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Spectrophotometry. Dr Ron Epping LQB381 Unit Coordinator 1991 - 2012. Spectrophotometry. Extraction/treatment dilutions, etc. Assay. ABSORBANCE READING C o l o u r. TEST SAMPLE clarified. ORIGINAL SAMPLE urine, plasma, etc. There are THREE ways to use spectrophotometry

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spectrophotometry
Spectrophotometry

Dr Ron EppingLQB381 Unit Coordinator

1991 - 2012

spectrophotometry1
Spectrophotometry

Extraction/treatment

dilutions, etc.

Assay

ABSORBANCE

READING

Colour

TEST

SAMPLE

clarified

ORIGINAL

SAMPLE

urine, plasma, etc

There are THREE ways to use spectrophotometry

Please familiarise yourself with the different approaches

and learn to recognise the difference

in your practical classes

spectrophotometry2
Spectrophotometry

Assay

ABSORBANCE

READING

Colour

TEST

SAMPLE

  • 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 litrexlcmx C mol
    • mol.cm litre
spectrophotometry3
Spectrophotometry

Extraction/treatment

dilutions, etc.

Assay

ABSORBANCE

READING

Colour

TEST

SAMPLE

clarified

ORIGINAL

SAMPLE

urine, plasma, etc

ABSORBANCE

READING

KNOWN

STANDARD

  • 2. Comparison to a single KNOWN STANDARD
    • COMMON IN CLINICAL ANALYSIS
    • Provided that the UNKNOWN (ORIGINAL SAMPLE) is treatedin 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 !
spectrophotometry4
Spectrophotometry

Extraction/treatment

dilutions, etc.

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.

Assay

ABSORBANCE

READING

Colour

TEST

SAMPLE

clarified

ORIGINAL

SAMPLE

urine, plasma, etc

STD A

STD B

STD C

ABSORBANCE

READINGS

lines curves of best fit1
LINES/CURVES of BEST FIT

1.6

1.4

1.2

1

0.8

absorabce @ 595nm

correct

0.6

0.4

0.2

0

0

20

40

60

80

100

120

ug of protein

lines curves of best fit2
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

0.8

absorabce @ 595nm

0.6

0.4

0.2

0

0

20

40

60

80

100

120

ug of protein

lines curves of best fit3

1.6

1.4

1.2

1

0.8

absorabce @ 595nm

0.6

0.4

0.2

0

0

20

40

60

80

100

120

ug of protein

LINES/CURVES of BEST FIT

There is an obvious linearity in this data.

In this case, LINEAR regression is appropriate

graphing results
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

A Plot of absorbance vs Glucose

Fig 1. Glucose Standard Curve

Absorbance at 420 nm

Absorbance

Glucose

Amount glucose (mmoles)

X Xcorrect