Indian Journal of Science and Technology, Vol 9(45), DOI: 10.17485/ijst/2016/v9i45/101163, December 2016
ISSN (Print) : 0974-6846
ISSN (Online) : 0974-5645
Measurement of Uncertainty Associated with
Quantification of Ethephon
Samarth I. Zarad*, Nitin R. Nimkar, Kishor R. Desai, Maheshwari S. Solanki, Devang M. Gandhi,
Harshal M. Gandhi, Aparna A. Khimani, Monali S. Desai and Macky N. Suraliwala
Department of Quality Control, Pollucon Laboratories Pvt. Ltd., Surat - 395003, Gujarat, India;
[email protected], [email protected], [email protected], [email protected],
[email protected], [email protected], [email protected], macky.suraliwala@gmail
Abstract
Objectives: Focus of the Research experiment is to evaluate the uncertainty in Ethephon determination. Validation of
analytical method and identify the factors effecting the uncertainty in measurement. Methods/Statistical Analysis: A
Titrimetric Method was used for determination of Ethephon content. A certified commercially formulated solution of
Ethephon 39.00 % (w/w) S.L has been taken as a sample for analysis. An estimation of uncertainty in quantification of
Ethephon was determined by identifying the following parameters, uncertainty influencing factors and its contribution
to measurement, Type A Uncertainty (UA), Type B Uncertainty (UB), Combined Uncertainty (UC), Expanded Uncertainty
(UE) and uncertainty budget. An Experimentally calculated value of combined uncertainty in preparation of 0.1 N NaOH
solutions is 0.132%. A value of relative standard uncertainty in repeatability test of Ethephon is 0.084%. Determined
% bias value of test method is 0.06%. An estimated value of combined uncertainty in Ethephon determination is 0.373
%. Obtained value of expanded uncertainty with coverage factor of 2.26 with 95% confidence is 0.843%. Obtained %
RSD of Ethephon test result is 0.26 %. LOQ value of analytical test method is 0.36%. A Measured uncertainty value in
Ethephon determination is 39.06 ± 0.33%. Findings: This paper produces Technique for analytical method validation
and uncertainty measurements in quantitative analysis of Ethephon. Application/Improvements: As per regulatory for
laboratory accreditation bodies, it is mandatory to evaluate the uncertainty in measurement. Presented method provides
extensive details regarding uncertainty measurements.
Keywords: Ethephon, Plant Growth Regulator, Uncertainty Measurement, Analytical Validation
1. Introduction
Uncertainty measurements provide an evidence of conformity of test results. As per the guideline of laboratory
accreditation bodies (ILAC, APLAC, NABL, ISO/IEC
17025 etc.) measurement of uncertainty in analytical
method is a mandatory requirement1. The evaluation of
uncertainty requires the analyst to look closely at all the
possible sources of uncertainty.
Ethephon (CAS No: 16672-87-0) is a plant growth
regulator belonging to phosphonate family. Ethephon
is absorbed by the plant and release ethylene which is
a Natural plant hormone. Ethylene influences various
*Author for correspondence
physiological processes like ripening, maturation and
stimulates the production of endogenous ethylene. It is
often used on wheat, cotton, tobacco, coffee and pineapple.
2. Material and Methods
2.1 Reagent and Chemicals
Sodium Hydroxide (NaOH), A.R. grade was purchased
from Merck chemicals India, primary standard potassium
hydrogen phthalate (C8H5KO4) (purity ≥99.5 % ± 0.05%)
Measurement of Uncertainty Associated with Quantification of Ethephon
was purchased from sigma-aldrich, Phenolphthalein
indicator was purchased from Rankem India, Thymol
blue 0.1 % w/v in alcohol indicator was purchased from
fisher scientific. Ethephon 39% S.L. (Soluble concentrate Liquid) solution was purchased from Hindustan
Agrotech Industries India. All the chemicals and reagents
were stored at 25oC ± 2oC and 40% relative humidity controlled room temperature.
2.2 Apparatus
Erlenmeyer flask 250 ml, Burette 50 ml ± 0.05 ml, Pipette
25 ml ± 0.03 ml and measuring cylinder 100 ml ± 0.5 ml
was used for the experiment. Calibrated Class ‘A’ grade
glassware of borosil used for the analysis. An Analytical
weight balance was calibrated using 2 g ± 0.01 g standard
weight.
2.3 Analysis Procedure
2.3.1 Preparation of 0.1 N Potassium Hydrogen
Phthalate Solutions (KHP)
0.2 g of Ethephon (39 % S.L. sol) sample was taken
in 250 ml Erlenmeyer flask, added about 80 ml of freshly
distilled water and stirred gently to dissolve the contents.
Added 2 drops of thymol blue indicator and titrate against
0.1 N NaOH solutions till blue color appears1,3. Repeat the
analysis process ten times and calculate the uncertainty.
Calculation: % Ethephon by (w/w) =V×N×7.225
M
Where, V= Vol. of in ml of NaOH consumed, N =
Normality of NaOH sol., M = Mass in g of sample taken.
3. Result and Discussion
3.1 Fish Bone Diagram of Uncertainty
Influencing Factors in Analysis
The objective of this step is to understand and identify
the major uncertainty sources and its contribution to the
uncertainty2-4. Description was given in Figure 1.
Dry about 25 g of Potassium hydrogen phthalate at 105oC
± 5oC for 2 hrs and allow cooling at room temperature.
Weigh 20.4223 g of dried KHP and dissolve in freshly distilled water and dilute to 1000 ml2.
2.3.2 Preparation and Standardization of 0.1 N
Sodium Hydroxide (NaOH) Solutions
Dissolve 5 g of NaOH in 100 ml of freshly distilled
water and dilute to 1000 ml with freshly distilled water.
Determine the normality of NaOH solution by titrating
25 ml of 0.1 N KHP solutions against NaOH solution
using phenolphthalein as an indicator. Observe end point
at color change from colorless solution to pink color.
Repeat the standardization process in triplicate and take
the mean value of Normal NaOH solution as a Normality
of NaOH2.
2.3.3 Determination of Ethephon Content in
Ethephon 39 % S.L. Solution
This method was based on the neutralization of 2-chloroethyl phosphonic acid in aqueous solution with sodium
hydroxide solution. Titration of Ethephon with 0.1 N
sodium hydroxide using Thymol blue indicator determines the 2-chloroethyl phosphoric acid originally
present2.
2
Vol 9 (45) | December 2016 | www.indjst.org
Figure 1. Fish bone diagram of Uncertainty influencing
factors.
3.2 Quantifying Uncertainty Components
3.2.1 Uncertainty in Weight Balance Calibration
(UB1)
An analytical weight balance was calibrated using certified
2 g ± 0.01 g of standard weight. Rectangular probability
distribution (a/√3) was used for determining standard
uncertainty of weigh balance .obtained value of standard
uncertainty of weight balance is 0.00577 g and % relative
standard uncertainty is 0.289%.
3.2.2 Uncertainty in Burette (UB2) and Pipette
(UB3) Calibration
A triangular probability distribution (a/√6) was used
for evaluation of standard uncertainty of burette and
Indian Journal of Science and Technology
Samarth I. Zarad, Nitin R. Nimkar, Kishor R. Desai, Maheshwari S. Solanki, Devang M. Gandhi, Harshal M. Gandhi, Aparna A.
Khimani, Monali S. Desai and Macky N. Suraliwala
pipette1,5-8. Calculated value of standard uncertainty
of burette was 0.02041 ml and for pipette the obtained
value was 0.01225 ml. As described in Table 1, % relative
standard uncertainty of burette and pipette was 0.041 %
and 0.049% respectively. Calculated value of Combined
Uncertainty of glassware calibration (UC2) was 0.064%.
UC2 =
As this expression is a sum of independent values, the
standard uncertainty of Molar mass of KHP is a simple
square root of the sum of the squares of the contributions:
Combined standard uncertainty in Molar mass of
KHP=
= 0.0038 g/mol
3.2.4 Uncertainty in NaOH Solution Preparation
(UA1
% UC2 =
= 0.064 %
3.2.3 Uncertainty in Purity (UB4) and Molar Mass
(UB5) of KHP
Purity of KHP is ≥ 99.5 %±0.05%. Rectangular probability distribution (a/√3) was used for evaluation of standard
uncertainty. Obtained value of % relative standard uncertainty in purity of KHP is 0.029 %. From the IUPAC table
current at the time of measurement6, the atomic weights
and listed uncertainties for the constituent elements of
KHP (C8H5O4K) were listed in Table 2 and 3.
Three readings were taken for standardization of NaOH
solution. Obtained normality of NaOH solution was
0.1007 N calculated % relative standard uncertainty was
0.129 % as seen in Table 4 and 5.
3.2.5 Combined Uncertainty in NaOH Solution
Preparation (UC1)
As described in Table 6, Obtained value of combined
uncertainty was 0.132%
UC1 =
%
UC1=
= 0.132 %
So, the Molar mass for KHP = 96.0856 + 5.0398 +
63.9976 + 39.0983 = 204.2212 g/mol.
Table 1. Summary of Equations for Uncertainty calculation.
Mean value (x)
Σ xi / n , Where i
Degree of freedom (Ɵ)
n-1 , where n= number of analysis
Standard deviation (σ)
-x)2 where N= number of analysis, x= mean value
,xi= individual analysis value
i
% Relative Standard Deviations (RSD)
, where σ= Standard deviation, x= Mean value
Standard Uncertainty (Ux)
,where n= number of analysis and σ = standard deviation
% Relative Standard Uncertainty
Ux×100/x, where Ux = standard uncertainty and x= mean value
Combined Uncertainty (Uc)
Where, UA= Type A unc and UB= Type B Unc.
Expanded Uncertainty (UE)
K×UC , where K= Coverage Factor and UC = Combined Uncertainty
% Bias
Mean value - Accepted true value
LOQ (Limit Of Quantification)
Va×N×7.225/M. Where Va= least burette reading in ml, N = Normality
of NaOH sol., M= mass in g of sample taken.
Vol 9 (45) | December 2016 | www.indjst.org
Indian Journal of Science and Technology
3
Measurement of Uncertainty Associated with Quantification of Ethephon
Table 2. Normality of NaOH solution.
No.
(n=3)
Normality
of
KHP(N2)
ml of
KHP
taken(V2)
ml of
NaOH
used(V1)
Normality of
NaOH obtained
N1=N2×V2/V1
Mean
(x)
Standard
deviation
(σ)
Standard uncertainty
( σ / √n)
1
0.1
25
24.8
0.1008
0.1007
0.0002
0.00013
2
0.1
25
24.9
0.1004
3
0.1
25
24.8
0.1008
Table 4. Standard uncertainty in g/mol of KHP
Table 3. Standard uncertainty of KHP elements
Element
Atomic
weight
Quoted
Uncertainty
(a)
Standard
Uncertainty
(a/√3)
C
12.0107
±0.0008
0.00046
H
1.00794
±0.00007
0.000040
O
15.9994
±0.0003
0.00017
K
39.0983
±0.0001
0.000058
Elements of
KHP
Calculation
Result
Standard
uncertainty
C8
8×12.0107
96.0856
0.0037
H5
5×1.00794
5.0397
0.00020
O4
4×15.9994
63.9976
0.00068
K
1×39.0983
39.0983
0.000058
Table 5. Summary of uncertainty factors in NaOH solution preparation
Description
Type
of unc
Distribution
Value (x)
Unc. Value
(a)
Standard
uncertainty u(x)
% Relative standard
uncertainty u(x)×100/x
Repeatability(UA1)
A
Normal
0.1007 N
**
0.00013
0.129
Purity of KHP(UB4)
B
Rectangular
(a/√3)
0.995
0.0005
0.00029
0.029
Molar mass of KHP(UB5)
B
Rectangular
(a/√3)
204.2212
g/mol
***
0.0038
0.002
Combined Uncertainty
(UC1)
A,B
-
-
-
-
0.132
Note: ** Refer Table 2, *** Refer Table 3 and 4
Table 6. Analysis results of Ethephon
4
No of sample
(n=10)
wt of
sample
in g
Burette
reading
in ml(A)
blank
reading
in ml (B)
A-B(ml)
%by m/m
Ethephon
(Xi – x)2
Σ(Xi – x)2
Standard
deviation (σ)
1
0.2014
10.9
0.1
10.8
39.02
0.002
0.0958
0.1032
2
0.2162
11.7
0.1
11.6
39.04
0.001
3
0.2690
14.5
0.1
14.4
38.95
0.014
4
0.2052
11.1
0.1
11.0
39.00
0.004
5
0.2257
12.2
0.1
12.1
39.01
0.004
6
0.2130
11.6
0.1
11.5
39.28
0.047
7
0.2184
11.8
0.1
11.7
38.98
0.008
8
0.2271
12.3
0.1
12.2
39.08
0.000
9
0.2083
11.3
0.1
11.2
39.12
0.003
10
0.2117
11.5
0.1
11.4
39.18
0.013
Mean(x)
39.06
Standard Uncertainty (σ/√n)
0.03
Vol 9 (45) | December 2016 | www.indjst.org
Indian Journal of Science and Technology
Samarth I. Zarad, Nitin R. Nimkar, Kishor R. Desai, Maheshwari S. Solanki, Devang M. Gandhi, Harshal M. Gandhi, Aparna A.
Khimani, Monali S. Desai and Macky N. Suraliwala
% relative standard deviation ( σ×100/x)
0.26
Degree of freedom (n-1)
9
%Relative standard uncertainty(Standard Unc×100./x)
0.084
% Bias (x-39.0)
0.06
Table 7. Summary of uncertainty budget
Description
Type of
Unc.
Distribution
Value
(x)
Unc. Value
(a)
Standard
uncertainty
u(x)
% Relative
standard
uncertainty
u(x)×100/x
Unc in Burette Calibration (UB2)
B
triangular
50 ml
0.05
0.0204
0.041
Unc in pipette Calibration (UB3)
B
triangular
25 ml
0.03
0.0122
0.049
% Combined Unc of glassware
calibration (UC2)
0.064
Description
Type of
Unc
Distribution
Value
%value
Combined Unc. in NaOH sol.
preparation(UC1)
A,B
Normal, Rectangular, triangular
0.00132
0.132
Unc in Repeatability(UA2)
A
Normal
0.0008
0.084
Relative Standard Unc of Bias(UBias)
A
Normal
0.00166
0.166
rectangular
0.0029
0.289
Balance uncertainty (UB1)
B
% Combined uncertainty (UC3)
0.373
% Expanded uncertainty (UE)
with coverage factor 2.26 and 95% confidence limit is 0.843
Measured Unc. value
39.06 ± 0.33%
3.2.6 Uncertainty in Repeatability of Ethephon
Test Results (UA2)
3.2.8 Expanded Uncertainty (UE)
Where, Va = least burette reading in ml, N = Normality
of NaOH sol., M = Mass in g of sample taken.
As per the T-distribution table1, with 9 degree of freedom,
value of Coverage factor (K) is 2.26 at 95% confidence
limit. Calculated Expanded Uncertainty in quantification
of Ethephon was 0.843%.
Degree of freedom= n-1, where n = Number of
Readings
= 10-1
=9.
Expanded Uncertainty (UE) = K×UC3
U = 2.26×0.373
% E
= 0.843 %
3.2.7 Combined Uncertainty (UC3)
3.2.9 Uncertainty in Ethephon Determination
For determination of precision in Test results, ten numbers
of experiments were carried out3. Obtained Bias value was
0.06%. Calculated LOQ value of test method was 0.36%.
Obtained % relative standard uncertainty value of repeatability was 0.084%. Data was described Table 7.
Limit of Quantification (LOQ):
% Ethephon=
by ( w / w )
Va × N × 7.225 0.1× 0.1007 × 7.005
=
= 0.36%
M
0.2
Obtained value of Combined Uncertainty of test method
was 0.373%
UC3
=
% UC3 =
= 0.373 %
Vol 9 (45) | December 2016 | www.indjst.org
An Estimated value of uncertainty in Ethephon determination was 39.06 ± 0.33%.
Uncertainty = x×UE
100
% Uncertainty = 39.06×0.843
100
= 0.33%
Indian Journal of Science and Technology
5
Measurement of Uncertainty Associated with Quantification of Ethephon
3.3 Uncertainty Budget
Budget represent the summary of all uncertainty components in tabular form3,6. Uncertainty budget gives quick
view at the totality of information associated with uncertainty measurements. An Estimated value of uncertainty
in Ethephon determination was 39.06 ± 0.33% as seen in
Table 7.
ments by virtue of Precision, LOQ and Uncertainty value
for determination of Ethephon.
5. Acknowledgement
We are Thankful to Pollucon Laboratories Pvt. Ltd, Surat,
India for Funding this project and supporting us during
the experiment of Uncertainty Measurements.
6. References
1. % Uncertainty in Repeatability of Ethephon determination (UA2)
was 0.084.
2. % combined Uncertainty of NaOH Solution preparation (UC1)
was0.132.
3. % Bias was (UBias) 0.166.
4. % Combined Glassware Uncertainty (UC2) was 0.064.
5. % Weigh Balance Uncertainty (UB1) was 0.289.
Figure 2. Calculated values of uncertainty influencing
factors in determination of Ethephon.
4. Conclusion
An estimation of uncertainty in analytical method was
calculated with the parameters such as uncertainty in
glassware calibration, Uncertainty in Analytical method
and uncertainty in reagent preparation. Uncertainty budget was prepared to summarize the measured uncertainty
value and its contribution towards uncertainty measurements description was show in Figure 1 and Figure 2.
Current Method is found suitable for Analytical measure-
6
Vol 9 (45) | December 2016 | www.indjst.org
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Indian Journal of Science and Technology