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Title: Ultra Violet Visible Spectroscopy.

Aim: To determine the iron content of a tablet by the visible spectrophotometry using the
external standard method.
Introduction/theory:
The external standard method
The external standard method uses standards that are prepared separately from the sample.
External standards are used to calibrate instruments and procedures when they are no
interference effects from matrix components in the analyte solution. A series of the external
standards of known concentration is prepared. In this experiment standard solution prepared
from ammonium ferrous sulphate and thiocyanate are used to form a complex which absorbs
light. This complex is read and has maximum absorbance at 490 nm and all absorbance readings
are measured at 490 nm.
To produce the coloured complex which absorbs light the ferrous ions that are present in the
tablet needs to be converted to the ferric form. Fe 3+ ions react with thiocyanate to form a bloodred colored complex which characteristically has a high molar absorptivity:
Fe3+(aq)

SCN- (aq)

[FeSCN]2+(aq)

The ferric ion is the analyte and to determine the amount present more easily it is converted to
the intensely colored thiocyanate complex. The ferric iron-thiocyanate complex is more stable
than what would have been formed by FeS

O4

, which

is present in the tablets. The

employment of nitric acid ensures that the ferrous ion would be oxidized to the ferric ion,
thereby allowing for a more stable complex to be formed with the thiocyanate.
Calibration is accomplished by obtaining the response signal (absorbance, peak height, peak
area) as a function of the known analyte concentration. A calibration curve is formed by plotting
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the data or using a suitable mathematical equation such as the slope-intercept form used in the
least square method. The step in this method is the prediction step, where the response signal
from the sample is used to find unknown analyte concentration from the calibration curve or best
fit equation. The concentration in the original bulk sample is then calculated from the
concentration of analyte by applying the appropriate dilution factors from the sample preparation
step.
When the external standard method is used it is assumed that the same responses will be obtained
when the same analyte concentration is present in the standard. Thus the calibration functional
relationship between the response and the analyte concentration must apply to the sample as
well. Usually, in determination the raw analytical response is corrected by measuring a blank. An
ideal blank is identical to the sample but without the analyte. In practice, with complex samples
it is too time consuming, or impossible to prepare an ideal blank and a compromise must be
made. Most often a real blank is either a solvent blank, containing the same solvent the sample is
dissolved in, or a reagent blank containing the same solvent and reagent used in the sample
preparation.
Reagents:
Concentrated nitric acid
Concentrated sulphuric acid
1M sulphuric acid
1M ammonium thiocyanate
Ammonium ferrous sulphate
Method:
Preparation of the Iron Tablet for analysis
Place iron tablet in a 100 ml beaker in a fume hood and use a measuring cylinder to add 5 ml of
concentrated nitric acid. Allow the tablets coating to break down and its contents to dissolve. You
may help this process by using a stirring rod to carefully crush the tablet and stir the solution. Do
not inhale any of the brown fumes that may be evolved. (NB: iron tablets sometimes contain
filler materials that may not fully dissolve in acid)

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Iron tablets usually contain ferrous sulfate, with iron present as Fe 2+ ions. Since Fe2+ does not
form a coloured complex with thiocyanate. The nitric acid is added to oxidise all the Fe 2+ to form
Fe3+ ions.
After any reaction subsides, add 5 ml concentrated sulphuric acid and transfer the iron solution to
a 250 ml volumetric flask, rinsing the beaker with distilled water a few times and transferring the
washings to the volumetric flask.
Make up to mark with distilled water, stopper the flak and mix well, and then allow any
undissolved matter to settle.
Use a pipette to transfer 20 ml of iron solution to a 100 ml volumetric flask. Add 10 ml 1M
sulphuric acid and make up to the mark with distilled water. This diluted solution will be used for
spectrophotometric analysis.
Preparation of standards
Using the given salt (ammonium ferrous sulphate) weigh out sufficient mass into a 100 ml
beaker to make 100 ml of a solution containing 1000ppm Fe. Place the beaker in the fume hood,
add 5ml concentrated nitric acid and stir until all the salt is dissolved. If brown fumes are
evolved, wait until they subside, then add 4ml concentrated sulphuric acid then quantitatively
transfer the solution to a 100 ml volumetric flask, and make up to mark with distilled water. This
is solA.
Pipette 10 ml of solution A into a 100 ml volumetric flask and dilute to mark with water. This is
solution B

External standard method


Prepare a blank and a series of standard by placing 0, 1, 2, 3 and 4 ml of solution B in separate
50 ml volumetric flasks. Using a measuring cylinder, add 5 ml of 1M sulphuric acid to each
flask.
Pipette 10 ml and 25 ml of diluted sample solution into separate 50 ml flasks. Add 5 ml of 1M
sulphuric acid to each.
Note the time and add 5ml of 1M thiocyanate to the first flask and make up to mark with water.
After 2 minutes add 5ml of thiocynate to the second flask and continue in this manner until
thiocyanate is added to each standard and sample.
Fifteen minutes after the first addition of thiocynate, measure the absorbance of each solution at
490 nm in the same 2 minute intervals.

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Results:
Table 1: The Absorbance Obtained for the Iron Standards and the Samples Made From the
Tablet.
Volume

Solutions
Blank
Standard 1
Standard 2
Standard 3
Standard 4
1.0 mL Diluted
Sample
1.0 ml

Diluted

Sample
1.0 ml

Diluted

Sample

of Concentration of Fe in solution Absorbance

Solution B
0 mL
1 mL
2 mL
3 mL
4ml

B (mg/L)
0.000
2.000
4.000
6.000
8.000

490nm
0.000
0.052
0.073
0.491
0.673

0 mL

3.240

0.190

0 ml

2.893

0.159

0 ml

3.240

0.190

Mass of the Iron Tablet:


Mass of beaker = 23.46g
Mass of beaker + tablet = 24.08g

Calculations:
Mass of iron tablet = Mass of beaker Mass of beaker + tablet
= 24.08g 23.46g
= 0.62g
So Fe(NH4)2(SO4)2. 6H20 = 392.15g/mol
Concentration of Solution A = 1000ppm of Fe

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at

= 1000mg of Fe
1 mole of Fe(NH4)2(SO4)2. 6H20 1 mole of Fe
392.15g 56g of Fe
x 100mg 0.1g
x 392.15g / 56g * 0.1g = 0.700g of Fe(NH4)2(SO4)2. 6H20
The concentration of Solution A is 1000ppm or 1000mg/L
Solution B:
M1V1 = M2V2
1000mg/L * 0.01L = M2 * 0.1L
M2 = 100mg/L
Concentration of Standards:
M1V1 = M2V2
For 1 ml 100mg/L * 0.00lL = M2 * 0.05L
M2 = 2 mg/L
For 2 ml 100mg/L * 0.002L = M2 * 0.05L
M2 = 4mg/L
For 3 ml 100mg/L * 0.003L = M2 * 0.05L
M2 = 6 mg/L
For 4 ml 100mg/L * 0.004L = M2 * 0.05L
M2 = 8mg/L

Diluted Samples:
The Least Square Regression Line of y on x is y = 0.08925x 0.0992
Where y is absorbance (nm) and x is the concentration (mg/L).
x = y + 0.0992 / 0.08925
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For 0.190nm x = 0.190 + 0.0992 / 0.08925 = 3.240 mg/L (4 significant figures)


The mass of Fe in the sample is concentration * volume 3.240 mg/L * 0.1L = 0.324 mg
For 0.159nm x = 0.159 + 0.0992 / 0.08925 = 2.893 mg/L (4 significant figures)
The mass of Fe in the sample is concentration * volume 2.893 mg/L * 0.1L = 0.2893 mg.
Therefore the average mass of Fe in Solution B is 0.324 mg + 0.2893mg + 0.324 mg / 3 = 0.9373
mg (4 significant figures).
There is 0.9373 mg of Fe in 20 mL of the original sample.
The mass of Fe in 1 mL of the original sample is 0.9373 mg / 20 = 0.046865 mg
The mass of Fe in 250 mL of the original sample is 0.046865 mg * 250 mL = 11.71625 mg or
0.01171625 g.
So 11.71625 mg of Fe / 0.62 g = 18.9 mg (3 significant figures) of Fe per gram of tablet.
The percentage mass of Fe in the tablet is 0.01171625 g / 0.62 g * 100% = 1.89% (3 significant
figures).
Mean of the concentrations of the diluted samples is 3.240 mg/L + 2.893 mg/L + 3.240 mg/L / 3
= 3.1243 mg/L (5 significant figures).
Standard Deviation of the diluted samples is ((x - ) 2 / (n 1)
Concentration (mg/L)
3.240
2.893
3.240

Deviation from mean


0.1157
-0.2313
0.1157

Therefore, (0.08027267 / 2) = 0.2003 (4 significant figures).

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(x - )2
0.01338649
0.05349969
0.01338649
(x - )2 = 0.08027267

GRAPH SHOWING CALIBRATION CURVE OF ABSORBANCE VS CONCENTRATION


A
B
S
O
R
B
A
N
C
E

0.8
0.7
0.6
0.5
0.4
0.3
0.2

( 0.1
n
0
m
0
)

CONCENTRATION (mg/L)

Discussion: Ultra Violet Visible spectroscopy refers to the absorption of light in the visible and
adjacent UV and infrared ranges or regions. The UV region falls in the range between 190nm
380nm and the visible region falls in the range between 380nm 750nm. The instrument that is
used in UV VIS spectroscopy is called a UV VIS spectrophotometer. The spectrophotometer
measures the intensity of light passing through a sample and compares it to the light intensity
before the sample undergoes UV VIS spectroscopy. The basic parts of a spectrophotometer are
the light source, a container for the sample, a diffraction grating and a monochromator. In UV
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VIS spectroscopy, the ultra violet and visible radiation interacts with matter which causes
electronic transitions from the ground state to a higher energy state whereby energy is absorbed
by an electron in an atomic orbital and is promoted to an anti bonding orbital such as * or *
orbital. UV VIS spectroscopy can only cause transitions from *, n * and n *
since more energy would be required to cause transitions to a higher energy state. In this
experiment, the iron content of a tablet was determined using visible spectroscopy using the
external standard method which is the preparation of standards that are prepared separately from
the sample under analysis. The external standards are prepared form ammonium ferrous sulphate
and thiocyanate to form a complex that absorbs light. The complex is red in colour. The purpose
of the thiocyanate is that some compounds such as the ammonium ferrous sulphate may be
colourless and the addition of a colouring agent such as the thiocyanate forms a highly coloured
complex which absorbs visible light. The thiocyanate attaches to the chromophore (a structural
feature of organic molecules which cause absorption of ultra violet and visible wavelengths) to
modify the wavelength and the intensity of the absorption. After the relevant calculations were
made to obtain the concentrations of the standards, a calibration graph was plotted of absorbance
against concentration and linear interpolation using the modified least square regression line of y
on x to predict values for the concentrations of the three diluted samples. This is an application
of Beer Lamberts Law which states that absorbance of a solution is directly proportional to the
concentration of the absorbing species in the solution and the path length. After the respective
concentrations were obtained, the mean and standard deviation were calculated. The standard
deviation was small which shows that there was a relatively small deviation for the mean. After
relevant calculations have been done, it was shown that the iron tablet contained 1.89% (3
significant figures) of Iron by mass and the tablet contained 18.9 mg of Iron for every gram of
tablet.
One limitation in this experiment is that the colouring agent may have absorbed some amount of
ultra violet visible radiation which may have altered the absorbance values obtained for the
samples that were analyzed thereby generating inaccurate data.
One source of error in this experiment is that the standards may have been incorrectly prepared
thereby causing inaccurate calculations for the concentrations of Iron in Solution B to be
obtained.
One assumption in this experiment is that the use of the least square regression equation is
limited to reactions or systems in which only a single complex is formed and for which Beer
Lamberts Law is obeyed.

Conclusion: It can be concluded that the iron content of a tablet was determined by the visible
spectrophotometry using the external standard method. When visible spectrophotometry was
used to determine the iron content of the tablet, it was calculated that the percentage mass of iron
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in the tablet was 1.89%. With the use of a calibration graph, Beers Law was applied using
interpolation by means of the least square regression equation to predict the concentrations for
the three diluted samples which allowed the conductor of the experiment to determine the iron
content of the tablet.

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