{"id":24981,"date":"2020-05-08T13:53:17","date_gmt":"2020-05-08T20:53:17","guid":{"rendered":"https:\/\/hepatochem.com\/standard-ferrioxalate-actinometer-protocol\/"},"modified":"2020-05-08T13:53:17","modified_gmt":"2020-05-08T20:53:17","slug":"standard-ferrioxalate-actinometer-protocol","status":"publish","type":"post","link":"https:\/\/hepatochem.com\/fr\/standard-ferrioxalate-actinometer-protocol\/","title":{"rendered":"A Standard Ferrioxalate Actinometer Protocol"},"content":{"rendered":"


\n<\/p>\n

[et_pb_section fb_built=\u00a0\u00bb1″ _builder_version=\u00a0\u00bb4.16″ global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][et_pb_row _builder_version=\u00a0\u00bb4.16″ background_size=\u00a0\u00bbinitial\u00a0\u00bb background_position=\u00a0\u00bbtop_left\u00a0\u00bb background_repeat=\u00a0\u00bbrepeat\u00a0\u00bb width=\u00a0\u00bb100%\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][et_pb_column type=\u00a0\u00bb4_4″ _builder_version=\u00a0\u00bb4.16″ custom_padding=\u00a0\u00bb|||\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb custom_padding__hover=\u00a0\u00bb|||\u00a0\u00bb][et_pb_text _builder_version=\u00a0\u00bb4.16″ header_3_font=\u00a0\u00bb|700|||||||\u00a0\u00bb background_size=\u00a0\u00bbinitial\u00a0\u00bb background_position=\u00a0\u00bbtop_left\u00a0\u00bb background_repeat=\u00a0\u00bbrepeat\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb]<\/p>\n

Before We Begin…<\/h3>\n

This post is an overview of how we perform a ferrioxalate actinometer protocol to determine photon flux in our various photoreactors. We try to go into as much detail as possible so you can replicate these steps in your own lab and with your own equipment. Please check out some of our earlier posts if you’re interested in the foundations of actinometry<\/a> or want some general background on how to measure light in photochemical reactions<\/a>. And if this post helps you successfully measure your own photochemical reactions or equipment, we would love to hear about it! Reach out to us using the contact form<\/a> or message us on Twitter @EvoluChem<\/a>.<\/p>\n

A Quick Description of The Ferrioxalate Protocol<\/h3>\n

This ferrioxalate actinometer protocol consists of irradiating a solution of ferrioxalate<\/a> (Fe3+<\/sup>) to measure the production rate of Fe2+<\/sup>. The solution of ferrioxalate is made indiluted H2<\/sub>SO4<\/sub>. The resulting solution is light sensitive, so care should be taken to keep the room as dark as possible while running the protocol.<\/p>\n

Samples are taken at specific points in timeand mixed with a AcONa (sodium acetate) buffer solution and phenanthroline solution to generate a phenanthroline Fe2+<\/sup> complex.<\/p>\n

The subsequent Fe2+<\/sup>concentration is then determined usingspectrometry measurement of the phenanthroline Fe2+<\/sup> complex absorption at 510 nm. A calibration curve is madeusing FeSO4<\/sub> solution and phenanthroline.<\/p>\n

Synthesis of Ferrioxalate K3<\/sub>Fe(C2<\/sub>O4<\/sub>)3<\/sub>.3H2<\/sub>O<\/h3>\n

In the dark (under a red light) and at room temperature, combine 50 mL of a 1.5 M aqueous solution of FeCl3<\/sub> (12.16 g) with 150 mL of a 1.5 M aqueous solution of K2<\/sub>C2<\/sub>O4<\/sub>.H2<\/sub>O (41.45 g). After 30 minutes, filter the solid off and recrystallize it 3 times in 50 ml of water. Store in an amber vial and place the solution in a desiccator overnight. Typically, 7-10 g are obtained. The resulting solid can be stored for months.<\/p>\n

Reagents:<\/h3>\n

Actinometric Solution<\/em><\/h4>\n

This solution must be made in the dark as once the complex is in solution it reacts to all light. The solution is made from 9.03 g of K3<\/sub>Fe(C2<\/sub>O4<\/sub>)3<\/sub>, 12.2 mL 1N H2<\/sub>SO4<\/sub> and 110 mL H2<\/sub>O. It should be stored at room temperature in a dark bottle wrapped in aluminum foil.<\/p>\n

AcONa Buffer Solution<\/em><\/h4>\n

Mix 12 mL of AcONa solution (8.2g\/100 mL), 7.2 mL 1N H2<\/sub>SO4<\/sub> and 0.8 mL of H2<\/sub>O.<\/p>\n

Spectrometric Solution<\/em><\/h4>\n

This solution is prepared for each sample by mixing 2.5 \u03bcL AcONa buffer, 100 \u03bcL of phenanthroline solution (0.1 g into 100 mL of H2<\/sub>O), and 892 \u03bcL H2<\/sub>O in a standard 96 deep well plate.<\/p>\n

Actinometric Measurement<\/h3>\n

In a dark room under red light, prepare your setup with a light source and set your vial. NOTE: For a quick discussion of how best to evaluate light sources, please click here<\/a>.<\/strong> Pipette the actinometric solution (in the tin foil bottle) into the vial. Irradiate the vials for exactly 5 or 10 second intervals taking 5 \u03bcL samples and transferring them to the clear 96 well plate containing the spectrometric solution. NOTE: You should be wearing protective orange goggles when the light is on. Once irradiation is over, transfer 200 \u03bcL of each sample to a reading 96 well plate. Wrap the plate in aluminum foil as it is still light sensitive. Measure the sample absorption with a plate reader at 510 nm.<\/p>\n

\"\"

Example of Fe-Phenanthroline complexe absorbance measurement of experiment timepoints. Six different vials (A, B, C, D, E, and F) at different position from a light source.<\/p><\/div>\n

Calibration Curve Preparation<\/h3>\n

Prepare a 0.4 mM ferrous solution, by adding 278.01 mg of FeSO4<\/sub>, 7 H2<\/sub>O (278.01 g\/mol, 1 mmol) in 1 mL of H2<\/sub>SO4<\/sub> (1 N) and dilute to 10 mL with H2<\/sub>O (0.1 M). Then transfer 80 \u03bcL into 2 ml of H2<\/sub>SO4<\/sub> (1 N) diluted into 20 mL (4 mM).<\/p>\n

Prepare buffer-phenanthroline solution, by adding 477.15 mg AcONa and 10.99 mg phenanthroline together, and then dissolve in 6 mL H2<\/sub>O. Add 3.6 mL 1N H2<\/sub>SO4<\/sub> and 0.4 mL H2<\/sub>O.<\/p>\n

Prepare the 9-point calibration in a 96 deep well plate by mixing the FeSO4<\/sub> solution, buffer-phenanthroline solution and H2<\/sub>O with the volumes described below. Let the solution sit for 30 minutes. Transfer 200 \u03bcL in a flat bottom transparent well plate. Measure the sample’s absorption on a plate reader at 510 nm.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
Fe2+ <\/sup>Concentration (mM)<\/strong><\/td>\nVolume buffer-phenanthroline solution<\/strong><\/td>\nVolume 0.4mM FeSO4<\/sub><\/strong><\/td>\nVolume H2<\/sub>O<\/strong><\/td>\n<\/tr>\n
0<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n0<\/span><\/td>\n650 \u03bcL<\/span><\/td>\n<\/tr>\n
0.02<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n50 \u03bcL<\/span><\/td>\n600 \u03bcL<\/span><\/td>\n<\/tr>\n
0.04<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n100 \u03bcL<\/span><\/td>\n550 \u03bcL<\/span><\/td>\n<\/tr>\n
0.06<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n150 \u03bcL<\/span><\/td>\n500 \u03bcL<\/span><\/td>\n<\/tr>\n
0.08<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n200 \u03bcL<\/span><\/td>\n450 \u03bcL<\/span><\/td>\n<\/tr>\n
0.1<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n250 \u03bcL<\/span><\/td>\n400 \u03bcL<\/span><\/td>\n<\/tr>\n
0.12<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n300 \u03bcL<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n<\/tr>\n
0.14<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n300 \u03bcL<\/span><\/td>\n<\/tr>\n
0.16<\/span><\/td>\n350 \u03bcL<\/span><\/td>\n400 \u03bcL<\/span><\/td>\n250 \u03bcL<\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

\"Chart

Typical calibration curve<\/p><\/div>\n

 <\/p>\n

Determine Photon Flux In Your Photochemical Experiments<\/b><\/h3>\n

To find out how to calculate the photon flux using your results, check out our post Determining Photon Flux Using Actinometry<\/a>.<\/p>\n

Did this post help you successfully measure your own photochemical reactions or equipment? We would love to hear about it! Reach out to us using the contact form<\/a> or message us on Twitter @EvoluChem<\/a>.<\/p>\n

 <\/p>\n

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We describe ferrioxalate actinometer protocol we use to determine photon flux in different reaction vials and photoreactors.<\/p>\n","protected":false},"author":1,"featured_media":24983,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"

Before We Begin...<\/h3>

This post is an overview of how we perform a ferrioxalate actinometer protocol to determine photon flux in our various photoreactors. We try to go into as much detail as possible so you can replicate these steps in your own lab and with your own equipment. Please check out some of our earlier posts if you're interested in the foundations of actinometry<\/a> or want some general background on how to measure light in photochemical reactions<\/a>. And if this post helps you successfully measure your own photochemical reactions or equipment, we would love to hear about it!\u00a0 Reach out to us using the contact form<\/a> or message us on Twitter @EvoluChem<\/a>.<\/p>

A Quick Description of The Ferrioxalate Protocol<\/h3>

This ferrioxalate actinometer protocol consists of irradiating a solution of ferrioxalate<\/a> (Fe3+<\/sup>) to measure the production rate of Fe2+<\/sup>. The solution of ferrioxalate is made in\u00a0diluted H2<\/sub>SO4<\/sub>.\u00a0 The resulting solution is light sensitive, so care should be taken to keep the room as dark as possible while running the protocol.<\/p>

Samples are taken at specific points in time\u00a0and mixed with a AcONa (sodium acetate) buffer solution and phenanthroline solution to generate a phenanthroline Fe2+<\/sup> complex.<\/p>

The subsequent Fe2+<\/sup>concentration is then determined using\u00a0spectrometry measurement of the phenanthroline Fe2+<\/sup> complex absorption at 510 nm. A calibration curve is made\u00a0using FeSO4<\/sub> solution and phenanthroline.<\/p>

Synthesis of Ferrioxalate K3<\/sub>Fe(C2<\/sub>O4<\/sub>)3<\/sub>.3H2<\/sub>O<\/h3>

In the dark (under a red light) and at room temperature, combine 50 mL of a 1.5 M aqueous solution of FeCl3<\/sub> (12.16 g) with 150 mL of a 1.5 M aqueous solution of K2<\/sub>C2<\/sub>O4<\/sub>.H2<\/sub>O (41.45 g). After 30 minutes, filter the solid off and recrystallize it 3 times in 50 ml of water. Store in an amber vial and place the solution in a desiccator overnight. Typically, 7-10 g are obtained. The resulting solid can be stored for months.<\/p>

Reagents:<\/h3>

Actinometric Solution<\/em><\/h4>

This solution must be made in the dark as once the complex is in solution it reacts to all light. The solution is made from 9.03 g of K3<\/sub>Fe(C2<\/sub>O4<\/sub>)3<\/sub>, 12.2 mL 1N H2<\/sub>SO4<\/sub> and 110 mL H2<\/sub>O.\u00a0 It should be stored at room temperature in a dark bottle wrapped in aluminum foil.<\/p>

AcONa Buffer Solution<\/em><\/h4>

Mix 12 mL of AcONa solution (8.2g\/100 mL), 7.2 mL 1N H2<\/sub>SO4<\/sub> and 0.8 mL of H2<\/sub>O.<\/p>

Spectrometric Solution<\/em><\/h4>

This solution is prepared for each sample by mixing 2.5 \u00b5L AcONa buffer, 100 \u00b5L of phenanthroline solution (0.1 g into 100 mL of H2<\/sub>O), and 892 \u00b5L H2<\/sub>O in a standard 96 deep well plate.<\/p>

Actinometric Measurement<\/h3>

In a dark room under red light, prepare your setup with a light source and set your vial. NOTE: For a quick discussion of how best to evaluate light sources, please click here<\/a>.<\/strong>\u00a0 Pipette the actinometric solution (in the tin foil bottle) into the vial. Irradiate the vials for exactly 5 or 10 second intervals taking 5 \u00b5L samples and transferring them to the clear 96 well plate containing the spectrometric solution. NOTE: You should be wearing protective orange goggles when the light is on. Once irradiation is over, transfer 200 \u00b5L of each sample to a reading 96 well plate. Wrap the plate in aluminum foil as it is still light sensitive. Measure the sample absorption with a plate reader at 510 nm.<\/p>[caption id=\"attachment_7516\" align=\"aligncenter\" width=\"623\"]\"\" Example of Fe-Phenanthroline complexe absorbance measurement of experiment timepoints. Six different vials (A, B, C, D, E, and F) at different position from a light source.[\/caption]

Calibration Curve Preparation<\/h3>

Prepare a 0.4 mM ferrous solution, by adding 278.01 mg of FeSO4<\/sub>, 7 H2<\/sub>O (278.01 g\/mol, 1 mmol) in 1 mL of H2<\/sub>SO4<\/sub> (1 N) and dilute to 10 mL with H2<\/sub>O (0.1 M). Then transfer 80 \u00b5L into 2 ml of H2<\/sub>SO4<\/sub> (1 N) diluted into 20 mL (4 mM).<\/p>

Prepare buffer-phenanthroline solution, by adding 477.15 mg AcONa and 10.99 mg phenanthroline together, and then dissolve in 6 mL H2<\/sub>O. Add 3.6 mL 1N H2<\/sub>SO4<\/sub> and 0.4 mL H2<\/sub>O.<\/p>

Prepare the 9-point calibration in a 96 deep well plate by mixing the FeSO4<\/sub> solution, buffer-phenanthroline solution and H2<\/sub>O with the volumes described below.\u00a0 Let the solution sit for 30 minutes.\u00a0 Transfer 200 \u00b5L in a flat bottom transparent well plate. Measure the sample's absorption on a plate reader at 510 nm.<\/p>
Fe2+ <\/sup>\u00a0Concentration (mM)<\/strong><\/td>Volume buffer-phenanthroline solution<\/strong><\/td>Volume 0.4mM FeSO4<\/sub><\/strong><\/td>Volume H2<\/sub>O<\/strong><\/td><\/tr>
0<\/span><\/td>350 \u00b5L<\/span><\/td>0<\/span><\/td>650 \u00b5L<\/span><\/td><\/tr>
0.02<\/span><\/td>350 \u00b5L<\/span><\/td>50 \u00b5L<\/span><\/td>600 \u00b5L<\/span><\/td><\/tr>
0.04<\/span><\/td>350 \u00b5L<\/span><\/td>100 \u00b5L<\/span><\/td>550 \u00b5L<\/span><\/td><\/tr>
0.06<\/span><\/td>350 \u00b5L<\/span><\/td>150 \u00b5L<\/span><\/td>500 \u00b5L<\/span><\/td><\/tr>
0.08<\/span><\/td>350 \u00b5L<\/span><\/td>200 \u00b5L<\/span><\/td>450 \u00b5L<\/span><\/td><\/tr>
0.1<\/span><\/td>350 \u00b5L<\/span><\/td>250 \u00b5L<\/span><\/td>400 \u00b5L<\/span><\/td><\/tr>
0.12<\/span><\/td>350 \u00b5L<\/span><\/td>300 \u00b5L<\/span><\/td>350 \u00b5L<\/span><\/td><\/tr>
0.14<\/span><\/td>350 \u00b5L<\/span><\/td>350 \u00b5L<\/span><\/td>300 \u00b5L<\/span><\/td><\/tr>
0.16<\/span><\/td>350 \u00b5L<\/span><\/td>400 \u00b5L<\/span><\/td>250 \u00b5L<\/span><\/td><\/tr><\/tbody><\/table>

\u00a0<\/p>[caption id=\"attachment_7517\" align=\"aligncenter\" width=\"539\"]\"Chart Typical calibration curve[\/caption]

\u00a0<\/p>

Determine Photon Flux In Your Photochemical Experiments<\/b><\/h3>

To find out how to calculate the photon flux using your results, check out our post Determining Photon Flux Using Actinometry<\/a>.<\/p>

Did this post help you successfully measure your own photochemical reactions or equipment?\u00a0 We would love to hear about it!\u00a0 Reach out to us using the contact form<\/a> or message us on Twitter @EvoluChem<\/a>.<\/p>

\u00a0<\/p>","_et_gb_content_width":"","_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[486,483,489,487],"tags":[502,517,504,518],"class_list":["post-24981","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-educational","category-feature-2","category-featured-articles","category-photoredox","tag-actinometry","tag-ferrioxalate","tag-photon-flux","tag-protocol"],"yoast_head":"\nA Standard Ferrioxalate Actinometer Protocol<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/hepatochem.com\/fr\/standard-ferrioxalate-actinometer-protocol\/\" \/>\n<meta name=\"twitter:label1\" content=\"\u00c9crit par\" \/>\n\t<meta name=\"twitter:data1\" content=\"admin\" \/>\n\t<meta name=\"twitter:label2\" content=\"Dur\u00e9e de lecture estim\u00e9e\" \/>\n\t<meta name=\"twitter:data2\" content=\"4 minutes\" 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