Dr Ron Epping LQB381 Unit Coordinator Spectrophotometry Dr Ron Epping LQB381 Unit Coordinator 1991 - 2012

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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

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

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 !

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.

LINES/CURVES of BEST FIT X X

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

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