COYAnaerobicChamber

Overview
Thechamberisaheavy
duty,clearpolyvinyl
chloride(PVC)enclosure
supportedbyanaluminum
frameandmountedona
plywoodbasecoveredwith
afoampadandheavyvinyl.
Itisequippedwitha
vacuumairlocksystemthat
allowsthetransferof
materialswhilemaintaining
aninternalanaerobic
atmosphere.
Theairlockisconnectedtotwogastankscontaining(i)100%N2and(ii)agasmix
of5%H2,85%N2,10%CO2.Standardsettingsfortheairlocksincludetwo
consecutivecyclesofvacuumandN2purgeforO2displacement,followedbyone
cyclevacuumandgasmixpurgetoequilibratetheatmospherewiththeinsideof
thechamber.
Vinylchambersaccuratelymaintainastrictanaerobic
atmosphereof05partspermillionO2.Internalatmosphereis
keptO2freewithacombinationofH2containinggasmixand
thepalladiumcatalyst.Thecatalystiscomposedofalumina
pelletscoatedwithathinlayerofpalladiumchloride.The
palladiumchlorideadsorbsH2fromthegasmixallowingitto
reactwithanyO2inthechambersatmospheretogenerateH2O,whichisthen
adsorbedbythealumina.Thecatalystiscontainedinametalwiremeshcontainer
(StakPak)thatishousedinafanboxtopromotegascirculation.
TheO2H2gasanalyzerconstantlymonitorsanddisplayslevelsin
thechambersatmosphere.Itiscalibratedtoproducevisibleand
audiblealarmswhenO2levelshaverisenaboveaset
concentrationorH2levelsareoutsideaconcentrationrange.

OperationandMaintenanceoftheGeneralUser
Transferringitemsintoandoutofthechamber:
1. Priortointroducingitemsintheairlock,checkthefollowing:
a. H2 levelsdisplayedonLEDoftheO2 H2 analyzerare~2%orabove
andO2levelsarewithindesiredrange(approachingorat0)
IfH2levelsarebelow2%orO2levelsareabove100ppmsee
GloveBoxMaintenancebelow.
b. Innerdoorofairlockiscompletelyclosedandlocked
2. Loadmaterialsthroughouterdoor
a. Sealedpackagesandtubesshouldbeopenedslightlytopermitgas
exchangeduringairlockcycles
b. Putobjectsinsideairlock,closeandlatchfrontdoor,andpressStart
button.Aftercycling(4exchanges),theairlockwillindicatethatitis
anaerobicandsafetoopentheinnerdooroftheairlock.
3. Transferringmaterialsoutofglovebox
a. Outerdoormustbelockedandairlockchambermustbeanaerobic
priortoopeninginnerdoor.Ifyouareunsureofthestatusofthe
airlock,performexchanges(i.e.,pressstart)tomakeitanaerobic.
b. Innerdoorcanbeopenedandmaterialscanbetransferredinto
airlockcabinet
c. Lockinnerdoortosealairlockcabinet
d. Outerdoorcanbeopenedtoremovematerials
e. ItisrecommendedthatouterdoortoairlockcabinetisLOCKEDAT
ALLTIMEStominimizeriskofO2intrusions
GeneralRulesforGloveBoxoperation:
1. Doors:Neveropenairlocksouterentrydoorbeforecheckingtoseeif
theinnerdoorissecurelyclosedandviceversa
2. Airlock:Alwayscheckthattheanaerobicdisplayisonbeforeopening
innerdoor
3. Sharps:Avoidusingsharpsinoraroundglovebox,ifnecessarykeep
Sharpsboxinchamber(Removeanysharpjewelrypriortouse)
4. Cleaning:useabsorbentbenchpaperinchambertocontainspillsand
debrisandtocleanworkspace(70%ethanolorotherdisinfectantscan
beused)
5. Emergency:Aroleofvinyltapeandscissorsshouldbekeptinornear
thechamberforemergencyrepair
6. Maintenance:
a. RegeneratecatalystStakPakinovenonceaweek
b. Periodicallyreplace~1/4ofthechambersatmospherewithgas
mixtomaintainoptimumH2levels
7. Economy:Minimizethenumberofchamberentriesbycoordinating

transferswithotherlabmembers
AsepticTechniquesinanAnaerobicChamberEnvironment
1. Noflamecanbeusedintheglovebox
a. LackofO2 preventsH2 ignition
b. IncandescentflamingdevicecanbepurchasedfromCoyif
necessary
2. Propercleaningofanaerobicchamberandairlocktoprevent
contamination
a. Priortowork,thechamberworkpad,instrumentationand
(especiallybetweenfingers)canbecleanedwith70%ethanolor
dampdisinfectantwipes.
b. Utilizedisposableitemswhenpossible(i.e.loops,spreaders,etc.)
c. Anyusedutensilsaredisposedofinawastecontainerandshould
beremovedfromtheglovebox,emptiedanddisinfectedafter
eachuse
3. Otherbasicrulesofsteriletechnique
a. Anymaterialshousedingloveboxforlongperiodsoftimeshould
beresterilizedfrequentlytopreventcontamination
b. Gloveboxshouldbecleanedperiodicallywithdisinfectantsaspart
ofitsregularmaintenance

AdditionalNotesforCoyUse
Gasanalyzeralarmgoesoff
1. LowH2 levels
a.ToincreaseH2concentrationinchamber,makesureinnerairlockis
anaerobic,openinnerairlockdoor,andtransferairlocktoManuel
modebypressingEnter/Menubutton,thenStartbutton.
b.Evacuate~1/4ofthechamberatmosphereusingthevacuum(^
button)
i. Deflateglovebox12inchesbelowthetopaluminumframe
ii. Replenishchambersatmospherewithgasmix(Start
button)untilinsidepressureissufficienttoinflatethebag
butnotthearmsleeves
iii.
RepeatasnecessarytoincreasetheH2levelsto23%
iv.
CloseinnerdooroftheairlockandpressEnter/Menu
buttontoexitManuelmodewhenfinished
c. Automaticgasinjectionsystemcanbeprogrammedtoreplacea
setvolumeofthechambersatmospherewithgasmixforafixed
timeperiodandatafixedfrequency
Timeouts
1.
Ifthereisatimeouterror,youcanusuallyidentifytheproblem
basedonwhatpartofthecyclingthetimeoutoccurred.

Iftheerrorhappenedduringavacuumcycle,oneofthedoorsis
probablyopen,socheckthatallouterandinnerdoorsare
completelyclosedandtryagain.
b. Ifthereisanerroronthepurgeorgasmixsteps,therespective
tankislikelyempty.Checkthelevelsindicatedonthetank
regulator,andifatankisclosetoempty,replaceit.
i. Thiswillalsohappensometimeswithanewtankif
someonehasforgottentoopenthetankortheregulator,so
checkthoseproblemsifthetimeoutpersists.
ii. Inmostcases,addressingthesepossibleissuesthen
turningtheCoyoffandbackonwillresolvetheproblem.
a.

Leaks
1.

IfthepressureinonesideoftheCoyisabnormallylow
a. Switchintomanualmodeandincreasepressureusingthegas
mixuntilpositivepressureisreached.
b. Ifthepressurehasdroppedthenextday,theremaybeasmall
holesomewhere,oftennearthegloves.
i. Usetheleakdetectorand/orsoapywatertoidentifythe
hole,thenpatchit.

Contamination
1. Openasolidifiedrichmediuminchambertocheckforpresenceof
contaminatingorganisms
i. Ifcontaminationissuspected,removeallequipmentand
cleanchamberwith50%IPA,12%Clorox
a. Routinelycleantherubbersealairlockdoorstoremovedustand
dirttopreventairleaks

LabJobDutiesforCoy
HeatcatalystplatesinovenunderCoy(150200oC)for>2hours,thenallow
platestocooltoapproximatelyroomtempandexchangewithplates
currentlyinsideCoy(weekly).
2. Checkoxygenlevelstomakesurethattheyareeitherat0ppmorare
steadilydecreasingtothat(daily).
a. Iflevelsaredecreasingtooslowly,exchangecatalystimmediately.
b. Ifoxygenstayshigh,checkforleaksinwallsofCoyanddoors.
3. Checkhydrogenlevelstomakesuretheyaremaintainedaround2.0%.
a. Iflevelsaretoolow,doanexchangetomakethetransferchamber
anaerobic,thenraisehydrogenlevelsbyswitchingCoyintomanual
mode,openingtheinteriordoorconnectedtothatside,and
repeatedlyalternatingcyclesofvacuumandgasmix.
b. Afteryouaredone,returnCoytostandardmodeandensurethat
1.

cyclingprogramisreturnedtoP1usingupanddownarrows(daily).
4. Throwawayleftoverplatesandcultures,especiallythosethatseemtobe
abandoned(crackedplates,driedoutcultures,>1monthold,etc.).
a. Ifyouwanttogivepeoplethechancetocleantheirownthings,give
themafewdaysnoticetomovethingstheywanttokeep,thentoss
anythingthatwasntmoved.Also,occasionallycheckandemptytrash
containers(asnecessary,everycoupleofmonths).
5. Checkhumidityindicatortoensurethathumidityis7080%.Ifnot,make
surethatdehumidifiersarentfull,andiftheyare,emptythem(asneeded,
everycoupleofmonths).
GloveBoxMaintenance
1. Catalyst
a. Palladiumcatalystiscontainedinametalwiremesh(StakPak)
i. Moisturecanberemovedbybakinginadryoven
(125200C)foraminimumof2hrs;regenerateoncea
week
ii. ReplaceStakPakevery1to2years
b. Testtomonitorcatalystperformanceistomeasuretheheat
producedduringthecatalysisreactionbetweenpalladium,H2and
O2
i. Placeatraycontainingthepalladiumcatalystinsidethe
airlock
ii. Placeathermometerindirectcontactwithcatalyst
iii.
Manuallyevacuategasatmospherefromairlock.
Vacuumpressureinsidetheairlockchamberisachieved
whenpressureinsidethechamberreads20in.mercury
units
iv.
Filltheairlockchamberwithgasmix(containingH2)
v. Ifcatalystisworkingcorrectly,thetemperaturewill
increaseatleast10Coveraperiodof1015min
2. Moisture
a. ExcessivemoisturedecreasesH2 levelsinchamberatmosphere
andsaturatesthecatalyst,increasinggasconsumption
b. Minimizehumidity
i. Coysareequippedwithdehumidifier
ii. Regeneratecatalystmoreoftenthanrecommended
schedule
iii.
Incubatesolidmediaplatesinairtightfoodcanisters
containingapetridishwithaluminadesiccantpellets
iv.
AvoidlongtermstorageofplatesandculturesinCoy
c. Ifmoisturedevelopsinthechamberandcondensationbecomes
noticeableonvinyl,removeimmediatelywithabsorbentpaperto

preventfungalgrowth
3. SharpsDisposal
a. Allsharpsshouldbedisposedofapprovedsharpscontainers
b. Operatorshouldremoveanysharpjewelrythatcouldpuncture
glovesand/orsleeves
4. Hygienicmeasures
a. Cleantheoutsidevinylofthegloveboxbeforeeachuse
b. 70%ethanolforworkpadandgloves
c. 50%I.P.A.or12%CloroxforPVC
GloveBoxTroubleshooting
Problem
O2H2analyzeralarm

PossibleCause
LowH2levels

Solution
ReplenishH2levelsinchambers
atmospherewithgasexchange

AirlockDoors

Ensuretheinnerandouterdoors
areproperlyclosedandthereis
nodirtbuilduponseals
Removeanyobjectsblockingthe
catalystand/orfan

Fanboxesareblocked
CatalystStakPak

ReplaceStakPaks(theyare
eithersaturatedwithmoistureor
dontwork)

Punctureinglovebox

Inflategloveboxwithgasmix
untilglovesarefullinflated.
CheckforleakswithH2detector
andrepairpunctures

Faultyregulators

Checkforleakswithsoapy
solution.Redoconnectionsor
replaceregulator

Leaksinconnectionstoairlock

Checkforleakswithasoapy
solutionorgasleakdetectorfor
H2containinggases

RegardlessofthesourceofO2,oncerepaired,purgeofthechambers
atmosphereandreplacewithgasmix.RepeatuntiloptimalH2levelsarereached.
Condensation/DeHumidifier

CatalystStakPak

DesiccantStakPak

ExchangecatalystStakPakwith
rejuvenatedones(theyeitherare
saturatedwithmoistureordont
work)
ReplacedesiccantStakPakwith

Experimentreleasingexcess
moisture

rejuvenatedones(theyareeither
saturatedwithmoistureordont
work)
Emptydehumidifiers
ReplacedesiccantStakPakwith
rejuvenatedonesandaddmore
desiccantStakPaksifneeded

Oncerepaired,purgeofthechambersatmosphereandreplacewithgasmix.
Repeatasnecessary.
Contamination

Improperlysterilizedmaterials

Resterilizeallsterilematerials
houseinchamber

Contaminatedcultures

Discardoldorcontaminated
cultures.Placeplatesinsealed
canisterstoprevent
crosscontamination

Disinfectthechamberandallsurfaceswithadisinfectant(I.P.A.,Clorox).Cleanwith
peraceticacidtosterilizechamber.
Bagpressuretoolowortoohigh Autoinjector

Airlockerror/Timeouterror

Testand/orreprogramthe
autoinjector.Ensurevacuumis
functioningproperly

Leaksinvinylbagorneoprene
rubbergloves

Detectleakswithgasleak
detector.Sealpunctures.
Replaceglovesifnecessary

Vacuum/gasexchangecycles

Usingtheairlock,evacuateor
injectgasasneededtobringthe
bagtotheappropriatepressure

Gastanks

Checkthatthetanksareturned
onandnotempty,andregulators
areonandfunctioningproperly
WorkingPSIshouldbe2025(on
regulator)

Airlockchamber

Ensuretheinnerandouterdoors
areproperlyclosedandthereis
nodirtbuildupontheseals

Vacuum

Ensurethevacuumisfunctioning
properly.Checkconnectionsto
vacuumpump.Replaceorfixas
needed

Onceproblemisdiagnosed,applyvacuumoraddgasasneededtoequalize
pressureintheairlocksothedoorscanbeopened.