Absorbance Demo Tutorial

1. Absorbance Demo Tutorial Working with Primary Colors . . .

2. Pathlength Correction: A Review …or Lets Go Over this One More Time

3. Pathlength Correction Feature... How Does it work ? • Uses Near IR absorbance of water • Water has a peak absorbance at 977nm • Commonly used reagent components do not absorb at977 nm • Absorbance at 977 nm can be used to calculate Pathlength Spectral scan of water in Near IR region

4. Pathlength Correction Feature...How Does it work ? • Measure absorbance of water in a 1 cm cuvette at 977 nm • Measure absorbance of water in a 1 cm cuvette at 900 nm as a reference 1 cm Light Detector

5. ?? Pathlength Correction Feature... How Does it work ? • Measure absorbance of sample in a microplate at 977 nm • Measure absorbance of sample in a microplate at 900 nm as a reference • Measure absorbance of sample at target wavelength (e.g.. 260 nm) Detector Microwell Light

6. Pathlength Correction Feature... How Does it work ? • O.D. = (e) x (Concentration) x (pathlength) - Beers Law • Concentration of Water is always the same (but not always 100%) • Extinction constant of Water is the same (at a given temp.) • OD of Water isaPathlength • Equation to Calculate Pathlength of Sample in Microplate well • (A977 - A900) Sample =Pathlength of Sample • (A977 - A900) 1.0 cm water

7. Pathlength Correction Feature... How Does it work ? • Having Calculated the Pathlength of the sample well we can correct the sample reading to 1.0 cm • Equation to correct sample OD’s to 1.0 cm pathlength OD Values • ((A260 Sample)- (A260 Blank)) = • Pathlength of Sample Absorbance of Sample Corrected to 1.0 cm

8. Demo Dye Solution • Secret formula: If I told you what was in it, I’d have to kill you… • Mixture of dyes (some yellow, some blue) • Looks green • Not entirely water based • “K” value not 0.180 • Spectral scans show several peaks • 413 nm and 630 nm most prominent

9. Absorbance Demo Dye Solution

10. Endpoint Reading • Dilution Series using Demo Dye and Demo Diluent • Linear Change in Absorbance with Raw Data • Slight Change in slope of line with pathlength correction • Dilution Series using Various volumes of Demo Dye • Linear Change in Absorbance with Raw Data • Pathlength Correction should correct all the wells to the same absorbance value

11. Hypothetical Procedure • Pipette various volumes of Demo Dye solution into wells of a microplate (4 wells per volume) • Volumes: 0, 150, 200, 250 µl • Adjust volume to 250 µl with Diluent in 2 wells per volume • Wells A1-B4 • Open Scan Protocol in KC4 • Scan well(s) from 300 nm to 700 nm • Discuss scan features • Endpoint Reads at peak(s) determined with scan • Choose 630 nm • Open Endpoint Protocol in KC4 • Demonstrate difference between samples that were diluted to 250 µl to those that were not

12. Solution Volumes (µl) 1 2 3 4 5 A 0 150 200 250 250 100 50 0 B 0 150 200 250 250 100 50 0 C 0 150 200 250 0 0 0 0 D 0 150 200 250 0 0 0 0 E Dye solution Diluent Pipetting Volumes

13. Uncorrected Values* *Approximate values at 630 nm

14. Tale of Two Dilutions

15. Pathlength Corrected Values* *Note that blank subtraction was performed

16. Wells Filled to 300 µl with Diluent:Raw Absorbance vs. Pathlength Corrected

17. Various Volumes of Dye Solution: Raw Absorbance vs. Pathlength Corrected

18. What’s So Special About Demo Dye Solution? • Does NOT contain 100% Water • Difference between Bio-Cell K value (approx 0.140) of diluent as compared to water only (0.180). False but believable results… • Proves that yellow and blue mixed together makes green • When used with a protocol for some sort of conversion of absorbance to concentration causes MD readers to fail

19. Water vs. Water-Methanol Mixture

20. Pathlength Incorrection! *Same plate with 200 µl samples

21. Fluorescence Test Plate only… No Dilutions Required

22. Fluorescence Procedure • Open Protocol • Requires fluorescein filter set (Standard) • Put plate in label side down with red wells to the right. • Read Plate • After Reading, Display Curve

23. Calibration Curve

24. Fluorescence Calibration Plate • Currently a project at Bio-Tek • Similar to Absorbance Cal Plates • Will NOT contain NIST traceable materials • Likely Features • Linearity • Alignment • Top and Bottom detector testing • Any Suggestions?

25. Questions?