micronutriends include the use of chelating agents like
DTPA (diethylenetriaminepentaacetic acid), EDTA (ethylenediamine tetraacetic acid), TEA
(triethanolamine), use of dilute salts (1 N KCl, 1 N NH4-Acetate) and acids (0.1 N HCl) or the
double acid (0.05 N HCl + 0.025 N H2SO4) method of extraction.
II. Objectives
The objective of this exercise is to determine the available Fe, Zn, and Cu levels in soils by
dilute acid extraction and atomic absorption/emission spectroscopy determination
III. Materials and Methods
A. Materials/Reagents
0.05 N HCl solution – exact volume from a 6 N HCl solution was diluted in a 250-mL volumetric
flask and was added with deionized-distilled water to mark.
0.1 N HCl solution – an exact volume from conc. HCl solution was diluted in a 2L volumetric flask
and was added with deionized-distilled water to mark.
Stock (1000 µg/mL) Solutions Fe, Zn, and Cu in 0.1 N HCl solution – previously prepared
Working (100 µg/mL) Standard Fe, Zn, and Cu in 0.1 N HCl solution – 10 mL aliquot from stock
solution was diluted to 100 mL total volume in volumetric flask.
Standard solutions for Fe L (0, 0.6, 2.6, and 6.0 µg/mL µg/mL) – diluted from the 100 µg/mL
working standard solution (in 25-mL volumetric flasks diluted with 0.1 N HCl solution).
Standard solutions for Cu and Zn: (0, 0.05, 0.5, and 1.0 µg/mL) – diluted from 10 µg/mL working
standard solution (in 25-mL volumetric flasks).
B. Procedure
1.) Dilute acid extraction
Fine soil sample (0.5 mm) was used and 1 g of the soil sample was weighed in a clean
P.E. bottle with water tight cover. The sample was added with 20.0 mL of 0.05 N HCl. The P.E
bottles with sample was shaken for 5 minutes in a horizontal shaker. The sample was filtered
through a Whatman No. 5 filter paper into a 20-mL test tube. The filtrate was added with 2 drops
of 6 N HCl. A blank was also ran for the extraction.
2.) Preparation of Standard Solutions
For the preparation of Fe solutions, 0, 0.15, 0.25, and 1.5 mL from the stock solution of
100 µg/mL were obtained and pipetted to a 25-mL volumetric flask in order to obtain the
concentrations 0, 0.6, 2.6, and 6.0 µg/mL, respectively. The solution was diluted to mark with 0.1
N HCl
For the preparation of Zn standards, 0, 0.125, 1.25, and 2.5 mL from the stock solution of
10 µg/mL were obtained and pipetted to a 25-mL volumetric flask in order to obtain the
concentrations 0, 0.05, 0.5, and 1.0 µg/mL, respectively. The solution was diluted to mark with
0.1 N HCl.
For the preparation of Cu standards, 0, 0.13, 1.25, and 2.5 mL from the stock solution of
10 µg/mL were obtained and pipetted to a 25-mL volumetric flask in order to obtain the
concentrations 0, 0.052, 0.5, and 1.0 µg/mL, respectively. The solution was diluted to mark with
0.1 N HCl.
3.) Atomic absorption Spectrophotometric Determination
The Zn, Cu, and Fe content of the extract and the blank was read in the atomic absorption
spectrophotometer (AAS) with the corresponding hollow cathode lamps and parameter settings
at specified λmax for each element.
IV. Data and Observations
Table 7.1. Data on the absorbance of standards for Fe solution.

Volume from Stock Final Volume of Concentration, ug/mL Absorbance

solution (100 ug/mL), dilution, mL
mL
0 25 0 0.0011
0.15 25 0.6 0.0329
0.65 25 2.6 0.1345
1.5 25 6.0 0.2081
Slope 0.03423770492
Y-int 0.01540327869
R2 0.9526089401
Equation of the line y= 0.03x + 0.015
Table 7.2. Data on the absorbance of standards for Cu solution.

Volume from Stock Final Volume of Concentration, ug/mL Absorbance

solution (10 ug/mL), dilution, mL
mL
0 25 0 0.0013
0.13 25 0.052 0.0064
1.25 25 0.5 0.0476
2.5 25 1.0 0.0960
Slope 0.09442852575
Y-int 1.186732008x10-3
R2 0.9998458587
Equation of the line y = 0.094x + 1.187x10-3

Table 7.3. Data on the absorbance of standards for Zn solution.

Volume from Stock Final Volume of Concentration, ug/mL Absorbance

solution (100 ug/mL), dilution, mL
mL
0 25 0 0.0030
0.125 25 0.05 0.0222
1.25 25 0.5 0.0939
2.5 25 1.0 0.1755
Slope 0.1678274209
y-int 8.616874401x10-3
R2 0.9966740554
Equation of the line y=0.168x + 8.617x10-3

Table 7.4 Determination of available micronutrient of the soil sample

Parameters Trial 1 Trial 2 Trial 3

Weight of Soil 1.0022 1.0397 1.0155
Determination of available Zn
Absorbance Sample 0.0471 0.0503 0.0487
Concentration Zn in 0.2293017756 0.2483689815 0.2388353785
sample, µg/mL
Absorbance blank 0.0098 0.0107 -
-3
Concentration blank, 7.049656086x10 0.01241230776 -
µg/mL
Volume of extractant 20 20 20
DF
Available Zn, µg 4.381775966 4.590516487 4.512149613
element/g
Average available Zn, 4.494814022 ± 0.075358704
µg element/g ± dev
Determination of available Cu
Absorbance Sample 0.0410 0.0462 0.0408
 Concentration Cu in 0.4216233143 0.4766914196 0.4195053102
sample, µg/mL
Absorbance blank 0.0028 0.0026 -
Concentration blank, 0.0170845407 0.01496653665 -
µg/mL
Volume of extractant, 20 20 20
mL
DF - - -
Available Cu, µg 8.094148386 b 8.861515455 c 7.94642583
element/g
Average available 8.300696557 ± 0.3738792653
Cu, µg element/g ±
dev
Determination of Available Fe
Absorbance Sample 0.0872 0.1148 0.0973
Concentration Fe in 2.097007422 2.903136222 2.392003831
sample, µg/mL
Absorbance blank 0.0058 0.0061 -
Concentration blank, -0.2804883888 -0.2717261192 -
µg/mL
Volume of extractant, 20 20 20
mL
DF - - -
Available Fe, µg 47.35810569 61.15693904 52.54773186
element/g
Average available Fe, 53.6875922 ± 4.979564562
µg element/g ± dev

V. Results and Discussion

The micronutrients Zn, Cu, and Fe in the Macolod soil sample were extracted and analyzed.
For the extraction method, dilute acid extraction method was used. The reagent used for the dilute
acid extraction was 0.05 N HCl. The microelements Zn, Cu, and Fe behave primarily as cations
in the soil. Fine sample was used for the experiment and 1 g of the sample was obtained. The
sample was added with the extractant (0.05 N HCl). The dilute acid extraction using 0.05 N HCl
extraction is a reliable, rapid, harmless, simple method of measuring the available Zn and Cu.
The sample with the extractant was shaken for 5 minutes. The shaking time can affect the
extraction of micronutrients Cu, Zn, Mn, and Fe. Only after 5 minutes of contact, solution
concentrations are still increasing at a rapid rate. Moderate deviation from the prescribed shaking
time for an extraction method has a potential to significantly change the final extract concentration
(Hoskins and Ross, 2009). The solution was filtered through a Whatman No. 5 filter paper. Also,
the type of filter paper can affect the filtration speed and influence overall contact time. After
filtration, the filtrate was added with 2 drops of 6 N HCl.
Ponnamperumu et al (1981) developed the dilute acid extraction procedure using 0.05 N HCl.
They used this method for the extraction of Zn, Cu, and B in rice soils. This method was adapted
to determine the available micronutrients for soils and crops. They established the critical limit for
Zn and Cu, which are 1.0 mg/kg and 0.1 mg/kg, respectively. Zn and Cu values below the critical
limits are considered deficient already (Ponnamperuma et al, 1981).
After extraction of the micronutrients, the available micronutrients were determined using AAS
method. Calibration curves were established first using the standard solutions prepared for every
element. The obtained R2 for Cu, Zn, and Fe are 0.9998458587, 0.9966740554, and
0.9526089401, respectively. After establishing the calibration curve, the concentration of the
micronutrient in the sample was determined via interpolation of the obtained absorbance to the
calibration curve. The concentration in the blank samples were also determined. The obtained
available Zn in the sample was 4.494814022 µg element/g. The obtained available Cu in the
sample was 8.300696557 µg element/g. Lastly, the obtained available Fe in the sample was
53.6875922 µg element/g. From the data, it can be observed that the sample has a high amount
of available Fe, followed by Cu, and lastly, the sample has Zn as the lowest amount of available
micronutrient. Based from the critical limit of each element, the soil is not deficient from any of the
micronutrient analyzed. The obtained available micronutrient far exceeds the critical limits
established for each element. According to Gupta and Potalia (1987), the established critical limit
of Fe is 4.5 ppm DTPA extractable Fe. The obtained available Fe far exceeds the established
critical limit.
The micronutrient Zn, Mn, Cu, and Fe form complexes with negatively charged surfaces. The
complexes form with organic compounds are more stable than complexes form with the inorganic
ones. The cations Fe, Cu, Mn, Ni and Zn also hydrolyze at pH values between 3 and 10 and
eventually precipitate as insoluble hydroxides (White, 2006). Zinc is an essential component of
RNA polymerase. Without Zn, the enzyme will be inactivated. The decrease in protein content of
zinc-deficient plants is also the result of enhanced rates of RNA degradation. Zn deficiency is
common in weathered and calcareous soils. Fe is required for the formation of chlorophyll in plant
cells. In aerobic soils, iron is in the form of oxides and hydroxides and is largely insoluble. Ferric
iron tends to be tied up in organic chelates. Hence, the concentration of free iron in the soil solution
is exceedingly low in many soils. Copper is an activator of several enzyme systems in plants and
functions in electron transport and energy capture by oxidative proteins and enzymes. A
deficiency interferes with protein synthesis (Lohry, 2007).
Errors in the experiment includes the extraction of the soil sample. As mentioned, a small
deviation from the prescribed shaking time can affect the concentration of the sample. Shaking of
the sample was not exactly 5 minutes so there could be a deviation as a result. Also, the dilute
acid extraction is suitable only for the extraction of Zn, Cu, and B. The method might not be
effective in extracting Fe compared to Zn and Cu.
VI. Summary and Conclusion
Micronutrients are essential elements needed by plants in a relatively small amount. Example
of these micronutrients are Zn, Fe, and Cu. In the experiment, the available micronutrients (Zn,
Fe, and Cu) were determined. Dilute acid extraction was used for the experiment. The extractant
for the dilute acid extraction was 0.05 N HCl. Shaking was done for 5 minutes. After filtering the
extract, it was added with 2 drops of 6 N HCl.
The filtrate was subjected to AAS to quantify the amount of available Zn, Fe, and Cu.
Calibration curve was established by determining the absorbance of the standard solutions. The
R2 obtained for Zn, Fe, and Cu were 0.9966740554, 0.9526089401, and 0.9998458587,
respectively. The obtained amount of available Zn, Fe, and Cu were 4.494814022 µg element/g,
53.6875922 µg element/g, and 8.300696557 µg element/g, respectively. All of the available
micronutrients exceed their critical limits. Errors in the experiment includes personal errors during
the extraction time, and the method is not suitable for extraction of available Fe.
VII. References
Butzen, S. (n.a.). Micronutrients for Crop Production. Pioneer. Retrieved on April 3, 2019 at
https://www.pioneer.com/home/site/us/agronomy/micronutrients-crop-production/
Gupta, V.K. and Potalia, B.S. (1987). Determination of Critical Limit of Fe in Soil For Predicting
Response of Sorghumto Fe Application In Aridisols. Annals of Arid Zon. 26(3): 139-142.
Hoskins, B., and Ross, D. (2009). Soil Sample Preparation and Extraction. Retrieved on April 3,
2019 at http://s3.amazonaws.com/udextension/lawngarden/files/2012/10/CHAP2.pdf
Lohry, R. (2007). Micronutrients: Functions, Sources and Application Methods. Indiana CCA
Conference Proceedings. Retrieved on April 3, 2019 at
https://www.agry.purdue.edu/cca/2007/2007/Proceedings/Raun%20Lohry%20-
%20CCA%20Proceedings_KLS.pdf
Ponnamperuma, F. N., Cayton, M. T., & Lantin, R. S. (1981). Dilute hydrochloric acid as an
extractant for available zinc, copper and boron in rice soils. Plant and Soil, 61(3), 297–
310. doi:10.1007/bf02182011

VIII. Sample Calculations

1.) Concentration of Standard Solution
C1V1 = C2V2
𝜇𝑔
10 ∗0.125𝑚𝑙
𝑚𝑙
𝐶2 = 25
= 0.05 𝜇𝑔/𝑚𝐿

2.) Concentration Zn of Sample (Trial 1)

Concentration, ug/mL Absorbance

0 0.0030
0.05 0.0222
0.5 0.0939
1.0 0.1755
Equation of the line: y=0.168x + 8.617x10-3
Concentration Zn= 0.0471x̅ = 0.2293017756
𝜇𝑔
3.) Available Zn (𝑔 𝑠𝑜𝑖𝑙)

𝜇𝑔
𝜇𝑔 (0.2293017756 𝑚𝐿) − 9.730981923x10^(−3) 𝜇𝑔/𝑚𝐿) 𝑥 20 𝑚𝐿
Zn ( ) =
𝑔 1.0022, 𝑔
𝜇𝑔 𝜇𝑔
Zn ( 𝑔 ) =4.381775966 𝑔
4.) Average of 3 trials
𝜇𝑔 4.381775966 + 4.590516487 + 4.512149613
Zn ( )=
𝑔 𝑠𝑜𝑖𝑙 3
𝜇𝑔
𝐴𝑣𝑒 Fe (𝑔 𝑠𝑜𝑖𝑙 ) =4.494814022