Microplate Spectrophotometer

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Alvin Hoover
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1 Microplate Spectrophotometer Features Benefits Applications Bali, October 1999

2 Optical System Xenon Flashing Lamp Order Sorting Filters Monochromator Data Light collection Light Transfer

3 Why are these Instruments So Good? Xenon Flashing Lamp

4 Xenon Flashing Lamp Continuous Spectrum from UV to IR Quality Results throughout the λ Range High Optical output More Energy Better Quality Results

5 Xenon Flashing Lamp : Relative Energy Xe Tg Wavelength

6 Xenon Flashing Lamp Continuous Spectrum from UV to IR Quality Results throughout the λ Range High Optical output More Energy Better Quality Results Exceptional Flash Stability Consistent Quality Precision

7 Xenon Flash Lamp : Expected Life : 1 Billion Flashes = 1,100,000 full plates min Energy 2 u seconds u Seconds

8 Xenon Flashing Lamp Continuous Spectrum from UV to IR Quality Results throughout the λ Range High Optical output More Energy Better Quality Results Exceptional Flash Stability Consistent Quality Precision Low Heat Radiation No Temperature effect on Assay Long Life No need to change Lamp No Warm Up Required

9 Why are these Instruments So Good? Diffraction Grating - Monochromator

10 Monochromator 999nm ~ 200nm Wavelength Selection from nm in 1nm increments Filters not required gives great convenience Customers can use any wavelength Saves customer money over time 5nm, 2.4nm or 2nm Bandpass Better than filters (10nm) Spectral Scanning Assays Increased Flexibility with New Applications

11 Why Pre filter the light? Order Sorting Filters 1 st Order 2 nd Order 1 st Order rd Order 2 nd Order Diffraction Grating 3rd Order

12 Monochromator : Input Slit Output Slit Concave Grating Motor with Lead Screw

13 Monochromator : Input Slit Output Slit Facts about the grating : 32 x 32 mm Square 1800 lines per mm 2.4 nm Bandwidth Concave Grating

14 Why are these Instruments So Good? Pathlength Correction

15 Pathlength Correction - Is it Important? Beer-Lambert Equation: O.D. = ( ) x (Concentration) x (pathlength) Required to directly link concentration to absorbance DNA and RNA concentrations can be calculated quickly Conventionally: Spectrophotometers use 1 cm pathlength Conversions ( ) based on 1 cm pathlength Microplates do not have a 1cm pathlength

16 Pathlength Correction - Is it Important? Horizontal photometry Light path at 90 angle to axis of solution Light pathlength determined by geometry of the vessel Light 1 cm Detector Vertical photometry Light passes though vertical axis of solution Detector Volume of liquid influences light pathlength?? Microwell Light

17 Pathlength Correction - How Does it Work? 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 Absorbance (blanked) Wavelength Spectral scan of water in Near IR region

18 Pathlength Correction - How Does it Work? Light 1 cm Detector Detector Measure Absorbance of water in a 1cm Cuvette at 977 nm Measure Absorbance of water in a 1cm Cuvette at 900 nm as a reference Measure Absorbance of Sample in a microplate at 977 nm?? Microwell Light Measure Absorbance of Sample in a Microplate at 900 nm as a reference Measure Absorbance of Sample at Target Wavelength eg 260 nm

19 Pathlength Correction - How Does it Work? O.D. = ( )( ) x (Concentration) x (pathlength) - Beers Law Concentration of Water is always the same Extinction constant of Water is the same (at a given temp.) OD of Water is Pathlength Equation to Calculate Pathlength of Sample in Microwell 0.12 (A977 -A900) Sample = Pathlength of Sample (A977 -A900) 1.0 cm water 0.65 cm 0.183

20 Pathlength Correction - 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 s to 1.0 cm pathlength OD Values 0.5 Absorbance of Sample ((A260 Sample)- (A260 Blank)) = Corrected to 1.0 cm Pathlength of Sample 0.65 cm 0.5 * 1.53 =

21 Pathlength Correction - How Does it Work? The 2 equations can be combined 0.76 Absorbance of Sample in Microwell Corrected to 1.0 cm = ((A260 Sample)- (A260 Blank)) x (A977 -A900) 1.0 cm water (A977 -A900) Sample 0.12 Corrects Reading Microplate Readings to True Spectrophotometer Values

22 Nucleic Acid Quantitation Protein Quantitation Lipoxygenase ELISA (Cell Viability/Cytotoxicity /Cytotoxicity)

23 Nucleic Acid Quantitation DNA RNA Oligonucleotides 3.5 Spectral Scan of DNA in Solution Peak Absorbance at 260nm Absorbance (ODs) Wavelength (nm)

24 Absorbance to Concentration Conversion for Nucleic Acids* Nucleic Acid Concentration for 1 Species A 260 unit ( g/ml) dsdna 50 (oligonucleotides) 33 ssrna 40 * Based on a 1 cm pathlength

25 A /A Ratio Phenol Contamination A 260 Ratio less than 1.0 suggests the presence of trace amounts of phenol 260 /A 230 Ratio EDTA Contamination Ratio less than 2.0 suggests the presence of EDTA contamination A 320 Scattering Values significantly above 0 suggests particulates in samples

26 A 280 Absorbance Measurements Colorimetric Determination Lowry Method (660 nm) Burton Method (595 nm) BCA Method (562 nm)

27 A 234 Measurements Lipoxygenase Cis, trans-conjugated diene A 268 Measurements Lipoxygenase Cis, trans-conjugated triene

28 Absorbance peak determinations nm in 1 nm increments

Compact Knowledge: Absorbance Spectrophotometry. Flexible. Reliable. Personal.

L A B O R A T O R Y C O M P E T E N C E Compact Knowledge: Absorbance Spectrophotometry Flexible. Reliable. Personal. The interaction of light with molecules is an essential and well accepted technique