1. Dr Ron Epping LQB381 Unit Coordinator
Spectrophotometry
Dr Ron Epping LQB381 Unit Coordinator

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3. Spectrophotometry Assay There are THREE ways to use spectrophotometry
Extraction/treatment
dilutions, etc.
Assay
ORIGINAL
SAMPLE
urine, plasma, etc
TEST
SAMPLE
clarified
ABSORBANCE
READING
Colour
There are THREE ways to use spectrophotometry
Please familiarise yourself with the different approaches
and learn to recognise the difference
in your practical classes

4. Spectrophotometry Assay Using the MOLAR ABSORPTIVITY COEFFICIENT (E)
TEST
SAMPLE
ABSORBANCE
READING
Colour
Using the MOLAR ABSORPTIVITY COEFFICIENT (E)
PROVIDED YOUR TEST SAMPLE IS PURE … i.e. no other molecules in the TEST SAMPLE or buffer absorb light at the wavelength used in the assay
The molar concentration is determined using the Lambert/Beer Law:
A = E litre x l cm x C mol
mol.cm litre

5. Extraction/treatment
Spectrophotometry
Extraction/treatment
dilutions, etc.
Assay
ORIGINAL
SAMPLE
urine, plasma, etc
TEST
SAMPLE
clarified
ABSORBANCE
READING
Colour
KNOWN
STANDARD
ABSORBANCE
READING
2. Comparison to a single KNOWN STANDARD
COMMON IN CLINICAL ANALYSIS
Provided that the UNKNOWN (ORIGINAL SAMPLE) is treated in EXACTLY the same manner as the KNOWN (STANDARD) sample throughout the entire procedure (including extractions)…
the ratio of absorbances at the end of the assay is directly proportional to their initial concentrations !
EASY ! Dilution factors are NOT required !

6. Extraction/treatment
Spectrophotometry
Extraction/treatment
dilutions, etc.
Assay
ORIGINAL
SAMPLE
urine, plasma, etc
TEST
SAMPLE
clarified
ABSORBANCE
READING
Colour
STD A
STD B
STD C
ABSORBANCE
READINGS
3. Using a SERIES of Standards (a “STANDARD CURVE”)
COMMON IN RESEARCH
A series of standards with known amounts or concentrations is prepared from a stock solution.
The standards and TEST SAMPLE are assayed at the same time.
Absorbances are plotted as a standard curve and the amount of material in the TEST SAMPLE is read from the standard curve.
DILUTION FACTORS may be required to calculate the concentration in the ORIGINAL SAMPLE if some extraction/dilution has taken place.

7. LINES/CURVES of BEST FITX X

8. LINES/CURVES of BEST FIT
1.6
1.4
1.2 1
595nm
0.8
correct
0.6
0.4
0.2 20 40 60 80
100
120
ug of protein

9. LINES/CURVES of BEST FIT
There is an obvious linearity in this data.
In this case, LINEAR regression is appropriate
1.6
1.4
1.2 1
595nm
0.8
0.6
0.4
0.2 20 40 60 80
100
120
ug of protein

10. LINES/CURVES of BEST FIT
There is an obvious linearity in this data.
In this case, LINEAR regression is appropriate
0.2
0.4
0.6
0.8 1
1.2
1.4
1.6 20 40 60 80
100
120
ug of protein
595nm

11. Graphing Results X X correct When graphing results:
Use a convenient scale – you do not have to fill the sheet of paper
The line/smooth curve should go through as many points as possible
STANDARD POINTS ONLY should be plotted, and they should be easy to see
The line does not have to go through zero - it depends upon what you used to zero the spectrophotometer
The figure should have a descriptive title, labeled axes and (UNITS)
When reading values from graphs, DONT DRAW EXRA LINES or plot test readings
A Plot of absorbance vs Glucose
Fig 1. Glucose Standard Curve
Absorbance
Absorbance at 420 nm
Glucose
Amount glucose (mmoles)
X X correct