Spectrophotometry Dr Ron Epping LQB381 Unit Coordinator 1991 - 2012 EPPING, LQB381 COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been copied and communicated to you by or on behalf of The Queensland University of Technology pursuant to Part VB of The Copyright Act 1968 (The Act). The material in this communication may be subject to copyright under The Act. Any further copying or communication of this material by you may be the subject of copyright protection under The Act. Do not remove this notice. 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