Dilution, pipetting and spectrophotometer
Par Junecooper • 26 Octobre 2018 • 873 Mots (4 Pages) • 438 Vues
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Final concentration Coomassie Blue (µg/mL)
Volume 50% ethanol (µL)
Volume Coomassie Blue stock solution (µL)
Total volume (µL)
Absorbance at λ=540 nm
20
1080
120
1200
0.396
40
960
240
1200
0.755
60
840
360
1200
1.128
80
720
480
1200
1.509
100
600
600
1200
1.870
Calculations
- Determining volumes of stock solution and ethanol required
Use c1v1 = c2v2
where c1 = concentration of stock solution (200 µg/mL)
v1 = volume of stock solution required
c2 = concentration of final solution
v1 = total volume of final solution (1200 µL (1.20 mL) for all samples)
example with 20 µg/mL:
200 µg/mL x v1 = 20 µg/mL x 1.20 mL
v1 = (20 µg/mL x 1.20 mL) ÷ 200 µg/mL
= 0.12 mL
convert to µL: 0.12 mL x = 120 µL[pic 7]
Calculate volume of 50% ethanol needed: Final volume- volume stock solution
1200 µL – 120 µL = 1080 µL of ethanol 50%
Table 3: Volumes of unknown solution and 50% ethanol used for 4 fold dilution of unknown solution and its absorbance at λ=540 nm.
Volume 50% ethanol (µL)
Volume unknown solution (µL)
Total volume (µL)
Absorbance at λ=540 nm
900
300
1200
0.784
Calculations:
Use vfinal = DF x vinitial
where vfinal = total volume of diluted solution
DF = dilution factor
vinitial = volume of unknown solution
Final volume chosen = 1200 µL
Volume of unknown required:
1200 µL = 4 x vinitial
vinitial = 1200 µL/ 4 = 300 µL of unknown solution
Volume of 50% ethanol required:
Final volume – volume unknown = 1200 µL – 300 µL = 900 µL 50% ethanol
Table 4: Wednesday section data of all groups for spectrophotometer exercise as well as average and standard deviation values at each concentration
[pic 8]
Calculations:
- Average = [pic 9]
Example with absorbance values at 40 µg/mL
Average =
= 0.8135 = 0.814[pic 10]
- Standard deviation :
[pic 11][8]
where s : standard deviation
N: total number of values
: each value in data set
: average of all values for given data set
∑: sum [pic 12][pic 13]
example with 40 µg/mL
σ = [pic 14][pic 15]
= 0.0844567 = 0.0845
[pic 16]
Results and discussion
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