Experiment 2

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

Objectives 

The student will be introduced to the operation of the Genesys20 visible spectrophotometer by performing a colorimetric experiment quantifying the concentration of iron in well water. An external standard calibration plot following Beer’s Law will be constructed at a wavelength corresponding to the absorbance maximum (λ_max). Multipoint regression analysis and single point method will be used to quantify the iron concentration (mg/ US liquid quart) 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

P

o

P

b

                                                                     

 

Figure 2: Absorption of Light by an Analyte Species in Solution

 

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: Determination of Wavelength 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 Law: 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.

 

 

 

 

Entrance

Slit

Stop

GRATING:

Micro-stepping

motor allows one

to

set

wavelength.

Grating

sends

horizontally

dispersed

polychromatic

collimated

light

back to the main

mirror

.

TURNING MIRROR:

Directs diverging beam to

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 the exit

slit.

Exit Slit

MONOCHROMATIC

RADIANT ENERGY

FALLS ON SAMPLE

nm

800

4

nm

00

The stop is a filter that

reduces the amount of

stray

light

in

the

monochromator.

Source

 

Figure 8: Diagram Illustrating how the Exit Slit is used to Select the Desired Wavelength

 

 

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:

 

                         

                        Fe²⁺   +   3 PhenH⁺   à  Fe(Phen)₃²⁺   +   3 H⁺

 

      Iron (II) +   3 (1,10-phenanthroline) à Iron(II)-1,10-phenanthroline complex

 

Figure 9: Iron(II)-1,10-phenanthroline complex

 

1,10- phenanthroline will not react with Fe³⁺ 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 2.000, 4.00, 6.00, 8.00 and 10.00 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 notebook (on the RHS) to record the data collected for this experiment. See Table 1- step 2 procedure for formatting.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Procedure: 

 

NOTE: The Genesys 20 spectrophotometer must be turned at the beginning of the experiment to warm-up.

NOTE:  The diluent for this experiment is RO water.  

 

A – Preparation of Fe²⁺Stock and Substock Solution

1. Using the analytical balance, weigh accurately 0.15 - 0.17 g (to the nearest 0.0001 g) of Fe(NH₄)₂(SO₄)₂* 6H₂O salt into a clean dry 50 mL beaker. Record the actual mass to 4 decimal places in your hardcover notebook.

 

2. To the 50 mL beaker containing the Fe salt, add approximately 20 mL of RO water. Swirl gently to dissolve. 

 

3. Analytically transfer the dissolved Fe salt solution to an analytically rinsed 100.00 mL volumetric flask. Rinse the beaker several times with small volumes of RO water and transfer the rinses to the volumetric flask. Make the solution up to the mark with RO water. Invert and shake 15 times. This is your iron salt stock solution.

 

4. Obtain a 0.500-5.000 mL micropipette and tip from the professor. Rinse the tip 3 times with stock solution. To prepare the Fe²⁺ substock solution, transfer 10.00 mL of the Fe²⁺ stock solution to a 100.00 mL volumetric flask. Make the solution up to the mark with RO water. Invert and shake 15 times.

B – Preparation of External Standard Fe²⁺ Calibration Solutions  

5. Prepare five calibration external standard solutions by using 0.500 -000 micropipettes to transfer the volumes of Fe²⁺ substock solution prepared in step 4 - procedure indicated in the table below into 200 mL analytically rinsed volumetric flasks. DO NOT DILUTE YET!

 

Table 1: Fe ²⁺ External Standard Calibration Solution Preparation

Standard #	Fe2+ Substock Solution Volumes pipetted (mL)	Fe2+  Calibration Standard Concentrations ( ppm)	Absorbance
1	1.000
2	2.000
3	3.000
4	4.000
5	5.000

 

 

 

 

6. Before making each calibration solution to the mark with RO water, add the following reagents using a transfer pump apparatus found in the fumehood to each 25.00 mL volumetric flask in step.  Follow the precise order as listed: 

            1.0 mL hydroxylamine chloride solution

            2.5 mL 1,10-phenanthroline solution

            4.0 mL sodium acetate solution

 

Dilute each calibration solution to the mark with RO water. Stopper invert and shake 15 times to ensure adequate mixing. Allow at least 15 minutes for the colour to develop before running the solutions using the spectrophotometer.

7. To prepare a blank solution, into a separate 25.00 mL volumetric flask, add each of the reagents in Step 6- procedure to the blank. Fill to the mark with RO water. Stopper invert and shake 15 times to ensure adequate mixing.

NOTE: The blank contains all the reagents used in the analysis except the analyte species. It is NOT RO water.

C - Sample Preparation of the Unknown Well Water Sample - Triplicate 

8. Obtain an unknown well water sample from the instructor. Record the sample number in your hardcover notebook.
9. Pipette using a volumetric pipette 2.000 mL of well water sample into three separate 25.00 mL volumetric flasks (triplicate solution).
10. To each of the three 25.00 mL volumetric flasks, add the following reagents in the precise order using the transfer pump apparatus located in the fumehood:

1.0 mL hydroxylamine chloride solution

2.5 mL 1,10-phenanthroline solution

4.0 mL sodium acetate solution

 

11. Dilute each unknown well water solution to the mark with RO water. Stopper invert and shake 15 times to ensure adequate mixing.
12. Allow 15 minutes for the colour to develop before running the solutions on the spectrophotometer. The Genesys20 visible spectrophotometer should be turned on for at least ten minutes before any measurements are recorded.

 

 

 

 

 

 

 

 

 

D – Absorbance Measurements of the External Standards and Unknown Sample Solutions

 

NOTE: See Appendix B for the operating procedure for the Genesys20 spectrophotometer. 

 

13. Obtain one cuvette and a cuvette rack. Using a Sharpie, mark a reference line on the cuvette.

 

14. Analytically rinse the cuvette with the blank solution. Fill this cuvette 2/3 full with the blank solution. Use this solution to set 100 %T on the Genesys20 spectrophotometer at 508 nm.  

 

NOTE: Use a Kimwipe to wipe the cuvette prior to insertion into the sample compartment. Insert the cuvette into the sample compartment the same way each time, with the reference line aligned to the mark on the sample compartment.

 

15. Remove the cuvette and dump the blank solution into a waste beaker. Now set the spectrophotometer to Absorbance mode.

 

16. Analytically rinse the cuvette with standard solution #1 three times. Fill the cuvette 2/3 full with standard solution #1 and measure the absorbance. Record your results in your lab notebook. Repeat this step for each of the Fe ²⁺ external standard solutions.

 

17. Once the absorbance measurements have been made for external standards #1-5, analytically rinse the cuvette with the well water sample #1 three times. Fill the cuvette 2/3 full and record the absorbance. Repeat this procedure for each replicate unknown well water sample solution.

 

18. Rinse the cuvette with RO water and return the cuvette to the front bench. 

 

19. Shut down the spectrophotometer using the power switch at the back of the unit. 

 

E - Disposal of External Standard and Sample Solutions

 

20. Discard all contents of the waste beaker including the stock, substock, external standards and sample solutions into the waste container designated by the technologist. The waste will be disposed of as hazardous waste by the technologist.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Laboratory Report Questions

 

A – Fe²⁺  External Standard Solution Preparation

 

1. Using the mass in grams of Fe salt and flask the size that was used in step 1-3 - procedure, calculate the actual Fe²⁺ stock solution concentration in ppm. Show the calculation.

 

2. Calculate the actual Fe²⁺ substock solution concentration in ppm prepared in step 4 - procedure. Show the calculation. 

 

3. Calculate the actual Fe²⁺ external standard solution concentration (ppm) for all external standard solutions prepared in steps 5 - procedure. Show one sample calculation for standard solution # 1 only. Record the Fe²⁺ concentrations in ppm and corresponding absorbance values in table format

 

4. Construct a computer generated diagram illustrating the preparation of the Fe²⁺ stock, substock and external calibration standard solutions. Include the volumes pipetted, volumetric flask sizes, reagents added and actual (calculated) concentrations of all solutions. 

 

5. Record the absorbance values for the replicate unknown well water samples in a new table. 

 

6. Construct a computer generated diagram illustrating the preparation of the well water samples. Include the unknown number, volumes pipetted, volumetric flask sizes and reagents added. 

 

B – Multipoint Method

 

7. Using EXCEL, construct a calibration curve of absorbance versus actual Fe²⁺ concentration in ppm. The linear regression equation and the correlation coefficient must be shown on the graph. 

 

NOTE: When creating the Excel graph, you must follow all of the graphing rules outlined in the lecture slides and in the lab techniques course. 

 

8. Using the linear regression equation, calculate the Fe²⁺ concentration in ppm in the diluted unknown triplicate well water samples. Show one sample calculation for replicate sample #1. Record your calculated results in a new table format. 

 

9. Since a dilution was made to the well water sample prior to measurement, back calculate the the Fe²⁺concentration in ppm for the original well water sample. Show one sample calculation for replicate sample #1. Record your calculated results in the same table as step 8 - report. 

 

10. Assuming that your sample came from a US liquid quart, calculate the Fe²⁺ concentration in mg/ quart for the original well water sample. Show one sample calculation for replicate sample #1. Record the final concentration (mg Fe²⁺ / quart) in the same table as step 8 - report. 

 

 

 

11. Calculate the mean Fe²⁺ concentration (mg Fe²⁺ / quart) in in the original well water sample absolute standard deviation, relative standard deviation and true value (µ) at a 95% confidence interval. Show one sample calculation for each statistic and include a statement about the meaning of your µ value. Record the statistical results in a new table

 

NOTE: Before calculating any statistics, Q-test, at 95% confidence, (only if necessary- real outlier) any suspect datum (show your work, if performed). 

 

C – Single Point Method

 

12. Compare the external standard absorbance values to those for the replicate unknown samples. Select and state the name of the external standard solution whose absorbance value is closest to that of the unknown replicate samples. Using the selected standard and the following equation, calculate the Fe²⁺ concentration in ppm in each diluted unknown well water sample.

????????  ??  ????????

                                                      ???????? = ????????  

 

Show the calculation for replicate sample #1. Record calculated single point method results for each replicate sample in a new table. 

 

13. For each replicate sample, repeat steps 10-11 - report to calculate the Fe²⁺concentration in (mg Fe²⁺ / quart ) in the original well water sample. Show one sample calculation for replicate sample #1 only. Record your results in the same table as step 12 - report. 
14. Calculate the mean Fe²⁺concentration (mg Fe²⁺/ quart) for the original well water sample, absolute standard deviation, relative standard deviation and true value (µ) at a 95% confidence interval. Before calculating any statistics, Q-test any suspect datum. Record your statistical results in a new table. 

 

NOTE: DO NOT SHOW ANY SAMPLE CALCULATIONS STATISTICS HERE. 

 

D – Summary of Results and Unknown Identification

 

15. In a new final summary table, compare the the mean Fe²⁺ concentration (mg Fe²⁺/ quart) for the original well water sample absolute standard deviation, relative standard deviation and true value (µ) at a 95% confidence interval for the multipoint and single point methods. Results must be summarized in one table. 

 

16. Using the multipoint method results only, create a table comparing your experimental mean   Fe²⁺concentration (mg Fe²⁺/ quart) result with all of the true values for the possible well water samples posted on SLATE. State the identity of your unknown well water sample. 

 

17. Calculate the relative error for the mean Fe²⁺concentration (mg Fe²⁺/ quart) in the well water sample for both single point and multipoint methods. Show one sample calculation for the multipoint method only. Report your results in table format. 

 

 

 

E – Discussion (~ 14   marks)

 

18. Fully explain the reason why the blank solution has to be analyzed first using the Genesys 20 spectrophotometer prior to making absorbance measurements for the 5 calibration standards and samples. 

 

19. Based on your experimental results step 15-report, explain which method, multipoint or single point, gave the most accurate results. Include numerical values in your answer. 

 

 

20. a) Examine the final experimental results table step-15 report and discuss the differences in precision between multipoint and single point methods.

 

1. 2. Fully explain one way you could improve your precise.

 

21. If the unknown well water samples were analyzed at 550 nm but the external standard solutions were analyzed at 508 nm, fully explain how this would influence your mean  Fe²⁺ concentration in the unknown well water sample.

 

22. a) If the colour of the unknown well water replicate samples was somewhat lighter than the colour of standard 1, the determination for the Fe²⁺concentration may not be accurate. Fully explain why.

 

1. 2. Fully explain two ways in which you could change the procedure to correct for this. 

 

 

                                                                                                  A P P E N D I X A | 1

 

Appendix A

 

Operating Procedure for Thermo Scientific Genesys20 Single Beam Visible Spectrometer

 

NOTE: When the Genesys20 is turned on a power-on sequence will be initiated.  The poweron sequence includes testing the software revision, initializing the filter wheel and the monochromator.  For this reason, the unit requires 30 minutes to warm up before use.

 

1. On/Off Switch
2. LCD display screen
3. Sample compartment door
4. Key pad
5. Printer (no connection) 6. Lamp compartment

 

Figure 1: Genesys20 Spectrometer 

 

 

NOTE: Be certain the lid of the sample compartment is closed whenever one adjusts the instrument or takes readings to eliminate as much stray light as possible.

 

NOTE: This operating procedure is based on matched cuvettes.   

 

1. The ON/OFF switch for the Genesys20 can be found at the back of the unit.  Turn the Genesys20 ON.  This should be done 30 minutes before measurements are made.  The cell compartment must be empty and closed before turning on the instrument.

 

2. To set the absorbance mode, press the A/T/C button until absorbance appears on the display screen.

                                                                                                  A P P E N D I X A | 2

 

3. Press the nm  or nm    button to select the wavelength.  Holding down either key will cause the wavelength to change more quickly.  

 

Setting the 100% Transmittance/Zero Absorbance

 

4. Obtain the reference cuvette and fill with blank or pure solvent.  Dry/wipe the outside of the cuvette with Kimwipe tissue.  Insert the cuvette into the sample compartment.  Position the cuvette with the aid of the index mark.  Close the lid of the sample compartment.

 

5. Press the 0 ABS / 100 %T button to set the blank to 0 absorbance and 100% transmittance.  The display screen will read 100 %T.

 

6. Open the sample compartment and remove the blank.  

 

Measuring Absorbance of a Standard or Sample Solution

 

7. Obtain a matched cuvette and fill with the sample solution.  Dry/wipe the outside of the cuvette with Kimwipe tissue.

 

8. Insert the matched cuvette containing the solution into the sample compartment making certain to align the tube to match the index mark.  Close the sample compartment door. The absorbance measurement will appear on the display screen.

 

Changing the Wavelength Setting and Resetting the 100 %T

 

9. If readings are to be taken at another wavelength, remove the sample cuvette and close the lid of the sample compartment.  Use the nm  or nm    button to set the desired wavelength.  

 

10. Insert the reference cuvette containing the blank solution into the sample compartment and close the lid.  Press the 0 ABS / 100 %T button, 100 %T will display on the LCD screen indicating the blank has been read.  

 

11. Now remove the blank solution and insert the matched cuvette with the standard or sample solution.  The absorbance reading for the sample will appear on the display screen.  

 

NOTE: The blank must be used to set the instrument to 100 %T each time the wavelength is changed.

 

 

 

 

 

 

Appendix B

Operating Procedure for Perkin Elmer AAnalyst 200, 400 and PinAAcle900F Atomic Absorption Spectrometers

NOTE: This SOP will apply to all AAS’s in the lab. The instrument will already be on and the lamp warmed up. If the software is running, start at step 5. If the software is not running, then start at step 1.

NOTE: Make sure you save all your data as a pdf file; email it to yourself, your partner and your professor; and save to your One Drive as a backup. It is YOUR RESPONSIBILITY to make sure you save your data. Data will NOT be stored and WILL be erased when you log off. 

1. The computer will be already logged on. If you become logged out, call a tech over for assistance to log back onto the computer.

 

2. To log into the Syngistix software, click on the Syngistix for AA icon on the taskbar or the desktop:

                                             or           

3. When the login screen pops up you will enter the corresponding login information depending upon the AAS being used:
   1. AAnalyst 200:   Login: AA200            PW: 123456
   2. AAnalyst 400:   Login: AA400            PW: 123456
   3. PinAAcle900F:   Login: PIN900F       PW: 123456

 

4. To open the Analysis Workspace for use:
   1. Click on the Syngistix icon at the top of the screen: 
   2. Hover over the Workspace command and choose open:

 

1. 3. Choose the correct workspace.  ex. Ca Standard Addition

 

5. The screen should now look like this:

 

6. a. The Method Editor Screen looks like this:

 

Click on the Calibration tab at the top.

 

b. Click on the Standard concentration tab and enter your concentrations into the appropriate cell for each concentration:

 

 

7. Click on the Analysis Window and be sure the Save data to Results Data Set is check marked off and then enter your name in the box as shown:

 

8. a. Click on the Flame control window. Make sure the green checkmark is shown. If it is not showing, have your professor check that the gas has been turned on. If it is still out after that, call a technologist over for help.

 

 

b. Click on the Flame On/Off button to start the flame.

 

9. In the Analysis window, you can analyze your blank, standards and samples  NOTE: If the external standards method is used, there will be samples to analyze

 If the standard addition method is used, there will be no samples to analyze

 

1. 1. The analysis screen looks as shown: The screen on the right side of the Analysis window will show the standards/samples after they have been analyzed.
   2. Analyze the blank with the Analyze blank button
   3. Analyze the standards with the Analyze standards button
   4. Analyze any samples (if any), with the Analyze samples button.

 

                             

 

1. 1. (IF NECESSARY) To remove or reanalyze a standard or sample click on the Results tab and select Edit Calibration:

 

1. 2. Uncheck the point that you wish to remove ‘not include in’ the calibration and the calibration will adjust itself accordingly. 

 

1. 3. To Reanalyze a standard, place a checkmark next to the standard to be reanalyzed in the reanalyze column, place the aspiration tubing into the standard and click on the Reanalyze button to reanalyze it.

 

 

10. To print results:
    1. To print the Calibration Curve, make sure the Edit Calibration window is selected and then click on the Syngistix icon at the top left of the screen and click on Print Active Window, Preview and then Print.

 

1. 1. 1. Make sure Microsoft Print to PDF is selected as the printer

  

 

1. 1. 2. Click on Ok. 

 

1. 1. 3. Select the Desktop on the side menu and then click on the Student Data W2021 folder:

 

 

1. 1. 4. Click on the Save button to save the file.

 

1. 2. To print the Results, click on the Results window to make it the active window and follow the steps above in 11 a. I - iii.

 

1. 3. To print the Method Editor, click on the Method Editor window to make it the active window and follow the steps above in 11 a. I - iii.

 

11. Open the browser Microsoft Edge. It will take you to the office.sheridancollege.ca page where you can email your data and save a backup copy to your One Drive.

                                                                   

1. 1. Open your One Drive program  to backup your results to your One Drive folders. Click on Upload and choose the files (results, calibration, method editor) to upload:

 

 

1. 2. Click on Desktop, then choose the Student Data W2021 folder to find your files to upload. Then click on the Open button at the bottom of the dialogue box to upload your files to your One Drive.

 

 

1. 3. Open your Outlook program to email yourself, partner and your professor ALL your results (results, calibration, method editor).

12. Click on the Flame On/Off button to shut off the flame.

 

13. Click on the Results tab at the top of the screen:

 

1. 1. Click on the Clear button in the Calibration window. Choose Clear Calibration Blank.

 

1. 2. Click on the New Calibration button to clear your calibration.

 

1. 3. Click on the Clear button in the Results window and choose Clear Results Display to clear your results.

 

14. Do NOT shut off the software. Leave the software running.

 

15. Do NOT shut off the computer.

 

Appendix C

 

Operating Procedure for Agilent Cary 100 UV-Vis Double Beam Spectrometer

 

A – Start Up

 

1. Turn on the computer and computer monitor while the instrument is off. The printer should already be on.

 

2. Turn ON the INSTRUMENT (switch on front left hand side of instrument). 

 

3. Double click to open the application CARY WINUV. 

        

B – Scan Application (Qualitative Spectrum)

 

4. In the CARY WINUV window, double click on the SCAN icon. The screen that opens should be divided into 2 parts. The top half shows a graph and the bottom half will display the peak report. The top left-hand corner of the screen should show Scan – Online. 

 

5. Click SETUP (top left-hand side of screen). Click on the CARY tab and enter the following parameters:
   • X Mode: Nanometers. 
   • Start wavelength: 320 nm (UV)
   • End wavelength: 200 nm (UV) Y Mode: Abs
   • All remaining default parameters should not be changed.

 

      NOTE: Cary scans from highest to lowest wavelength.         

 

6. While in SETUP, click on the Reports tab.

                    

 In the Operator Name box, type your name.  In the Operator Comments box, type the analyte name.

 

 Under Options, select Parameters, User Data Form and Graph. Deselect AutoPrint and Company Logo. For %Page Height enter a value of 75.  

                                

 Under Peaks Table, select Maximum Peak and ensure that All Peaks is deselected. Under X-Y Pairs Table, ensure that Include X-Y Pairs Table is deselected.

 

7. To exit SETUP, click OK. 

 

8. Obtain two quartz cuvettes from your professor or technologist. These cuvettes have 2 transparent sides and 2 frosted sides. Handle cuvettes carefully – do NOT drop the cuvettes!

 

 

 

NOTE: Prior to each measurement, the cuvette must be rinsed 3X with the solution to be analyzed and wiped with a Kimwipe. Ensure there are no air bubbles in the solution. Do not scratch the cuvettes, and do not touch the transparent faces of the cuvette with your fingers.

 

9. Slide the cell compartment lid open and place cuvettes filled with BLANK solution in both the reference and sample compartments. The transparent face of the cuvette must face the direction of the light source. Slide the lid shut.

 

10. Click ZERO (upper left hand side of screen). A ‘Zero’ dialog box will appear. Click OK. After zeroing, the absorbance reading in the top left hand corner of the screen will read 0.000 in RED. 

 

11. Slide the cell compartment lid open and remove the cuvette from the sample compartment. Dump the blank solution into a waste beaker. Fill a cuvette with the highest concentrated standard solution, insert the cuvette into the sample compartment and slide the lid shut.

 

NOTE: The cuvette filled with blank solution remains in the reference compartment at all times. Do not remove.

 

12. Click START (green light) to begin the scan. A ‘Save As’ dialog box will appear, prompting you to save your file. Click on desktop, open the Student folder and then open the folder corresponding to your lab section. Type your name, date and the word ‘Scan’ for the name of your file and click OK. (If the ‘Save As’ box does not appear, save your scan before exiting the software by selecting FILE, SAVE AS.)

 

             A ‘Sample Name’ dialogue box will appear. Type the standard solution name and click OK. 

 

13. Once the spectrum is complete, a ‘Sample Name’ dialog box will appear again. Click Finish. 

 

 The absorbance maximum on the spectrum can be labelled before printing. Move the cursor to the peak maximum and right click. Select Add Label.  The peak maximum will be labelled.

 

14. Click on GRAPH, GRAPH PREFERENCES. Change Trace Width from 1 pixel to 4 pixels. Click OK.

 

15. To print the spectrum and peak table report, click PRINT (bottom left hand side of screen). Click OK to print your results.

 

16. To exit the SCAN application, select FILE, EXIT. This application will shut down.

 

C – Concentration Application: Analysis

17. In the CARY WINUV screen, double click on the CONCENTRATION icon. The screen that opens should be divided into 2 parts. The top half shows a graph and the bottom half will display the report. The top left-hand corner of the screen should show Concentration – Online. 

 

18. Select SETUP (top left-hand side of screen). Click on the CARY tab and enter the following parameters:
    • Wavelength: Absorbance maximum obtained from the UV spectrum in Part B 
    • Replicates: 1
    • All remaining default parameters should not be changed.

 

19. While in SETUP, click on the STANDARDS tab. Enter the following parameters:
    • Select Calibrate during run
    • Units: mg/L
    • Standards: 5
    • Enter the ACTUAL concentration of each calibration standard solution.
    • Fit Type: Select linear
    • Min R²: 0.9

 

20. While in SETUP, click on the SAMPLES tab. Decrease the number of samples to 3.  

 

21. While in SETUP, click on the REPORTS tab.

        

 In the Operator Name box, type your name.  In the Operator Comments box, type the analyte name, analysis wavelength and unknown #.

 

 Under Options, select Results, Graph and Parameters. Deselect Standards, AutoPrint and Company Logo. For %Page Height enter a value of 75.  

         

22. To exit SETUP click OK. An empty graph should be displayed on the computer screen of absorbance versus concentration.  

 

NOTE: Prior to each measurement, cuvette must be rinsed 3X with the solution to be analyzed. Ensure there are no air bubbles in the solution and wipe the cuvette dry with a Kimwipe.

 

23. Slide the cell compartment lid open and place the cuvettes filled with BLANK solution into both the reference and sample compartments. The transparent face of the cuvette must face the direction of the light source. Slide the lid shut. 

 

24. Click ZERO (upper left hand side of screen). A ‘Zero’ dialog box will appear. Click OK. After zeroing, the absorbance reading in the top left hand corner of the screen will read 0.000 in RED. 

 

25. Slide the cell compartment lid open and remove the cuvette from the sample compartment. Dump the blank solution into a waste beaker. Fill a cuvette with the lowest concentrated standard solution and insert the cuvette into the sample compartment. 

 

NOTE: The cuvette filled with blank solution remains in the reference compartment at all times. Do not remove. 

 

26. Click on START (green light) to begin the absorbance readings of each calibration standard and unknown sample solutions.
27. After clicking on START, a ‘Standard/Sample Selection’ dialog box will appear showing a list of 5 calibration standards and 3 unknown samples. The list should appear on the right hand side of the dialog box under the heading ‘Selected for Analysis’. Highlight the entire list.  The solutions are now selected for analysis. Click OK.

 

28. A ‘Save As’ dialog box will appear, prompting you to save your file. Click on desktop, open the Student folder and then open the folder corresponding to your lab section. Type your name, date and the word ‘Calibration’ for the name of your file and click Save.

 

29. Follow the onscreen instructions.  A ‘Present Standard’ dialog box will appear, prompting you to Present Std 1". Click OK only after the 1^st standard solution has been placed in the sample compartment with the lid shut.  

 

NOTE: It is important to measure standards beginning with the lowest concentration calibration standard to the highest in ascending order.

 

30. Continue to follow on-screen instructions to complete measurement of your calibration standards AND unknown samples.

 

NOTE: If you have to re-read a particular standard (or unknown sample), click Reread (upper left hand side of screen) and a dialog box will appear. Highlight the standard or group of solutions that needs to be re-read and click on the arrow > key to make the solutions available for analysis (right hand side of dialog box). Click OK and follow the onscreen instructions.

 

31. To remove a point from the calibration curve, select RECALCULATE (bottom left hand corner of screen). To remove a point, highlight Yes next to the point you want to remove and re-type No. Click OK.

 

32. Click on GRAPH, GRAPH PREFERENCES. Change Trace Width from 1 pixel to 4 pixels. Click OK.

 

33. Remove the two cuvettes from the cell compartment and rinse with RO water. Place the cuvettes back in the appropriate case.

 

 

D- Printing Reports from the Cary 100 UV-VIS Spectrometer

 

34. To print the desired report:
    1. Click on the Print button. 
    2. Make sure Primo PDF is selected as the printer.

 

 

  

 

1. 1. Click on OK. 
   2. This screen will appear    Click on create PDF.

 

1. 3. Select the Desktop on the side menu and then click on the Student Data W2021 folder and select the folder for the day of your lab:

 

 

1. 4. Name your data file with your name and the type of data.
   5. Click on the save button to save the file.

 

 

35. Once all reports have been saved to a pdf file, open the browser Microsoft Edge or Google Chrome. It will take you to the office.sheridancollege.ca page where you can email your data and save a backup copy to your One Drive.

 

  

1. 1. 1. Open your One Drive program  to backup your results to your One Drive folders.

 

1. 1. 2. Click on upload and choose the files to upload:

 

 

1. 1. 3. Click on Desktop, then choose the Student Data W2021 folder to find your files to upload. Then click on the open button at the bottom of the dialogue box to upload your files to your One Drive.
      4. Open your Outlook program to email yourself,  your partner and your professor ALL your results.

 

 

 

Appendix D

 

Operating Procedure for Agilent Cary Eclipse Fluorescence Spectrometer

 

A – Start Up

 

1. Turn on the computer and computer monitor. The printer should already be on.

 

2. Turn on the instrument by pressing the Power Switch ON (bottom right on the front of instrument).
3. Open the software by double clicking on Cary Eclipse icon on the desktop. Double click on the CONCENTRATION icon. All work on this instrument will be completed using the

Concentration application. The top half of the screen displays the graph, while the bottom half of the screen displays results.

 

4. After initialization of the instrument, the traffic light next to the Start button (top center of the screen) should turn green and ‘Concentration – Online’ is shown in the top left corner of the screen.

 

B –Concentration Application:  Prescan (Determination of the Instrumental Settings)

 

5. Obtain one quartz cuvette from your professor or technologist. All sides of this cuvette are transparent. Handle the cuvette carefully – do NOT drop the cuvette!

 

NOTE:Prior to each measurement, the cuvette must be rinsed 3X with the solution to be analyzed and wiped with a Kimwipe. Ensure there are no bubbles in the solution. Do not scratch the cuvette, and avoid touching the transparent faces of the cuvette with your fingers (hold the cuvette near the top).

 

6. Carefully pour the external standard solution prepared for prescan analysis into the quartz cuvette. Make certain there are no air bubbles inside the cuvette. Wipe the outside surfaces of the cuvette with a Kimwipe.

 

7. Slide the cell compartment lid open and place the cuvette filled with the external standard solution in the cell holder. Slide the lid shut.

 

8. Click on SETUP. Click on the Cary tab. The Excitation slit width (nm) should be 5.0 nm. If necessary, change the Emission slit width to 5.0 nm. To exit SETUP click OK.

 

9. Click on PRESCAN (top left corner), to generate an excitation and emission spectrum for the analyte. A ‘Prescan’ dialog box will appear with the instruction to insert sample. Click OK.

 

10. A ‘Prescan’ dialog box will appear to select the type of scatter for which to search. Select Rayleigh scatter and click OK.

 

 

 

 

 

 

 

 

11. An excitation and emission spectrum will be generated. An ‘Accept Changes’ dialog box then appears that asks if you want to update the current method with the new prescan settings. Record the excitation and emission wavelengths in your lab notebook. Click OK.

 

12. The maximum of the excitation and emission spectrum should be labelled with the wavelengths. If the labels are not present, move the cursor to the maximum (top of the peak), and right click and select Add label. An ‘Add label’ dialog box will appear. Click OK. The wavelength and corresponding intensity value will appear on the graph. Single click on the label and a box will appear around the values. Click and drag this box next to the peak.  

 

C – Concentration Application : Analysis

13. Single click on SETUP (top left corner of the screen).

 

14. Click on the Cary tab and ensure the following parameters are entered.

 

• • Data mode: Fluorescence
  • Ex. wavelength & Em. wavelength: Values determined by the prescan should already be entered here. Ask the professor or the technologist to check these values to ensure they are correct.
  • Ex. slit width: 5 nm
  • Em. slit width: 5 nm

 

15. Click on the Options tab. A PMT voltage will already be entered here as determined from the prescan. Do not change any of the default settings.
16. Click on the Standards tab and enter the following parameters.

 

• • • Units: µg/L
    • Standards: 5
    • Replicates: 1
    • In the table, type in the ACTUAL concentration of each of the standard addition solutions. Std 1 refers to U (0.0 ppb), Std 2 refers to U+1, Std 3 refers to U+2, etc. Remember that the concentration entered for.
    • Fit type: Linear (click on corresponding graph)
    • Min R²: 0.8

 

17. Click on the Samples tab. Enter 0 (zero) for No. of samples.

 

18. Click on the Reports tab and enter the following parameters:

 

• • • Name: Type your names and the date.
    • Comments: Type the name of analyte and unknown #
    • Options: Deselect Auto-print and Company logo. Ensure that Parameters, Results and Graph are selected. Enter a value of 50 for % Page height.

 

19. To exit SETUP click OK.

 

 

 

 

 

 

 

20. Carefully pour the BLANK solution into the quartz cuvette. Make certain there are no air bubbles inside the cuvette. Wipe the outside surfaces of the cuvette with a Kimwipe.

 

21. Slide the cell compartment lid open. Place the cuvette filled with the BLANK into the cell compartment and slide the lid shut. Select ZERO (top left side of the screen) to zero the instrument on the blank. Results of the zero will be displayed on the bottom half of the screen upon completion.

 

22. Slide the cell compartment lid open and remove the cuvette. Dump the blank into a waste beaker. Analytically rinse the cuvette with the first solution (U). Fill the cuvette with U (unknown) solution. Place the cuvette in the sample holder and slide the lid shut.
23. Click on START (top center of the screen). A ‘Standard/Sample Selection’ dialog box will appear. Stds 1 – 5 should all appear under the column heading Selected for Analysis. Click OK.

 

24. A ‘Warning’ dialog box will appear with the message that data in the graphics window will not be saved. Click OK.

 

25. A ‘Present Standard’ dialog box will appear with the instruction to Present Std 1 (U). Click OK. The fluorescence intensity of Std 1 (U) will be measured.
26. The ‘Present Standard’ dialog box will appear again with the instruction to Present Std 2. Slide the cell compartment open. Dump the U solution into the waste beaker. Analytically rinse the cuvette with Std 2 (U+1) and fill the cuvette with the solution. Place the cuvette back in the cell compartment, slide the lid shut, and click OK in the Present Standard box.

 

27. Continue reading the other standard additions in the correct order by following the onscreen procedure.

 

NOTE: If you have to re-read a standard addition solution, click REREAD (top left corner) and a ‘Standard/Sample Selection’ dialog box appears. Stds 1 – 5 should appear under the column heading Solutions Available. Click to highlight the standard that needs to be re- read and then click on the > arrow to move the standard under the Selected for Analysis heading. Click OK and follow the onscreen instructions.

 

28. To save your data, click on FILE, SAVE AS to save your data as a Batch (.FBCN) file. Click on the Desktop icon, open the Student folder, and then open the folder corresponding to your lab section. Type your name and the date. Click Save.

 

29. To clear the calibration curve, click on the icon of a garbage can with a graph. To clear the report/results, click on the Clear Report in the bottom left-hand corner of the screen.

 

30. Repeat steps 20-29 to analyze the U_ii series.

 

31. Remove the cuvette from the cell compartment and rinse with Type 1 ASTM water. Place the cuvette back in the appropriate case.

 

 

 

 

D- Printing Reports from the Cary Eclipse Fluorescence Spectrometer

 

32. To print the desired report:
    1. Click on the Print button. 
    2. Make sure Primo PDF is selected as the printer.

  

 

1. 1. Click on OK. 
   2. This screen will appear    Click on create PDF.

 

1. 3. Select the Desktop on the side menu and then click on the Student Data W2021 folder and select the folder for the day of your lab:

 

 

 

1. 4. Name your data file with your name and the type of data.
   5. Click on the save button to save the file.

 

33. Once all reports have been saved to a pdf file, open the browser Microsoft Edge or Google Chrome. It will take you to the office.sheridancollege.ca page where you can email your data and save a backup copy to your One Drive.

 

1. 3. Open your One Drive program  to backup your results to your One Drive folders.

 

 

1. 4. Click on upload and choose the files to upload:

 

 

 

1. 5. Click on Desktop, then choose the Student Data W2021 folder to find your files to upload. Then click on the open button at the bottom of the dialogue box to upload your files to your One Driv
   6. Open your Outlook program to email yourself,  your partner and your professor ALL your results.