Spectrophotometry Dr Ron Epping LQB381 Unit Coordinator 1991 - 2012

Spectrophotometry
Dr Ron Epping
LQB381 Unit Coordinator
1991 - 2012
EPPING, LQB381
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EPPING, LQB381
Spectrophotometry
Extraction/treatment
dilutions, etc.
Assay
ORIGINAL
SAMPLE
TEST
SAMPLE
ABSORBANCE
READING
urine, plasma, etc
clarified
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
EPPING, LQB381
Spectrophotometry
Assay
TEST
SAMPLE
ABSORBANCE
READING
Colour
1. 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
EPPING, LQB381
Spectrophotometry
Extraction/treatment
dilutions, etc.
Assay
ORIGINAL
SAMPLE
TEST
SAMPLE
ABSORBANCE
READING
urine, plasma, etc
clarified
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 !
EPPING, LQB381
Spectrophotometry
Extraction/treatment
dilutions, etc.
Assay
ORIGINAL
SAMPLE
TEST
SAMPLE
ABSORBANCE
READING
urine, plasma, etc
clarified
STD A
STD B
STD C
Colour
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.
EPPING, LQB381
LINES/CURVES of BEST FIT
Protein Standard Curve
1.6
absorabce @ 595nm
1.4
1.2
1
X
0.8
0.6
0.4
X
0.2
0
0
20
40
60
80
100
120
ug of protein
EPPING, LQB381
LINES/CURVES of BEST FIT
1.6
absorabce @ 595nm
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
corre
ct
80
100
120
ug of protein
EPPING, LQB381
LINES/CURVES of BEST FIT
There is an obvious linearity in this data.
In this case, LINEAR regression is appropriate
1.6
absorabce @ 595nm
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
ug of protein
EPPING, LQB381
LINES/CURVES of BEST FIT
There is an obvious linearity in this data.
In this case, LINEAR regression is appropriate
1.6
absorabce @ 595nm
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
ug of protein
EPPING, LQB381
Graphing Results
 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
Absorbance
Glucose
X
Fig 1. Glucose Standard Curve
Absorbance at 420 nm
A Plot of absorbance vs Glucose
Amount glucose (mmoles)
X
EPPING, LQB381
EPPING, LQB381