Difference between revisions of "Team:Aachen/Notebook/Protocols"
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{{Team:Aachen/Protocol|title=Nash Assay|id=Nash_Assay|text=<span style="display:none;">spacer</span> | {{Team:Aachen/Protocol|title=Nash Assay|id=Nash_Assay|text=<span style="display:none;">spacer</span> | ||
− | === | + | ===Principle of detection=== |
− | The colorimetric and fluorometric assay first described by T. Nash in 1953 <ref>Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. ''Biochemical Journal'', '''55'''(3), 416-421.</ref> | + | The colorimetric and fluorometric assay first described by T. Nash in 1953<ref>Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. ''Biochemical Journal'', '''55'''(3), 416-421.</ref> detects the presence of formaldehyde. Two molecules of acetylacetone react with formaldehyde and ammonia to form the strong yellow and fluorescing diacetyl-dihydro lutidine which can be detected via absorption at a wavelength of 412 nm and via fluorescence at an excitation wavelength of 410 nm and an emission wavelength of 510 nm. |
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* mix Nash reagent : reaction buffer : sample in the ratio of 20 : 19 : 1 in a MTP/cuvette | * mix Nash reagent : reaction buffer : sample in the ratio of 20 : 19 : 1 in a MTP/cuvette | ||
* Measurement | * Measurement | ||
− | ** measure | + | ** measure absorbance at 412 nm |
** measure fluorescence with an excitation wavelength of 410 nm and an emission wavelength of 510 nm | ** measure fluorescence with an excitation wavelength of 410 nm and an emission wavelength of 510 nm | ||
** measure hourly starting directly after mixing for at least 5 hours | ** measure hourly starting directly after mixing for at least 5 hours | ||
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# Add one third of the total volume of potassium sodium tartarte solution (40 % wt/vol) to samples, to stabilyze the color | # Add one third of the total volume of potassium sodium tartarte solution (40 % wt/vol) to samples, to stabilyze the color | ||
# Cool samples down to 25 °C | # Cool samples down to 25 °C | ||
− | # Analyze samples at 540 nm | + | # Analyze samples at 540 nm absorbance and substract the absorbance value of color blank each time |
}} | }} | ||
Latest revision as of 03:37, 19 September 2015