48 0 obj <> endobj h�b```"yɰ���x�bL,�W0��D��p��� The spectrophotometer uses light emitting diodes (LEDs) with a dominant wavelength peak of 645nm and 663nm respectively. chlorophyll is desired) or at 750, 665, 663, 645, and 630 nm if both corrected and uncorrected chlorophyll are desired). The sample is clarified through centrifugation. Courses. �?�[���O�̪e��u�a���j�ϭ틘�֐�>?�Lߚ���D$@��S�^ @��6�[��k�% �uT�>a�� wavelength. H�\��n�0Ee�)P���e� Po*Y�L�,$Ґ�������6#����w�=� �9�M�QpG�QB����V"���X+�XK�$���Dp<. stream 3.0 SAMPLE PRESERVATION, CONTAINERS, HANDLING AND STORAGE Tissue samples will be placed in aluminum foil immediately following collection. Each of these wavelengths respresent the peak absorption ranges for chlorophyll a and b. �g����^?p��:�{��J�n6�� �������|t�h�VFg��ߝ����]���t����k��/x�Y>3 This process is time consuming, destructive to the plant material and above all, impractical in applied situations. What wavelength of light best distinguishes chlorophyll b from a and carotenoids? ; spectrophotometer: An instrument used to measure the intensity of electromagnetic radiation at different wavelengths. ChemSurvival 305,104 views. h�25Q0P���wJ,Nu��+��H�)K-�LN�u��I�w�K�O��K���s�+΄�K�J*R�C��!��鷳0 v�� Chlorophyll aabsorbs light at 433 nm and 666 nm and chlorophyll babsorbs light at 462 nm and 650 nm. The Thermo Scientific™ Orion™ AquaMate™ 8000 UV-Vis spectrophotometer is a good choice for this testing and meets the requirements for accepted chlorophyll . The LTER samples should be run at 750, 665, 664, 647, and 630 nm. This can help us iden- tify a pigment's role in a plant. [�b���({�A�Q�緧V6���y�������A},/�n���Y{�ޯ��)����Y˻�5���v|��T����y���٘�������({��K=����_2�"�䧢�+/��G�ơ��Y�=G�W_��ɍc�JG��[�R�������>;�����/~ƴ�! The NanoDrop One/OneCUV-Vis application can be used to observe the spectrum of each chlorophyll. Use a spectrophotometer of 2 nm maximum bandwidth and stoppered matched glass or quartz cuvettes with path-length of 1cm. a. testing methods, such as listed above. Chlorophylls a and b are thereby measured as their pheophytins. chlorophyll forms, as well as accessory pigments, carot-enoids (carotenes and xanthophylls) [21]. A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. p}�E��M_� chlorophyll a and b? 7.6.1 Use a shorter or longer cell as necessary to maintain absorbance between approximately 0.1-1.0 at 663 nm. Place the cuvette into spectrophotometer and check the absorbance and transmittance of Chlorophyll at different wavelengths:630 nm, 646 nm, 665 nm, 750 nm Note: Whenever the wavelength is changed, its blank should be checked and corrected. Spectrophotometry Fall, 2010 Dr. Robert A. Robergs, Ph.D., FASEP, EPC 5 Genesys 10 Spectrophotometer. �6^Ǻ�f}���؟b� ��3�U{y7�N������~7o9����2�9, �?� ����r��"��p��p7; ��3�V�k?������߭��Τw�8~^�dMf��LA��~ ��a���Is�c�I��C��m�i7K�g�f�1f5��а����f]�`�6r�"?3�wb�3�p�O0�d��q��:?�:�]��}�>�K��GN��5s����rY�Y>!����g���QӅ������_�Q���?�����v�����t�����?�a�]�����v������~��9��x��_�0����v�]M+�]�hC�eu���>�t��Y]�a��1_]p��g�~�P��]�@�.����sF�ϥ�Z�:�l�R�p�=�h{pvxk�g��G�-K�*����vY�Y홬2���8훭 ���j*vb�2�Uڙ�SpOo���c~���N3���~u������3x:�l�g $��8M�'K a�/�Z2g���V@ܝ��:*7�[����@�n��faxR�v��x�dܼ>\+���揆�fOZ����t����R���������a/��㡵C�_ �;�i���ߧ�w�j;��{�����^�-�k~b�8�}�����m�Şz�jқ�~r�̬�}?3n��w��sĉ���%^]��t�[WX��|/1�m�aϚ��� �]o1i���{��ّxc���#I��_k=l4���3V5ս_e|�S��Ӟ�A���/�N������e��.i#���3}�E�(�#$���q ���7z��2=���:��Ս��vqgD氠W���O�ƪ����nZ�����9hS��4T뤀i��@��ڽ �A��9ʽ�u�|�[M�k���s�]��w�G+��f���{�íGg��L�>�s���]mIi�kܟ��'��zןwv�/|�Y= ~ 675 nm ~ 520-530 nm ~ 650 nm Spectrophotometers . The extract is measured spectrophotometrically for absorbance at 664 nm (A664b) wavelength. wavelengths and chlorophyll a and b concentrations are calculated. endstream endobj startxref Concentration of chlorophyll a and b and total carotenoids in obtained extracts was measured spectrophotometrically (721-2000 SPECTROPHOTOMETER, China) with equations presented below (Table 1)[11], according to these measurements, each of chlorophyll a, b and total carotenoids content in fresh materials were determined. Learn about chlorophyll and light, why they make plants green, and why they matter to photosynthesis. The samples were tested using 90% acetone method by using spectrophotometer (model HITACHI U-1900) to record the absorbance values with selected wavelengths namely 750 nm, 664 nm, 647 nm, and 630 nm The results showed that the chlorophyll-a concentration in microalgae increased when the absorbance was increased. The raw chlorophyll extract was diluted to a concentration that would absorb the proper amount of light to read within the limits of the spectrophotometer T/A meter scales. %�쏢 Absorbance is a measure of the light absorbed by a solution. ��Χ�)Y�ۛ�Ӈoy�x?x�����n�X�_O,�P�b�F��k��G��v{H-ق��� ��u h�bbd`b`�bb`Hab`\ī���012�g���` � Equipment for testing chlorophyll a by spectrophotometer . }�|z@_K����K���Sm߬�,���A���7�&o���7\��/7�R��aP��|>�`P~�����κ>�����m/UM��'����O�0�Sc�;����52�[�k�S*�/�έ��2��3�&�}ĤK�sk�ݻ��o�}P(�䵵��t�}��ʭ�1�=��؃�n⽛���|c�'�M��N�6��#����Q�i�Mq:W9�r`�4F�}��e>�9qy��n�պ�Y/0}THЎ �f5c���dFc�Fc��whj�BBS���z����G�|7S��a����C�w�33lU��{p��I�`�|w�Q�/�O�JJ��.��0���u�:�����4��Bw�+eA�e�ysS�G����2q�~09����5��"mK� }�@���X#��QJR���bL� Describe your procedure as well as how you would show your results. The a* and b* for the calibration data range from − 3.7 to 42.87 and 5.51 to 47.67 with CV of 0.55 and 0.39 respectively and 1 to 43.92 and 6.4 to 36.95 with CV of 0.52 and 0.41 for validation data respectively. Chlorophyll is the most dominate of these pigments, absorbing specific wavelengths of the visible light when collecting energy from the sun. Given a pure sample of chlorophyll b and a spectrophotometer, how would you measure the ability of the pigment to absorb various wavelengths? ��?w��8��UIfȬ�,3�p ���qG���\��,� ��^��������z���Ł�|��ׯ�5��z� ��.��=c��kX}����W˵ֲ��w���_�[�;s{��/o>)��kv{���y�x ��S��s��k�KM{p��� ����n/=]�O�h��4�k^/�z�����p]����/�PYV^��z]BZ��߷`�5�ןj쿼q����^��_h��+��Oo���5�ן���������G"���Zr��(�%%���7��p�>��߿�d����^��p ���Aa��w������L�/m$���/�*�mG��5���noJ�:w��+�k���r]�xq.�K�+a�+�7_���G�_��ǣ�v���?���-���B&��7�Z�2z�=����k�����|��#�j)M?._C��Q�w�rԏ�9\b"ݬKh���ꉩ�'5m���1v�TO��ś|�a]��n�Dk���E�:((#T��4x�����f_��' 2H�=DN�.��>��!���#_S�F�ί��f���K�uU�կ/5�.�����WE����[.�]O�Onח�C�6�'���S���z- w^���^9>m;��ri7S[؞�J+eW�^ښc�ݓ>��K1�����'������@䟽�$_}��_�q5�%��9��� The sample was diluted 1 in 20 in 90% acetone and the spectrum measured over the range 600 to 700nm. UV-260 spectrophotometer. �v���s`��S�%�.sr���2f���>�d��y�v�sS�j���L��A�G�i�.S���Ɨ[uM`|ܲɝO���:$~��ֆ�ǘ�㐚�Z7��/f�0f�6/���ɋ��&]��ˠ/�o'�o��e��|&���7'���� ��|��Nƃ7��fn��_S����M�ث�:�����e�0�_>��|���[��6��=+�,�˥��n��3xàbf"�τL�P All of the readings at all wavelengths should be within .001 of 0. Switch on spectrophotometer and leave to warm up for 45 min before using �K]c0=��F�-�������Ϛ�n1�}�{ʏ���݈�dj)\J�U�W�Ki��i>�7�q1C�B��j:��ZEw;�4S�b.Ʈ&C��� ��K1v��:�;�w7���)x�J���/ :�K}r��>=�Xď��t7�`]�-�7Z���N{e��p��a~�_�'� ,f��O����ݬ���77cč�����x����l f��2\秨�_���t��a���V�b1f0��D�hE��f�W~���Csc�m.�Bc2���p}&ɷ��E���{Ii-�XI�Q#�`�!���7�j$M֤����y�S�d�r\:9f���â�g�Ú��]F[M�����Ϙf���;L�F�t_�YWp��@LE�S�~i��w��G�Mi}��=�l�ќn�X���3|B�=/sl�ӧSe�eΖ�U���W������eXP�=�^J�|7Ey�؏d����}�Sl�q��!�� %%EOF If you're seeing this message, it means we're having trouble loading external resources on our website. Thus, the variability arising from manipulations involving separations, pheophytiniza-tion uponextraction, and allomerization ofthe chloro-phylls, is minimized. The sample is then acidified so all chlorophyll a is converted to pheophytin a by eliminating the … babsorb light at slightly different wavelengths. %PDF-1.6 %���� ]�c�#3�=d�o3#����ftG�c����:Yc����k�?1���,��x`�G�E�����Y��~dч�ɢ{�c�>�鬜�#�>f����,�07�;d���\y/�^�è_>+|O�^�1���R��A�qdԔ�h���ǃ��>d 2�tVNNʹ`����ǃ��܄��a�z ���{u|�~���=�:>c�9�g���86��Ȩ)��Q7��n�F},�5�>4�?�������J�v�m�Wݯt�&t��b����2j�F����{�ƨ���t�R�adԔ�Ш��Q7��>�ݍ�r���.�1���>f�o3C���>����d��� 80 %-ethanolic leaf extracts at two wavelengths. ; carotenoid: Any of a class of yellow to red plant pigments including the carotenes and xanthophylls. Then the absorbance of the chlorophyll sample was measured at a series of wavelengths (colors) across a range of visible light wavelengths from 400nm (violet) to 700nm (deep red). Keywords: Chlorophyll, high throughput, microplate, spectrophotometer, methanol INTRODUCTION The chlorophyll content of plants is a fundamental measurement that underpins many branches of the plant sciences. The chlorophyll fraction – the puri-fied mixture of various chlorophyll forms (e.g. How chlorophylls and other pigments absorb light. Properties of light. … A path-length of up to 5cm can be used for dilute cultures. 5 0 obj It was recorded that Chlo-rophyll a showed the maximum absorbance at 662 nm, chlorophyll b at 646 nm and total caroten at 470 nm and the amount of these pigments was calculated according to the formulas of Lichtentaler and Wellburn (1985) (14). The computer test showed 553"5 nm to be the best wavelength for estimating chloro- phyll a, 642 nm for chlorophyll b and 606 nm for chlorophyll c concentrations. If you're behind a web filter, please make sure that the domains *.kastatic.org and *.kasandbox.org are unblocked. � A�AQ�4?à͙��Z�7r��S���oGU���-���\-��c����-�'�ꓦHJ�5�i����M��^i�� �8U�Q������'����'i U;�r��jeS.��E�d�)�/�&P�k���~��C�a~�}�7�i_���g-��:��go�5tMs����G䯆_�y�Z��\BZ7��z@yg��8���c43bgu�W��V=Uh�w�z������`tu��4�xQ���B��ħ�̯�!� �b����%6~���QU�Zm�w=�!�����j�ˡ% �s� ��㌥�;�%[� [��ħ]w1L�d�}sc���\2*o����nY{�^�/ߔk�ˢUaً��쫙"�*V��؜�ۻ��?�ëK��S��o�zw%�����u�׵�gښ��~f����v�5�����N�{�b�{� ���봠�i�t_������׃�zf' CM�����'��i����(y~� {�ļ�4�p]Ɉ�O��^R���ڇz^���W%_���{}Vsr ��˚������ͧ�Z~��<��7e�#4���bh_m?~�y�����ێk��ѣ��j��>�Z�}��o��7�᫕��a�t�U,����3�����G棘w���n��i��y��O*�׉=>Wv�B��h��;�-y���:�̽{������{y�\�]��ݏ�|�a�;郏��`��G����1�<>�r_��w�n?6������э�w~������ixP?m�0��D��9�f������h�y�Ş��uW/��>��D,i�����}���#�/x�,�֟B5����B����/O�?3#��/�~��I�'�l��������{��7p.���'$�}��F��KK�tz���X2맅�� ���>̈́�����s���W��ܛ�p�.�I8����7+��Fa�u) OQj����Ok�a����zh���8}n���u�g�ꎦ_'�'U�L�f�(���}��Ɠ��W�|��y)���I������-�Y_9�����+�#j��O���q��䭿p� Ix; ���j�? Inside the mind of a master procrastinator | Tim Urban - … Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. F�7��O v�j~�� ��o'���Q�d(������O�FOqo槚���Y��2?�ow눋Ϛ��ȣ���D�������#��˽�.^�c���1)�c~n|ҽ�����d&o�;�œ����ؿ�K6y-��wo������lb�g��A��z���f�~�,E}^�l:����n�_��%\� �.�[������m��s��+��Es�nZ��oo�Y�ܰ? tissue grinder and steeped in 90% acetone to extract chlorophyll from the algal cells. Chlorophyll-a, b and c Plymouth Marine Laboratory Version 30/06/2014 4 4.3 Measurement. %PDF-1.4 Interferences 2.2.1. Make sure that the correct program is being run by checking the wavelengths. The calculated chlorophyll a and b concentrations were 0.91mg/l and 0.19mg/l respectively, therefore the ratio of chlorophyll a to b in this sample is 1:0.21. Instead of the wave-lengths determined by these ;b�;v�ћ����nY۰�=ǹ��>p��.ZާH�֤�„����Z�E�&���2sܭ�G��� kH�*�ZƐk��4��5�oX�گ>[s� vP-�{�U��I�R����W�nK�Ǻi���ktnfǸ$�J��k{YS�ȏ5��`�E^��w�ѩ���~�S���{�$�jRȞ�Mᖘ���$w��e!���%;�u��H��}+��}n��̃�ϰ���fp\��MF3�����O�E��܄���uK[�����6�hNޙf~U��O�����^涾Ǧ��?�����F���o�B�i^�����&�F�Z@X�3��S��adߔ�о��}7��>���r�������UMp����okl?eaOs�C��1�5� e��oy���5]7+������>�R�ͺ|@��%?c�=űY@fM�ͺ��uc?0�c�ݬ)��Y?�����!�~��~d�G���Øu*x1����`���z��v��p�?��v�x8�G���ȱl���Ñ?���⢶����1�����Sv�47�;d������u� v���]�h�.���uOqlׇ�]S�C�n�v����Xv�k�}h�~:+g�'A��1����~�s�C������C�����{ٵ��v�9~l�=ű]@vM�����uc?��c�ݮ)��]?��W�H%�[�~�n�����]�=�����]���s�>����]�3�;�5��[u(����pb���'��ɇֻ+I��Y/�`��w�{\@�^�]&����sݲ��z���>�ݬ�s���b�? Chlorophyll fraction. Quantitative Measurement of Leaf Chlorophylls by Spectrophotometry of Their Pheophytins in Aqueous Alcoholic Extracts 1, 2 J. L. Wickliff and S. Aronoff Department of Biochemistry & Biophysics, & the Institute for Atomic Research, Iowa State University, Ames chlorophyll: Any of a group of green pigments that are found in the chloroplasts of plants and in other photosynthetic organisms such as cyanobacteria. ���nַ7�&�̳w��V�oUvy?��n/ݚ��ۜv���Q;��R�痶_�#�C��;��Ӡ;,s�!c��6�YJC2�7����ɽՃ��ɽչc��m�]�]���N��L��K\��4UA�vb��C�6�]���a�#�q� In this lab we will use the spectrophotometer to accurately determine the chlorophyll concentration in leaves. Color and chlorophyll content traits of Sassafras tzumu leaves. >A���I�T����p0H���A�4���9D^���G���Q��>,���� ��F�b������3/��ֈ��ܰ���98ԯ� �kW5�������L/2��^Tx�����P��Ë��$`�U��:K3L>��4\� W��+�"�~#�=�r�t���Y�#7��a�^ޟ���M��U���!ǻ����=�r��C��~�'����{�F��շ���?Gğ��<7����:^��\�2�7^f�7_q�\]��_�/rO���?}c�x�㆛s�����}^e�p�s�>��S�W���k�? Chlorophyll content was traditionally measured in the laboratory by extracting the chlorophyll itself from a leaf sample using acetone before calculating the chlorophyll concentration by spectrophotometrically measuring absorption at 663 nm and 645 nm. 55 0 obj <>stream How chlorophylls and other pigments absorb light. Search. Tissue samples must be frozen at approximately -56.6 C to prevent chlorophyll breakdown. N:��t�4wW�"�W֡ȣ�c��)�z���c���KW�O=�{�2�˴c�y��1���I�5u��Cә�]V�M^:��S����n��є���̿Gq��8���OZ�7�翛����+�X0��d��ɿi���mo���� L{�aᦗ�/NLWH�!�a��1?�����~�7�n��of�K���>ai_N>���g����g�gNF���'�vD�d��X¼S1��%᝕S7h���M&��_N�M=ڴ�d���_4�u�ɢ���"�����k����Ͱ�x�I�;�K��^:����hkX���lz��X�.�˺��WL7@f��s�g���2)t�*��-m���OQG�L?����.d�������g�U�|'��/l]��[��+�5"�r�\��m�W�7ev�A�3���;i��ݛ��og>1��p^����H�������?�[5_Ζ{�'��K���m)��w߄��U�G{�3QnG���{�`�`�Ÿ��i;��O��H�����dx��K�p��8Iyg�����y'iU���ɨ�����iE�T��. The absorbance of the clarified extract is then measured on a spectrophotometer at 750,665,664,647 and 630 nm wavelengths before and after a 90 second HCl acidification step. The light falls onto the light dependent resistors (LDRs) which decreases in resistance as more light falls upon it causing an increase in voltage as measured by the Arduino. The foil-wrapped sample will then be placed in a resealable plastic bag and held in darkness. ����da b`�^����l�rNFU� �(C\�f"]� B�� y�n�ݳ���� {��{��{/|�X��R\z����s܍�/�����������+d����ށ�;���]��c��$�� �n����rw#�Pz���Wk������]�P�E]��vX !��f���oO>�O؛ͮ�_��[w�������Z���%��%(���T���%�{��6�w��[pє��nZ����k����1��$�T�����T�ݨ-�R�u�=�'���5Ž (�\[Ǩ�x�%�V���߻�����:�6����i�4[cX��Į��6�߷V5�t͵���ү�ܿ`e���Y���զ�]-��׿i�uo ������_kP��[�f��[�H�jl)�� ��>�r{��7�\���d�����n��i7w*��5R~�f��_�����K��lAߨ�}O2]h�L�ه[m���݆��uo Key Terms. Chl a and b contents were calculated using molar absorptivities and formulas from Lichtenthaler (1987). 6:48 . An absorption spectrum is a plot of absorbance vs. wavelength. 53 0 obj <>/Filter/FlateDecode/ID[<33E2E552E603C2E1C44100FC30C9F630><0F0301B68649994F8C7FA59EDB7AC393>]/Index[48 8]/Info 47 0 R/Length 41/Prev 23168/Root 49 0 R/Size 56/Type/XRef/W[1 2 0]>>stream ��2GM�ݪ�&d�n�&�:+�vI��ru��R�{X�{Ϊ���U/�U�V�ćV�ćV�ćV}(��z������rR������[c?���܄��a������C|ƨ��6��;��窓/�uOqlԇ�QS�C�n�F����Xv7j�}h�~:+'��3�cv�m1=�)�&t��c�k��|���?ڪ��ߨ:&������x�� �j�}�����?���DZ�nՔ�p���Og�d����[_�>���܄��a�����齬:�h�N�a������bOq� ��܇������~૏ew��܇���O�,z�i ����2�XM� 6. To avoid … spectrophotometer estimates of chlorophyll concentrations. Title: Microsoft PowerPoint - Spectrophotometry.ppt [Compatibility Mode] Author: Robert Robergs Created Date: 8/23/2010 3:28:15 … aand chlorophyll band identify major absorbance peaks (Figure 1). 1 Received Jan. 2, 1962. Using a spectrophotometer to measure chlorophylls in solvents is not difficult and you can look at my papers on it or look at the papers by Porra. If this is not the case, remove the suspect cuvette and rinse, wipe, add acetone, and rerun it. Running the Spectrophotometer. �dt7;�gC���vf���S����~a�s�����/״b��g�f�7�_��ޤ��CRXK�W�V���b�s/~�zum=� ��]���s흰���KW�w˫�o� �6�'o����T�-�׆L�mn�L�82Ib��#c>C�ڏ\�e��i�� ��3G|��Z����&�-�[�?�8(-����Ysή=�����qK."�������p�;��3f�c�T�! x����%9r&Fi�k=�ٌM&��t� hcc3���E��f\p��ʺ5YQ����M�ch'3��6z�� The formulas were showed in Table 1. Chlorophyll pigment is sensitive to heat and light. 7.6.2 Note: Operate the spectrophotometer at a slit width no wider than 2 nm for maximum resolution. 0 endstream endobj 49 0 obj <> endobj 50 0 obj <>/ProcSet[/PDF/Text]>>/Rotate 0/Type/Page>> endobj 51 0 obj <>stream }�{tPQ�u���5k iM�&�����&�H_L��c�E��;b����s;׷���rt0L���W���tz��j�9bk�o��I��z׭q؍���vP�� 2.2. The absorbance of cooled crude extracts was measured on a photodiode‐array UV‐Vis spectrophotometer (HP 8453; Hewlett‐Packard, Les Ulis, France) from 190 to 1100 nm. How a Simple UV-visible Spectrophotometer Works - Duration: 6:48. The instrument has the necessary narrow band pass, covers the required wavelengths, accommodates sample cells from 1 to … endstream endobj 52 0 obj <>stream As the absorption spectra of 80% acetone, 90% acetone, and 95% ethanol were similar in a given wavelength (Porra 2002, Li et al. First, we need to consider how spectroscopy works. The chlorophyll content in each solution was measured by reading optical density (OD) on a spectrophotometer. Check the performance and accuracy of your spectrophotometer right in your own laboratory using the Thermo Scientific™ SPECTRONIC Standards. ���j�}Me5�X�m��9`J����$���l͵�ۭ���X��A�{�ћS��*�H��R�e{�{h%�1���@"o'�~��|�v��V�{��+'أ���O����ӟ�՟����_\x�ӿ��W?�ӿ��Ky�ӿ��_~������?��o���|���~��e���7�o����w�����|��?������������G������^���.w ~������������q}���>|�_>���������������������݋�AG#z֖}�p3�(�B���x��}�RrX��A*����o��}`�8��;�$�}��؟���ޫ���!�m�o�PM�_R�K\M�o9�>����4��'X��L����u�_��_�E)��� [����hr�Ը��Dg����)m���~K���$K�|[�A�[��`>���A6J�/����+7�s�mmD[���V!f��g�@�[U The wavelengths not absorbed are transmitted to our eyes, leading to the observation of green colored leaves. Donate Login Sign up. chlorophyll a as it absorbs in the same region of the spectrum. <> With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. 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