ࡱ> 796 )bjbj .*||$79cccWcchb}yK:4m0<,Ihh|h Wcccc| : Ashing/Acid Digestion for Plant Tissue (based on M. Arthur lab, U. of Kentucky, updated by Corrie on October 5, 2009, amended by Chuck Schirmer 10/8) Day 1 Pulverize samples in the ball mill (this can be done before or after drying the samples, but ideally would be done before). this step may be optional depending upon sample type and amount of sample. Place samples in oven over night to dry (with lids unscrewed slightly) Day 2 Place samples in desiccator while waiting to be weighed Retrieve clean acid washed crucibles from drawer by the ovens in the soils lab (make sure they are not the soil crucibles as these are not acid washed). Place them right side up in a clean tray (or upside down on a tray with paper towel in the tray to keep them from becoming contaminated) Weigh samples: Record crucible number Place empty crucible on scale, record weight Tare scale Add 0.25 gram of pulverized sample (+/- .005 g) Record weight of sample Include one duplicate sample Include one crucible with no sample (a digestion blank) Include at least one Standard Reference Material (SRM) The total number of samples that fits well in a muffle furnace is 40, so a run will usually consist of 37 samples, one duplicate, one blank, and one SRM. Place crucibles in the cool muffle furnace (currently furnace 1 is out of commission) If you are running root samples, place these nearest to the oven opening so that they can be retrieved first and put in the dessicator to cool and be weighed again Begin ashing at 200 C (follow temperature increase procedure posted near muffle furnaces, it goes basically like this): Increase temperature 5 C every 20 min until 250 C is reached This is the most important step to prevent the samples from igniting Continue to increase temperature 10 C every 20 min until 300 C is reached Continue to increase the temperature 25 C every 30 minutes until 400 C is reached Turn to 470 C and leave to ash over night (For the latter part of these increments it is not as important to be exact as it is for the first 50 C.) Weigh centrifuge tubes for tomorrow, write weight on the label of each tube. Day 3 Turn off the furnace and allow to cool for 10-15 minutes (take out root samples and allow to cool in dessicator, then reweigh sample). Remove samples and place into dessicator Reweigh samples steps 15 and 16 are most important when digesting root tissue. Once samples have cool and been weighed add ~ 2 mL of distilled deionized water (DDI) to moisten the sample Do this with care, using a squeeze bottle and spraying down the side of the crucible - the ash is fragile and we are trying to stabilize it and not loose any sample by blowing it out with the squeeze bottle Add 10 mL of 6 N nitric acid (HNO3) to each sample 10 mL is already measured out by an automatic pipeter located next to the hot plate. Double check you are using nitric acid. Place samples on the hot plate in groups small enough to monitor (~15 at a time) Turn on the hot plate. Watch samples, once they start to a slow bubbling point (or where you can see the steam coming off of them) continue to heat for at least 15 minutes, but do not allow to boil dry While they are heating agitate the samples so that the ash does not creep up the side of the crucible and form a ring of material Try to keep the solids in the acid solution Remove from hot plate and allow the samples to cool slightly While keeping an eye on the hot plate set up a number of pre-weighed centrifuge tubes, with funnels, and Whatman # 42 filter paper on lab benches. Rinse the filter paper with DDI water to rinse and also keep the filter paper in the filter Filter sample through filter paper into 50 mL centrifuge tubes Rinse the crucible 3 times, keeping the crucible at 45 degree angle over the filter Let the sample filter Rinse the filter paper 3 times, starting at the top and working your way down the filter After all of the solution has drained through the filter, bring tubes up to volume with DDI. Shake centrifuge tubes. Weigh centrifuge tubes for the second time (now with the digestion mixture). Clean and acid wash all crucibles, glassware and funnels Acid washing procedure is located above acid washing station Notes: In the event that Chuck is not tNQnt e f  ? (,nF@Aǿӿӫëyuh.h.h.6hqhahxb h*H*h*h*>*h* h.5hO>hph\h hcWh>h2QhFh/hh4hF5h4h45hFh/5 hF5 hq5 h5 h/5hFhF5.'f  3 ` k  ? /  & FgdF & Fgd2Q & Fgd2Qgd4 & FgdF & Fgd$a$gd/$a$gdF p:f7u@} & Fgdxb & Fgdxb & Fgd*gd4 & Fgdp & Fgdp & Fgd\lm1j(J(K((() hqH*Uhqh.hqh/hxblo1j8(9()gdqgdxbgdq & Fgd. & Fgd/ & Fgdxbhere, or you run out of acid and need to make your own: To prepare 6N HNO3: Using a 1 liter volumetric flask, add 378ml of concentrated HNO3 to approximately 500ml of distilled deionized water. Allow solution to cool and bring up to volume with DDW. Transfer solution to the dispensing container in the fume hood. Measuring and pouring of the acid should also be performed in the fume hood. Be safe: always wear a lab coat, gloves and eye protection when working with acids. ,1h/ =!"#$% ^ 2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA D Default Paragraph FontRiR  Table Normal4 l4a (k (No List PK![Content_Types].xmlj0Eжr(΢Iw},-j4 wP-t#bΙ{UTU^hd}㨫)*1P' ^W0)T9<l#$yi};~@(Hu* Dנz/0ǰ $ X3aZ,D0j~3߶b~i>3\`?/[G\!-Rk.sԻ..a濭?PK!֧6 _rels/.relsj0 }Q%v/C/}(h"O = C?hv=Ʌ%[xp{۵_Pѣ<1H0ORBdJE4b$q_6LR7`0̞O,En7Lib/SeеPK!kytheme/theme/themeManager.xml M @}w7c(EbˮCAǠҟ7՛K Y, e.|,H,lxɴIsQ}#Ր ֵ+!,^$j=GW)E+& 8PK!Ptheme/theme/theme1.xmlYOo6w toc'vuر-MniP@I}úama[إ4:lЯGRX^6؊>$ !)O^rC$y@/yH*񄴽)޵߻UDb`}"qۋJחX^)I`nEp)liV[]1M<OP6r=zgbIguSebORD۫qu gZo~ٺlAplxpT0+[}`jzAV2Fi@qv֬5\|ʜ̭NleXdsjcs7f W+Ն7`g ȘJj|h(KD- dXiJ؇(x$( :;˹! I_TS 1?E??ZBΪmU/?~xY'y5g&΋/ɋ>GMGeD3Vq%'#q$8K)fw9:ĵ x}rxwr:\TZaG*y8IjbRc|XŻǿI u3KGnD1NIBs RuK>V.EL+M2#'fi ~V vl{u8zH *:(W☕ ~JTe\O*tHGHY}KNP*ݾ˦TѼ9/#A7qZ$*c?qUnwN%Oi4 =3ڗP 1Pm \\9Mؓ2aD];Yt\[x]}Wr|]g- eW )6-rCSj id DЇAΜIqbJ#x꺃 6k#ASh&ʌt(Q%p%m&]caSl=X\P1Mh9MVdDAaVB[݈fJíP|8 քAV^f Hn- "d>znNJ ة>b&2vKyϼD:,AGm\nziÙ.uχYC6OMf3or$5NHT[XF64T,ќM0E)`#5XY`פ;%1U٥m;R>QD DcpU'&LE/pm%]8firS4d 7y\`JnίI R3U~7+׸#m qBiDi*L69mY&iHE=(K&N!V.KeLDĕ{D vEꦚdeNƟe(MN9ߜR6&3(a/DUz<{ˊYȳV)9Z[4^n5!J?Q3eBoCM m<.vpIYfZY_p[=al-Y}Nc͙ŋ4vfavl'SA8|*u{-ߟ0%M07%<ҍPK! ѐ'theme/theme/_rels/themeManager.xml.relsM 0wooӺ&݈Э5 6?$Q ,.aic21h:qm@RN;d`o7gK(M&$R(.1r'JЊT8V"AȻHu}|$b{P8g/]QAsم(#L[PK-![Content_Types].xmlPK-!֧6 +_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!Ptheme/theme/theme1.xmlPK-! ѐ' theme/theme/_rels/themeManager.xml.relsPK]  *) )8@0(  B S  ?z@B=*urn:schemas-microsoft-com:office:smarttags PlaceType G# jlW`(-y3333NQente% Z zzNQente% Z zz/z5 `^`o(. ^`hH. pLp^p`LhH. @ @ ^@ `hH. ^`hH. L^`LhH. ^`hH. ^`hH. PLP^P`LhH./z5         4Nq*/.xbp\>qO>2Q4F}d(cW a@o4X@XX(@XP@UnknownG* Times New Roman5Symbol3. * ArialA BCambria Math"qhd&d& ! !!24d~~2QHP ?F2!xx&Ashing/Acid Digestion for Plant TissueHeather EngelmanHeather Engelman Oh+'0 8D d p |(Ashing/Acid Digestion for Plant TissueHeather Engelman Normal.dotmHeather Engelman3Microsoft Office Word@Ik@;yK@ؑ_yK՜.+,0 hp   Ƶ! ~ 'Ashing/Acid Digestion for Plant Tissue Title  !"#$%'()*+,-/0123458Root Entry FyK:1TableWordDocument.*SummaryInformation(&DocumentSummaryInformation8.CompObjy  F'Microsoft Office Word 97-2003 Document MSWordDocWord.Document.89q