UV-VIS Spectroscopy - Lab report of the detection of analytes from environmental samples using UV PDF

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Lab report of the detection of analytes from environmental samples using UV -VIS spect...

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UV –VIS Spectroscopy Analyte Detection Aim The various objectives of this experiment are; perform a quality control check on the UV-VIS spectrometer to ensure the machine is in working order, determine the iron concentration from the formation of an orange-red iron (II)-phenanthroline complex in a dilution serious using UV-VIS spectrometry and therefore calculate the stoichiometry of iron-phenanthroline complex using the mole-ratio method. Determine the amount of iron in mg/L present in a liquid iron supplement and compare the results with the information provided with the supplement. Determine the amount of quinine in a series of dilutions solutions by measuring fluorescence intensity and also determine the optimum excitation and emission wavelength for a 0.4mg/L quinine standard solution. Using the results from the quinine measurements determine the amount of quinine in a beverage. The final section of the experiment is designed to show how quenching reduces the intensity of fluorescence, using chlorine, bromine and iodine ions. A series of dilutions of the halides with a constant quinine concentration are made and the fluorescence intensity measured, graphs are constructed to note whether an expected value of 1 is achieved from the results obtained.

Part A: 

Quality control for UV-VIS spectrometer

It is vital to ensure equipment in the laboratory is in working order and calibrated correctly. A method for ensuring the equipment is within an acceptable range is the use of the Shewhart chart, which indicated if the results obtained are within the warning or action lines.

The UV-VIS spectrometer is turned on and set to wavelength of 800nm. The spectrometer is zeroed with distilled water. The absorbance of 0.01M Cu(NO3)2 is measured in triplicate. This exact process is redone 30minutes later again.

Absorbance results of 0.1M Cu(NO3)2 at wavelength 800nm Measurement Read 1

Read 2

Read 3

Average

Standard

1 2

0.1140 0.1170

0.1136 0.1175

0.1144 0.1181

deviation 0.00035 0.00048

0.1156 0.1197

Calculation of molar absorption coefficient using Beer-Lambert equation A= εcl Read 1 1. (0.1156) = (ε)(0.01)(1) ε = 11.56 ε = 11.40 ε = 11.36

Read 2 1. (0.1197) = (ε)(0.01)(1) ε = 11.97 ε = 11.7 ε = 11.75

Note that the molar absorption coefficient values for the first set of readings are below the warning line on the Stewhart chart, indicating that the UV-VIS spectrometer is ‘out of control’. The second set of values fall within the target mean on the Stewchart chart indicating that the spectrometer in now ‘in control’. It is most likely that the machine requires time to warm up when it is first switched on to give accurate absorbance readings of a sample.

Part B: 

Determination of iron using UV-VIS spectrophotometry.



Determination of the stoichiometry of the iron-phenanthroline complex



Anaysis of an iron supplement using UV-VIS spectrophotometry.

The presence of iron in a solution can be identified by the formation of an orange-red iron (II)-phenanthroline complex. Formation equation: Fe2+ + xPhH+ > Fe(Ph)x2+ + xH+

During sample preparation for the dilution series excess hydroxylamine hydrochloride, a reducing agent, is added in order to keep the iron in the Fe2+ form and therefore keep the solution stable for longer. An optimum pH is 4.5 and keeping the solution around this range will yield the most accurate results for iron determination.

Reagent Preparation: 10% solution of hydroxylamine hydrochloride is prepared in a 25ml volumetric flask. 2.5ml hydroxylamine hydrochloride made to the mark with water. Prepare 0.1mg/L Fe in 250ml volumetric flask. MW Fe = 55.85g/mol MW FeSO4.(NH4)2SO4.6H20 = 392.12g/mol 392.12g/mol = 55.85g X = 0.25g X = (0.25)(392.12)/(55.85) = 1.7552g needed 0.2ml of sodium citrate needed for colour change (yellow to blue) of standard iron solution.

Sample preparation: Calculate mole ratio (0 to 6) iron-phenanthroline complex in 5ml of standard iron solution. n = m/MW n = 0.0005g Fe/55.85g Fe n = 0.0000089 moles/5ml standard iron solution

(i)Dilution series (0 to 6) preparation: 1:0 will contain no phenanthroline. 1:1 MW phenanthroline = 198.33g/mol (0.0000089moles)(198.33g/mol) = 0.0018g 100ml = 0.3g Xml = 0.0018g Xml = (0.0018g)(100ml)/(0.03g) = 0.6ml 1:2 (0.0000089)(2)(198.33) = 0.0035g 100ml = 0.3g Xml = 0.0035g

X = 1.18ml 1:3 = 1.79ml 1:4 = 2.43ml 1:5 = 2.98ml 1:6 = 3.57ml

(ii)Iron supplement preparation: Make a 5 fold dilution of the liquid iron supplement in a 25ml volumetric flask Adjust the pH of the diluted supplement with 0.85M sodium citrate. 0.8ml sodium citrate needed for colour change. Add 5ml fresh diluted supplement, 0.5ml 10% hydroxylamine hydrochloride, 1.5ml 0.3% phenanthroline solution, 0.8ml sodium citrate and fill to mark with water in 50ml volumetric flask. Note that the supplement in is 50ml volumetric flask whereas dilution ratio iron solutions in 100ml.

Using the most concentrated iron solution (1:6) measure the absorbance spectrum in the UVVIS spectrometer using scan mode to determine the maximum wavelength. Max wavelength = 500nm Using single wavelength mode the absorbance values of the dilutions series was measured after the instrument was zeroed with distilled water.

Absorbance results at 500nm Dilution

Read 1

Read 2

Read 3

Average

Standard

1:0 1:1 1:2 1:3 1:4 1:5 1:6 X(supplement

0.0015 0.2884 0.6130 0.9213 0.8240 0.9696 0.9211 1.8042

0.0017 0.2894 0.6137 0.9210 0.8231 0.9696 0.9216 1.8056

0.0018 0.2905 0.6138 0.9207 0.8218 0.9698 0.9214 1.8026

0.0016 0.2894 0.6135 0.9210 0.8229 0.9697 0.9214 1.8035

deviation 0.000005 0.000350 0.000145 0.000100 0.000369 0.000003 0.000008 0.000597

)

1.2 1 f(x) = 0.2 x − 0.2 0.8 Absorbance

0.6 0.4 f(x) = − 0.04 x + 0.39 0.2 0 0

1

2

3

4

5

6

7

Phenanthroline:Iron (moles)

Fig.1 Mole ratio plot of absorbance V mole ratio of phenanthroline to iron. The point of intersection between the two lines is the point where all the iron has been complexed by the ligand molecules.

A) Stoichiometric ratio for Iron-phenanthroline complex Equation of the line: y= 0.308x + 0.005 Average absorbance value = 1.8035 (1.8035) = (0.308) X (x) + 0.005 X = 5.84:1 mole ratio of phenanthroline to iron

B) Extinction coefficient of the complex wavelength max using Beer-Lambert law A= εcl Max wavelength of 0.4mg/L phenanthroline solution fluorescenvce intensity = 463.7073 ? = (ε) X (0.4mg/L) X (5cm) ε=

C) Fomation constant for the complex when 0.01% iron remains unreacted 2+

Fe + xPhH+ > Fe(Ph)x2+ + xH+ [Fe(Ph)x2+][ xH+]/[ Fe2+][ xPhH+]

D) Calculate mg/L in the liquid iron supplement: 1 in 5 dilution of supplement

Another 1 in 10 dilution of supplement Dilution series of iron (0 to 6) in 100ml flask whereas supplement in 50ml volumetric flask; 1 in 2 dilution factor. 5.84:1 mole ratio of phenanthroline to iron 5:1 = 0.000883g 6:1 = 0.010591g 5.84:1 =

Part C: 

Quinine determination using spectrofluorimetry

Quinine is a strong fluorescent compound when in the presence of a low acidic solution such as H2SO4. When the concentration of quinine is low its florescence intensity is directly proportional to the concentration and should produce an intercept of 1 when plotted.

Reagent preparation: Prepare 10mg/L quinine stock solution in 100ml volumetric flask from 100mg/L solution M1V1 = M2V2 (100mg/L)(V1) = (10mg/L)(100ml) V1 = 10ml needed of 100mg/L quinine.

Sample preparation: (i)Prepare dilution series (0.05, 0.1, 0.2, 0.3, 0.4mg/L) in 50ml volumetric flask. 0.05 M1V1 = M2V2 (10mg/L)(V1) = (0.05mg/L)(50ml) V1 = 0.25ml needed 1.1 = 0.5ml 1.2 = 1ml 1.3 = 1.5ml 1.4 = 2ml

(ii)Beverage sample preparation: 250 fold dilution in 25ml flask = 0.01ml of bitter lemon

Using the most concentrated quinine solution (0.4mg/L) perform excitation and emission scan to determine the optimum wavelength and slit parameters. Optimum excited wavelength = 350nm Optimum emission wavelength = 450nm Slit size = 5cm Set instrument to simple scan and blank with 0.05M H2SO4. Blank fluorescence intensity = 10.232 Fluorescence intensity results @ Ex wavelength 350nm/Em wavelength 450nm/slit size 5cm. Concentratio Read 1 Read 2 Read 3 Average Standard n (mg/L) deviation 0.05 66.530 64.986 67.736 67.084 0.203 0.1 123.243 122.567 121.789 122.533 0.243 0.2 187.532 189.253 187.111 187.965 0.378 0.3 245.873 245.300 244.933 245.369 0.158 0.4 463.048 462.691 465.383 463.707 0.487 Beverage 112.816 114.764 115.384 114.321 0.447 500 450 400

f(x) = 1025.56 x + 1.96 R² = 0.92

350 300 Fluorscence intensity

250 200 150 100 50 0 0

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Quinine concentration (mg/L)

Fig.2 Calibration plot of mean fluorescence intensity V quinine concentration (mg/L). The average beverage fluorescence intensity value was 114.3213. This value corresponds to roughly 0.9 mg/L of quinine. The beverage was diluted by a factor of 250. (0.9mg/L) X (250) = 225mg/L The fluorescence intensity of the beverage was also measured in a 25ml volumetric flask whereas the 0-40mg/L solutions were measured, therefore the concentration value obtained will be multiplied by a factor of 2. (225mg/L) X (2) = 450mg/L quinine in bitter lemon. Part D  Quenching of quinine fluorescence by chloride, bromide and iodine ions.

Quenching is the reduction in the intensity of fluorescence which is caused by interference of halides (Cl, Br, I ions). These halides will be added in various concentrations in the form of salts to solutions containing a constant quantity of quinine. The fluorescence intensity will then be measured and plotted to investigate whether the presence of the halide ions caused quenching and consequently do not produce a linear graph. Sample preparation: Prepare 100ml stock solution of 4000mg/L concentration for NaCl, KBr and KI. MW NaCl = 58.5g/mol Na (23) + Cl (35.5) 58.5g = 35.5g X g = 0.4g X = (0.4)(58.5)/(35.5) = 0.6666g in 100ml will give 4000mg/L concentration KBr = 0.5953g KI = 0.5237g Prepare dilution series (0, 40, 80, 800, 2000mg/L) in 50ml volumetric flask. 0mg/L = No NaCl is needed 40mg/L M1V1 = M2V2 (4000mg/L)(V1) = (40mg/L)(50ml) V1 = 0.5ml 80mg/L = 1ml 800mg/L = 10ml 2000mg/L = 25ml Note the same volumes will be needed for KBr and KI. Fluorescence intensity results (Cl) Concentratio Read 1 Read 2 n (mg/L) 0 999.807 999.803 40 802.443 806.920 80 810.152 808.756 800 251.070 252.479 2000 114.787 114.627 Fluorescence intensity results (Br) Concentratio Read 1 Read 2 n (mg/L) 0 917.050 915.984 40 908.370 907.382 80 706.526 706.717 800 391.575 391.009 2000 179.923 179.886

Read 3

Average

998.341 807.036 808.926 253.925 114.191

999.317 805.466 809.278 252.491 114.535

Read 3

Average

915.271 906.919 705.541 389.860 180.122

916.102 907.557 706.261 390.815 179.977

Standard deviation 0.282 0.873 0.254 0.476 0.103

Standard deviation 0.298 0.247 0.210 0.291 0.043

Fluorescence intensity results (I) Concentratio Read 1 Read 2 n (mg/L) 0 922.524 922.012 40 723.974 723.100 80 620.474 618.580 800 359.608 358.927 2000 170.712 170.561

Read 3

Average

924.422 724.491 619.338 356.613 170.386

922.986 723.855 619.4464 358.383 170.553

Standard deviation 0.423 0.234 0.318 0.523 0.054

0mg/L of each halide = I0 10 9 8 7 f(x) = 2.02 x − 3.27 R² = 0.75

6 (I0)/(I)

5 4 3 2 1 0 0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Cl ion concentration (mg/L)

Fig.3 Relative fluorescence intensity (I0)/(I) V ion concentration for Chloride. Note in Fig.3 the fluorescence intensity value for 80mg/L phenanthroline was not included in the calibration curve as the value was not in keeping with the sequence of the other halide ions and was most likely prepared inaccurately.

6 5 4 (I0)/(I)

f(x) = 1.15 x − 1.51 R² = 0.88

3 2 1 0 0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Br ion concentration (mg/L)

Fig.4 Relative fluorescence intensity (I0)/(I) V ion concentration for Bromine. 6 5 f(x) = 1.21 x − 1.49 R² = 0.88

4 (I0)/(I)

3 2 1 0 0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

I ion concentration (mg/L)

Fig.5 Relative fluorescence intensity (I0)/(I) V ion concentration for Iodine. The intercept on the graphs (Fig.3-Fig.5) Chloride = 250 Bromine = 480 Iodine = 600 20% quenching effect Chloride = 799.4536 or less fluorescence intensity has 20% quenching effect 800/2000mg/L both give over 20% quenching effect. Bromine = 732.8814 or less 80/800/2000mg/L give over 20% quenching effect. Iodine = 738.3888 or less 40/80/800/2000mg/L give over 20% quenching effect.

Therefore Iodine does in fact have the strongest quenching effect. The expected intercept value was 1; therefore there is a significant difference in the values obtained. The fact that the intercept values are so different is due to the quenching effect caused by the presence of the halide ions. The lowest intercept value was 250 which was Cl, from the calculations about it was determined that Cl has the lowest percentage quenching effect. It can be concluded that the higher the intercept value the higher the percentile quenching effect. Conclusion: In part A the UV-VIS spectrometer was checked for quality control by measuring the absorbance value of 0.01M copper nitrate, calculate the absorption coefficient and determine where the values falls on the Shewhart chart when the machine was first turned on and then again after 30 minutes. The results were that the values obtained on the second set of data points where closer to the target value section of the shewhart chart than the first set of data points, concluding that the UV-VIS spectrometer produced more accurate results when it was turned on for half an hour to warm up. Part B included a dilution series (0 to 6) of phenanthroline to iron, a graph produced from the absorbance readings and the determination of the stoichiometric ratio for the ironphenanthroline complex, which resulted in 5.84:1 when the equation of the line was filled in. This ratio value is the point at which no more complex is being formed at the addition of more ligand (phenanthroline). The amount of iron in mg/L in a liquid supplement was to be calculated. The amount of iron present in the supplement was not calculated but if it had been it would have been compared with the mg/L of iron information provided with the supplement to check if the calculated value was similar to the value provided. Part C measured the fluorescence intensity of a dilution series of quinine (0 to 0.4mg/L). A calibration curve was produced and the concentration of quinine beverage of bitter lemon was found to be 450mg/L. The actual concentration of quinine in the bitter lemon was not provided but it likely wrong as the recommended concentration of quinine in non alcoholic beverages is less than 85mg/L in USA and Germany (Updated BfR health assessment, 2008). Part D measures the fluorescence intensity of various halide ion dilutions with a constant quinine concentration, the results were plotted and the intercept determined by extrapolating the linear line. When measuring the fluorescence intensity of quinine if the concentration is low, which it was in this experiment, the fluorescence intensity should be directionally proportional to the concentration giving an intercept of 1. The results obtained gave intercept readings of 250/480/600 for Cl/Br/I respectively. This shows how the addition of the halide ions caused a quenching effect which reduced the fluorescence intensity and how each ion has a stronger or weaker quenching effect. References: 1. Updated BfR health assessment; http://www.bfr.bund.de/cm/349/quinine_containing_beverages_may_cause_health_pr oblems.pdf 9th May 2008. Date accessed: 14/02/16.

Questions: 1. 2. 3. 4. 5. Quenching is the reduction in fluorescence intensity which is caused by reaction of halide ions with the sample being measured. When these halide ions are present in a sample the accuracy of the spectrofluoremeter is compromised due to the reduction in the fluorescence intensity that is being measured....