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Chapter 5: Quality Assurance and Calibration Methods 5.0 - The need for Quality Assurance -

River water was tested for lead content by different l a bo r a t o r i e st oa s s e s st her e l i a b i l i t yoft h e i r a na l y s e s .

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Re s ul t swe r ev a r i e da n ds omeoft he m we r eo u t s i d et h ec e r t i fie dr a n g e

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Th i se x a mpl ei l l us t r a t e st ha tt he r ei snogua r a nt e et h a tr e s ul t sa r er e l i a b l e ,e v e ni ft he ya r eob t a i ne db y “ a c c r e di t e d ”l a b or a t or i e su s i n ga c c e pt e dpr o c e du r e s . Ag oo dwa yt oa s s e s st h er e l i a b i l i t yo fal a bwor ki n g f ory oui st opr o vi det hel a bwi t h“ b l i nd”s a mp l e s —s i mi l a rt oy ourun kno wns —f o rwh i c hy oukn o wt he “ r i g ht ”a ns we rb utt hea n a l y s td oe snot .I ft h el a bd o e sno tfin dt hek no wnr e s u l t ,t he r ei sapr ob l e m. Pe r i o di cbl i ndc he c ks a mpl e sa r er e q ui r e dt od e mo ns t r a t ec ont i nui n gr e l i a b i l i t y .

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Qua l i t ya s s ur anc e :i swha twedot og e tt her i ghta ns we rf orou rpu r p o s e .Th ea n s we rs ho ul dha v e s uffic i e n ta c c u r a c ya n dpr e c i s i o nt os up por ts u b s e q u e ntde c i s i o n s . Th e r ei snop oi n ti ns pe n di n ge xt r a mon e yt oo bt a i namor ea c c ur a t eo rmor epr e c i s ea n s we ri fi ti sn otn e c e s s a r y .

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Data quality standards:

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Get the right data

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Get the data right

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Keep the data right

5.1 – Basics of Quality Assurance - spaghetti sauce analogy each tasting is a sampling event with a quality control test; at factory: taste few a day !

Ra wd at a :i n di v i du alme a s u r e me nt s ;a r ei nd i vi dua lv a l u e so fame a s ur e dq ua nt i t y ,s uc ha spe a ka r e a sf r om a c h r o ma t o g r a m orv ol ume sf r o m ab ur e t t e .

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-T r e a t e dd at a:a r ec on c e nt r a t i on sora mou nt sf o undb ya ppl yi n gac a l i b r a t i o np r oc e du r et ot h er a wda t a . -Re s u l t s :a r ewha tweul t i ma t e l yr e p or t , s uc ha st h eme a n,s t a nda r dd e vi a t i on ,a n dc on fid e nc ei nt e r v a l , a f t e ra p pl yi n g s t a t i s t i c st ot r e a t e dd a t a . -Use objective: states purpose for which results will be used

- Speci fic a t i o ns :s t a t e sh o wg o o dt hen umb e r sn e e dt ob ea ndwh a tp r e c a u t i o n sa r er e q ui r e di nt h ea na l y t i c a l p r o c e d u r e .Ho ws h a l ls a mp l e sb et a k e na n dh o wma n ya r en e e d e d ?Ar es pe c i a lp r e c a u t i o n sr e q u i r e dt op r o t e c t s a mpl e sa n de n s u r et ha tt he ya r en o td e gr a de d ?Wi t hi npr a c t i c a lr e s t r a i nt s ,s u c ha sc o s t ,t i me ,a n dl i mi t e d a mou n t so fma t e r i a la v a i l a b l ef ora n a l y s i s ,wh a tl e v e lo fa c c u r a c ya n dp r e c i s i o nwi l ls a t i s f yt h eu s eo b j e c t i v e s ? Wh a tr a t eo ff a l s ep o s i t i v e so rf a l s en e g a t i v e si sa c c e p t a b l e ?Th e s eq ue s t i o n sn e e dt ob ea n s we r e di nd e t a i l e d s p e c i fic a t i o ns . -Sp e c i fic a t i on smi g hti n c l u de :  s a mpl i n gr e q ui r e me n t s  a c c u r a c ya n dp r e c i s i o n

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r a t eo ff a l s er e s u l t s s e l e c t i vi t y s e ns i t i v i t y a c c e p t a bl eb l a n kv a l u e s r e c o v e r yoff or t i fic a t i on c a l i br a t i onc h e c ks q ua l i t yc o nt r ols a mpl e s

False positive vs False negative: Supposeyoumustcer t i f yt ha tac on t a mi na nti ndr i n ki n gwa t e ri sbe l o wa l e g a ll i mi t .Af al s epo s i t i v es a y st h a tt h ec o nc e nt r a t i one x c e e dst hel e g a ll i mi twhe n ,i nf a c t ,t hec onc e nt r a t i oni s b e l o wt hel i mi t .Af a l s ene g a t i v es a y st ha tt hec onc e nt r a t i oni sb e l o wt hel i mi twhe ni ti sa c t u a l l ya b ov et hel i mi t . Ev e nwe l l e x e c ut e dp r oc e d ur e sp r odu c es o mef a l s ec o n c l us i o nsb e c a us eoft h es t a t i s t i c a ln a t ur eofs a mpl i n ga nd me a s ur e me nt .Fo rdr i nki n gwa t e r , i ti smor ei mp or t a n tt oha v eal o wr a t eoff a l s ene ga t i v e st h a nal o wr a t eoff a l s e p os i t i v e s . I twou l dbewo r s et oc e r t i f yt ha t c on t a mi n a t e dwa t e ri ss a f et ha nt oc e r t i f yt ha t s a f ewa t e ri sc o nt a mi n a t e d . -

Se l e c t i vi t y :( a l s oc a l l e ds pe c i fi c i t y )me a n sb e i n ga bl et od i s t i n gui s ha na l y t ef r om o t h e rs p e c i e si nt h e s a mp l e( a v o i d i n gi nt e r f e r e nc e ) .

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Se ns i t i v i t y:i st hec a pa bi l i t yo fr e s pon di n gr e l i a bl ya ndme a s ur a b l yt oc h a n g e si na na l yt ec o nc e n t r a t i o n. Th e de t e c t i o nl i mi t ofa na na l yt i c a lme t h odmu s tb el owe rt h a nt h ec onc e nt r a t i o nst obeme a s ur e d .

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Se n s i t i v i t y=s l op eoft h ec a l i br a t i o nc ur v e=c h a n g ei ns i g na l /c h a n g ei na n a l yt i c a lc o nc e n t r a t i o n

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Bl a n ksa c c o untf o ri nt e r f e r e n c eb yo t h e rs p e c i e si nt h es a mpl ea ndf o rt r a c e sofa na l yt ef ou ndi nr e a g e nt s us e df o rs a mpl epr e s e r v a t i o n,p r e pa r a t i on,a n da na l y s i s . Fr e qu e ntme a s ur e me nt so fbl a nksde t e c twhe t he r a na l y t ef r ompr e v i o uss a mp l e si sc a r r i e di nt os ub s e q ue n ta n a l y s e sb ya d he r i n gt ov e s s e l sori n s t r u me n t s .

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There are different types of blanks to account for different situations: o

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Method blank:  All components except analyte  Taken through all steps of the analytical procedure  Subtract the response of the method blank from the response of sample before calculating the quantity of analyte Reagent blank:  Similar to a method blank, but it has not been subjected to all sample preparation procedures Field blank:  Indicates if analyte is inadvertently picked up by exposure to field conditions  I ss i mi l a rt oame t ho dbl a nk ,b uti tha sb e e ne x po s e dt ot h es i t eo fs a mpl i n g .

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s p i k er e c ov e r y :s o me t i me s ,r e s pon s et oa na l y t ec a nbede c r e a s e dori nc r e a s e db ys ome t hi n ge l s ei nt h e s a mp l e .

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Mat r i x:r e f e r st oe v e r y t hi n gi nt hes a mpl eo t he rt h a na n a l yt e .

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Spi ke :a l s oc a l l e daf or t i fic at i on, i sakn o wnq ua n t i t yofa n a l yt ea dde dt oas a mp l et ot e s twh e t h e rt he r e s p ons et oas a mpl ei st h es a mea st ha te xp e c t e df r omac a l i br a t i onc u r v e .

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c al i br a t i o nc he c k:a n a l yz es o l u t i o n sf or mul a t e dt oc ont a i nkn o wnc onc e nt r a t i on sofa na l y t e . Thi spr a c t i c e he l p sv e r i f yt ha tt h ei ni t i a lc a l i b r a t i ons t a nd a r dswe r ema depr o pe r l y .

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Pe r f or manc et e s ts a mpl e s :( a l s oc a l l e dq u al i t yc on t r o ls a mpl e sorbl i n ds a mp l e s )a r eaq ua l i t yc o nt r ol me a s ur et ohe l pe l i mi na t ebi a si nt r o duc e db ya na na l y s twhokno wst h ec on c e n t r a t i o noft hec a l i b r a t i on c h e c ks a mp l e .

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St a ndar dope r a t i ngpr o c e dur e ss t a t i n gwha ts t e p swi l lb et a k e na n dho wt h e ywi l lbec a r r i e dou ta r et h e bul wa r kofqu a l i t ya s s u r a nc e .Fo re x a mpl e ,i far e a g e nth a s“ g o neb a d”f ors omer e a s o n,c ont r ole x pe r i me nt s bui l ti n t oy o urnor ma lp r oc e d ur e ss h ou l dde t e c t t h a ts o me -t hi n gi swr on ga ndy ou rr e s ul t ss h oul dno tb e r e por t e d.

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To gauge accuracy: calibration checks, fortification recoveries, quality control samples and blank

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To gauge precision: replicate samples and replicate portions of same sample

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As s e s s me nti st h epr oc e s sof :

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Col l e c t i n gda t at os ho wt ha ta na l y t i c a l pr oc e du r e sa r eo pe r a t i n gwi t h i ns pe c i fie dl i mi t sa nd

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Ve r i f y i n gt h a tfina lr e s ul t sme e tu s eob j e c t i v e s .

5.2 – Method Validation -

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Me t ho dv al i dat i o ni st h epr oc e s so fpr o vi n gt h a ta na na l y t i c a lme t ho di sa c c e pt a bl ef o ri t si nt e nd e d 4 pur p os e . I npha r ma c e ut i c a lc he mi s t r y ,me t h odv a l i da t i o nr e q ui r e me nt sf orr e gul a t or ys u bmi s s i o ni n c l u de s t udi e so fme t h ods pe c i fi c i t y , l i ne ar i t y , ac c u r ac y , pr e c i s i o n,r a ng e , l i mi to fd e t e c t i o n, l i mi to fq uan t i t a t i o n, a ndr ob us t ne s s .

Method is the standard operating procedure Method validation: do an assessment; LOD = Limit Of Detection; the lower limit of detection the better we see things (1 ppm vs 1 ppt); you see something but don't know how much it is LOQ = Limit Of Quantitation; bigger than LOD

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i s hea b i l i t yofa na n a l y t i c a lme t ho dt odi s t i n gu i s ha na l y t ef r om e v e r y t hi n ge l s et h a t mi g htb e Spe c i fic i t y :t i nt h es a mp l e . El e c t r op hor e s i s :i sa na na l yt i c a lme t ho di nwhi c hs u b s t a n c e sa r es e pa r a t e df r omonea n o t h e rb yt he i r di ffe r i n gr a t e sofmi g r a t i o ni nas t r o n ge l e c t r i cfie l d . El e c t r op he r o gr ami sagr a p hofde t e c t o rr e s pon s ev e r s ust i mei na ne l e c t r op ho r e t i cs e p a r a t i on .

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Bas e l i n es e pa r at i onme a n st h a tt hede t e c t o rs i gn a lr e t u r n st oi t sb a s e l i neb e f o r et hene x tc ompou ndr e a c he s t hed e t e c t o r .

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Li ne a r i t yme a s ur e sh o wwe l lac a l i br a t i o nc ur v ef o l l o wsas t r a i g htl i ne , s h o wi n gt ha tr e s po ns ei s pr op o r t i o na lt ot hequ a nt i t yofa na l y t e .

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Ac c ur a c y :i s“ ne a r n e s st ot h et r ut h. ”Wa y st od e mon s t r a t ea c c u r a c yi nc l ud e :

Ana l y z eac e r t i fie dr e f e r e n c emat e r i ali nama t r i xs i mi l a rt ot h a to fy ou run kn o wn .You rme t ho d s h oul dfin dt h ec e r t i fie dv a l u ef o ra na l yt ei nt her e f e r e n c ema t e r i a l ,wi t h i nt h epr e c i s i o n( r a nd om unc e r t a i nt y)ofy o urme t h od . o Compa r er e s ul t sf r om t woo rmo r edi ffe r e n ta n a l yt i c a lme t ho ds .Th e ys h oul da g r e ewi t h i nt h e i r e xpe c t e dpr e c i s i on . o Ana l y z eabl a n ks a mpl es p i k e dwi t hakn o wna dd i t i o nofa na l y t e .Thema t r i xmu s tbet h es a mea s y ou run kno wn. Wh e na s s a yi n gama j o rc o mpon e nt ,t hr e er e p l i c a t es a mp l e sa te a c hoft hr e el e v e l s r a n gi n gf r om 0 . 5t o1. 5t i me st hee xp e c t e ds a mpl ec on c e nt r a t i ona r ec us t o ma r y .Fo ri mp ur i t i e s , s p i k e sc oul dc o v e rt h r e el e v e l ss pa n ni n ga ne xpe c t e dr a n g eo fc o nc e nt r a t i ons ,s u c ha s0. 1t o2wt %. o I fy ouc a nn o tpr e pa r eab l a n kwi t ht h es a mema t r i xa st h eunk no wn,t he ni ti sa pp r o pr i a t et oma k e s t an dar da ddi t i on s( Se c t i on5 3)ofa na l yt et ot heu nkn o wn .Ana c c ur a t ea s s a ywi l lfindt hekn o wn a mou nto fa n a l yt et h a twa sa dd e d . Sp i ki n gi st h emo s tc ommonme t ho dt oe v a l ua t ea c c ur a c ybe c a us er e f e r e nc ema t e r i a l sa r eno tu s u a l l y a v a i l a b l ea n das e c o nda n a l yt i c a lme t h odma ynotb er e a di l ya v a i l a b l e .Sp i ki n ge n s ur e st ha tt h ema t r i x r e ma i n sn e a r l yc on s t a n t . Pr e c i s i o ni sho wwe l lr e pl i c a t eme a s ur e me nt sa gr e ewi t hon ea no t h e r , us ua l l ye x pr e s s e da sas t a nd a r d de vi a t i o n. o I ns t r u me ntp r e c i s i o n, a l s oc a l l e di n j e c t i o npr e c i s i o n,i st her e pr o duc i bi l i t yobs e r v e dwh e nt hes a me q ua n t i t yo fon es a mpl ei sr e p e a t e dl yi n t r o du c e d( i me s )i n t oa ni ns t r u me nt .Va r i a bi l i t yc ou l d �10t a r i s ef r om v a r i a t i o ni nt hei n j e c t e dqu a nt i t ya n dv a r i a t i o nofi n s t r ume ntr e s p on s e . o

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I nt r a a s s ayp r e c i s i o ni se v a l ua t e db ya na l yz i n ga l i q uo t sofah omo g e n e ou sma t e r i a ls e v e r a lt i me s b yonep e r s o nono neda ywi t ht h es a mee qui p me n t . Ea c ha n a l y s i si si n d e pe n de n t , s ot hei nt r a a s s a ypr e c i s i o ni st e l l i n gush o wr e pr o duc i b l et h ea na l y t i c a lme t h odc a nbe . I nt r a -a s s a yv a r i a bi l i t y i sg r e a t e rt ha ni n s t r u me n tv a r i a b i l i t y ,b e c a u s emor es t e p sa r ei n v o l v e d .Exa mpl e so fs pe c i fic a t i ons mi g htb et h a ti n s t r ume ntp r e c i s i o ni s�1 %a n di n t r a a s s a ypr e c i s i oni s�2%.

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I nt e r me di at ep r e c i s i on ,f or me r l yc a l l e dr ug g e dn e s s , i st hev a r i a t i ono bs e r v e dwhe na na s s a yi s pe r f o r me db yd i ffe r e ntpe o pl eo ndi ffe r e nti n s t r u me nt so ndi ffe r e ntd a y si nt h es a mel a b .Ea c h a na l y s i smi g hti nc o r po r a t ef r e s hr e a g e nt sa n ddi ffe r e ntc h r o ma t o gr a ph yc o l u mn s .

o

I nt e r l ab or at o r ypr e c i s i on ,a l s oc a l l e dr e p r o duc i b i l i t y , i st hemos tg e ne r a lme a s u r eo f r e pr od uc i b i l i t yo bs e r v e dwhe na l i q uot soft h es a mes a mpl ea r ea na l y z e db ydi ffe r e ntp e op l ei n di ffe r e ntl a b or a t or i e s .I n t e r l a b or a t o r ypr e c i s i o nbe c ome sp oor e ra st hel e v e lo fa n a l yt ede c r e a s e s ( Bo x5 2) .

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Ra ngei st hec o nc e nt r a t i o ni nt e r v a lo v e rwhi c hl i ne a r i t y ,a c c u r a c y , a n dpr e c i s i ona r ea l la c c e pt a bl e .

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Li n e a rr a n g e :c on c e n t r a t i o nr a n g eo v e rwh i c hc a l i b r a t i o nc u r v ei sl i n e a r

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Dyna mi cr a n g e :c onc e nt r a t i onr a n g eo v e rwhi c ht h e r ei sme a s u r a bl er e s p ons e

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de t e c t i onl i mi t( a l s oc a l l e dt hel owe rl i mi tofd e t e c t i on )i st hes ma l l e s tq u a nt i t yofa na l y t et ha ti s “ s i gn i fic a n t l yd i ffe r e n t ”f r om t h ebl a nk .

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Thec o e ffic i e ntofv a r i a t i o ni st hes t a nd a r dde vi a t i o nd i v i d e db yt h eme a n ,u s ua l l ye x p r e s s e da sa pe r c e nt a g e :CV(%)= 1 00x s /x( b ar ). Thes ma l l e rt hec o e ffic i e ntofv a r i a t i on ,t hemor ep r e c i s ei sas e to f me a s u r e me nt s .

Detection limit. Distribution of measurements for a blank and a sample whose concentration is at the detection limit. The area of any region is proportional to the number of measurements in that region. Only �1% of measurements for a blank are expected to exceed the detection limit. However, 50% of measurements for a sample containing analyte at the detection limit will be below the detection limit. There is a 1% chance of concluding that a blank has analyte above the detection limit (false positive). If a sample contains analyte at the detection limit, there is a 50% chance of concluding that analyte is absent because its signal is below the detection limit (false negative). Curves are Student’s t distributions for 6 degrees of freedom and are broader than the corresponding Gaussian distributions.

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Ther e por t i ngl i mi ti st h ec o nc e n t r a t i o nbe l o wwhi c hr e gul a t i o nss a yt ha t ag i v e na na l y t ei sr e por t e da s “ no td e t e c t e d, ”whi c hdo e sn otme a nt h a ta n a l yt ei sn oto b s e r v e d .I tme a nst h a ta na l y t ei sbe l o wap r e s c r i b e d l e v e l

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Ro bus t ne s si st h ea bi l i t yo fa na na l y t i c a lme t h odt ob eu na ffe c t e db ys ma l l ,d e l i be r a t ec ha n g e si nop e r a t i n g pa r a me t e r s .Fore x a mpl e ,ac hr oma t o gr a p hi cme t h odi sr obu s ti fi tgi v e sa c c e pt a bl er e s ul t swhe ns ma l l c h a n g e sa r ema dei ns o l v e n tc o mpo s i t i on,pH,bu ffe rc onc e nt r a t i on ,t e mpe r a t ur e , i n j e c t i onv o l u me ,a nd de t e c t o rwa v e l e n gt h .

5.3 – Standard Addition -

I ns t anda r daddi t i o n, kno wnq ua n t i t i e sofa na l y t ea r ea dde dt ot heun kno wn .Fr o mt hei n c r e a s ei ns i gn a l , wede d uc eh o wmu c ha n a l yt ewa si nt heo r i g i n a lun kno wn. Th i sme t hodr e q ui r e sal i n e a rr e s p ons et o a na l y t e . Asi nt i t r a t i o ns ,h i g he rpr e c i s i o nc a nb ea c hi e v e dwh e ns t a n da r d sa r ea d de db yma s si n s t e a do f v ol ume .St a nd a r da d di t i oni se s pe c i a l l ya p pr op r i a t ewh e nt hes a mp l ec o mpos i t i oni sun kno wnorc o mp l e x a nda ffe c t st hea na l yt i c a ls i g na l .

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St a nda r da dd i t i o n:d e t e r mi nec o nc e nt r a t i onofun kno wnb ya d di n gakno wns t a n da r dc o nc e nt r a t i onofou r a na l y t et oou rs a mp l e .

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Mat r i xi se v e r y t hi n gi nt heun kno wn, ot he rt h a na n a l yt e( t hea na l yt e ’ se n v i r on me n t )

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Mat r i xe ffe c ti sac ha n g ei nt h ea na l y t i c a ls i gna lc a u s e db ya n y t h i n gi nt h es a mp l eo t he rt ha na na l yt e .

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The matrix affects the magnitude of the analytical signal. In standard addition, all samples are in the same matrix.

How much would we have to remove from the initial sample with no standard added to get the signa l down to zero - In graphical procedure for standard addition to a single solution(adding small volumes of concentrated standard to an unknown solution and measuring signals one at a time): the extrapolated value at y=0 (X-intercept) will give the initial concentration of unknown [X]i - In graphical procedure for multiple solutions with constant volume (Equ a lv ol ume sof unk no wna r epi pe t t e di n t os e v e r a lv ol u me t r i cfla s ks . I n c r e a s i n gv ol ume so fs t a n da r da r ea d de dt oe a c hfla s k a nde a c hi sdi l ut e dt ot hes amefina lv o l u me .Ev e r yfla s kc on t a i nst hes a me c on -c e nt r a t i onofun kno wna n ddi ffe r i n gc on c e n t r a t i o nso fs t a n da r d .Fo re a c h fla s k ,ame a s ur e me ntofa na l yt i c a ls i gn a l , I , i s t h e n m a d e ) : t h e x i n t e r c e p t S+X i st hefin alc onc e nt r a t i o nofu nkn o wn ,[ X f]

5.4 – Internal Standards - Internal standard: a known amount of a compound (not analyte) that is added to the unknown. Signal from the analyte is compared to the signal from the internal standard to find out how much analyte is present. I nt e r n a ls t a nda r d sa r ee s p e c i a l l yu s e f u lf o r a na l y s e si nwhi c ht heq ua nt i t yofs a mp l ea na l yz e d, ort hei ns t r u me ntr e s p ons e v a r i e ss l i gh t l yf r o mr unt or u n. o Ac a l i br a t i onc u r v ei son l ya c c u r a t eu nde rt hec o nd i t i o nsi twa sma de . o I nt e r n a ls t a nda r d sa r eus e di nc hr oma t o gr a ph yb e c a u s et h es ma l l q ua n t i t yo fs a mpl ei n j e c t e di n t ot hec hr oma t o gr a phi sn ot r e pr od uc i b l e . o I nt e r n a ls t a nda r d sa r ede s i r a b l ewhe ns a mp l el os sc a no c c u rd ur i n g s a mp l ep r e pa r a t i o ns t e p spr i o rt oa na l y s i s .I fakno wnq ua nt i t yo f s t a nd a r di sa dd e d...