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Phenol Sulphuric Acid Assay (To al car!ohydra e analysis"

(Analytical Biochemistry 339 (2005) 69-72, Masuko et al. !ar"ohy#rate analysis "y a $henol-sul%uric aci# metho# in micro$late %ormat)

Any accidents must be reported immediately to Carolyn Schultz 37342 (office) 37392 (lab) or Gary aylor e!t 3727" General # This protocol requires the use of sulphuric acid and phenol, you should be familiar with the MSDS of these chemicals before you start. $azards %henol & Class 6.1 To ic. Secondary Class !. "oison, combustible and to ic #ia in$estion, inhalation and s%in absorption. Corrosi#e & Causes burns Sulphuric Acid & Class ! ' corrosi#e To ic by inhalation, causes se#ere burns 'is( Control )easures %henol* (se "henol in a fumehood. "henol is easily absorbed throu$h the s%in) fatalities ha#e been documented from s%in absorption #ia a relati#ely small surface area. (se double $lo#es, made of nylon,rubber or neoprene. Safety $lasses, $o$$les and face shield should be worn. *ear a laboratory coat. +f a splash potential e ists, wear a rubber or neoprene apron. Sulphuric acid* (se in a fumehood *ear nitrile $lo#es, safety $lasses or face shield and a lab coat. +n,ineerin, - .entilation Controls ,ocal e haust #entilation -e$ fume hood. recommended Stora,e 'e/uirements %henol & Store away from o idi/ers such as chlorine, bromine and hypochlorite. Sulphuric Acid # Store away from flammables0combustibles and o idisers. 0irst Aid - Spill Control %rocedures %henol* *ash off immediately with copious amounts of 1lycerol -or cold water if 1lycerol is una#ailable.. 2inse a$ain with copious amounts of cool water -at least 13 minutes.. 4ll laboratories ha#e access to safety showers. "henol is absorbed readily

throu$h the s%in so follow the cold water washin$ with a thorou$h washin$ with warm water and soap. Contaminated clothin$ should be remo#ed as soon as possible and thorou$hly washed. +n case of contact with eyes, immediately flush eyes with copious amounts of water for at least 15 minutes -eye wash.. See% medical attention. "oison +nformation Centre phone 161176 Sulphuric Acid* +ye* Continue flushin$ until ad#ised to stop by the "oisons +nformation Centre or a doctor, or for at least 15 minutes. S(in* +mmediately flush body and clothes with lar$e amounts of water, usin$ safety shower if a#ailable. 8uic%ly remo#e all contaminated clothin$, includin$ footwear. *ash s%in and hair with runnin$ water. Continue flushin$ with water until ad#ised to stop by the "oisons +nformation Centre. See% Medical attention. "oison +nformation centre "hone 161176 Small spill9 Do not attempt clean up if you feel unsure of your ability to do so or if you percei#e the ris% to be $reater than normal laboratory operations. (se #ermiculite or "oly/orb spill absorbent material to soa% up spill. "lace in labelled waste container ready for disposal. :lood area with water then co#er with caustic soda ash to neutralise the residual material. ,ar$e Spill9 ;otify others in the area of spill. <#acuate area. ;otify Dept Safety =fficer -1ary Taylor 6>7>5., Carolyn Schult/ 6>6?7 -office. 6>6@7 -lab. and Campus Security -6>733.. Aarricade area with tape -in Spill Bits. to pre#ent entry until arri#al of response personnel. "ro#ide assistance and information to spill clean up crew. 1aste 4ll waste to be collected followin$ the (ni#ersity of 4delaide *aste Disposal procedure. Collect liquid and solid waste separately. Contact Carolyn Schult/ 6>6?7 -office. 6>6@7 -lab. for current waste disposal instructions. Applicable Standards and 'e,ulations 4 computer based chemical mana$ement system -Chemwatch. is a#ailable throu$h the (ni#ersity webpa$es. The address is ;ational Code of "ractice for the Control of *or%place Ca/ardous Substances D;=CSC9733> -1@@?E. =ccupational Cealth and Safety -Ca/ardous Substances. 2e$ulations 1@@@ 4ustralian Standard 4S 77?6.13 -Safety in laboratories & Stora$e of chemicals..

Phenol Sulphuric Acid Assay (To al car!ohydra e analysis"

(Analytical Biochemistry 339 (2005) 69-72, Masuko et al. $henol-sul%uric aci# metho# in micro$late %ormat) !ar"ohy#rate analysis "y a

&'ea# ()* an# rele+ant M(,( $rior to startin- $rotocol&

'ea,ents D-F.&1alactose D&1lucose "henol has been dissol#ed in water. Sulphuric 4cid @!I we ha#e in lab. )aterials* Microtitre plate, polystyrene Microtitre plate reader *ater Aath

Si$ma 1&3>53 Merc% -melb protocol ' Si$ma 1&5333. 4nalytical rea$ent -solid ' will try GelleHs that 4nalytical rea$ent -will test old sulphuric acid

Sarstedt !7.15!7.331 Aarley $roup0Mares ,ab @3JC static

1or(in, 'an,e "aper claims 1&153nmol for mannose. Melb protocol 3&133$ -appro 16 #olumes. )ethod* Standard Cur2e 1. "repare 7m$0m, solution of $lucose or $alactose in water -73m$ F 13m, water. 7. Dilute to !3$0, - ?, F @@6, water. 6. 4liquot into wells a ran$e from 3&?3$ su$ar standard and ma%e up to 53, with water 4bs $ su$ar , of Std , water ?@3nm 3 3 53 1 1.75 ?!.>5 5 6.75 ?6.>5 13 17.5 6>.5 73 75 75 ?3 53 3 ?. 5. 6. >. !. @. "repare samples to 53, 4dd 153, of concentrated sulphuric acid +mmediately add 63, of 5I phenol +ncubate for 5 min at @3JC in a static water bath -carefully float plate.K Cool to room temp for 5 min in water bath at 2TK *ipe dry and measure on microtitre plate reader

K;otes 16.7.3> ' + found enou$h colour de#elops without the need for heatin$. So Lust read on microtitre plate reader after 13&15 min.