Quantitative Analysis of Iron in Well Water by External Standard Method using a Visible Spectrophotometer

 

Objectives

 

The student learn how to operate the Genesys20 visible spectrophotometer by performing a colorimetric experiment for the determination of iron in well water. An external standard calibration graph following Beer’s Law will be plotted at the wavelength corresponding to the absorbance maximum (λ_max). Multipoint regression analysis and single point method will be used to quantify the iron concentration (mg Fe/ 1.0 US gallon jug) in well water. Statistical data handling methods will be employed to evaluate the quality of the final results. The student will determine the identity of the well water by comparing their final experimental results with those posted on Slate.  

 

Introduction

 

I- Absorption of Light

 

When a chemical species such as an organic molecule absorbs a photon, the energy of the species is increased and the species is promoted to a high energy excited state (E₁) as illustrated in Figure 1.

 

E

1

????

=

?

??

=

E

1

–

E

0

PHOTON

E

0

Figure 1: Excitation of an Analyte by UV

-

visible Light

 

 

If a chemical species emits a photon, its energy is lowered and the species is said to return to its ground state (E₀ - lowest energy state).

 

When incident light (P_o) is absorbed by an analyte species in solution, the radiant power of the incident beam of light (P) decreases. Radiant power refers to the energy per second per unit area of the light beam. Figure 2 shows a schematic diagram of light absorption by an analyte species in solution with concentration “c”.

 

 

 

Cuvette containing absorbing analyte with concentration “C”

b = path length

Figure 2: Absorption of

Light by an Analyte Species in Solution

P

o

P

b

 

 

Incident light of all wavelengths emanating from a continuous radiation source is passed through a monochromator (a grating and series of mirrors) in order to select one wavelength. Monochromatic light of a particular wavelength, with incident radiant power (P_o) strikes analyte in solution with a cuvette path length (b) and the resulting radiant power is attenuated (P) emerging from the other side of the cuvette due to absorption of light by the analyte, such that P < P_o. The transmittance (T) is defined as the fraction of the original light that passes through the analyte solution.

    

                                                             

 

Transmittance has a range of zero to one, and by multiplying transmittance by 100, percent transmittance (%T) ranges between 0-100%. 

 

                                                       

If light is not absorbed by the solution relative to the blank, then the %T is 100% (P = P_o) and absorbance is zero (A=0). If all of the light is absorbed by analyte in solution then none is transmitted to the detector causing %T to be 0. 

 

II- Beer Lambert Law 

 

The laws of Lambert, Bouger, and Beer state that at a given wavelength (monochromatic light- λ_max ), the proportion of light absorbed by a transparent medium is independent of the intensity of the incident light and is proportional to the number of absorbing analyte species through which the light passes. The combined Beer-Lambert Law may be expressed mathematically as:

 

 

where         P_o - power or intensity of incident light                    P -  power or intensity of transmitted light                    T -  transmittance of the analyte solution and   A -  absorbance at λ_max

       ε  -  molar absorptivity or molar extinction coefficient  (L cm^-1 mol^-1)             b  -  path length of cell which light passes through (cm)             c  - concentration of analyte species in solution (mol/L)  

It can be seen that absorbance is directly proportional to the analyte concentration (c) and can be expressed in moles/L with the molar absorptivity (ε) having units of “L cm^-1 mol^-1”. Molar absorptivity is a constant for a particular absorbing analyte in a particular solvent at a particular wavelength (λ_max). The cuvette width or pathlength (b) is also constant for a particular analysis and with units of “cm”. Thus, if the analyte concentration doubles, the absorbance will also double and if the concentration is cut by half then absorbance will be cut by half. 

 

One basic assumption when applying Beer's Law is that monochromatic light is used. In experimental situations, this is not the case, but rather a band of radiation is passed (bandwidth) the width of which depends on the dispersing grating and exit slit width. The absorption spectrum of an absorbing analyte solution as seen in Figure 3 shows that different wavelengths are absorbed to different degrees; that is, the molar absorptivity changes with wavelength. At a wavelength corresponding to a fairly broad maximum on the qualitative absorption spectrum, the band of wavelengths will all be absorbed to nearly the same extent. The Genesys20 spectrophotometer bandwidth spans 8 nm compared to more sophisticated instruments that range from 0.5-4.0 nm.

 

Figure

3

:

D

etermination

of W

avelength

Maximum

 

 

The band of wavelengths (or bandwidth) emerging from the exit slit (monochromatic wavelength value set on the instrument) produce large variations in the absorbance value if measurements are recorded on the shoulder of an absorption peak as illustrated in Figure 4 – Band B. When wide bandwidths are used for quantitative analysis, the Beer’s Law calibration plots curve dramatically due to a departure from Beer’s Law. 

 

 

 

 

 

 

 

 

Figure

4

:

Deviations from Beer's

L

aw: Band A

-

No Deviation, Band B

-

Negative Deviation

 

  

Before determining the concentration of an analyte species in a sample solution using visible spectrophotometric methods, it is necessary to find a suitable wavelength band where deviation from Beer's Law will be almost negligible (i.e. broad absorbance maximum).  To achieve this goal, the analyst must first run a qualitative spectral plot of absorbance as a function of changing wavelength to determine the absorbance maximum.  The visible absorbance spectrum is unique for each absorbing analyte species and in the case of the ferrous 1,10-phenantroline complex shown in Figure 5, the absorbance maximum appears at approximately 508 nm.

 

Figure

5

:

Qualitative Visible Spectrum of

Ferrous

1

,

10

-

phenantroline

 

 

 

Beer's Law plots are prepared by measuring the light absorbed at 508 nm by a series of iron external standard solutions of known concentration as shown in Figure 6. In this case, the analyte will be iron (II) complexed with 1,10-phenantroline to produce an orange colour. The absorbance of each solution will be measured and a linear calibration curve can be constructed as illustrated in Figure 6 exhibiting the equation:  

                                                               ??=????+??

 

Figure

6

:

A Beer's Law Plot

-

Dashed Line Indicates Concentration

 

 

To determine the concentration of iron in the unknown well water sample, the sample must be diluted, acidified and complexed with excess 1,10-phenantroline. The absorbance of the resulting orange solution will be measured and the concentration determined by rearranging the linear calibration equation:

                                                       

 

When it is impossible to prepare a series of external standard calibration solutions due to time constraints, it is possible to estimate the analyte concentration in an unknown sample using a mathematical single-point approach. One only needs to prepare one single calibration standard of known concentration in addition to the unknown sample solution. The absorbance values of both solutions are measured and by using the Beer Lambert Law single point equation, the analyte concentration in the unknown sample can be calculated:

 

 

 

In order to use this method, it is assumed that the linear dynamic range is known and that the standard used for the calculation falls within that range. The major drawback of the single point approach is that one single calibration solution is used instead of a series of external standard solutions to evaluate the analyte concentration in an unknown sample. If the single standard is not prepared accurately, the calculated analyte concentration will be erroneous and accuracy is lost.

 

III-Thermo Scientific Genesys20 Visible Spectrophotometer

 

One of the most common ways to measure absorbance of visible light is to use a low cost single beam spectrophotometer shown in Figure 7 called a Genesys20. Within this instrument, the light source is an ordinary tungsten halogen lamp whose emission covers the entire visible spectrum, extending somewhat into the ultraviolet and infrared regions (325 - 1100 nm). The lamp is mounted very close to the entrance slit of the monochromator to ensure continuous energy output. An optical stop situated between the entrance slit and the turning mirror reduces the amount of stray light in the instrument. Diverging light projected onto the turning mirror is directed to the main mirror which in turn reflects the diverging light to parallel beams onto the grating.

 

Figure

7

:

Schematic Diagram of the Optical System of a

Genesys20

Spectrophotometer

 

 

The Czerny-Turner type monochromator uses a 1200 line/mm reflective grating as a wavelength dispersing element. The grating disperses the collimated light into its component wavelengths back to the main mirror where it is reflected the exit slit. When white light falls on the reflection grating it is dispersed into a fan of light beams with the short wavelengths (visible (violet) - UV) at one end and the longer wavelengths (red and infrared) at the other end. Figure 8 illustrates the how the overall spectrophotometer disperses the polychromatic source radiation into component wavelengths with the reflection grating which in turn focuses the monochromatic light onto the exit slit of a fixed bandwidth.  The exit slit acts as a blocking filter allowing only a certain bandwidth of wavelengths to pass onto the sample cuvette while blocking the rest of the entire visible spectrum. The grating position determines which band of wavelengths emerge from the exit slit.

 

Any radiation not absorbed by the analyte solution (transmitted radiation) falls on the solid state photodiode array detector located in the front section of the instrument immediately following the sample compartment. The mounting plane of the detector is angled with respect to the incoming light beam in order to minimize back reflections into the sample compartment. The detector converts the incoming light signal to an electrical signal which is then amplified. The readout device registers either percent transmittance or absorbance on an LED display.

 

 

 

 

 

Figure

8

:

Diagram Il

l

ustrating how the Exit Slit is used to Select the Desired Wavelength

Entrance

Slit

Stop

GRATING:

Micro

-

stepping

motor allows one

set

to

wavelength.

Grating

sends

horizontally

dispersed

polychromatic

collimated

light

back to the main

mirror

.

TURNING MIRROR:

to

Directs

diverging

beam

main mirror.

MAIN MIRROR:

Converts

diverging beam to

parallel

light

and

directs

it

to

the

grating.

MAIN MIRROR:

Main mirror directs

selected

from

wavelength

grating

to

exit

the

slit.

Exit Slit

MONOCHROMATIC

RADIANT ENERGY

FALLS ON SAMPLE

nm

800

4

00

nm

The stop is a filter that

of

amount

reduces

the

stray

the

in

light

monochromator.

Source

 

 

 

IV- Iron in Water Analysis

 

Iron present in natural waters is usually in the form of ferric or ferrous salts and can be attributed

to the weathering of rocks and minerals, acidic mine water drainage, landfill leachates, sewage effluents and iron-related industries. The iron concentration in drinking water is normally below

1.0 ppm where most water treatment plants remove insoluble iron, the major form found in aqueous environments. The established Canadian guidelines for iron in drinking water is less than 0.3 ppm.  

In this experiment, a rapid and simple spectrophotometric method for the determination of ferrous iron Fe²⁺ with the ligand 1,10-phenanthroline will be studied. Pure iron does not absorb light in the visible region (360 - 900 nm) of the spectrum. In order to use the Genesys20 spectrophotometer to determine the maximum absorbance wavelength for quantification, iron must first be treated with 1,10 -phenanthroline to form an orange-red complex that does absorb  light. 1,10-phenanthroline has two pairs of unshared electrons that can be used to form coordinate covalent bonds creating a coordinate covalent complex (brightly colored with central metal atom).

 

The complexation reaction forming an orange

-

red species is described by the equation:

Fe

2+

+

3 PhenH

+

à

Fe(Phen)

3

2+

+

3 H

+

Iron (II) +

(

3

10

1

,

-

phenanthroline

)

à

Iron(II)

-

10

,

1

-

phenanthroline complex

Figure 9: Iron(II)

-

1

10

,

-

phenanthroline complex

1

,10

-

phenanthroline will not react with Fe

3

+

and

must

first

be treated with hydroxylamine to reduce

 

Fe³⁺ to Fe²⁺. For the complex to form, iron must be in the +2 oxidation state The hydroxylamine will keep iron in the +2 state. Since dissolved oxygen in water can oxidize Fe²⁺ to Fe³⁺ an excess amount of hydroxylamine reducing agent is added to ensure iron remains as Fe²⁺. Sodium acetate buffer solution is also added to the blank, standards and unknown solutions to maintain the pH to 3.5 in order to ensure that Fe²⁺ does not oxidize to Fe³⁺.

 

The 1,10-phenanthroline complex of iron(II) illustrated in Figure 9 is an example of a chargetransfer complex, which consists of an electron-donor group bonded to an electron acceptor. When the complex absorbs radiation, an electron from the donor is transferred to an orbital that is largely associated with the acceptor. The excited state is thus the product of a kind of internal oxidation/reduction process. In this complex the metal ion serves as the electron donor. Charge-transfer absorption by complexation is important for spectrophotometric quantitative analyses because molar absorptivities of these types of complex ions are usually large (ε_max > 10,000 L/mg * cm), a circumstance that leads to high sensitivity.

 

 

 

 

 

 

 

 

 

 

 

 

Prelab Questions

1. Calculate the theoretical concentration of Fe²⁺ in ppm (mg/L) if 0.734 grams of Fe(NH₄)₂(SO₄)₂ * 6H₂O salt was weighed on an analytical balance and dissolved in a 1000.0 mL volumetric flask which was made to the mark.
2. Using the Fe²⁺ stock solution, calculate the theoretical concentration in ppm when 10.00 mL of stock solution (question 1) is transferred to a 100.00 mL volumetric flask and made to the mark.
3. Using the Fe²⁺ substock solution (question 2), calculate the theoretical concentrations in ppm for all Fe ²⁺ external calibration solutions when 1.000, 000, 5.000, 7.000 and 9.000 mL is transferred to five 25.00 mL volumetric flasks respectively.
4. Describe the preparation of the blank solution. What is the role of each chemical in the blank? Explain why it is not RO water.
5. Explain why a transfer pump rather than a volumetric pipette can be used to add 1,10phenanthroline, sodium acetate and hydroxylamine chloride solution to all the standards, unknowns and blank solutions
6. Explain why 1,10-phenanthroline is added to the external calibration solutions as well as the unknown solutions.
7. Explain the purpose of adding hydroxylamine and sodium acetate buffer to the external calibration solutions as well as the unknown solutions.
8. For the unknown sample, determine the dilution factor that you are diluting the unknown to.
9. State the analysis wavelength used for this experiment.

 

10. Set up tables in your lab hardcover lab notebook in order to record the data collected for this experiment. See Table 1- step 2 procedure for formatting.