Page 1 of 11 v 2004

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:;;[Link]."[Link];chesbay;6+0P;g%[Link]%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 [Link]"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((,------------

1. *+,,OLN( 28.0 F O7 $00OE+K0 0OLJI*$3( 9N+: ?9E54:B0ODO24
.
452O# M% ./01234 56M(7A +E 400
0L O7 *+,3+LL(* -$3(6 +E $E $C+*1-$,5(* 800 0L (56L(E0(J(6 7L$,R.
2. *+,,OLN( 1.28 F O7 $00OE+K0 0(3$N$E$*$3( ?E54NO>4 M% ..4283 56M(7A IJ 5($3+EF 3O
IO+L+EF +E >00 0L *+,3+LL(* -$3(64 +E $E $C+*1-$,5(* 1.00 L NOLK0(36+C 7L$,R. COOL 3O 63
>. 35(E4 +E +EC6(0(E3, -+35 ,3+66+EF C$6(7KLLJ $** >>0 0L O7 COEC(E36$3(* 5CL 3O 35(
$ION(
,OLK3+OE 9H2:. COOL. PL$C( ,(3 KP +E $ IKCR(3 KE*(6 5OO* -5(E $**+EF 5CL.
4. POK6 35( $00OE+K0 0OLJI*$3( ?N+A ,OLK3+OE +E3O 35( $00OE+K0 N$E$*$3( ,OLK3+OE4 $E*
*+LK3( 3O 1.00 L.
8. 9(7(R 9(,*: (R!$5*

PP
3O 0$R( KP ,3OCR P5OP,5$3( ,OLK3+OE: I$ TH* H((,-

1. *+,,OLN( 0.08>4 F 9EO3( (S$C3 0$,, K,(*LLLL: O7 ,O*+K0 5J*6OF(E P5O,P5$3(
?E$25PO4.520:
0-: .0:288 56M(7A +E C00 0L O7 *+,3+LL(* -$3(6 +E $E $C+*1-$,5(* 1.00 L NOLK0(36+C 7L$,R4
$E*
35(E *+LK3( 3O 1.00 L. 6(CO6* *;!9T 9($9*$TR!TI($ OE 7L$,RL
2. 9(7(R 9(,*: R*,
02 M!TH: M!SS (< S(7I,6 M% (< S(7I, ; 8=,8:56M(7 >M% (< PH(SPH!T* ($7?@ABBBBBP(=
567IT*R
BBBBBBBB567IT*R ; .C
4
)565 ; .76.CCCM7 =______UG/ML(LABEL A!"

DDD T( M!E* )!7IT? 9($TR(7 S(7)TI($: I$ TH* H((,-
1. *+,,OLN( .0214 F6$0, 9EO3( (S$C3 0$,,LLLL: O7 ,O*+K0 5J*6OF(E P5O,P5$3(
?E$25PO4.520: 0-: .0:288 56M(7A +E C00 0L O7 *+,3+LL(* -$3(6 +E $E $C+*1
-$,5(*
1.00 L NOLK0(36+C 7L$,R4 $E* 35(E *+LK3( 3O 1.00 L. 6(CO6* (S$C3
COEC(E36$3+OE OE
7L$,RL
2. 9(7(R 9(,*: B7)*
02 M!TH: S!M* !S !B(V*

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'[Link].e'";chemistry;C5(0 10>;[Link] 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*.