CHEM 213 Quantitative Analysis II Introduction to Instrumental Analysis.
CHEM 213 Instrumental Analysis
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Transcript of CHEM 213 Instrumental Analysis
CHEM 213Instrumental Analysis
Lab Lecture – Fluorescence and Calculations
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Fluorescence Determination of Quinine in Tonic Water
M + hexcitation M* M + hemission
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Fluorescence Spectrometer
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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.
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(emission)
Key features of instrument
• Two monochromators
• Emission spectrum usually collected at 90° angle to excitation beam
Fluorescence Spectrometer
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• 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
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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
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2
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10
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0.0 0.5 1.0 1.5 2.0 2.5
[glucose] mg/mL
PL c
urre
nt (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
mgmLmLmg 57550
10100
2162.1139.1Glucose 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
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6
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0.0 0.5 1.0 1.5 2.0 2.5
[glucose] mg/mL
PL c
urre
nt (n
A)
Analyte Graph LSQ (all) LSQ(outlier)
Glucose 575 546 568
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Sucrose Data
Sucrose Calibration
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5
10
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20
25
30
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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
[sucrose] mg/mL
Diff
PL c
urre
nt (n
A)
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
mgmLmLmg 176050
10100
2654.3385.3Sucrose Total
Glucose Data
Sucrose Data
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Sucrose Calibration
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5
10
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20
25
30
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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
[sucrose] mg/mL
Diff
PL c
urre
nt (n
A)
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 Abs
1 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
Standard Average Abs
Average 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
Abso
rban
ce
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 6835.11
1000150
ggmgmL
For calibration curve, plot
AVERAGE CORRECTED ABSORBANCE
for each standard.
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DM·HBr by Least Squares1234567891011121314151617
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