CHEM 213Instrumental Analysis

Lab Lecture – Fluorescence and Calculations

2

Fluorescence Determination of Quinine in Tonic Water

M + hexcitation M* M + hemission

3

Fluorescence Spectrometer

4

Definitions

• Emission spectrum – graph of fluorescence intensity vs. wavelength.– Obtained using a fixed excitation wavelength.

• Excitation spectrum – graph of fluorescence intensity (observed at a constant emission wavelength) vs. the excitation wavelength. – Closely related to absorbance spectrum.

5

(emission)

Key features of instrument

• Two monochromators

• Emission spectrum usually collected at 90° angle to excitation beam

Fluorescence Spectrometer

6

• Must calibrate 1.0mL micropipette before making solutions– Similar procedure as for Cu

• Measure excitation and emission spectra to optimize wavelengths

• Be very careful to avoid fingerprints on cuvette walls• Use Standard Addition Calibration

• Only have 40 min on instrument so plan ahead and keep on top of things.

Tips on Lab Procedure

7

Glucose Experiment

Mass Sucrose (g): 6.0179Mass Glucose (g): 2.0164

Volume of solution (mL): 250

[sucrose] (g/L): 24.0716[glucose] (g/L): 8.0656

Working Standards:

Standard#Vol Std.

(mL)Final Vol

(mL)[glucose] [sucrose]

1,2 10 100 0.8066 2.40723,4 15 100 1.2098 3.61075,6 20 100 1.6131 4.81437,8 25 100 2.0164 6.0179

Glucose / Sucrose Stock Standard Unknown:

Dilute to 50 mL

Take 10 mL aliquot, Dilute to 100 mL for analysis

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Glucose Calibration

0

2

4

6

8

10

12

14

16

0.0 0.5 1.0 1.5 2.0 2.5

[glucose] mg/mL

PL

cu

rren

t (n

A)

Glucose Data

1.139 mg/mL 1.162 mg/mL

StandardPL

current (nA)

1 7.613 10.845 12.487 14.82

UnknownsPL

current (nA)

Unknown G 9.66Unknown G 9.52

mgmLmL

mg57550

10

100

2

162.1139.1Glucose Total

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1234567891011121314151617

A B C D Eyou enter x you enter y enter unkn y unk x (calc) RESULTS

0.80656 7.61 9.66 1.1476312791.61312 12.48 9.52 1.1241833762.0164 14.82 #N/A

#N/A#N/A#N/A

ave unk x = 1.135907327unk error = 0.0084543

slope = 5.970683254intercept = 2.807857143

slope error = 0.058288048intercept error 0.091039911

Glucose by Least Squares1234567891011121314151617

A B C D Eyou enter x you enter y enter unkn y unk x (calc) RESULTS

0.80656 7.61 9.66 1.1034309241.20984 10.84 9.52 1.0791682681.61312 12.48 #N/A2.0164 14.82 #N/A

#N/A#N/A

ave unk x = 1.091299596unk error = 0.077812929

slope = 5.770184487intercept = 3.293

slope error = 0.531962205intercept error 0.788232516

Using all Data Points

Omitting Outlier

Glucose Calibration

0

2

4

6

8

10

12

14

16

0.0 0.5 1.0 1.5 2.0 2.5

[glucose] mg/mL

PL

cu

rren

t (n

A)

Analyte Graph LSQ (all) LSQ(outlier)

Glucose 575 546 568

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Sucrose Data

Sucrose Calibration

0

5

10

15

20

25

30

35

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

[sucrose] mg/mL

Dif

f P

L c

urr

ent

(nA

)

3.385 mg/mL 3.654 mg/mL

StandardPL

current (nA)

Diff PL current

2 13.83 6.224 22.44 11.66 37.81 25.338 47.44 32.62

StandardPL

current (nA)

1 7.613 10.845 12.487 14.82

UnknownsPL

current (nA)

Unknown G 9.66Unknown G 9.52

UnknownsPL

current (nA)

Diff PL current

Unknown S 23.09 13.43Unknown S 24.87 15.35

mgmLmL

mg176050

10

100

2

654.3385.3Sucrose Total

Glucose Data

Sucrose Data

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Sucrose Calibration

0

5

10

15

20

25

30

35

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

[sucrose] mg/mL

Dif

f P

L c

urr

ent

(nA

)

Sucrose by Least Squares1234567891011121314151617

A B C D Eyou enter x you enter y enter unkn y unk x (calc) RESULTS

2.40716 6.22 13.43 3.498580283.61074 11.6 15.35 3.7472484824.81432 25.33 #N/A6.0179 32.62 #N/A

#N/A#N/A

ave unk x = 3.622914381unk error = 0.281611146

slope = 7.721131956intercept = -13.583

slope error = 0.904419885intercept error 3.999557538

1234567891011121314151617

A B C D Eyou enter x you enter y enter unkn y unk x (calc) RESULTS

2.40716 6.22 13.43 3.337615844.81432 25.33 15.35 3.5970355376.0179 32.62 #N/A

#N/A#N/A#N/A

ave unk x = 3.467325688unk error = 0.16074879

slope = 7.401134235intercept = -11.27214286

slope error = 0.465645951intercept error 2.17058313

Using all Data Points

Omitting Outlier

Analyte Graph LSQ (all) LSQ(outlier)

Sucrose 1760 1811 1734

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DM·HBr in Cough Syrup Experiment

Mass DM HBr (g): 0.0502Mass Unknown (g): 1.6835

Volume of Stock DM HBR (mL): 100Volume of Unknown (mL): 50

DM HBr Stock 1 (µg/mL): 502DM HBr Std Stock 2 (µg/mL): 25.1

Working Standards:

Standard#Vol Stock 2

(mL)Final Vol

(mL)[DMHBr] (µg/mL)

1 3.5 50 1.75702 6.5 50 3.26303 11 50 5.52204 16 50 8.0320

Standard Abs.Corrected

Abs1 0.102 0.0962 0.187 0.1813 0.316 0.314 0.45 0.444

Blank 0.006 01 0.096 0.0942 0.182 0.183 0.31 0.3084 0.447 0.445

Blank 0.002 01 0.099 0.0952 0.185 0.1813 0.314 0.314 0.449 0.445

Blank 0.004 0

StandardAverage

AbsAverage

corrected Abs

1 0.099 0.0952 0.185 0.1813 0.313 0.3094 0.449 0.445

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Results Graphical Method

DM HBr Calibration

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

0.500

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

[DM HBr] µg/mL

Ab

sorb

ance

6.896 µg/mL6.948 µg/mL6.983 µg/mL

Unknown Abs.Corrected

Abs

Run 1 0.39 0.384Run 2 0.39 0.388Run 3 0.393 0.389

Conc DM HBr (measured) (µg/mL): 6.942Conc DM HBr in 50 mL Syrup sample (µg/mL): 34.71

Conc DM HBr in Syrup mg/g: 1.031

× 50/10 – Dilution from original solution

sample 6835.1

1

1000

150

gg

mgmL

For calibration curve, plot

AVERAGE CORRECTED ABSORBANCE

for each standard.

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DM·HBr by Least Squares

1234567891011121314151617

A B C D Eyou enter x you enter y enter unkn y unk x (calc) RESULTS

1.757 0.096 0.384 6.9122774283.263 0.181 0.388 6.9839701975.522 0.31 0.389 7.0018933898.032 0.444 #N/A1.757 0.094 #N/A3.263 0.18 #N/A5.522 0.3088.032 0.4451.757 0.095 ave unk x = 6.9660470053.263 0.181 unk error = 0.0273526835.522 0.318.032 0.445 slope = 0.055793632

intercept = -0.001661064

slope error = 0.000263638intercept error 0.001374585

Use Corrected Absorbance

Use ALL numbers (not averages)- Makes a big difference in precision of

measurement (here error ~doubles)- Makes no difference to actual reported

value

Order that numbers are entered does not matter- But ensure that the X:Y pairs are correct!!!

1234567891011121314151617

A B C D Eyou enter x you enter y enter unkn y unk x (calc) RESULTS

1.757 0.095 0.384 6.9122774283.263 0.18066667 0.388 6.9839701975.522 0.30933333 0.389 7.0018933898.032 0.44466667 #N/A

#N/A#N/A

ave unk x = 6.966047005unk error = 0.042455498

slope = 0.055793632intercept = -0.001661064

slope error = 0.000550511intercept error 0.002870321

Here only averages used – NOT a good idea

Graphical Least Squares

Conc DM HBr (measured) (µg/mL): 6.942 6.966Conc DM HBr in 50 mL Syrup sample (µg/mL): 34.71 34.83

Conc DM HBr in Syrup mg/g: 1.031 1.034

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A class exercise…

Using the following data calculate [X] in the unknowns by both the graphical method and by least squares.

For the least squares, also perform the calculations using only the data from Reading 1 and Reading 2 columns.

Try to do it by yourself, especially the graphical method.

This is set up as an Assessment for you to practice with on the course website. It is available until Sunday evening.

Standard# [X] (mg/mL)

1 2.9752 5.5253 9.3504 13.6005 17.000

Standard Reading 1 Reading 2 Reading 31 0.113 0.116 0.1152 0.212 0.210 0.1933 0.354 0.358 0.3564 0.517 0.542 0.5155 0.646 0.646 0.647

Corrected Abs

Reading 1 Reading 2 Reading 3Unknown 1 0.133 0.130 0.129Unknown 2 0.486 0.482 0.484

Corrected Abs