Macquarie

University
Faculty of Science and Engineering
Department of Chemistry and Biomolecular Sciences
CBMS308 / CBMS825
Chemical Analysis II
2018
Model Solutions to Assignment 2

Question 1:

Part (a)

In a standard, the measured [Cu] in HNO3 was determined to be very similar to the actual
[Cu] with a small relative standard deviation of 1.1%. Similar result was observed when
Cu was detected in HNO3 in a reference sample. However, when Cu was detected in HNO3
and glycerol, the measured [Fe] was much lower than that the actual [Cu] with a larger
relative standard deviation.

Part (b)

The observation in (a) could be caused by the complexation between for copper and
glycerol, reducing the quantity of free copper atoms to be detected. The presence of
glycerol would have also increased viscosity of the solution, and in turn the surface
tension, reducing the efficiency of nebulisation, making atomisation harder to occur.

Part (c)

Three possible methods of overcoming the problem include
1. adding a releasing agent that forms more stable complexes with sulfate than
iron formed;
2. adding a protective agent, such as EDTA, that forms a stable, but volatile
complex with iron;
3. using a higher temperature flame.




Question 2:

(a) The higher result in Experiment 2 compared to Experiment 1 is likely to have caused
by the effect of diluting the interfering species, so they do not interfere as much in
Experiment 2 as in Experiment 1. Dilution lowers the concentration of species that
might react with Li or generate smoke that scatters light. In Experiment 3,
interference is present to the same extent as in Experiment 2, but the standard
addition procedure corrects for the interference. The whole point of standard
addition is to measure the effect of the complex interfering matrix on the response
to known quantities of analyte.

(b) Experiment 4-6 use a hotter flame than Experiment 1-3. High temperature appears
to eliminate most of the interference observed at lower temperature. Dilution has
only a minimal effect on the results.

(c) In Experiment 1-3, standard addition gives a more realistic / representative result,
we surmise that Experiment 3 and 6, and possibly Experiment 5, are within
experimental error of each other. The more realistic / representative result can
therefore be the mean of Experiment 3 and 6 (81.4 ppm). It might also be
reasonable to take the average of Experiment 3, 5 and 6 (80.8 ppm).




Question 3:

La3+ is a well-known releasing agent. It will preferentially react with PO3-4 , which would
otherwise have complexed with Pb, making the detectable quantity of Pb less than
expected. Therefore, La3+ is deliberately added to minimise matrix interference of Pb
during the analysis of bone samples.




Question 4:

The dissociation energy of YC is greater than that of BaC. Therefore, the equilibrium
BaC + Y Ba + YC
is driven to the right, increasing the concentration of free Ba atoms in the gas phase.

Page 2 of 15
Question 5:

A standard addition calibration was used in this experiment. Accordingly, we have
obtained the following calibration plot.

We need to initially prove that the above is an acceptable linear relationship.

(1) Physically plotting the graph. We have duly done so above and the graph appears to
be linear.

(2) Testing for the statistical significance of the correlation coefficient.

Null hypothesis – Absorbance and VCa2+ are not linearly related.

Alternative hypothesis – Absorbance and VCa2+ are linearly related.

r n # 2 0.9996 8 − 2
Test statistic - = = 86.58
1# r 2 1− (0.9996) 2
Critical value – Based on a two-sided t-test, the critical value is 2.45 for 6 degrees of
freedom at the 95% confidence level.
Decision – As 86.58 > 2.45, we will reject the Null hypothesis.
Conclusion - Absorbance and VCa2+ are linearly related.

Page 3 of 15
(3) Examination of the residual plot using runs test.

The residual plot is constructed based on values tabulated in the following table.

VCa2+ / mL ExptA PredictedA Residual

0.00 0.151 0.158 -0.00690
1.00 0.185 0.186 -0.00109
3.00 0.247 0.242 0.00453
5.00 0.300 0.299 0.00115
8.00 0.388 0.383 0.00458
10.00 0.445 0.440 0.00520
15.00 0.572 0.581 -0.00875
20.00 0.723 0.722 0.00130

10

5
3
Residual-x-10

VCa2+-/-mL

5 10 15 20 25

!5

!10


The residual plot clearly shows randomly distributed data points, supporting a linear
relationship between and absorbance and VCa2+ .

The signs of the set of results: – – + + + + – + ⇒ N1 = 3 negatives and N2 = 5 positives.

Null hypothesis – A curve is not a better fit between Absorbance and VCa2+ .
Alternative hypothesis – A curve is a better fit between Absorbance and VCa2+ .
Test statistic – The above results gave a total of 4 runs.
Critical value – For N1 = 3 and N2 = 5 runs test table indicates a range between 1 and 8.
Decision – As 4 is between 1 and 8, we will accept Null hypothesis.
Conclusion – A curve is not a better fit between Absorbance and VCa2+ .

Page 4 of 15
(4) Conducting ANOVA.

VCa2+ (AbsPred – (AbsPred –
AbsExpt AbsPred Residual (Residual)2
Absmean) Absmean)2
0.00 0.151 0.158 -0.00690 4.761E-05 -0.2184 0.0477
1.00 0.185 0.186 -0.00109 1.188E-06 -0.1904 0.0362
3.00 0.247 0.242 0.00453 2.052E-05 -0.1344 0.0181
5.00 0.300 0.299 0.00115 1.323E-06 -0.0774 0.0060
8.00 0.388 0.383 0.00458 2.098E-05 0.0066 0.0000
10.00 0.445 0.440 0.00520 2.704E-05 0.0636 0.0040
15.00 0.572 0.581 -0.00875 7.656E-05 0.2046 0.0419
20.00 0.723 0.722 0.00130 1.690E-06 0.3456 0.1195
Absmean Σ= Σ = 0.2734
= 0.3764 0.0001969

Sources of variation Degrees of freedom Sum of squares Mean square F
Regression 1 0.2734 0.2734
Residuals 8–2=6 0.0001969 3.2817×10-5 8331.05
Total 8–1=7

Null hypothesis – There is not a linear relationship between Absorbance and VCa2+
Alternative hypothesis – There is a linear relationship between Absorbance and VCa2+
Test statistic – 8331.05
Critical value – From a one-sided F-test with 1 and 6 degrees of freedom at 95%
confidence level, the critical value is 5.99
Decision – As 8331.05 > 5.99, we will reject the Null hypothesis
Conclusion – There is a linear relationship between Relative Peak Area and
Concentration


After proving a linear relationship between absorbance and VCa2+ , we can now confidently
determine the x-intercept: 0 = 0.1579 + 0.02819 VCa2+ ⇒ x-intercept = 5.60 mL.

As the Ca standard used = 20.0 µg mL-1, the x-intercept value corresponds to
5.60 mL × 20.0 µg mL-1= 112.0 µg.

This is the mass of Ca in 5.00 mL of unknown. The total volume of unknown was 100.0
100.0$mL
mL, the mass of Ca in total unknown = ×112.0$µg = 2240 µg.
5.00$mL

Page 5 of 15
 2240 ×10%6 g
Hence, ×100 = 0.429%- w/w
0.5216 g
S y/x 1 y2
V
Also, the standard deviation of the extrapolated Ca2+ sx = +
E slope n (slope)2 2
∑ xi ! x ( )
i

We will perform all the necessary calculations in the table below.

VCa2+ / mL y ŷ y - ŷ (y - ŷ )2 x!x ( x ! x )2

0.00 0.151 0.158 -0.00690 4.761E-05 -7.75 60.0625
1.00 0.185 0.186 -0.00109 1.188E-06 -6.75 45.5625
3.00 0.247 0.242 0.00453 2.052E-05 -4.75 22.5625
5.00 0.300 0.299 0.00115 1.322E-06 -2.75 7.5625
8.00 0.388 0.383 0.00458 2.098E-05 0.25 0.0625
10.00 0.445 0.440 0.00520 2.704E-05 2.25 5.0625
15.00 0.572 0.581 -0.00875 7.656E-05 7.25 52.5625
20.00 0.723 0.722 0.00130 1.69E-06 12.25 150.0625

x = 7.75 y = 0.3764 Σ=19691×10-4 Σ=343.5

2
∑( y # ŷ )
i i
0.00019691
Therefore, S y/x = i
= = 5.72873×10−3
n$2 8−2
5.72873×10−3 1 (0.376375)2
sx = + = 0.2032 × 0.80246 = 0.163
E
0.02819 8 (0.02819)2 × 343.5

In other words, the x-intercept is 5.60 mL with a standard deviation of 0.16 mL.

⇒ Standard deviation of mass of Ca = 0.163 × 20 µg = 3.26 µg.
100.0$mL
⇒ Standard deviation of mass of Ca in total unknown = 3.26 × = 65 µg.
5.00$mL
65×10%6 g
⇒ Standard deviation of %weight = ×100 = 0.012%+
0.5216 g
Accordingly, the 95% confidence interval = t6 × 0.012 = 2.45 × 0.012 = 0.029%

Hence, 0.429% ± 0.029% w/w.

Page 6 of 15
Question 6:

We clearly need to initially establish a calibration plot based on the standards used.

70

60

50
Emission intensity

40

30

20

-1
10 Emission intensity = 3.18 + 9.20 x VK / µg mL
2Cr 2O7

Correlation coefficient = 0.9996

0 2 4 -1
6 8
Concentration of K2Cr2O7 / µg mL


We should prove that the above is a statistically acceptable linear relationship before
progressing.

(1) Physically plotting the graph. We have duly done so above and the graph appears to
be linear.

(2) Testing for the statistical significance of the correlation coefficient.

Null hypothesis – Emission intensity and VK Cr O are not linearly related.
2 2 7

Alternative hypothesis – Emission and VK Cr O are linearly related.

2 2 7

r n # 2 0.9996 5− 2
Test statistic - = = 61.2
2 2
1# r 1− (0.9996)
Critical value – Based on a two-sided t-test, the critical value is 3.18 for 3 degrees of
freedom at the 95% confidence level.
Decision – As 61.2 > 3.18, we will reject the Null hypothesis.
Conclusion – Emission intensity and VK Cr O are linearly related.
2 2 7

Page 7 of 15
(3) Examination of the residual plot using runs test.
1.5

1.0

0.5

-1
[K2Cr2O7] / µg mL

Residual
2 4 6 8

-0.5

-1.0

-1.5

Despite the small number of data points, the residual plot appears to show randomly
distributed data points, suggesting a linear relationship.

The signs of the set of results: – – + – + ⇒ N1 = 3 negatives and N2 = 2 positives.
Null hypothesis – A curve is not a better fit between Emission intensity and VK Cr O .
2 2 7

Alternative hypothesis – A curve is a better fit between Emission intensity and VK Cr O .

2 2 7

Test statistic – The above results gave a total of 4 runs.

Critical value – For N1 = 3 and N2 = 2 runs test table indicates a range between 1 and 6.
Decision – As 4 is between 1 and 6, we will accept Null hypothesis.
Conclusion – A curve is not a better fit between Emission intensity and VK Cr O .
2 2 7

(4) Conducting ANOVA.

[Cr2O7]2- / (IPred – (IPred –
IExpt IPred Residual (Residual)2
µg mL-1 Imean) Imean)2
0 3.1 3.18 -0.08 0.0064 -36.8 1354.24
2 21.5 21.58 -0.08 0.0064 -18.4 338.56
4 40.9 39.98 0.92 0.8464 0.00 0.00
6 57.1 58.38 -1.28 1.6384 18.4 338.56
8 77.3 76.78 0.52 0.2704 36.8 1354.24
Imean
Σ = 2.768 Σ = 3385.6
39.98

Page 8 of 15
 Sources of variation Degrees of freedom Sum of squares Mean square F
Regression 1 3385.6 3385.6
Residuals 5–2=3 2.768 0.9227 3669.4
Total 5–1=4

Null hypothesis – There is not a linear relationship between [Cr2O7]2- and Emission

intensity
Alternative hypothesis – There is a linear relationship between [Cr2O7]2- and Emission

intensity
Test statistic – 3669.4
Critical value – From a one-sided F-test with 1 and 3 degrees of freedom at 95%
confidence level, the critical value is 10.13
Decision – As 4892.49 > 10.13, we will reject the Null hypothesis
Conclusion – There is a linear relationship between [Cr2O7]2- and Emission

intensity


We can now progress to analysis of the given results.

The results for the three samples are tabulated below.

Emission intensity
Blank Sample A Sample B Sample C
Replicate 1 5.1 28.6 40.7 73.1
Replicate 2 4.8 28.2 41.2 72.1
Replicate 3 4.9 28.9 40.2 Spilled

Mean 4.9 28.6 40.7 72.6

[K2Cr2O7]* /
0.1906 2.7594 4.0783 7.5457
µg mL-1
Blank
subtracted / 2.5688 3.8877 7.3551
µg mL-1
* [K2Cr2O7] obtained by interpolation on the calibration plot.

Therefore, for Sample A, [K Cr O ] = 2.5688 µg mL-1 = 2.5688 × 10-6 g mL-1
2 2 7

Before diluting sample to 100.0 mL, [K2Cr2O7] = 2.5688 × 10-6 × 100 g = 2.5688 × 10-4 g

Using the molar mass of K2Cr2O7 = 294.1846 g mol-1 and
the molar mass of Cr2O3 = 151.9904 g mol-1

Page 9 of 15
 151.9904
Percentage weight of Cr2O3 = × 2.5688 ×10−4 ×100 = 0.0133%
294.1846
We obtain the percentage weight in Sample B and Sample C in a similar way.

Sample A Sample B Sample C
%weight Cr2O3 0.0133 0.0201 0.0380

We will next compute the standard deviations and then the confidence intervals.

x y ŷ (y - ŷ )2 (x - x )2
0.0 3.1 3.18 0.0064 16
2.0 21.5 21.58 0.0064 4
4.0 40.9 39.98 0.8464 0
6.0 57.1 58.38 1.6384 4
8.0 77.3 76.78 0.2704 16
Σ(y - ŷ )2 =
x = 4 y = 39.98 Σ(x - x )2 = 40
2.768

2
∑( y # ŷ ) i i
2.768
Accordingly, S y / x = i
= = 0.960555394
n$2 5− 2
2
S y/x
1 1 Y!y ( )
Using ( ) SD = + + 2

x slope m n (slope)2 ∑ x ! x
( )
i

2
0.960555394 1 1 28.567 ! 39.98 ( )
Sample A: ( ) SD = + + = 0.07895
x 9.20 3 5 (9.20)2 × 40

As [Sample A] = [K2Cr2O7]A – [K2Cr2O7]Blank, we can use propagation of errors to work
out that
2 2
2 2 ⎛ ∂SD ⎞ 2⎛ ∂SD[Sample A] ⎞
( ) (
SD[Sample A] ≈ SD[K Cr O ] ⎜⎜
2 2 7 A
∂[K
[Sample A]

Cr O ]
)
⎟⎟ + SD[K Cr O ]
2 2 7 Blank
⎜⎜
∂[K Cr O ]
⎟⎟ ( )
⎝ 2 2 7 A⎠ ⎝ 2 2 7 Blank ⎠
2 2 2 2
(
= SD[K Cr O ]
2 2 7 A
) (1) + (SD [K 2Cr2O7 ]Blank ) (1)
2 2
= (SD [K 2Cr2O7 ]A ) ( + SD[K Cr O ] )
2 2 7 Blank

2 2
(
⇒ SD[Sample A] =) (SD [K 2Cr2O7 ]A ) + (SD ) Blank
= (0.07895)2 +(0.09884)2 = 0.1265 µg mL-1

Page 10 of 15
We then proceed in the same way in calculating the percentage weight shown above.

⇒ 0.1265 × 10-6 × 100 g = 1.265 × 10-5

151.9904
⇒ ×1.265×10−5 ×100 = 0.00065% w/w
294.1846

In a similar way, we obtain

Sample A Sample B Sample C
SD %weight 0.00065 0.00064 0.00071

To estimate the 95% confidence level, we use t2 × SD. This gives us

Sample A – 4.30 × 0.00065 = 0.0028 ⇒ 0.0133% ± 0.0028%

Sample B – 4.30 × 0.00064 = 0.0028% ⇒ 0.0201 ± 0.0028%

But, for Sample C, we will use t1 × SD,
Sample C – 12.71 × 0.00071 = 0.0090% ⇒ 0.0380 ± 0.00903%




Question 7:

(a µep is inversely proportional to the analyte size.

m2 m2
(b) For phenol: µ A" = "1.53×10"8 and for trichlorophenol: µ A" = "2.85×10"8
V ⋅s V ⋅s
But only a fraction of HA is ionised ⇒ µpredicted is only a fraction of µ A" .

(c) Ethylphenol ≈ methylphenol < phenol < trichlorophenol

Page 11 of 15
Question 8:

At pH 10, the wall of the bare capillary is negatively charged with –Si-O- groups and there
is strong electroosmotic flow (+3.1 mm s-1) towards the cathode.

At pH 2.5, the wall is nearly neutral with –Si-OH groups and there is almost no
electroosmotic flow. The few –Si-O- groups left give slight flow (+0.2 mm s-1) towards the
cathode.

The aminopropyl capillary also has a positive flow at pH 10, but the rate is (+1.8 mm s-1)
only about half as great as that (+3.1 mm s-1) of the bare capillary. The negative charge
might be reduced because there are fewer –Si-O- groups (because some of them have
been converted to –Si-CH2CH2CH2NH2) or because some of the aminopropyl groups are
protonated )-Si-CH2CH2CH2 NH+3 ) at pH 10.

At pH 2.5, all of the aminopropyl groups are protonated. The net charge on the wall is
positive and the flow (-1.3 mm s-1) is reversed.


Question 9:

(a) A sketch of the apparatus described:
Computer

Separation capillary
Detector

+ -
Buffer Buffer
reservoir reservoir

30 kV
Power Supply


(b) Based on the information given, we cannot deduce if the net charge carried by the
compound is positive or negative. This is because we are not given no information
about the electroosmotic flow (EOF) rate of the buffer, which is needed to compare
to the flow rate of the compound of interest. A velocity less than that of EOF would
indicate that the charge on the compound is negative, due to the attraction to the
positive terminal. If the compound were neutral, it would migrate at the same rate
as the EOF. If the compound were positively charged, it would have a velocity
greater than the EOF, as migration is influenced positively by the electrophoretic
mobility and the EOF.

Page 12 of 15
(c) To calculate the apparent electrophoretic mobility of the compound:

L t 90 cm 600 s
µ app = d = = 5.0 ×10−4 cm 2 V −1 s−1
V L t 30000 V 100 cm

(d) As the neutral compound moves at the same rate as EOF, the mobility of the
compound is equal to the mobility of the electroosmotic flux:

L t 90 cm 300 s
µ EOF = d = = 1.0 ×10−3 cm 2 V −1 s−1
V L t 30000 V 100 cm

(e) The electrophoretic mobility of the compound with a migration time of 10 minutes
can be determined by subtracting µ EOF from µapp :

µ EP = µ app −µ EOF
= 5.0 ×10−4 −1.0 ×10−3
2
= −5.0 ×10 cm V s
−4 −1 −1

(f) The negative character of µ EP implies that it originates from a species carrying a net
negative charge. The compound will migrate more slowly than a neutral marker.

(g) If the internal lining is rendered neutral, there would be no more electroosmotic
flux. As a result, a compound that is generally expected to migrate towards the
cathode will reappear in the anode compartment.

(h) If the pI is 4 and the pH<4, the compound will be in the form of a cation. In this case,
the migration time will normally be shorter than for a neutral marker.

(i) To calculate the number of theoretical plates:

N=
(=
−4 2
)(
µ app V 5.0 ×10 cm V s 30000 V
−1 −1
)
= 375000=3.8 ×105
2D (
2 2 ×10 cm s
−5 2 −1
)

(j) A small molecule generally diffuses faster than a larger one, ie a smaller molecule
µ app V
will have a larger diffusion coefficient. Based on the relationship, N = , a
2D
smaller molecule will give rise to a smaller number of theoretical plates, indicating a
poorer separation.

A narrower capillary will facilitate a more efficient dissipation of heat generated
from the application of a large voltage across in the system. Moreover, a narrower
capillary will also reduce zone broadening, leading to sharper peaks in the
electropherogram.

Page 13 of 15
Question 10:

Description of “electroosmosis” – see page 731 of Harris, 9th Edition.

Significance of “electroosmosis” – contributes to separation in electrophoresis and aids in
achieving less broadening in electrophoresis.




Question 11:

(a) Structure of sodium dodecyl sulfate (SDS):

This structure clearly shows that SDS consists of a polar terminal with a sulfate
group (the left hand side) and a non-polar terminal with hydrocarbons (the right
hand side). As polar groups like polar groups, and non-polar groups like non-polar
groups, the polar sulfate groups are interacting with the aqueous polar groups of the
buffer, while the non-polar groups reside in an inner region, after being placed in a
buffer. Such an SDS structure is termed a micelle.

(b) In the absence of micelles, the expected order of elution is (1) cations, (2) neutrals
and (3) anions. Accordingly, thiamine < (niacinamide + riboflavin) < niacin.

(c) As thiamine is eluted last, it must be most soluble in the micelles.

Page 14 of 15
Question 12:

The most influential parameters is (i) pH.

Although all four parameters affect the electrophoretic mobility, and hence the separation
of proteins, some will have similar effects on all the proteins in the sample.

pH has a very significant effect on the magnitude of the charge carried by a protein
molecule (consider the relation between pH and pKa). As pH affects the protonation and
ionisation of the side-chains of amino acids, of which a protein is composed, it affects
different proteins to different extents, because the amino acid composition and sequence
of every protein is unique.

Ionic strength affects the charge characteristics of protein molecules, as charged
macromolecules become surrounded by a “shield” of counter-ions that effectively
decreases the net charge of the molecule and so affects its electrophoretic mobility. The
size of this effect depends on the nature and charges of the ionisation groups on the
surface of the macromolecule. Therefore, the effect is likely to be different for each
protein in a sample. However, the effects of ionic strength are not as great as those of pH,
which affects amino acid side-chains inside the molecules, as well as those on their
surface.

Changes in temperature can have very significant effects on the electrophoretic separation
of protein molecules. Temperature can alter several of the parameters of an
electrophoretic system (e.g. convection current, diffusion, evaporation, and viscosity), but
these changes are not highly specific for individual protein molecules, and all the
molecules in a sample will be affected in much the same way, although there will be small
individual differences.

Changing the current will generally have similar effects on all the protein molecules in a
sample, although individual molecules may be affected slightly differently.

Page 15 of 15