Photo of Browns River at Mills River Park, Jericho VT
Photo by Jack Cameron How Much Stuff Is In There?: uantitative !nal"sis of Phos#hate In $atural %aters Objectives: . Learn how to erform a !"antitative sectrohotometric assay# $na%y&e 'ata "sing the statistica% an' grahing f"nctions of ()ce%# *etermine the %eve% of free hoshate in a on' water sam%e.
+ntro'"ction: ,cientists ask a %ot of !"estions. -e ask: .%hat kin& of stuff is there in this iece of the "niverse we are st"'ying/ +s that a ne"trino being emitte' from the s"n/ -hat como"n's are resent in 'rinking water/ $re there so'i"m ions in this softene' water/ +s there water on 0ars/ $re there anabo%ic steroi's in the "rine of major1%eag"e baseba%% %ayers/ -hat e%ements are resent/ +s merc"ry resent in this %ake erch/ +s this shiny n"gget go%' or iron/2 3his is qualitative analysis. Once yo" know what st"ff is resent4 the ne)t !"estion yo" want to ask is: .How 'uch stuff is there in this iece of the "niverse we are st"'ying/ 5ow many ne"trinos are being emitte' from the s"n/ 5ow m"ch so'i"m ion is resent in the softene' water/ 5ow m"ch hoshate is going into the %ake from the new fish hatchery/2 3his is quantitative analysis, from the Latin quantus?4 .5ow m"ch/2 +n this %ab yo" wi%% !"antitative%y 'etermine the amo"nt of hoshate in a water sam%e taken from a on' bor'ering the ,0C cam"s or the -inooski 6iver. ($ PH(SPH(R)S I$ %!T*R: Phoshor"s4 the e%ement4 is non1meta%%ic4 an' e)ists in three forms4 or a%%otroes: 3hese are re'4 white an' b%ack hoshor"s. (%ementa% hoshor"s e)hibits some rather interesting chemistry. 7or e)am%e4 when white hoshor"s is %ace' in the 'ark an' is e)ose' to air4 it g%ows green an' gives off white smoke. +n warm4 h"mi' air it wi%% sontaneo"s%y b"rst into f%ames. +ngestion of as %itt%e as 801100 mg of white hoshor"s may ca"se 'eath. 3hat being sai'4 hoshor"s1containing o+i&es4 phosphates4 9P ) O y n1 : are abso%"te%y necessary for %ife as we know it. $s water r"ns over an' thro"gh rocks it carries off sma%% amo"nts of hoshates. 3hese inorganic hoshates 9hoshates not attache' to carbon1containing como"n's: are a %ant n"trient an' are taken in by %ants with water an' incororate' into organic hoshate como"n's. $nima%s obtain hoshor"s from hoshates in water an' %ant materia%. C%ean streams an' %akes have a hoshor"s concentration of aro)imate%y 0.02 "g;L 9arts er mi%%ion4 m:. Phoshates are essentia% for a%gae an' %ant growth4 an' at the 0.02 "g;L %eve%4 they are the limiting reagent for %ant growth. $'' more Page 2 of 11 v 2004 nitrogen of otassi"m 9a%so essentia% for %ant growth: an' %ant an' a%gae %eve%s 'on<t change. $'' more hoshate4 an' yo" en' " with a %ot more green st"ff in the %ake. $''ing hoshates to %akes an' stream increases e"trohication = that is4 e)cessive growth of a%gae an' %ants4 res"%ting in the 'e%etion of o)ygen in the water. ("trohication can %ea' to fish ki%%s4 'egra'ation of habitat4 an' %oss of secies. Large mats of a%gae can form an' sometimes com%ete%y cover sma%% %akes or sha%%ow bays. ,o"rces of e)cess hoshate inc%"'e h"man sewage4 agric"%t"ra% r"n1off from cros4 sewage from anima% fee'%ots4 fish hatcheries4 the aer in'"stry4 vegetab%e an' fr"it rocessing4 chemica% an' ferti%i&er man"fact"ring4 an' 'etergents. Phoshor"s an' ,"sen'e' 0ateria% Phoshor"s is resent in streams an' %ake waters in severa% 'ifferent forms: orthohoshate4 PO 4 >1 # o%yhoshates ?P n O >n@1 9n@2:1 A an' metahoshates ?9PO > : n n1 A# an' organic hoshor"s. Orthohoshate is the most biochemica%%y avai%ab%e form of hoshor"s in nat"ra% waters4 an' microorganisms cata%y&e the hy'ro%ysis of con'ense' hoshates to orthohoshate. 5owever4 >0 to B0 ercent of the hoshor"s resent in many nat"ra% waters is bo"n' to organic materia%. P O O- O- NH C N C C C C N O NH2 N C C C O C H H H CH2 H H H -O P O O O- -O P O O- O- -O P O O O- P O O O- P O O- O- -O P O O- O- Phoshate ion4 .orthohoshate2 $ o%yhoshate P O P O P O O- -O O- O- O- O- -O P O O O- P O O O- P O O- O- -O P O O- O- > 5y'ro%ysis of a o%yhoshate to orthohoshate 5>O @ Page > of 11 v 2004 One may ana%y&e hoshor"s content in water severa% ways4 an' get very 'ifferent res"%ts. $ 3ota% Phoshor"s ana%ysis4 for e)am%e4 wi%% meas"re hoshate4 o%yhoshates4 metahoshates4 an' organic hoshates4 both 'isso%ve' in the water an' a'sorbe' onto s"sen'e' so%i's. 3his is a somewhat time1cons"ming roce'"re4 re!"iring reaction of the sam%es with strong aci's4 an' heating. +n this e)eriment4 we wi%% test on%y for so%"b%e orthohoshate4 that is4 PO 4 >1 'isso%ve' in the water sam%e. ,ee: 0erck +n'e)4 C th ('ition4 1CDB. 0. -in'ho%t&4 e'. 0erck an' Comany4 6ahway4 EJ4 Fro"n'water Contamination: ,o"rces4 Contro%4 an' Preventative 0eas"res. 1CGC. Chester *. 6ai%4 e'. 3echnomic P"b%ishing Co.4 +nc.4 Lancaster4 P$. htt:;;va.water."sgs.gov;chesbay;6+0P;g%ossary.htm%Hconcentration ,tan'ar' 0etho's 7or 3he ()amination4of -ater an' -astewater4 14 th e'. 1CD8. 6an'4 0.C.4 -PC74 Chair4 Joint ('itoria% Ioar'. P"b%ishe' by $P5$1$--$1-PC74 -ashington4 *C.
$ cyc%ic metahoshate Page 4 of 11 v 2004 P5O,P5$3( $E$LJ,+, P6O3OCOL: 3his metho' fo%%ows the rotoco% for orthohoshate ana%ysis "b%ishe' in: ,tan'ar' 0etho's 7or 3he ()amination4of -ater an' -astewater4 14 th e'. 1CD8. 6an'4 0.C.4 -PC74 Chair4 Joint ('itoria% Ioar'. P"b%ishe' by $P5$1$--$1-PC74 -ashington4 *C. Orthohoshates 9PO 4 >1 : are "se' as ferti%i&ers4 an' enter %akes an' streams by storm r"n1off an' me%ting snow. *O EO3 6(7(6(EC( 35+, KEL(,, JOK FO F(3 +3 $E* 6($* +3 9$E* JOK E((* EO3 *O 35$3L: +n this e)eriment yo" wi%%: 1. Obtain a water sam%e from the sma%% on' behin' the townho"ses4 or from a stream or river# 2. 3reat the on' water with 'eco%ori&ing charcoa%4 an' fi%ter it# >. 0ake " severa% so%"tions containing known amo"nts of so'i"m hy'rogen hoshate 9Ea 2 5PO 4 :# 4. Obtain a .M"a%ity Contro%2 hoshate so%"tion# 8. 6eact a%% sam%es with mo%yb'ate1vana'ate reagent an' obtain absorbance rea'ings at 400 nm. B. Constr"ct a stan'ar' c"rve of absorbance vs. ?PO 4 >1 A "sing 'ata from the known hoshate sam%es# D. *etermine ?PO 4 >1 A in yo"r on' water an' .M"a%ity Contro%2 sam%es. Jo" wi%% nee' the fo%%owing g%assware an' e!"iment: P5O,P5O6OK, ,(3 KP: ?er air "n%ess note' otherwiseA ,am%e bott%e4 aci' washe'4 2 'ram 9or scinti%%ation via%: 1 er st"'ent 1 L bott%e to make " 1 0 5C% @ stoer ?Kn'er sinks in %abA 1 (hr%enmeyer f%ask4 128m% 9for fi%tering into: 1 No%"metric 7%ask4 10.00 mL D No%"metric iet4 1.00 mL 2 orange tae on one of the two No%"metric iet4 2.00 mL 1 No%"metric iet4 >.00 mL 1 No%"metric iet4 8.00 mL > 6e' O b%"e tae on one of the > I%"e iet b"%b;han'%e 2 F%ass f"nne%4 %ong stemme' 2 7i%ter aer4 !"a%itative 9to fit above: 2 bo)es er riser -aste J"g 9.Phoshoro"s2: 1 in 5oo' 6ing ,tan' w; 1 ring 9to ho%' f"nne%: 1 ,at"%a 1 er charcoa% set " $n organic hoshate4 a'enosine ><1 monohoshate 8<1'ihoshate Page 8 of 11 v 2004 Jo" wi%% nee' the fo%%owing reagents: C5(0+C$L P6(P: 12 0 5C% G>m% er air of concentrate' 120 5CL 0o%yb'ate1vana'ate reagent 0.02 0 ammoni"m mo%yb'ate 9N+:;0.01 0 ammoni"m metavana'ate in 4.0 0 5C% P 10 mL er air: 1 set " er hoo': 2)128m% f%ask w; CC 10m% gra' cy%. O6$EF( ,tock: >B.D8"g;mL ?PO 4 A 20 m% er air 1 set " er hoo' 2) 280m% f%ask w; CC 10m% gra' cy%. 6(* M"a%ity Contro%: 14.D0 "g;m% 10 m% er air 1 er hoo' 2 ) 3a%% form beaker w;CC 10m% gra' cy%. *eco%ori&ing charcoa% 2 80m% beaker: Q f"%% w; a sat"%a P%ace on back bench on aer towe%s. Page B of 11 v 2004 ,3OCR6OO0 ,3$77 P6(P *+6(C3+OE,: 3O 0$R( KP 0OLJI*$3(1N$E$*$3( 6($F(E3: I$ TH* H((,------------
Page D of 11 v 2004 PR*7!B: $. (BT!I$ ! %!T*R S!MP7*2 F?ou will nee& aGout 1 '7 of water2 9ollect the water Hfill the vial co'#letel"-I in the clean, aci&J washe& flask #rovi&e&, seal it, an& laGel it with "our na'e, the &ate an& ti'e of collection, an& the site of collection2 ,escriGe the e+act ti'e, location, an& con&itions un&er which "ou collecte& "our sa'#le2 HThis is #art of "our Kra&e-I e2K2 LThe water sa'#le I oGtaine& was fro' the %inooski River &elta at the BurlinKton Bike Path Bic"cle <err" lan&inK2 ,ue to severe &rouKht Honl" C2C: L of rain in the last 0C &a"s, accor&inK to www2$(!!2Kov2 M BrinK "our sa'#le an& collection &ata to laG2 I2 <ilterinK an& ,ecoloriNinK P6OC(*K6(: +f yo"r water sam%e has co%or to it4 a'' a sma%% sat"%a1ti f"%% of activate' charcoa% to it = 35+ER 3+EJ $0OKE3 9abo"t 20 mg. *O EO3 -(+F5 OE 35( I$L$EC(:. ,wir% it aro"n'4 %et it sit for 8 min. an' fi%ter the sam%e thro"gh a moistene' fi%ter to remove the carbon. II2 The Phos#hate !ssa" To Ge &one G" T!: Make a stock solution of #hos#hate accor&inK to the #roce&ure on #aKe 12 Recor& the e+act concentration on the container2 To Ge &one G" the stu&ent: R*9(R, the e+act concentration of the #hos#hate stan&ar& solution in "our noteGook2 Before "ou co'e to laG: 9alculate the volu'e of ./ M H9l "ou will nee& to &ilute to . 7 to 'ake a . M solution2 !9I, %!SH !77 ?()R 57!SS%!R* %ITH . M H97, an& then rinse with &istille& water2 $ow that "ou have sOueak" clean &ishes: MakinK caliGration stan&ar&s P solutions for anal"sis: .2 (Gtain : .C2CC '7 volu'etric flasks2 /2 7aGel the volu'etric flasks QCQ throuKh Q4Q2 02 !&& 1 '7 of &eioniNe& water to the QCQ flask22 =2 To flask Q.Q carefull" #i#et in .2CC '7 of the stock #hos#hate solution usinK a volu'etric #i#et2 12 <ollowinK the chart on the followinK #aKe, a&& /2CC '7 of the stock #hos#hate to flask /, 02CC '7 of the stock #hos#hate to flask 0, an& so on2 To flask 1 a&& 12CC '7 of the ualit" 9ontrol solution2 To flask 4 a&& 12CC '7 of "our water sa'#le2 42 !&& .2CC '7 of the vana&ateJ'ol"G&ate reaKent to each flask, P &ilute to the 'ark on the neck with &eioniNe& water2 The Gotto' of the 'eniscus shoul& Ge restinK on the 'ark2 Re'e'Ger to thorouKhl" 'i+ each flask2 Page G of 11 v 2004 :2 7et the "ellow color &evelo# for .C 'inutes or 'ore2 Rea& an& recor& the aGsorGance of each sa'#le at a wavelenKth of =CC nM2 Page C of 11 v 2004 S*T )P TH* <(77(%I$5 9H!RT I$ *;9*7 B*<(R* ?() 9(M* T( 7!B2 PRI$T (<< ! H!R, 9(P? !$, BRI$5 IT %ITH ?()2 $ssay H mL stock PO 4 >1 7ina% vo%"me 9mL: ?stock PO 4 >1 A "g;mL $ 4G0 0 0 10.00 1 1.00 10.00 2 2.00 10.00 > >.00 10.00 4 8.00 10.00 8 !"a%ity contro% 8.00 10.00 B water sam%e 8.00 10.00 7or best res"%ts4 ca%ibration stan'ar's sho"%' be reare' fresh each time a batch of sam%es is ana%y&e'. Page 10 of 11 v 2004 9alculations: Ca%c"%ate the ?PO 4 >1 A in each of yo"r stan'ar's. Fenerate a stan'ar' c"rve of absortion vs. concentration for yo"r stan'ar's. +n'icate what wave%ength yo" "se'. Ca%c"%ate the best fit straight %ine for yo"r 'ata. ,ee chater 4 in the ()ce% 0an"a% to fin' o"t how to 'o this4 at htt:;;aca'emics.smcvt.e'";chemistry;C5(0 10>;weekT2.htm Ksing yo"r stan'ar' c"rve best fit %ine4 ca%c"%ate the ?PO 4 >1 A in yo"r water sam%e. $%so ca%c"%ate ?PO 4 >1 A in yo"r !"a%ity contro%. Ca%c"%ate a U abso%"te error between what yo" REO- yo"r !"a%ity contro% ?PO 4 >1 A content is an' yo"r e)erimenta% va%"e. -$,5 O 6(3K6E $LL FL$,,-$6(L
3he e!"ation of the best fit straight %ine is: J V m) @ b4 where J V absorbance4 m V s%oe4 ) V ?PO 4 1> A4 an' b V J1intercet. 3h"s4 if yo"r water sam%e has an absorbance of 0.0181 at 400 nm4 the ?PO 4 >1 A is: 0.0181 V 4.111 ) 10 1B 9?PO 4 >1 A: @ 2.B> ) 10 1>
?PO 4 >1 A V 90.0181 = 2.B> ) 10 1> :; 4.111 ) 10 1B V >0>0 "g;L ,%oe of best fit straight %ineV 4.111 ) 10 1B J =int V 2.B> ) 10 1> 400 nm Page 11 of 11 v 2004 Jo" origina%%y "se' 8.00 mL of water 90.008 L: an' 'i%"te' it to 10.00 mL 90.0100 L:. 3h"s4 the origina% ?PO 4 >1 A in yo"r sam%e was: 9>0>0 "g;L :90.0100 L;0.008 L: V B0B0 "g;L Jo" can "se this as a s"ggeste' format to set " yo"r 'ata in ()ce%WEotice that this 'ata is 'ifferent than yo"rsL $LL -$,3( 76O0 35+, (SP(6+0(E3 FO(, +E 35( JKF L$I(LL(* .P5O,P5$3( -$,3(2 +E 35( 5OO*.