Tutorial Discussion for C 21082 21st Saturday Sept

(1) Answer all parts.

(a) Describe briefly how you would construct a valid calibration curve in instrumental
methods of chemical analysis. ( 15 marks)
(b) An analyst collected the following data for construction of an external calibration curve
required for a flame atomic absorption determination of Cd in aqueous samples .

Concentration of Cd Average
standards in dil. absorbance
HNO3/ ppm
Reagent blank 0.008
2.5 0.125
5.0 0.250
7.5 0.375
10.0 0.500
12.5 0.625

(i) Construct an external calibration curve using the above data. Calculate the slope
of the calibration curve and sensitivity of the measurements in absorbance units as well as
in concentration units. (20 marks)

(ii) The sample standard deviation of absorbance for the reagent blank was 0.003.
Calculate the instrument detection limit (IDL) and practical quantitation limit (PQL) in
absorbance units and in concentration units according to convention adopted by analytical
chemists in USA. (15 marks)

(iii) The averages of absorbances observed for three aqueous solutions A, B and C
with matrices similar to those of the standards are given below.

Sample Average
absorbance
A 0.014
B 0.025
C 0.150

Taking into consideration of the IDL and PQL values calculated above, comment on the
validity of the concentration values of Cd in samples A, B and C read from the above
external calibration curve above. (15 marks)

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 (c) The following data also were collected by the analyst for the purpose of estimating the
spike recovery corresponding analysis of Cd in a soil extract, S, having a matrix different from
that of standards.

• Average absorbance for the soil extract, S = 0. 435

• Average absorbance for a 100.0 ml soil extract, S
spiked with 10.0 µL of 1000.0 ppm Cd standard = 0.450
• Average absorbance for a 2.0 ppm Cd standard = 0.400

(i)Using the above data, calculate the % spike recovery corresponding to determination of Cd
in soil extract, S. (25 marks)

(ii) Using the % recovery value obtained at step (c) (i) above, calculate the true concentration
of Cd in soil extract, S. Discuss the validity of the concentration determined by matrix
correction in this problem. (10 marks)

(2) (a) An analyst had to determine the concentrations of lead in blood of a group of people
exposed to vehicle emission over an extended period of time. He decided to use furnace atomic
absorption spectrometry for this analysis and 2.00 ml blood samples collected from each person
were processed according to a recommended method which involved digestions, filtrations,
extractions and transfers and dilutions of extracts. To minimize matrix effects, he treated all
extracts with a matrix modifier. Through out this process the final volume of each 2.00 ml blood
sample had been brought to 10.00 ml. Since considerable matrix effects were still prevalent in
the extracts even after processing, he decided to use the standard addition technique to further
minimize the matrix effects on the final results.

The data collected for plotting of a standard addition curve for determination of lead in
blood extract labeled as “a” is given below.

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 Standard Volume of Volume of Volume of Final
Addition Blood Extract Volume/m Absorbance
10 ppb lead Distilled
mixture “a” / ml water/ml l
standard
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##mixture 2.00 0.00 3.00 5.00 0.140
2 2.00 /ml
0.50 2.50 5.00 0.300
mixture
3 2.00 1.00 2.00 5.00 0.450
mixture
4 # 2.00 1.50 1.50 5.00 0.600
5 2.00 2.00 1.00 5.00 0.750

i) Calculate the concentration of the standard lead in each of the standard addition mixtures
(05 marks)
ii) Plot a "standard addition curve" using the data calculated in step (i) above.
(10 marks)
iii) Using the graph, estimate the concentration of Pb present in any of the standard
addition mixtures that belongs to the blood extract “a”.
( 05 marks)

iv) Calculate the concentration of blood lead in blood extract “a” in units of ppb,
( Note 2.00 ml of blood extract were diluted to 5.00 ml in addition mixtures)
(05 marks)
v) Calculate the concentration of lead in the untreated blood sample corresponding blood
extract “a” in units of ppb (Note: 2.00 ml of blood sample during preparation diluted to
10.00 ml)
(05 marks)
(b )
i) Construct an "extended standard addition curve" on the same graph paper you used
to construct the "standard addition curve". (10 marks)
ii) The average absorbance of a blood extract “P” measured directly from the instrument
was found to be 0.250. Using the extended standard addition curve, read the
concentrations of Pb in the blood extract “P” in units of ppb.
(05 marks)
iii) Calculate the concentration of lead in the blood sample used to prepare blood extract,
” P” in units of ppb (05 marks)

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 (c) For estimation of losses or contamination of lead during the sample preparation stage, a
known amount of a standard was added to each of the five 2.00 ml aliquots of a blood
sample Y for the total lead concentration of these samples to increase from 8.75 ppb to
12.5 ppb. These spiked samples were subjected to the entire preparational procedure. The
average absorbance of 0.350 was observed for these spiked samples.

i) Calculate the percentage loss or gain of lead that occurs during the above sample
preparation stage. (15 marks)
ii) Taking into account of the loss or gain determined above, correct the concentration of
lead estimated for blood extract of P you determined using the extended standard addition
curve in step (b) above. (10 marks)
iii) Taking into account the losses or contamination, calculate the concentration of lead in
the blood sample used to prepare blood extract P in units of ppb. (10 marks)
iv) State briefly, how you would estimate the concentration of lead in the other blood
samples and how you would correct the values for contaminations and/or losses of lead
at the preparation stage. (15 marks)

( 3). The data relating to an atomic absorption determination of lead in an aqueous solution of
unknown concentration using standard addition technique is given below.

Volume of Volume of std. Total Absorbance

solution solution (Vs)/ ml volume(Vt) / ml
unknown(Vx)
/ml
10.00 0.00 100.00 0.163
10.00 1.00 100.00 0.240
10.00 2.00 100.00 0.319
10.00 3.00 100.00 0.402
10.00 4.00 100.00 0.478

Vs – volume of the standard

Cs - concentration of the standard
Vx - volume of the unknown
Vt - total volume
Cx – concentration of the unknown

The concentration of the standard (Cs)was 1000.00 µg/ml.

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 i. Plot absorbance as a function of volume of standard, Vs.

ii. Derive an expression relating absorbance to the concentration of standard (Cs), the
volume of the standard (Vs), the volume of the unknown (Vx) and the total volume
(Vt) and other characteristic costants.

iii. Derive an expression for the slope ( m) and the intercept (b) of the straight line obtained
in (I) in terms of the variable listed in (II) and other relevant constants.

iv. Show that the concentration of the analyte is given by the relationship
Cx = b Cs / m Vx

v. Calculate the concentration of lead in the unknown aqueous solution in µg/ml using the
relationship given in (iv).

(04) (a).The linear dispersion of a monochromator is 10+7. The width of the slit is 2.0 mm.
Calculate the spectral band pass of the mnochromator at this width of the slit.

( b ) The monchromator has been adjusted to a peak wavelength , 𝜆𝑝𝑒𝑎𝑘 of 550 nm at this
slit setting. What is the interval of wavelengths found in the monochromator output.
(c ) Calculate the resolving power of the monochromator at this peak wavelength and at this
slit setting.
(15 marks)

( 5) Provide reasons briefly for the following.

(i) VGAAS provides sensitivities in the ppb range with flame atomic absorption
spectrophotometers.
(ii) ICP-OES provides a linear range from nearly one hundredth ppb to nearly 100 ppm
concentration range.
(iii) GFAAS is near 100 to 1000 times more sensitive than FAAS.
(iv) Monochromator is always placed between atom cell and the detector in AAS
instruments.
(v) Physical properties such as viscosity, surface tension , vapor pressure of standard
solutions and samples should nearly match to each other.

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 (vi) It is the resonance radiation which is used frequently for AAS.
(vii) Radiation source is never placed along axis where atom cell and detector are present
in fluorescence spectrometers.

Answers with marking Scheme

(1) a)
• Prepare a minimum of three calibration standard solutions with concentrations within the
linear range specified by the manufacturer for the particular analysis.
• Let Cmax, Cmid and Cmin are the concentration sof these standards. Cmax is the highest
standard. Cmid = ½ Cmax , Cmin = ½ Cmid
• Measure the signal in replicates starting from the lowest concentration and plot the
calibration curve. R2 for the calibration curve must be equal or greater than 0.999. If this
condition is satisfied the first requirement foe acceptability is fulfilled. If failed need to
repeat until it is met.
• Measure the signal for one of the calibration standards , usually the Cmid again. The
deviation of the new signal value from the previous one cannot be larger than 5 %. The
selected standard is called the continuous calibration standard ( CCS).
• Prepare the middle standard using another source of chemical. The signal value deviation
cannot be larger than 10 % from the first value. This standard is called the calibration
verification standard (CVS).
( 15 marks)

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b) i)

(15 marks for the graph)

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 0.500−.375 0.125
Sensitivity = = = 0.05 𝑝𝑝𝑚−1
(10.0−7.50) 𝑝𝑝𝑚 2.5

Sensitivity in atomic absorption spectrometry is given in terms of the concentration that gives
0.0044 absorbance units for FAAS, .
1𝑝𝑝𝑚
Therefore , sensitivity according to that definition = 𝑥 0.0044 = 0.088 𝑝𝑝𝑚
0.05

( 20 marks)

(ii) IDL = 𝑋̅ + 3σ (2 marks)

= 0.008 + 3 x 0.003
= 0.017 (2 marks)
1 𝑝𝑝𝑚
In concentration units 𝑥 0.017 = 0.34 𝑝𝑝𝑚 ( 3 marks)
0.05

PQL = 𝑋̅ + 10σ (3 marks)

= 0.008 + 10 x 0.003
=0.038 (2 marks)
1 𝑝𝑝𝑚
In concentration units 𝑥 0.038 = 0.76 𝑝𝑝𝑚 ( 3 marks)
0.05

(iii ) Absorbance for sample A, 0.014 is below the value of IDL, 0.017.
Therefore analyte may or may not be present in sample A. Neither qualitative nor quantitative
analysis is possible with the instrument in use ( 4 marks)
Absorbance for sample B is in between the IDL and the PQL.
Analyte is definitely present in sample B but its concentration cannot be accurately determined
with the present instrument. (4 marks)
Absorbance for Sample C is greatr than the PQL.
Therefore its concentration can be accurately determined . ( 4 marks)

( c ) (i)

Let the concentration of the standard Cd present in the spiked mixture is X,

𝑚𝐿
X ppm x 100 ml = 1000 ppm x 10 𝜇 𝑙 𝑥 10−3 𝜇𝐿

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X = 0.1 ppm
Note the assumption : the volume remains almost at 100 ml even after addition on10 micro liters
of standard . The error negligible.
Now we can compare the absorbances for 0.1 ppm standard Cd when it is present in the mixture
and when it is present in the standard solution alone.
Consider the situation when it is in the standard solution .
The absorbance for 2.00 ppm standard = 0.400 (refer data given)
0.400
Therefore absorbance for 0.1 ppm Cd in the standard solution = 𝑥 0.1 𝑝𝑝𝑚 = 0.02
2.00 𝑝𝑝𝑚

Now what is the absorbance due to 0.1 ppm Standard lead when it is in the mixture

Soil Cd alone had an absorbance of 0.300 (refer Data)

The volume of it remained the same even after adding 10 microliters of standard Cd
(assumption)
Also if we assume that regent blank (dilute acid present in 10 microliters of the standard) has no
influence on the absorbance due to soil Cd,
the absorbance of soil Cd in the mixture = 0.435.
The absorbance in the mixture is the sum of the absorbance due to soil Cd present in it and the
added Cd standard in it.
i.e 𝐴𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 = 𝐴𝑠𝑜𝑖𝑙 𝐶𝑑 + 𝐴𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐶𝑑
0.450 = 0.435 + 𝐴𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐶𝑑
Therefore absorbance due to 0.1 ppm standard in the mixture = 0.450 -0.435 = 0.015
Now compare the absorbance for 0.1 ppm Cd when it is in standard solution and when it is in
thesoil matrix.
In soil matrix 0. 1 ppm Cd has the absorbance = 0.015
In standard solution 0. 1 ppm Cd has the absorbance = 0.02
Absorbance is proportional to concentration of Cd atoms in the vapor phase.

The percentage of Cd atoms going to vapor phase from Standard solution is higher than that
going from soil matrix.

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It can be calculated using the observed absorbances as below
.015
% spike recovery in he soil matrix = 0.02 𝑥 100 = 75

( 25 marks)
Important : This level of explanations are not expected for an examinations. The purpose here
was to explain for learning.

Note : This is poor recovery.

Usually it has to be around 90.
In certain situation you may get more than 100 % recovery. (think).
Also remember the % R depends on the relative proportions of the analyte and the matrix. Other
approaches are available to estimate it. The purpose of the above estimation is to get a feeling of
the level of matrix effect.
(ii) The absorbance for the soil sample = 0.435
Since soil matrix is giving 75 % of Cd atoms to the vapor phase compared to the matrix of the
standard
100
The absorbance for the soil Cd if it were measured in the standard matrix = 𝑥 0.435 = 0.580
75
2.0 𝑝𝑝𝑚
There fore the concentration of Cd in soil extract = 𝑥 0.580 = 2.90 𝑝𝑝𝑚
0.400

( 10 marks)

(02) a) i)
Concentration of the standard/ppb Absorbance
10ppb × 0.00 0.140
𝑥= =0
5
10ppb × 0.50 0.300
𝑥 = =1
5
10ppb × 1.00 0.450
𝑥 = =2
5
10ppb × 1.50 0.600
𝑥 = =3
5
10ppb × 2.00 0.750
𝑥 = =4
5
(05 marks)

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Notice detail calculations are not required Show it only for one step
ii) On graph paper will be attached to Google class)
( 10 marks)
iii) Concentration of Pb in the blood extract in the addition mixture = 0.93 ppb ( 05 marks)
iv) Concentration of Pb in the blood extract = 0.93 x 5/2 ppb
= 2.33 ppb (05 marks)
v) Concentration of Pb in the blood = 2.33 x 10/2 ppb
= 11.65 ppb( 05 marks)
b) i) On the graph paper ( 10 marks)
ii) Concentrations of Pb in the blood extract P = 1.63 ppb ( 05 marks)
iii) Concentrations of Pb in the blood sample = 1.63 x 10/2 ppb
= 8.17 ppb ( 05 marks)
Because 2.0 ml blood samples diluted to 10.0 ml during pretreatment.
c) i) According to the graph,
Concentration corresponding to 0.350 absorbance = 2.3 ppb
Before the preparational procedure,
Concentration of lead in blood sample (with the standard) = 2.3 x 10/2
= 11.5 ppb
(Because there was a 5 fold dilution of blood during pretreatmet)
11.5 − 12.5
percentage loss = x 100%
12.5
(15 marks)
=8%
ii) 100
concentration of lead in blood extract of P = 1.63 𝑝𝑝𝑏
82

= 1.77 ppb ( 10 marks)

concentration of lead in blood sample = 8.17x 5

iii)

= 40.85ppb
(10 marks)
iv)
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 • Measure the absorbance of each of the blood extracts.

• Using interpolation from the extended standard addition curve obtain the concentration

of lead in each extract.

• Determine the concentration of lead in the blood samples taking into account of the

percentage loss determined during the preoperational procedure.

• Correct for dilution during ( 15 marks)

(3)

The graph of absorbance vs. volume of

standard(Vs )

0.6 y = 0.0792x + 0.162

0.5
Absorbance

0.4
0.3
0.2
0.1
0
0 2 4 6
Volume of standard / ml

II. A= ε C l
A – absorbance
C – concentration
L - path length
ε - absorption coefficient

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 A =εl Cs Vs + Cx Vx
Vt

III. A= εl Cs Vs + ε l Cx Vx
Ct Ct

Y = mx + b

Cx Cs
IV. εl Vx = b εl =m
Ct Ct
 l  l m
 C x Vx = b =
 Ct  Ct C s
m
C V =b
Cs x x
Cx = bCs / mVx

V. m = 0.0792 ml-1
b = 0.0828
Cx = bCx / mVx
Cx = 0.0828 x 1000 µg ml-1 / 0.0792 ml-1 x 10 ml
Cx = 104.54 µg ml-1

𝑑𝑙
(a) (4) Linear dispersion = 𝑑𝜆 = 10+7
𝑑𝜆
Spectral band pass at any slit width W = xW
𝑑𝑙
1 𝑛𝑚
= 𝑥 2.00 𝑚𝑚 𝑥 10+6
10+7 1 𝑚𝑚
= 0.20 𝑛𝑚 ( 05 marks)

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 (b) When a monochromator is set to a peak wave length the wavelength interval coming out
form the monochromator is from 𝜆𝑝𝑒𝑎𝑘 − 𝑏𝑎𝑛𝑑 𝑝𝑎𝑠𝑠 𝑡𝑜 𝜆𝑝𝑒𝑎𝑘 + 𝑏𝑎𝑛𝑑 𝑝𝑎𝑠𝑠.
Therefore the wavelength interval = (550.0-0.2 nm )to (550.0 + 0.2 nm).

i.e from 549.8 nm to 550.2 nm (05 marks)

(c ) Resolving power ( R ) can also defined as below

𝑝𝑒𝑎𝑘 𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 500
R = = 𝑛𝑚 = 2500 ( 05 marks)
𝑏𝑎𝑛𝑑 𝑝𝑎𝑠𝑠 0.2 𝑛𝑚

Notice: the theory parts to do parts (b) and (c) were not done
before.
( 5)
( a) Like in normal FAAS no sample lost to the drain in VGAAS. Also no dilution in the flame
due to expansion of vapor to temperatures more than 2000 K.

(b). Losses to the drain in the ICP-OES nebulization is negligible as the nebulization is much
effective in this system. This makes a higher fraction going to plasma giving opportunity to
detect lower concentration. Since the temperature in analytical zones n around 8000 K ecited
atoms can populate higher energy levels No saturation of levels occur unless solution
concentrations are higher than hundreds of ppm.

(iii). In GFAAS entire volume though very small ( few microliters) is atomized forming a dense
atom cloud. In FAAS more than 70 percent of analyte lost to the drain ( big aerosol particles ) .
Further once in the flame the atomic vapour further expanded at flame temperature

(iv) This arrangement minimizes amount of flame spontaneous emission reaching the
detector ( though the detector does not recognizes it due to lock in amplification it still increases
the noise at the detector.

(v) Solution uptake rate is control by the viscosity of the solution. Droplet size by the surface
tension of the solution. Rate of evaporation by the vapor pressure of the solution. Therefore it
minimizes err0rs if those properties closely matched for sample solutions and standard solutions
in FAAS

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