Absorbance Demo Tutorial Working with Primary Colors . . .

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

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

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

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

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 is a Pathlength Equation to Calculate Pathlength of Sample in Microplate well (A977 - A900) Sample = Pathlength of Sample (A977 - A900) 1.0 cm water

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

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

Absorbance Demo Dye Solution

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 Pathlength Correction should correct all the wells to the same absorbance value

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

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

Uncorrected Values* Plate Map 1 2 3 4 A 0.025 0.463 0.614 0.757 B 0.024 0.457 0.602 0.759 C 0.023 0.459 0.777 D 0.460 0.610 0.760 *Approximate values at 630 nm

Tale of Two Dilutions

Pathlength Corrected Values* 1 2 3 4 A 0.003 0.684 0.899 1.117 B 0.002 0.674 0.889 1.106 C 0.000 1.174 1.118 1.100 D 0.024 1.133 1.096 1.098 *Note that blank subtraction was performed

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

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

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

Water vs. Water-Methanol Mixture

Pathlength Incorrection! Wavelength Bio-Cell Microplate*K=0.139 Microplate* K=0.180 405 nm 1.282 1.271 1.672 413 nm 1.339 1.329 1.748 450 nm 1.059 1.042 1.370 630 nm 2.137 2.145 2.819 *Same plate with 200 µl samples

Test Plate only… No Dilutions Required Fluorescence Test Plate only… No Dilutions Required

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

Calibration Curve

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?

Questions?