Chem 137.

1 1L | UPLB 1
Exercise No 1 | 2019

Exercise No 1
Sampling in Chemical Analysis

Marinella Q. Escano*, Mary Jean Barranco, Renz Danan, Rubemar Ordonio

*mqescano@up.edu.ph

Abstract
As analytical methodology improves and instrumental methods allow, or often require, the use of smaller
and smaller analytical test portions, the error in the sampling operations becomes increasingly significant. Also,
heterogeneity of trace components can introduce major sampling problems. Sampling errors cannot be controlled by
use of blanks, standards, or reference samples and so are best treated independently. Variance, which is the square
of the standard deviation (s2), is used because, unlike standard deviations, variances are additive. Thus, the variance
of a method is the sum of the variances of its component operations In this exercise, nested design was used to
experimentally analyze the different factors affecting the variance of a chemical analysis. Based on the results, Level
I (sampling), which have the largest variance of 1.47E-05, is the weakest link of the analysis. Moreover, the Ingamell’s
sampling constant was also determined. The calculation showed that the average Ingamell’s sampling constant using
different nominal masses was 48.3. Ingamell’s sampling constant, Ks, is useful for predicting the sampling variance
because it is equivalent to the mass of sample giving a percent relative standard deviation due to sampling of 1%.

Keywords: Sampling, variance, Nested design, Ingamell’s sampling constant, UV-vis spectrophotometry

I. Introduction
Chemical analysis involves three major operations—sampling, sample preparation, and measurement.
Although powerful instrumental techniques such as ICP-OES tend to focus attention on measurement, the other two
operations are also critical to die quality of the data obtained from the analysis (Settle and Pleva, 1999). The quality
of the data can be no better than the least precise operation in the method; thus, while the precision of the measurement
may be high, the overall data quality may be much lower because of imprecise sampling or sample preparation
techniques.
Each step of the chemical analysis can contribute to random errors denoted as standard deviation or variance.
Variance, which is the square of the standard deviation (s2), is used because, unlike standard deviations, variances are
additive. Total variance, σ2total, is the sum of the variances in each step of chemical analysis which can be consists of
the variance for the preparation of the samples, σ2prep, the variance for the measurements, σ2meas, and the variance for
obtaining samples, σ2samp. In a spectrophotometric analysis there are variance due to the spectrometer’s source,
detector, and optics, σ2spect, and variance to the partitioning of the sample cell within the spectrophotometer, σ 2pos. A
simple determination of σ2total does not provide enough information to partition the overall variance into its component
parts.
𝜎 2 𝑡𝑜𝑡𝑎𝑙 = 𝜎 2 𝑠𝑎𝑚𝑝𝑙𝑒 + 𝜎 2 𝑝𝑟𝑒𝑝 + 𝜎 2 𝑚𝑒𝑎𝑠 Equation 1.1

𝜎 2 𝑡𝑜𝑡𝑎𝑙 = 𝜎 2 𝑠𝑎𝑚𝑝𝑙𝑒 + 𝜎 2 𝑝𝑟𝑒𝑝 + 𝜎 2 𝑝𝑜𝑠 + 𝜎 2 𝑠𝑝𝑒𝑐𝑡 Equation 1.2

Nested design, sometimes referred to as a hierarchical design, is used for experiments in which there is an
interest in a set of treatments and the experimental units are sub-sampled, and one of the approaches that can be used
to find values of individual variances (Harvey, 2002). It consists of several levels, with the number of levels equal to
the number of parameters you wish to evaluate. In this experiment, the total variance of a quantitative
spectrophotometric analysis will be determined using four levels: the contribution of sampling, sample preparation,
the positioning of the sample cell, and the spectrometer. Figure 1.1 was shown below to illustrate the relationship
between samples at different levels. Samples are coded using a Roman numeral for Level I, an uppercase A or B for
Level II, the number 1 or 2 for Level III, and a lower case a or b for Level 4.
The objective exercise is to identify the weakest or least precise operation in an analysis and evaluate the
magnitudes of the variance for each of the four operations. In addition, the sampling constant should be determined.
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Figure 1. Four-level nested design showing the relationship between samples at Levels I, II, III, and IV (Harvey, 2002)

II. Methodology
This experiment uses a four-level nested design to determine the variances due to sampling, sample
preparation, the spectrometer, and the sample cell’s positioning in the spectrometer (Figure 1.1). The first level
consists of four samples collected randomly from the gross sample. After grinding the Level I samples, duplicate
samples are obtained from each and diluted to volume in volumetric flasks, providing the eight Level II samples. Each
Level II sample is divided in half, yielding the 16 Level III samples. Finally, each Level III sample is placed in the
spectrometer and its absorbance is measured twice without repositioning the sample, providing the 32 Level IV
samples. The concentration of congo red is determined spectrophotometrically at a wavelength of 526 nm, providing
a rapid analysis using commonly available instrumentation. The % w/w congo red is calculated for each using Beer’s
law with an absorptivity of 0.0916 ppm-1 cm-1. The % w/w congo red for each Level I–III sample is the average result
for the corresponding Level IV samples. The result for sample IA, for example, is the average result for samples IA1a,
IA1b, IA2a, and IA2b.
For evaluating the sampling constant, six replicate samples of the gross sample are prepared for each
condition (nominal mass and volumetric flask) listed in Table 1.6. Samples are analyzed spectrophotometrically and
the % w/w congo red calculated. The absorbance of last replicate of nominal mass of 1.0 g was measured 10 additional
times using different aliquots of the solution for the determination of variance due to measuring absorbance.

III. Results and Discussion

The issue of obtaining a representative sample is significant. In heterogeneous materials, the variance
associated with the sampling component is expected to be the largest source of uncertainty in an analytical method.
The procedures for sampling solid materials include considerations of the minimum mass, particle size, and sample
splitting (Settle, 1984). Nested design is a statistical approach used in the experiment to find the values for individual
variances. Different levels contributes to the total variance of the analysis. The variance for the level IV, 𝑠 2 𝐼𝑉 , is
influenced only by the spectrometer’s variance and calculated as
∑ 𝑖(𝑑𝐼𝑉 )2 𝑖
𝑠 2 𝐼𝑉 = 𝑠 2 𝑠𝑝𝑒𝑐𝑡 = Equation 1.3
8𝑛

where dIV is the difference between related Level IV samples, and n is the number of Level I samples. Table 1.1
showed the calculated % w/w congo red and its variance for level IV.

Table 1.1. Data results on Level IV for nested design.

Sample ID Absorbance at 526nm Mass of sample % (w/w) congo red Variance
IA1a 0.157 0.2547 0.0343 1.78E-08
IA1b 0.159 0.2547 0.0347
IA2a 0.163 0.2547 0.0356
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IA2b 0.165 0.2547 0.0360

IB1a 0.148 0.257 0.0320
IB1b 0.147 0.257 0.0318
IB2a 0.149 0.257 0.0323
IB2b 0.149 0.257 0.0323
IIA1a 0.142 0.2567 0.0308
IIA1b 0.143 0.2567 0.0310
IIA2a 0.148 0.2567 0.0321
IIA2b 0.147 0.2567 0.0319
IIB1a 0.148 0.2523 0.0326
IIB1b 0.148 0.2523 0.0326
IIB2a 0.149 0.2523 0.0329
IIB2b 0.149 0.2523 0.0329
IIIA1a 0.17 0.2555 0.0370
IIIA1b 0.17 0.2555 0.0370
IIIA2a 0.173 0.2555 0.0377
IIIA2b 0.173 0.2555 0.0377
IIIB1a 0.199 0.2512 0.0441
IIIB1b 0.199 0.2512 0.0441
IIIB2a 0.201 0.2512 0.0445
IIIB2b 0.201 0.2512 0.0445
IVA1a 0.174 0.2536 0.0382
IVA1b 0.174 0.2536 0.0382
IVA2a 0.172 0.2536 0.0377
IVA2b 0.173 0.2536 0.0380
IVB1a 0.159 0.253 0.0350
IVB2b 0.159 0.253 0.0350
IVB2a 0.158 0.253 0.0348
IVB2b 0.158 0.253 0.0348

The variance for Level III 𝑠 2 𝐼𝐼𝐼 , includes contributions from the spectrometer and the positioning of the
sample cell. It is computed as
𝑠 2 𝑠𝑝𝑒𝑐𝑡 ∑ 𝑖(𝑑𝐼𝐼𝐼 )2 𝑖
𝑠 2 𝐼𝐼𝐼 = 𝑠 2 𝑝𝑜𝑠 + = Equation 1.4
2 4𝑛

where dIV is the difference between related Level III samples. The factor of 2 in the term for the spectrometer s variance
accounts for the two Level IV samples used to determine the result for each sample in Level. The variance 𝑠 2 𝐼𝐼𝐼 has
2n degrees of freedom. In the experiment, the % congo red was determined by getting the average of the associated
results for Level IV. Table 1.2 showed the calculated % w/w congo red and its variance for Level III.

Table 1.2. Data results on the Level III for nested design.
Sample ID % (w/w) erythrosin B Variance
IA1 0.0345 2.39E-07
IA2 0.0358
IB1 0.0319
IB2 0.0323
IIA1 0.0309
IIA2 0.0320
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IIB1 0.0326
IIB2 0.0329
IIIA1 0.0370
IIIA2 0.0377
IIIB1 0.0441
IIIB2 0.0445
IVA1 0.0382
IVA2 0.0378
IVB1 0.0350
IVB2 0.0348

The variance for the Level II samples, 𝑠 2 𝐼𝐼 , includes contributions from the spectrometer, the positioning of
the sample cell, and the sample preparation; thus
𝑠 2 𝑝𝑜𝑠 𝑠 2 𝑠𝑝𝑒𝑐𝑡 ∑ 𝑖(𝑑𝐼𝐼 )2 𝑖
𝑠 2 𝐼𝐼 = 𝑠 2 𝑝𝑟𝑒𝑝 + + = Equaton 1.5
2 4 4𝑛

where dII is the difference between related Level II samples. The factors of 2 and 4 in the term for the variances due
to the sample cell’s positioning and the spectrometer, respectively, account for the two Level III samples and the four
Level IV samples used to determine the result for each Level II samples. The variance, 𝑠 2 𝐼𝐼 , has n degress of freedom.
The % congo red was also determined the same manner for Level III. The values of % congo red and variance were
shown in Table 1.3.

Table 1.3. Data results on the Level II for nested design.

Sample ID % (w/w) congo red Variance
IA 0.0352 8.69E-06
IB 0.0321
IIA 0.0314
IIB 0.0328
IIIA 0.0374
IIIB 0.0443
IVA 0.0380
IVB 0.0349

Lastly, the Level I variance, 𝑠 2 𝐼 , is determined using the standard equation for the variance, and includes
contributions from sampling, sample preparation, the positioning of the sample cell, and the spectrometer.
𝑠 2 𝑝𝑟𝑒𝑝 𝑠 2 𝑝𝑜𝑠 𝑠 2 𝑠𝑝𝑒𝑐𝑡 ∑ 𝑖(𝑋𝑖 −𝑋)2
𝑠 2 𝐼 = 𝑠 2 𝑠𝑎𝑚𝑝 + + + = Equation 1.6
2 4 8 𝑛−1

where Xi is the result for each Level I sample and x̅ is the average result for all Level I samples. The factors of
2, 4, and 8 in the terms for the variances due to sample preparation, the positioning of the sample cell, and the
spectrometer, respectively, account for the two Level II samples, four Level III samples, and eight Level IV
samples used to determine the result for each Level I sample. The variance 𝑠 2 𝐼 has n – 1 degrees of freedom.
Same calculation with Level II and III was done to obtain the % w/w congo red. Table 1.4 showed the calculated
values of % congo red and its variance.

Table 1.4. Data results on the Level I for nested design.

Sample ID % (w/w) congo red Variance
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I 0.0336 1.47E-05
II 0.0321
III 0.0408
IV 0.0364

The difference between the results for samples IA1a and IA1b in Level IV are influenced only by
indeterminate errors since the source of variance is only from instrument used, the spectrometer. The same is true for
the difference between IA1 and IA2 for Level III because it concerns only from the spectrometer and the positioning
of the sample cell. However, samples IA and IB, or samples I and II are influenced by systematic error due to the
sampling preparation and sampling such as inaccurate measurement of samples, and bias dilution in the volumetric
flasks. Based on the calculated variances for each level, Level I has the largest variance among of them, which means
it is the weakest link of the analysis. Factors contributing to these differences are the distribution of the salt within the
sample, the method of sampling, and the efficiency of the extraction process (Settle and Pleva, 1999). The overall
standard deviation for the analysis can be improved by working on the accuracy and precision of the measurement
done for the analysis. The data from the four-level nested design can be also used to evaluate the accuracy of the
analysis. The best experimental estimate of the % w/w congo red is the average result for the Level II sample. It is
because most of uncertainty obtained in the sample preparation for the analysis.

Table 1.5. Calculated variances using the nested design.

Parameter Values
σ2samp 1.47E-05
σ2prep 8.69E-06
σ2pos 2.39E-07
1.78E-08
σ2spect
2.36E-05
σ2total

When the variance in sampling is large, a highly precise analysis is wasted, since s2sampling >> s2meas and
s sampling >> s2prep. Thus, s2total ≈ s2sampling. Generally, the variances due to the analysis and the sampling steps should be
2

similar in order to minimize analysis costs; i.e. it is not cost effective to perform a highly precise analysis when the
sampling variance is high. Since sampling is the weakest link of the experiment, the sampling plan should be designed
to minimize its contribution to the overall variance.one way to address this is by increasing the gross sample’s
homogeneity before collecting individual samples. For instance, grinding the gross sample will help to decrease the
average particle size (Kratochvil, 1984). Making a composite sample is another approach that can be used. This can
be done by collecting several portions of the gross sample and mixing them together before they are analyzed. To
calculate its total variance, equation 1.7 can be used, as shown below

𝜎 2 𝑡𝑜𝑡𝑎𝑙 = 𝜎 2 𝑠𝑎𝑚𝑝 + 𝜎 2 𝑚𝑒𝑡 ℎ Equation 1.7

𝜎 2 𝑠𝑎𝑚𝑝 𝜎 2 𝑚𝑒𝑡 ℎ
𝜎 2 𝑡𝑜𝑡𝑎𝑙 = + Equation 1.8
𝑛 𝑛

where σ2samp is the sampling variance for a particular sample size, and σ2met h is the variance due to the analysis.
Equation 1.8 is used if we collect and analyze n separate samples of the same size. However, if a composite sample is
formed by mixing together these n samples and removing k identical samples for the analysis, the total variance is
computed using equation 1.9.
𝜎 2 𝑠𝑎𝑚𝑝 𝜎 2 𝑚𝑒𝑡 ℎ
𝜎 2 𝑡𝑜𝑡𝑎𝑙 = + Equation 1.8
𝑛𝑘 𝑛
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Using the results for σ2samp and σ2met h, the total variance for the analysis of four separate sample taken from
the gross sample was 8.18E-12 while the total variance for the analysis of two portions of a composite sample formed
by combination of n and k is 3.14E-06

For the second part of the experiment, three sources of variance were evaluated for the analysis of a solid
mixture. Each nominal mas has six replicates and the absorbance of each replicate was measured using
spectrophotometer as shown in table 1.6 and the % w/w congo red were summarized in Table 1.7. The variances of
each nominal mass were calculated as shown in Table 1.8. These variances are the total variance for the analysis of a
given nominal mass, and include the variances in sampling, variances in preparing samples, and variances in
measuring absorbance.

Table 1.6. Absorbances of the samples for each nominal mass.

Nominal Mass of sample Absorbance at 526 nm
mass 1 2 3 4 5 6 1 2 3 4 5 6
0.25 g 0.2517 0.2506 0.2545 0.2574 0.2531 0.2514 0.300 0.357 0.339 0.321 0.363 0.414
0.50 g 0.5086 0.5082 0.5083 0.5073 0.509 0.5045 0.252 0.359 0.282 0.309 0.294 0.316
1.00 g 1.0468 1.0354 1.0306 1.0279 1.0485 1.012 0.341 0.317 0.293 0.290 0.340 0.311

Table 1.7. Percent (w/w) congo red of the samples.

Nominal mass % (w/w) congo red
1 2 3 4 5 6
0.25 g 0.0332 0.0396 0.0371 0.0347 0.0399 0.0458
0.50 g 0.0276 0.0393 0.0309 0.0339 0.0321 0.0349
1.00 g 0.0363 0.0341 0.0316 0.0314 0.0361 0.0342

Table 1.9. Determination of the total variance.

Nominal mass (g) Total variance
0.25 2.03497E-05
0.50 1.57424E-05
1.00 4.36105E-06
For the determination of variance due to measuring absorbance, 10 absorbance measurements for the last
replicate sample of nominal mass 1.00 g were used, and this is the same for all nominal masses. The calculated values
were shown in table 1.10 wherein the variance is 1.49 x 10-7. Moreover, the variance due to sample preparation was
obtained by using a propagation error. Upon calculation, the variances obtained are shown in table 1.11. Since there
are values for the total variances, the variance due to the measurement of absorbance, and variance due to sample
preparation, the variance due to sampling for each nominal mass can be obtained as shown in table 1.12.

Table 1.10. Determination of the variance due to measuring absorbance.

Replicate Absorbance Mass sample % (w/w) Variance
1 0.325 1.012 3.57E-02 1.49E-07
2 0.323 3.55E-02
3 0.329 3.62E-02
4 0.329 3.62E-02
5 0.330 3.63E-02
6 0.332 3.65E-02
7 0.332 3.65E-02
8 0.333 3.66E-02
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9 0.333 3.66E-02
10 0.333 3.66E-02

Table 1.11. Determination of the variance due to sample preparation.

Nominal mass Average mass Ave. % w/w congo uncertainty Variance
(g) red Mass volume
0.25 0.2531 0.038379 0.0001 0.03 2.581E-09
0.50 0.5077 0.033106 0.0001 0.05 8.506E-17
1.00 1.0335 0.033942 0.001 0.08 2.894E-09

Table 1.12. Determination of the variance due to sampling.

Nominal mass (g) Variance
0.25 2.019810515E-05
0.50 1.5592486E-05
1.00 4.214590346E-06

Table 1.13. Determination of Ks.

Nominal Average mass, Average Standard deviation Ingamell’s sampling Average Ks
mass, g g %(w/w) constant, Ks
congo red
0.25 0.2531 0.038379 4.494230207E-03 34.70915671 48.24682727
0.50 0.5077 0.033106 3.948732202E-03 72.22147134
1.00 1.0335 0.033942 2.052946747E-03 37.80985376

Ingamell’s sampling equation, as shown below, is used in sampling statistics for many well mixed samples due to its
consistency. Ingamell’s sampling constant, K s, is useful for predicting the sampling variance because it is equivalent
to the mass of sample giving a percent relative standard deviation due to sampling of 1% (Harvey, 2002).

𝐾𝑠 = 𝑚𝑅2 Equation 1.9

m= sample’s mass R= percent relative stanad deviation Ks= Ingamell’s sampling constant

Figure 1.2. Results used to compile the information in Table 1.13. The solid lines represent boundaries of one
standard deviation about the overall mean of 0.035% w/w using an average K s of 48.
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The analysis is dominated by sampling uncertainty and that the experimentally determined total variance
provides a good estimate for σ2samp. As noted by Guy et al. (1998), it can be verified by using a propagation of error
to estimate σ2prep and estimating σ2meas by measuring the absorbance of any sample several times. Values of Ks are
determined using equation 1.9, which gives an average value of 48.2. The solid curves in Figure 1.2 represent
boundaries of one standard deviation, calculated using equation 1.9 and the average value for Ks, around the overall
mean concentration of 0.035 % w/w congo red.

IV. Conclusion
The goal of a quantitative analysis is to determine the amount of analyte in a sample with accuracy and
precision. An unusually large variance occurs when the analyst fail to reproducibly carry out the steps of an analysis,
and that results that are consistently too high or too low indicate the presence of a significant determinate error. The
sources of variance in an analysis are additive; thus, the total variance, σ2total, for an analysis can be partitioned into
that due to sampling, σ2samp,that due to sample preparation, σ2prep, and that due to the measurements, σ2meas. In this
exercise, nested design was used to experimentally analyze the different factors affecting the variance of a chemical
analysis. Based on the results, Level I (sampling), which have the largest variance of 1.47E-05, is the weakest link of
the analysis. Moreover, the Ingamell’s sampling constant was also determined. The calculation showed that the
average Ingamell’s sampling constant using different nominal masses was 48.3. Ingamell’s sampling constant, K s, is
useful for predicting the sampling variance because it is equivalent to the mass of sample giving a percent relative
standard deviation due to sampling of 1%.

V. References
 RD Guy, L Ramaley, and PD Wentzell. 1998. An Experiment in the Sampling of Solids for
Chemical Analysis J. Chem. Educ. 75, 1028–1033.
 DJ Harvey. 2002. Two experiments illustrating the importance of sampling in a quantitative
chemical analysis. J Chem Educ. Vol. 79. No.3, 360-363.
 B Kratochvil, D Wallace, and J Taylor. 1984. Sampling in chemical analysis. Analytical Chemistry
Vol.56 (5), 113-129
 FA Settle and M Pleva. 1999. The weakest link exercise. Anal. Chem. 71, 538A-540A.