Intergovernmental
Oceanographic
Commission
Manuals and guides
12
CHEMICAL METHODS
FOR USE IN MARINE
ENVIRONMENTAL MONITORING
1983 Unesco
TABLE OF CONTENTS
Pages
..................................................
Preface
2.
.......................
Determination of Dissolved Oxygen and Oxygen Saturation ....
3.
Determination of hydrogen sulphide
4.
Determination of dissolved inorganiz phosphate
Potentiometric determination of pH
5.
6.
7.
........................
............
Determination of total phosphorus .........................
Determination of reactive silicate ........................
i
1
6
11
16
23
23
Direct determination of ammonia with the indophenol blue
method ...................................................
29
a.
Determination of nitrite
...................................
36
9.
Determination of nitrate
...................................
40
10.
Simultaneous persulphate oxidation of phosphorus and
nitrogen containing compounds in water ....................
48
Figure
1
................................................
5
Figurz
2
................................................
19
Figure
3
................................................
43
................................................
53
References
i
This Manual was prepared by the Intergovernmental Oceanographic
Commission ( I K ) of Unesco, with the collaboration of a consultant,
Mr. Stig R. Carlberg, of the Institute o f Hydrographic Research, National
Swedish Board of Fisheries, Gothenburg, at the request of the United Nations
Environment Programme (UNEP), for its Co-ordinated Mediterranean Pollution
Monitoring and Research Programme (MED POL), whose support in this regard
is gratefully acknowledged.
The Manual contains descriptions of chemical methods for analysis
of parameters of general interest in programmes for chemical oceanography,
as well as for marine environmental mcnitoring. It is intended for use
by marine science institutes that are or will become involved in such
activities, particularly within the Regional Seas Programme of UNEP.
For preparation of practical manuals a supply of good background
material is absolutely essential. In this regard, the use of three earlier
publications (see Carlberg 1972, FAO 1975 and Grasshoff 1976 in the list
of references) is gratefully acknowledged.
The methods presented in this report were produced for the purpose
of serving as reference methods for work performed under M D POL. However,
since they have much wider applicability than for just one regional area,
they are published for the use of interested marine scientists throughout
the world.
1 . POTENTIOMFTRIC DFTFRMINATION OF pH
1.1.
SCOPE AND FIELD OF APPLICATION
The determination o f pH d e s c r i b e d h e r e i n i s performed e l e c t r o m e t r i c a l l y , using a g l a s s e l e c t r o d e , w i t h c a l o m e l e l e c t r o d e a s
reference. F o r the d e t e r m i n a t i o n o f pH w i t h t h e h i g h e s t p r e c i s i o n a n d
accuracy, which represents normal practice i n oceanography, the full
d e t a i l s of t h i s methods should be f o l l o w e d . I f the d e m a n d s a r e l e s s
stringent (precision and a c c u r a c y of 5 0.1 p H unit o r l e s s ) a
simplified p r o c e d u r e , o m i t t i n g the c l o s e d m e a s u r i n g c e l l a n d t h e
constant temperature b a t h , may be a p p l i e d . T h e c a l i b r a t i o n p r o c e d u r e
is , however, generally applicable.
1.2. PRINCIPLE
pH is defined a s t h e n e g a t i v e l o g a r i t h m o f t h e h y d r o g e n i o n
activity (aH+) and t h u s pH
-log aH+ is a direct measure o f acidity
a c c o r d i n g t o the Brijnsted d e f i n i t i o n of a c i d s a s s u b s t a n c e s t h a t
are a b l e to donate hydrogen i o n s , and bases a s s u b s t a n c e s that a r e
a b l e t o c o m b i n e with hydrogen i o n s : a c i d s ----base + h y d r o g e n
7----ion.
=
The principle for the p o t e n t i o m e t r i c d e t e r m i n a t i o n s i s t h a t
the potential of the e l e c t r o d e c h a i n c o n s i s t i n g o f a n i n d i c a t o r
electrode sensitive t o the h y d r o g e n i o n a c t i v i t y a c c o r d i n g t o t h e
Nernst e q u a t i o n - and a pH-independent r e f e r e n c e e l e c t r o d e , both
immersed i n t h e test s o l u t i o n , i s measured by m e a n s o f a s u i t a b l e
galvanometer (pH meter). T h e m e a s u r e d p o t e n t i a l i s t h e n c o m p a r e d
w i t h the electrode c h a i n potential o b t a i n e d w h e n a buffer s o l u t i o n o f
k n o w n pH i s used as test s o l u t i o n . T h e a c t u a l pH v a l u e i s found
u s i n g the formula for " O p e r a t i o n a l pH" :
pH(X)
=
pH(S)
+
,%(X)
-
E(SVT
/R'T
where :
=
pH(X) and pH(S)
pH o f test s o l u t i o n and b u f f e r s o l u t i o n respectively
E(X) and E(S) = electrode c h a i n p o t e n t i a l i n t e s t s o l u t i o n a n d b u f f e r
respectively. R'T
t h e Nernst f a c t o r
(R'
a constant, T
the a b s o l u t e temperature).
=
=
A correctly c a l i b r a t e d pH m e t e r a u t o m a t i c a l l y c o n v e r t s the
measured potential to give direct r e a d i n g i n pH u n i t s .
1.3. REAGENTS
As pH values a r e t e m p e r a t u r e d e p e n d e n t , t h e pH v a l u e s o f the
following buffer s o l u t i o n s a r e g i v e n i n T a b l e I. For a proper c a l i b r a t i o n a t l e a s t two o f the s o l u t i o n s a r e r e q u i r e d . T h e c h o i c e of s o l u t i o n s is governed by the pH r a n g e o f t h e s a m p l e s .
- 2 -
1.3.1. Bates 1:l buffer.
P o t a s s i u m d i h y d r o g e n phosphate, K H 2 P 0 4 ;
3 . 3 9 g and d i s o d i u m h y d r o g e n p h o s p h a t e , Na2HP04, 3 - 5 3 g are dissolved
i n d i s t i l l e d w a t e r f r e e o f c a r b o n a t e s and diluted t o 1 000 ml.
P o t a s s i u m dihydrogen p h o s p h a t e , KH2P04;
1.3.2. Bates 1 : 3.5 buffer.
1 . 1 7 9 g and d i s o d i u m h y d r o g e n p h o s p h a t e , Na2HP04, 4 . 3 0 g are dissolved
i n d i s t i l l e d w a t e r f r e e o f c a r b o n a t e s and diluted t o 1 000 m l .
1.3.3. Palitzsch borax buffer
S o d i u m tetraborate, Na2B407. 10H20,
9 . 5 5 4 g , boric a c i d , H B O , 6 . 2 0 2 g , and s o d i u m c h l o r i d e , N a C 1 ,
1.462 g a r e d i s s o l v e d ?n J i s t i l l e d w a t e r free o f c a r b o n a t e s a n d
d i l u t e d t o 1 000 m l .
1.3.4. Bates 0.01 M borax-___
buffer.
S o d i u m t e t r a b o r a t e , Na2B407. 10H 0 ,
3 . 8 1 4 g i s dissolved in distilled w a t e r free o f carbonates a n d diluzed
t o 1 000 ml.
T h e sodium and potassium s a l t s should be dried before weighing.
It i s a l w a y s a d v i s a b l e t o k e e p a s u p p l y o f these s a l t s in a desiccator
c o n t a i n i n g silica g e l ; t h u s , t i m e - c o n s u m i n g drying i n an oven i s avoided.
S o m e a u t h o r s r e c o m m e n d r e c r y s t a l l i z a t i o n o f borax b e f o r e use. This
s h o u l d be done from w a t e r below 5OOC. T o prevent partial dehydration
o f b o r a x , i t s h o u l d be k e p t o v e r a saturated solution o f s u g a r or s o d i u m
c h l o r i d e o r s l i g h t l y m o i s t s o d i u m b r o m i d e , i n a closed container. he
boric acid c o u l d be k e p t i n t h e s a m e manner.
D i s t i l l e d w a t e r f r e e o f c a r b o n a t e s i s prepared by boiling
d i s t i l l e d w a t e r f o r a w h i l e a n d , p r e f e r a b l y , passing nitrogen gas
t h r o u g h d u r i n g t h e c o l i n g . T h e water should then i m m e d i a t e l y be used
f o r the p r e p a r a t i o n o f s o l u t i o n s .
Table I
------Standard p H v a l u e s o f t h e buffer solutions
a~ For P a l i t z s c h buffer o n l y t w o v a l u e s a r e given because a t other
t e m p e r a t u r e s t h e v a l u e s a r e u n c e r t a i n , a s found in e.g. ICES
i n t e r c a l i b r a t i o n exercises(Gieskes 1969).
- 3 1.4.
APPARATUS AND EQUIPMENT
1.4.1. Water b a t h , t h e r m o s t a t e d , w i t h pump for e x t e r n a l c i r c u l a t i o n
o f t h e thermosted water. S h o u l d be c a p a b l e o f h o l d i n g a s e l e c t e d
temperature t o within + 0.5'.
-
1.4.2. Measuring cell and h e a t e x c h a n g e r , o f g l a s s , c o n s t r u c t e d
a s i n Figure 1. These i t e m s a n d c o n n e c t i n g t u b e s s h o u l d be a s s m a l l
a s possible i n order t o r e a c h t h e r m a l e q u i l i b r i u m q u i c k l y , a n d
should not r e q u i r e too m u c h s a m p l e w a t e r for w a s h i n g and m e a s u r e m e n t .
The e l e c t r o d e s should be k e p t in s e a - s u r f a c e water between
measurements. Only d u r i n g l o n g e r s t o r a g e should d i s t i l l e d w a t e r
be u s e d .
1.4.3. Electrode (glass e l e c t r o d e a n d c a l o m e l r e f e r e n c e e l e c t r o d e )
any c o m m e r c i a l type o f g o o d q u a l i t y m a y be used. (W. I n g o l d A . G . ,
Z u r i c h , S w i t z e r l a n d , h a s a g l a s s e l e c t r o d e called L O T 201, w h i c h
may be a good choice because i t h a s a f a s t reponse a n d w o r k s
properly even a t low temperature).
1.4.4. pH m e t e r of good q u a l i t y . An i n s t r u m e n t w i t h a g a l v a n o m e t e r
should be provided w i t h s c a l e e x p a n s i o n f a c i l i t i e s . F o r s h i p b o a r d
use a n instrument with d i g i t a l d i s p l a y i s p r e f e r r e d , a s t h e
galvanometer needle i s i n f l u e n c e d by t h e m o v e m e n t s o f the s h i p .
The pH value must be displayed w i t h at l e a s t t w o d e c i m a l s .
1.5.
SAMPLING PROCEDURE
I t is important t o m i n i m i z e i n t e r a c t i o n b e t w e e n the s a m p l e
and t h e carbon dioxide in t h e a i r . H e n c e the s a m e c a r e and p r e c a u t i o n s
a s i n oxygen sampling a r e a d v o c a t e d . B e c a u s e o f t h i s s t a t e m e n t O2
a n a l y s e s probably should preceed pH. T h e s a m p l e i s d r a w n v i a a h o s e
from t h e w a t e r sampler ( i m m e d i a t e l y a f t e r the o x y g e n s a m p l e ) i n t o a
100 m l glass bottle with a g r o u n d s t o p p e r . T h e s a m p l e s h o u l d be
a n a l y s e d w i t h i n one h o u r a f t e r s a m p l i n g .
1.6.
ANALYTICAL PROCEDURE
For t h e exact h a n d l i n g o f the pH m e t e r , t h e i n s t r u c t i o n
manual supplied with t h e i n s t r u m e n t s h o u d be c o n s u l t e d .
The p H meter i s s t a n d a r d i z e d w i t h B a t e s 1:l p h o s p h a t e b u f f e r
( 1 . 3 . 1 . ) at t h e chosen c o n s t a n t t e m p e r a t u r e (20 or 25OC). B e c a u s e
most electrode pairs a r e not e n t i r e l y l i n e a r o v e r t h e e n t i r e pH
s c a l e , the s l o p e (mV/pH) o f t h e e l e c t r o d e s should be c o r r e c t e d .
I m m e r s e the electrodes i n B a t e s 0.01 M borax buffer ( 1 . 3 . 4 . )
(rinse well). I f a slope c o r r e c t i o n i s provided o n t h e i n s t r u m e n t ,
this is used t o get the p r o p e r pH r e a d i n g a c c o r d i n g t o t h e v a l u e s i n
T a b l e I. (In t h i s case t h e t e m p e r a t u r e c o m p e n s a t i o n i s set a t t h e
m e a s u r i n g temperature and not s h i f t e d a f t e r c a l i b r a t i o n o f t h e
instrument). If no s l o p e c o r r e c t i o n i s a v a i l a b l e , t h e t e m p e r a t u r e
compensator c a n b e used because i t w o r k s s i m i l a r l y . As a f i n a l
check B a t e s 1:3.5
(1.3.2), b u f f e r c a n be r e c o m m e n d e d .
- 4 I n s t e a d o f B a t e s 0.01 M borax buffer ( 1 . 3 . 4 1 , Palitzsch
borax buffer (1.3.3) c a n be used. T h e advantage o f this i s that
t h e pH v a l u e a t room t e m p e r a t u r e i s c l o s e to t h e maximun! pH value
o f sea water.
R i n s e t h e m e a s u r i n g c e l l w i t h s e a water several times
t o r e m o v e a l l t r a c e s o f buffer s o l u t i o n s . Keep t h e cell filled up
w i t h s u r f a c e s e a water.
1.6.2.
Measurement
T h e s a m p l e i s brought t h r o u g h t h e heat e x c h a n g e r into the
c e l l w i t h t h e aid of a s i p h o n , a r u b b e r tube connected t o the heat
e x c h a n g e r . R i n s e t h e c e l l t h r e e to four times w i t h the s a m p l e . F i l l
t h e c e l l c o m p l e t e l y a n d let t h e s a m p l e reach constant temperature.
(A s m a l l t h e r m o m e t e r s h o u l d extend i n t o the cell a s in F i g u r e I).
For h i g h p r e c i s i o n and a c c u r a c y , t h e temperature d i f f e r e n c e
between c a l i b r a t i o n a n d m e a s u r e m e n t s h o u l d not exceed 0 . 5 O C . Make
a n o t e o f t h e pH and t h e m e a s u r e m e n t t e m p e r a t u r e , and r e m o v e the
sample.
E x p e r i e n c e h a s s h o w n t h a t n o problem i s encountered when
g o i n g from a sample w i t h a high pH t o a sample w i t h a l o w pH (as i s
t h e case a t the halocline). H o w e v e r , a f t e r measurement o f a sample
w i t h a l o w p H , a d d i t i o n a l r i n s i n g m a y be required.
1.7.
CALCULATION AND EXPRESSION OF RESULTS
T h e measured pH v a l u e i s not t h e true pH v a l u e (in s i t u
o f the s a m p l e d water. T h e measured -or apparent- value h a s to
c o r r e c t e d for the t e m p e r a t u r e d i f f e r e n c e between measurement
c o n d i t i o n s a n d t h o s e a t t h e s a m p l e d e p t h in the water. If the
w a s o b t a i n e d from about 500 m or d e e p e r , a further correction
t h e d i f f e r e n c e i n h y d r o s t a t i c p r e s s u r e must be a p p l i e d .
value)
be
sample
for
T h e f o r m u l a f o r c a l c u l a t i o n o f t h e pH i n situ is t h e n :
-------
P H------in situ
where : t l
t2
=
=
=
PHmeasured
+
0.0118(t2
-
t,)
t e m p e r a t u r e __----in Situ
measurement t e m p e r a t u r e
T h i s f o r m u l a i s valid f o r work i n areas w i t h water depths
n o t e x c e e d i n g 500-1 000 m ( d e p e n d i n g o n the accuracy desired).
T h e complete correction formula i s :
1
1
4
6
i-
i
3
Figure
;:1
7
1 9
1 . Thennostated pH-cell and Lrat-c>xcharLgPr
7
3)
5)
electrodes
ttennome ter
heat exchange- (a bout :[rim b o
sample inlet
teflon valve ( 3 w a y - k e y )
6 ) s n m p l e overflow
7) t:irrnmostaterl hater inlet
8)) t h e n o c t a t e d water outlet
(9) to waste
- 6 T h e last t e r m B. Z i s a c o r r e c t i o n f a c t o r for t h e difference in
h y d r o s t a t i c p r e s s u r e between the s a m p l i n g depth and the laboratory
a t the s e a s u r f a c e . Z i s the s a m p l i n g depth in m e t r e s a n d the
8 - v a l u e i s o b t a i n e d from T a b l e 11.
1.8.
ESTIMATION OF PRECISION AND ACCURACY
It i s o b v i o u s that p r e c i s i o n and accuracy a r e dependent not
o n l y on the h a n d l i n g o f the s a m p l e s a n d the quality of buffer
s o l u t i o n s . The pH meter and i t s e l e c t r o d e s are important factors.
I t i s a b s o l u t e l y e s s e n t i a l t h a t these a r e of good quality and that
t h e y are m a i n t a i n e d and used a c c o r d i n g t o instructions given
by the m a n u f a c t u r e r . With good q u a l i t y equipment the reproducibility
can be a s good a s + 0 . 0 0 2 pH u n i t s and standard deviation between
d i f f e r e n t i n s t r u m e n t s may be a s l o w a s kO.005 pH units (Gieskes 1969).
For n o r m a l r o u t i n e work a p r e c i s i o n a n d accuracy around 0.02 pH
u n i t s m a y be e x p e c t e d with p r o p e r equipment.
2. . -DETERMINATION
OF DISSOLVED OXYGEN
-- AND OXYGEN- SATURATION
2.1. SC-OPE AND
FIELD OF APPLICATION
The m e t h o d d e s c r i b e d is the c o m m o n Winkler m e t h o d , modified
f o r sea w a t e r b y C a r r i t t and C a r p e n t e r .
2.2. PRINCIPLE
T h e method i s . b a s e d on the f o l l o w i n g principle : Manganous
i o n s are p r e c i p i t a t e d i n a l k a l i n e m e d i u m , forming manganous
h y d r o x i d e . T h i s i s o x i d i z e d b y the dissolved o x y g e n :
2 M r 1 ( 0 H ) ~+ 02- 2MnO(OHI2.The m a n g a n e s e hydroxide is dissolved with
a c i d and r e d u c e d by i o d t d e i o n s :
MnO(OH)2 + 4 H O + + 31---+Mn2+
+ I
'
3
3
+
7H20. T h e I - i o n s produced
3
are d e t e r m i n e d by t i t r a t i o n with thiosulphate i o n s :
2s2023
+
I3
.S40E-
+
31-
- 7 2.3.
REAGENTS
S t o r e in a plastic bottle. (MnS04.4H20, 670 g , or MnCL 2 in
equivalent a m o u n t may be u s e d . 1.
_____________
2.3.2. S o d i u m iodide, N a I . 600 g i s d i s s o l v e d by w a r m i n g i n t h e
s m a l l e s t possible volume o f d i s t i l l e d w a t e r .
________
d r o x i d e , N a O H . 320 g i s dissolved i n the s m a l l e s t
2.3.3. Sodium h y_-----possible v o l u m e of d i s t i l l e d water. G r e a t care s h o u l d be e x e r c i s e d
i n d o i n g t h i s a s the h y d r o x i d e p r o d u c e s c o n s i d e r a b l e heat w h e n
it d i s s o l v e s . A d d the h y d r o x i d e p e l l e t s , portion by p o r t i o n , i n t o
the w a t e r , s t i r r i n g c o n t i n u o u s l y .
____________
2.3.4. S o d i u m azide 9 NaN3.
10 g i s dissolved i n t h e s m a l l e s t
possible v o l u m e of distilled w a t e r . ( T h i s r e a g e n t i s used i n o r d e r
to destroy n i t r i t e in s t a g n a n t waters).
2.3.5.
Alkaline iodide r e a g --ent
________________---
T h e s e three s o d i u m r e a g e n t s ( 2 . 3 . 2 . - 2.3.4) a r e m i x e d
together and diluted t o one l i t r e w i t h distilled w a t e r . S t o r e i n a
plastic bottle. These r e a g e n t s a r e v e r y concentrated and g e n e r a l l y
will not b e c o m e clear u n t i l t h e y a r e d i l u t e d to the full v o l u m e .
If c r y s t a l s a r e f o r m e d , t h e c l e a r l i q u i d i s decanted i n t o
a n o t h e r vessel.
2.3.6.
Sulphuric a c i d , H2S04 ( 1
+
1)
solution about 9 moles/liter.
Concentrated a c i d s m i x e d w i t h t h e s a m e v o l u m e o f distilled w a t e r .
The a c i d i s slowly poured i n t o t h e w a t e r u n d e r c o n s t a n t m i x i n g and
cooling.
The reason for u s i n g s u l p h u r i c a c i d instead o f the c o m m o n l y
recommended concentrated phosphoric acid i s because d u r i n g f i e l d
work there i s a great r i s k o f C o n t a m i n a t i n g s a m p l e s for the p h o s p h a t e
a n a l y s i s when phosphoric a c i d i s used.
-
______
0-.5H20, 0.02 mo-les/liter.
Sodium thiosulphate , Na2S-2-3
2 3.7
__
25 g is dissolved in distilled water. 50 ml isobutylalcohol is added and the
solution is diluted to 5 litres.
~
_______________
2 . 3 . 8 . ' S t a r c h solution. 1 g s o l u b l e s t a r c h i s dissolved in 100 m l
distilled w a t e r by w a r m i n g u n t i l t h e s o l u t i o n b e c o m e s c l e a r .
T h e s o l u t i o n may be p r e s e r v e d by t h e a d d i t i o n o f 0 . l g s a l i c y l i c
acid (HOCOC6H4COOH).
__ _______________
2 . 3 . 9 . 'ihyodene indicator ( C a m p b e l l W i l l i a m s & C O , 14 S t . N e o t s R o a d ,
A b b o t s l e y , H u n t s , England). T h i s i n d i c a t o r i s better than t h e s t a r c h
s o l u t i o n , because the s a m p l e r e m a i n s c l e a r d u r i n g t h e whole t i t r a t i o n
and t h e end point is e a s i l y d e t e c t e d . W h e n using t h e s t a r c h s o l u t i o n
the s a m p l e mostly becomes t u r b i d . T h i s i n d i c a t o r a l s o can be used
a s a s o l u t i o n , prepared a c c o r d i n g to t h e i n s t r u c t i o n s on t h e c o n t a i n e r
- 8 2.4.
APPARATUS AND EQUIPMENT
2 . 4 . 1 . Calibrated b u r e t t e , f o r i n s t a n c e a 12 m l Derona burette
w i t h a n a u t o m a t i c z e r o a d j u s t e r , o r a motor-driven piston burette.
2 . 4 . 2 . Automatic s y r i n g e p i p e t t e s , p i s t o n pipette or s i m i l a r . As
t h e r e a g e n t s have t o be a d d e d s w i f t l y , common t y p e s o f pipettes
w i t h o r without s u c t i o n d e v i c e s s h o u l d be avoided.
2 . 4 . 3 . W i n k l e r b o t t l e s , v o l u m e about 100-130 m l , carefully
c a l i b r a t e d . T h e s e m a y be s u b s t i t u t e d by common 50 ml g l a s s bottles
w i t h g r o u n d s t o p p e r s , i f t h e s e s t o p p e r s a r e long enough t o extend
t h r o u g h t h e bottle n e c k a n d i n t o t h e body of t h e bottle, thus
e l i m i n a t i n g the r i s k o f t r a p p i n g a i r bubbles in t h e bottle.
2.5.
SAMPLING AND PRE-TREATMENT
T h e s a m p l e should i f possible be drawn from water s a m p l e r s
o f plastic or plastic-coated metal. T h i s should be done a s soon a s
possible a f t e r the h y d r o g r a p h i c cast i s brought o n board (sampling).
T h e o x y g e n s a m p l e s h o u l d be t h e first sample d r a w n from t h e water
s a m p l e r . For t h i s p u r p o s e a r u b b e r t u b i n g is fitted to t h e outlet o f
t h e s a m p l e r . A g l a s s t u b e a b o u t 10 cm l o n g is connected t o the
t u b i n g . T h e g l a s s t u b e i s i n s e r t e d t o t h e bottom O f the Winkler bottle
a n d at l e a s t one v o l u m e of s a m p l e w a t e r i s allowed to f l o w through
t h e bottle before f i l l i n g with t h e s a m p l e . The w a t e r should f l o w at
a m o d e r a t e s p e e d , a v o i d i n g a i r bubbles. (Air bubbles trapped in the
r u b b e r t u b i n g a r e r e m o v e d by squeezing). The t u b e i s then carefully
removed from the bottle w h i c h i s filled t o overflowing. With
a u t o m a t i c s y r i n g e p i p e t t e s t h e r e a g e n t s manganous sulphate ( 2 . 3 . 1 )
a n d a l k a l i n e - i o d i d e ( 2 . 3 . 5 ) a r e a d d e d simultaneously. T h e syringes
s h o u l d h a v e l o n g p l a s t i c t i p s , e x t e n d i n g deep i n t o the bottle.
1 ml of e a c h reagent is a d d e d t o t h e s a m p l e v o l u m e 100-130 ml.
( T o the s m a l l e r bottle 0.5 ml o f e a c h i s added). T h e w i n k l e r bottle
is careful-ly closed w i t h the s t o p p e r a v o i d i n g t h e trapping o f a i r
b u b b l e s , a n d the bottle is v i g o r o u s l y shaken w i t h a s n a p p i n g motion
o f t h e wrist.
2.6.
ANALYTICAL PROCEDURE
2.6.1.
---A n a l y --sis
W h e n the p r e c i p i t a t e h a s s e t t l e d to at l e a s t about half
w a y d o w n t h e b o t t l e , 1 ml s u l p h u r i c a c i d (2.3.6) i s added with a n
a u t o m a t i c s y r i n g e . The bottle i s closed a g a i n , a v o i d i n g a i r bubbles,
a n d s h a k e n until the p r e c i p i t a t e i s dissolved. N o w the bottle should
be stored i n a dark p l a c e and t i t r a t e d after a maximum t i m e o f o n e
h o u r . If the a n a l y s i s h a s to be d e l a y e d , the bottles should be stored
w i t h the o x y g e n precipitated. T h e a c i d should by n c meanste added
i f the bottles have t o be stored for a l o n g time. I f d i s s o l v e d ,
t h e i o d i n e will e v a p o r a t e , passing the glass s t o p p e r . If the s a m p l e s
h a v e t o be stored for s e v e r a l d a y s , or until the ship a r r i v e s at
t h e home p o r t , the W i n k l e r bottles should be stored in g l a s s or
plastic j a r s filled w i t h s a m p l e water. T h e bottles s h o u l d be
c o m p l e t e l y s u b m e r g e d , a n d the j a r s c l o s e d ( i t is a good i d e a t o
s t o r e the bottles u p s i d e - d o w n in the jars).
- 9 The dissolved s a m p l e i s q u a n t i t a t i v e l y t r a n s f e r r e d i n t o a
wide-necked 300 m l E r l e n m e y e r f l a s k a n d titrated w i t h s t a n d a r d i z e d
thiosulphate solution ( 2 . 3 . 7 . ) , u n t i l a very pale s t r a w c o l o u r
remains. The titration must be c a r r i e d out a t a n e v e n speed.
Three drops o f starch s o l u t i o n (2.3.8.)
freshly prepared
(or Thyodene indicator ( 2 . 3 . 9 ) are a d d e d a n d the t i t r a t i o n i s
continued u n t i l t h e s o l u t i o n i s c o l o u r l e s s . If the t h i o s u l p h a t e i s
added too s l o w l y at the e q u i v a l e n t p o i n t , t h e r e w i l l a l w a y s be
a s l i g h t c o l o u r in the s a m p l e due to o x i d a t i o n .
-
2.6.2.
-
Standardization o f the t h i o s u l p _____________
hate solution.
___-______________________
The exact titre o f t h i s s o l u t i o n c a n be d e t e r m i n e d by
t i t r a t i o n a g a i n s iodate i o n s in a c i d s o l u t i o n :
IO; + 51- + 6 H O+
3
U
21- + 9 H 2 0
3
T h u s 1 mole i o d a t e is e q u i v a l e n t t o 6 m o l e s t h i o s u l p h a t e .
Analytical g r a d e potassium i o d a t e (KIO
molecular weight
2 1 4 . 0 4 ) , i s dried at 18OOC to c o n s t a n t w e i g h t ? ’ E x a c t l y 1.1891 g
KIO i s dissolved in d i s t i l l e d w a t e r a n d diluted t o 1 000 rnl.
5 m? o f this 0.00555 moles/litre s o l u t i o n i s added t o a g l a s s stoppered Erlenmeyer f l a s k (250 m l ) c o n t a i n i n g 40 m l d i s t i l l e d w a t e r ,
1 ml o f the sulphuric a c i d ( 2 . 3 . 6 ) s o l u t i o n , 1 ml m a n g a n o u s a n d
1 m l sodium reagent. After five m i n u t e s t h e solution i s t i t r a t e d
with the thiosulphate s o l u t i o n a s described for the s a m p l e s . T h e
r e a g e n t blank i s obtained by o m i t t i n g the a d d i t i o n of i o d a t e a n d t h e n
repeating the procedure. The a d d i t i o n of s t a r c h s h o u l d g i v e n o ,
or o n l y a f a i n t , blue c o l o u r .
M
=
the c o n c e n t r a t i o n o f the t h i o s u l p h a t e s o l u t i o n , moles/litre
a
rnl KIO
y
the c o n c e n t r a t i o n o f the KIO
b
=
3
solution
3
s o l u t i o n , moles/litre
ml thiosulphate s o l u t i o n c o n s u m e d , c o r r e c t e d f o r blank
2.7.
10
-
C a l c u l a t i o n and R e p o r t i n g
__________-________-____
o f Results
___c-__-___
T o c a l c u l a t e t h e c o n c e n t r a t i o n o f dissolved oxygen , use
t h e f o r m u l a below.
a
v o l u m e o f t h e t h i o s u l p h a t e consumed i n m l
M
c o n c e n t r a t i o n o f t h e t h i o s u l p h a t e s o l u t i o n , moles/litre
V
=
then
v o l u m e of t h e W i n k l e r b o t t l e i n ml
O2
ml/l
=
a.M.22393
4 (V
fo:.
-
2)
the c a l c u l a t i o n s i t m a y be p r a c t i c a l t o calculate the term
a c o n s t a n t f a c t o r ( w h i c h i s close to 112 for a s o l u t i o n
4
t h a t c o n t a i n s 0,02 moles/litre
_--___M.22393 as
If t h e s m a l l s a m p l e b o t t l e (50-6Onl)is used the v o l u m e o f
e a c h a d d e d r e a g e n t is 0 . 5 m l . I n t h i s c a s e the ( V - 2 ) in t h e formula
i s r e p l a c e d by ( V - 1 ) .
At a n y g i v e n t e m p e r a t u r e a n d salinity a w a t e r is s a t u r a t e d
w i t h o x y g e n w h e n it h a s d i s s o l v e d a c e r t a i n , specified quantity.
T h i s q u a n t i t y i s referred t o a s t h e s a t u r a t i o n c o n c e n t r a t i o n o r
100 percent s a t u r a t i o n . T h u s , a n y o x y g e n concentration f o u n d by
a n a l y s i s c a n be expressed a s a p e r c e n t a g e o f the s a t u r a t i o n value
i n s t e a d o f ml/1. T h e s a t u r a t i o n c o n c e n t r a t i o n o f t h e water i s obtained
f r o m a s t a n d a r d t a b l e w h e n t h e t e m p e r a t u r e and s a l i n i t y o f the
s a m p l e a r e known.
T h e main s o u r c e f o r s y s t e m a t i c errors in a n a l y s i s o f
d i s s o l v e d o x y g e n i s i n t h e s a m p l i n g . I f t h i s is l z f t out o f the
d i s c u s s i o n t h e i n d i v i d u a l r e p r o d u c i b i l i t y + can be
0.02 ml/litre
for o x y g e n c o n t e n t s b e l o w 2 m l / l i t r e a n d -0.04 ml/litre a t higher
c o n c e n t r a t i o n s . T n t e r c a l i b r a t i o n e x e r c i s e s between five different
l a b o r a t o r i e s , e a c h u s i n g i t s o w n m o d i f i c a t i o n of t h e m e t h o d ,
d e m o n s t r a t e d a s t a n d a r d devia-tion o f + 0.03 ml/litre (Grasshoff 1976).
-
-
2.9.
Remarks
-_-____
T h e c o n c e n t r a t i o n o f s o d i u m i o d i d e has t o be at l e a s t 600 g/l
in- t h e r e a g e n t , a c c o r d i n g to C a r r i t t a n d Carpenter. Potassium
i o d i d e m a y a s well be u s e d , but m a y ' b e more difficult to dissolve
i n t h e c o n c e n t r a t i o n used.
P o t a s s i u m hydrogen i o d a t e , KH(I0
may a s well be used
i n e q u i v a l e n t c o n c e n t r a t i o n a s s t a n d a r d 2iZl.e substance.
I t i s v e r y i m p o r t a n t t h a t the entire s a m p l e volume i s t i t r a t e d .
D u e t o t h e l o s s e s o f i o d i n e , it i s impossible to t a k e out a repres e n t a t i v e a l i q u o t for t h e t i t r a t i o n a s described i n some procedures.
3.
11
-
DETERMINATION OF HYDROGEPJ SUL,PHIDE
3.1.
SCOPE AND FIELD QE APPI.ICATION
S u l p h i d e is found in a n o x i c w a t e r s , w h e r e it i s formed by
microbiological reduction o f s u l p h a t e i o n s . T h e method d e s c r i b e d h e r e ,
depending on the formation o f m e t h y l e n e blue from d i m e t h y l p - p h e n y lene d i a m i n e , i s a s i m p l e a p p l i c a t i o n o f a w e l l e s t a b l i s h e d
colorimetric method for s u l p h i d e . T h e method i s a s n e a r l y s e n s i t i v e
a s i s theoretically p o s s i b l e , and i s f o r d i r e c t d e t e r m i n a t i o n
applicable t o concentrations up t o a b o u t 100 moles/liter. At h i g h e r
c o n c e n t r a t i o n s of hydrogen s u l p h i d e t h e s a m p l e has t o be d i l u t e d
with o x y g e n - f r e e distilled water prior t o t h e a n a l y s i s .
3.2.
PRINCIPLE
The method is based o n the f o l l o w i n g principle : the a c i d i f i e d
s a m p l e i s a l l o w e d to react w i t h d i m e t h y l p-phenylene d i a m i n e , w i t h
ferric ions a s catalyst. A c o m p l e x o x i d a t i o n a n d s u b s t i t u t i o n t a k e s
p l a c e , r e s u l t i n g in t h e q u a n t i t a t i v z i n c o r p o r a t i o n o f a n y s u l p h i d e sulphur present into a h e t e r o c y c l i c d y e called m e t h y l e n e blue. T h e
absorption o f light by the s a m p l e i s m e a s u r e d before or a f t e r
dilution in 1 , 5 or 10 cm c e l l .
3.3.
REAGENTS
3.3. 1 .
___________________
N , N - d i m e t h y l p-phenylene
_____
di
_a
_m_i_
n_
e _d_i_h_
yd
_r
_o_c_
h_
l o_r-i-de _ _ _ _ _ vL _ ~ ~ ~ .
1 g i s d i s s o l v e d i n 500ml 6M hydrochloric.
(CH
. 3)2 NC6Hq.NH72HC1( 1 , 4 ) .
This acid may be prepared by diluting concentrated HCI (37 perce-t,
1.19)
with an equal amount of distilled water.
3.3.2. _______________L_El_.
F e r r i c chloride
a 7 FeCl . 8 g is dissolved in hydrochloric
acid (6 moles/liter) (prepared a s a a o v e ) to 500 m l .
3-3.3.
__O x y g_______________________
e n - free di st i 1 led water
A s u i t a b l e v o l u m e o f d i s t i l l e d w a t e r i s boiled f o r 3 0 m i n u t e s
Nitrogen g a s may be bubbled t h r o u g h t h e w a t e r d u r i n g t h e b o i l i n g . As
the water c o o l s down t o room t e m p e r a t u r e , n i t r o g e n g a s must be
continually bubbled t h r o u g h t h e w a t e r . T h i s i s c o n t i n u e d a s l o n g a s
water i s needed for t h e c a l i b r a t i o n .
3.3.4.
S u l p h i d e stock s o l u t i o n .
_______________________
( A b o u t 3 . 1 2 pmoles/mlS2-). . A s o l u t i o n o f s o d i u m s u l p h i d e i s
prepared w i t h oxygen-free distilled w a t e r . C r y s t a l s of Na2S.9H 0
2
p.a. a r e q u i c k l y washed w i t h distilled water by s q u i r t i n g from a
w a s h i n g bottle. The c r y s t a l s a r e dried w i t h f i l t e r p a p e r and p l a c e d
in a p r e - w e i g h e d , g l a s s s t o p p e r e d , w e i g h i n g g l a s s . 0.750 g i s w e i g h e d
on a n a n a l y t i c a l balance a n d dissolved i n o x y g e n - f r e e d i s t i l l e d
w a t e r (added with aid o f a s i p h o n ) t o 1 000 rnl i n a v o l u m e t r i c f l a s k .
-
12
2
--_
S u l p ___-______h i d e w o r k i n g______-__
s o l u t i o n (about 0,156 !Jmoles/ml S 7
3.3.5.
S i p h o n a s u i t a b l e v o l u m e o f oxygen-free distilled water i n t o
a 5 0 0 ml v o l u m e t r i c f l a s k . I n t o t h i s i s pipetted 25 ml of the sulphide
s t o c k s o l u t i o n a n d o x y g e n - f r e e distilled water i s added t o the
m a r k . F o r the g r e a t e s t a c c u r a c y , t h e concentration i s determined
by t i t r a t i o n a s described below. T h e solution i s stable f o r only
15-30 minutes.
T h e s a m e s o l u t i o n a s f o r the determination o f dissolved
o x y g e n . 5 g Na S 0 .5H20 p.a. i s dissolved in 1 000 ml distilled
w a t e r . Add 5 mf ?sJbutyl a l c o h o l before diluting t o the full volume
o f t h e v o l u m e t r i c flask.
3.3.7.
Potassium i o d a t e s o l u t i o n ( 0 . 0 0 5 5 5 moles/litre)
W e i g h o u t a c c u r a t e l y 1.1891 g KIO
analytical g r a d e (molecular
w e i g h t 2 1 4 . 0 4 ) , dried a t 18OOC f o r one h o a r . D i s s o l v e i n distilled
w a t e r a n d d i l u t e t o 1 000 m l . T h i s s o l u t i o n is t h e same a s used
f o r t h e o x y g e n d e t e r m i n a t i o n s a n d i t i s very s t a b l e .
T h e s a m e s o l u t i o n a s f o r t h e dissolved o x y g e n determination.
O n e v o l u m e o f a c i d i s c a r e f u l l y mixed w i t h one v o l u m e o f distilled
water under constant cooling and mixing.
3.3.9.
Starch solution
1 g per 100ml distilled water or Thyodene indicator as for
the dissolved o x y g e n d e t e r m i n a t i o n .
3.3.10.
Potassium iodide
K I , p.a. c r y s t a l s .
3.4.
APPARATUS AND EQUIPMENT
3 . 4 . 1 . Winkler b o t t l e s , volume calibrated o r volumetric flasks
of 100rnl volume.
3 . 4 . 2 . S p e c t r o p h o t o m e t e r o f filterphotometer w i t h filter at or
c l o s e t o 670 nm. I c m a n d 5cm o r 10cm c e l l s .
3.4.3.
Automatic s y r i n g e pipettes.
3.5.
13
-
SAMPLING
The s a m p l e s must be o b t a i n e d with plastic w a t e r s a m p l e r s . If
these are not a v a i l a b l e , m e t a l l i c o n e s may be used provided t h a t
the inner s u r f a c e s are p l a s t i c - c o a t e d .
3.6.
ANALYTICAL PROCEDURE
3.6. 1.
Calibration
----------_
3.6. 1 . 1 .
S t a n d a r d i z a t i o n o f the thiosul-phate _solution.
P r o c e e d a s described i n s e c t i o n 2.6.2. a b o v e .
3.6.1.2.
S t a n d a r d i z a t i o n o f th.e s u l p h i d e working s o l u t i o n .
T h i s is carried out w i t h i n m i n u t e s a f t e r p r e p a r a t i o n o f
the working solution ( 3 . 3 . 5 ) and a t the s a m e time a s the p r e p a r a t i o n
of t h e photometric s t a n d a r d s a m p l e s (3.6.1.3).
T a k e six E r l e n m e y e r f l a s k s with ground g l a s s s t o p p e r s a n d
add t o each about 10 ml distilled w a t e r a n d 1-2 g potassium i o d i d e
(3.3.10). P i p e t t e i n t o e a c h f l a s k 10.00 m l i o d a t e s o l u t i o n (3.3.7).
A d d 1.0 ml sulphuric a c i d (3.3.8) i n t o e a c h flask. I n t o t h r e e o f
the flasks pipette 5 0 ml o f the s u l p h i d e w o r k i n g s o l u t i o n a n d t o t h e
other three a d d about 50 m l d i s t i l l e d w a t e r . S e t a l l flasks a s i d e
i n a cool p l a c e whilst t h e c o l o r i m e t r i c s t a n d a r d i z a t i o n i s b e g u n
t h e n titrate the c o n t e n t s of the f l a s k s w i t h t h i o s u l p h a t e u s i n g
the starch indicator (3.3.9).
A
=
B
M
mean of the t i t r a t i o n s o f t h r e e s o l u t i o n s with n o
added s u l p h i d e , i n m l .
m e a n of the t i t r a t i o n s o f the t h r e e s o l u t i o n s c o n t a i n i n g
sulphide, in ml.
=
concentration o f the t h i o s u l p h a t e s o l u t i o n , moles/litre
(the individual t i t r a t i o n s c o n s t i t u t i n g a t r i p l i c a t e s h o u l d
agree t o within 0.05 ml).
3.6.1.3.
14
-
P h o t o m e t r i c s t a n d a r d samples.
From t h e w o r k i n g s o l u t i o n (3.3.5) the following standard
series is prepared.
Too 100 ml v o l u m e t r i c f l a s k s or volume calibrated Winkler
bottles the f o l l o w i n g v o l u m e s o f w o r k i n g solution are a d d e d by means
o f a pipette or burette ( T a b l e 1 1 1 ) :
T h e s e c o n c e n t r a t i o n s c o r r e s p o n d to a s u l p h i d e working
s o l u t i o n w i t h precisely 0 , 1 5 6 p m o l e s / m l . Thus t h e y have t o be
c o r r e c t e d a c c o r d i n g t o the t r u e v a l u e found b y titration.
I f W i n k l e r bottles are used the concentration values
g i v e n a b o v e have to be r e c a l c u l a t e d accordingly for the calibration
g r a p h described below.
With the a i d of a s i p h o n the bottles a r e filled up with
o x y g e n - f r e e d i s t i l l e d w a t e r t o the 100 ml mark, o r in c a s e of
c a l i b r a t e d Winkler b o t t l e s , to the neck. I n the l a t t e r c a s e a
c o r r e c t i o n for the v o l u m e has to be made f o r each bottle.
As s o o n a s a bottle i s f i l l e d , 1 m l o f each r e a g e n t i s
a d d e d w i t h a n a u t o m a t i c s y r i n g e p i p e t t e a n d the c o n t e n t s of the
bottle a r e mixed well. After 60 m i n u t e s , the s a m p l e s are measured
a g a i n s t the blank, a t 6 7 0 nm i n 1 cm and/or 5 cm c e l l s , a s suitable.
F r o m the r e s u l t s a c a l i b r a t i o n g r a p h for each o f the cell lengths
i s prepared on m i l l i m e t e r paper. T h e g r a p h should b e a straight l i n e
and g o through origin.
If higher c o n c e n t r a t i o n s a r e analysed, the graph will
s t a r t d e v i a t i n g f r o m the s t r a i g h t l i n e at 40-50 p m o l e s / l i t e r . The
e.xact point for the s t a r t o f the d e v i a t i o n depends on the quality
o f the a m i n e s o l u t i o n . H o w e v e r , i f t h i s i s taken i n t o account when
c a l c u l a t i n g the a n a l y t i c a l r e s u l t s , the graph
a n d thus the working
r a n g e o f the method
can be extended UP to about 100 pmoles/liter.
-
-
3.6.2.
15
-
---Analxsis
_--
An ordinary W i n k l e r b o t t l e i s filled w i t h t h e s a m p l e i n e x a c t 1
the s a m e m a n n e r as d e s c r i b e d f o r the d i s s o l v e d o x y g e n determination
(2.5.). The reagents d i a m i n e and ferric ( 3 . 3 . 1 . and 3 . 3 . 2 ) c h l o r i d e
are i m m e d i a t e l y added w i t h pipettes ( p r e f e r a b l y a u t o m a t i c s y r i n g e
pipettes) t o the s a m p l e . Let the p i p e t t e t i p s e x t e n d d e e p i n t o t h e
s a m p l e bottle. The s t o p p e r i s n o w i n s e r t e d a v o i d i n g a i r b u b b l e s .
The blue c o l o u r s t a r t s d e v e l o p i n g i n a f e w m i n u t e s a n d t h e s a m p l e
is r e a d y for measurement in a p h o t o m e t e r a f t e r 30 m i n u t e s . H o w e v e r ,
i f t h e s a m p l e contains high c o n c e n t r a t i o n s o f h y d r o g e n s u l p h i d e ,
one hour must be a l l o w e d for f u l l c o l o u r d e v e l o p m e n t . T h e c o l o u r
intensity m a y be regarded a s c o n s t a n t f o r a t l e a s t 24 h o u r s .
The colour i n t e n s i t y o f t h e s a m p l e i s m e a s u r e d a g a k n s t
distilled w a t e r (or, i f n e c e s s a r y , c o m p e n s a t e d f o r by r e a g e n t b l a n k s ,
a s described i n the n o t e below) a t 670 nm u s i n g 1 o r 5 cm c e l l s
a s required.
I f t h e sample c o n t a i n s higher c o n c e n t r a t i o n s o f h y d r o g e n
s u l p h i d e t h a n 100 p m o l e s / l i t e r , it h a s t o be d i l u t e d prior t o t h e
analysis. T h i s is done by p i p e t t i n g a s u i t a b l e v o l u m e o f t h e s a m p l e
i n t o a m e a s u r i n g flask o r W i n k l e r bottle c o n t a i n i n g s o m e o x y g e n - f r e e
distilled w a t e r . The pipette t i p should e x t e n d b e l o w t h e s u r f a c e
of t h e water. Then m o r e o f t h e d i l u t i n g w a t e r i s a d d e d by m e a n s
o f a siphon u p to the c a l i b r a t e d v o l u m e of t h e f l a s k o r bottle. T h e
reagents a r e added a n d t h e s a m p l e i s then t h o r o u g h l y mixed. A c c o u n t
has t o be t a k e n of t h e d i l u t i o n f a c t o r w h e n c a l c u l a t i n g the r e s u l t
of the analysis.
According to S t r i c k l a n d a n d P a r s o n s (1968) it may be n e c e s s a r y
to c o m p e n s a t e the a b s o r p t i o n v a l u e o f e a c h s a m p l e for t h e a b s o r p t i o n
v a l u e of a reagent b l a n k . T h i s l a t t e r v a l u e i s o b t a i n e d by a d d i n g
reagents t o filtered s u r f a c e w a t e r a n d m e a s u r i n g t h i s a g a i n s t t h e
same filtered water c o n t a i n i n g no r e a g e n t s . T h e a b s o r p t i o n v a l u e
should not e x c e e d 0.5 i n a 10 c m c e l l and s h o u l d preferably be l e s s
than 0.25. H o w e v e r , t h e blank v a l u e s a r e u s u a l l y n e g l i g i b l e , e v e n if a
discoloured a m i n e s o l u t i o n i s used.
Note : It is difficult to prepare a standard solution of sulphide with a high
degree of accuracy. Therefore a systematic error may be included in the
analysis. Using the method described, this error will be below 2%.
3.7.
CALCULATION AND REPORTING OF RESULTS
For the routine analysis a calibration factor is calculated. Select from
the calibration graph a corrected absorbance value (e.g. 0,500)and find its
corresponding concentration figure (e.g. 20,2 pmoles/litre) and calculate the
factor F for the cell length that was used for the measurement :
F
20,2
0,500
which gives F
=
40,4
Then the concentration of the sample, inmnoles/litre, is obtained by
multiplying its abosrbance with the factor F.
3.8.
I6
-
ESTIMATION OF PRECISION AND ACCURACY
A t a c o n c e n t r a t i o n level o f 25 p m o l e s / l i t e r , the precision
~ d be
n e x p r e s s e d a s +0.3%lnu;;;F;er o f r e p l i c a t e s a m p l e s .
-
3.9.
REMARK
I t s e e m s i m p o s s i b l e to o b t a i n a diamine that is not more
or l e s s d i s c o l o u r e d . H o w e v e r , t h i s d o e s not seem t o affect the
r e s u l t s . O c c a s i o n a l t e s t s u s i n g a o n e - y e a r old brownish diamine
s o l u t i o n produced r e s u l t s w h i c h differed only slightly from
t h e r e s u l t s o b t a i n e d w i t h a f r e s h l y prepared s o l u t i o n .
DETERMINATION OF DISSOLVED INORGANIC PHOSPHATE
4.1. SCOPE AND FIELD OF APPLICATION
T h i s i s a m o d i f i c a t i o n o f t h e w e l l known S i n g l e solution method
by M u r p h y a n d Riley (1962). T h e u s e o f t w o different solutions m a k e s
the reagents more stable.
N o e r r o r from s a l i n i t y w i l l o c c u r i n the d e t e r m i n a t i o n s a s t h i s
d e v i a t i o n is l e s s t h a n 1 percent. No i n t e r f e r e n c e s from c o p p e r , i r o n
a n d s i l i c a t e o c c u r a t c o n c e n t r a t i o n s s e v e r a l t i m e s higher than t h o s e
i n s e a water. Arsenate r e a c t s g i v i n g a similar c o l o u r , but i s regarded
a s e x i s t i n g i n m i n i m a l a m o u n t s i n s e a water. Recent results by
J o h n s o n ( 1 9 7 1 ) i n d i c a t e , h o w e v e r , that t h e concentration o f arsenate
i s higher t h a n w a s believed. I n periods o f high primary production
- a n d hence l o w c o n c e n t r a t i o n s o f p h o s p h a t e a r s e n a t e a n d phosphate
l e v e l s a r e s a i d t o be e q u a l a n d s o m e t i m e s arsenate may be
dominating?
-
T h e d e t e r m i n a t i o n , on a n unfiltered s a m p l e , gives t h e concent r a t i o n o f d i s s o l v e d i n o r g a n i c phosphate ions i n t r u e s o l u t i o n and
probably a l s o i n c l u d e s a s m a l l f r a c t i o n o f these i o n s w h i c h are
a b s o r b e d i n t o p a r t i c l e s and a r e s u b s e q u e n t l y dissolved by the acid
i n the mixed r e a g e n t . T h e l a t t e r f r a c t i o n i s not included i f the
a n a l y s i s i s performed o n a filtered s a m p l e .
4.2. PRINCIPLE
T h e p h o s p h a t e in water i s allowed t o react w i t h ammonium
m o l y b d a t e , f o r m i n g a c o m p l e x h e t e r o p o l y acid. T h i s acid i s reduced
by a s c o r b i c a c i d t o a blue-coloured c o m p l e x , the light a b s o r p t i o n
of which i s m e a s u r e d in a p h o t o m e t e r . Normally, t h i s reduction i s
s l o w , b u t by a d d i n g a c a t a l y s t - i n this case antimonyl tartrate t h e r e d u c t i o n proceeds s w i f t l y .
-
17
-
This i s a modification o f the well k n o w n s i n g l e s o l u t i o n method
b y Murphy and Riley (1962). T h e use o f t w o different s o l u t i o n s m a k e s
the reagents more stable. According t o e x p e r i e n c e from i n t e r c a l i b r a tions it i s necessary to measure t h e s a m p l e s o f s e a water a g a i n s t
water from t h e same depths i n order t o c o m p e n s a t e f o r the v a r y i n g
turbidity. This turbidity i s s o m e w h a t by the acid in the m o l y b d a t e
r e a g e n t . The turbidity blank s h o u l d f o r t h i s r e a s o n be o f t h e s a m e
acidity as t h e sample. Therefore it i s convenient t o use t w o r e a g e n t
solutions - o n e containing a l l c o m p o n e n t s except t h e a s c o r b i c a c i d
and a second which i s the a s c o r b i c acid s o l u t i o n .
REAGENTS
4.3.
4.3. 1
-
4 75
__-___________L_-------L_______________
Sulphuric acid about
moles/litre
253 m l o f concentrated s u l p h u r i c acid ( d e n s i t y 1.84) i s ,
under cooling and continuous m i x i n g , carefully poured i n t o a s u i t a b l e
volume (600 m l ) of distilled water. After cooling t h e v o l u m e i s
adjusted to 1 000 ml with distilled water.
Dissolve 9.0 g o f the salt (NH4!6M~ 024.4H 0 i n distilled
water and dilute to 100 ml. A n e w s o l u t i o n zhould 6 e made i f a n y
precipitation occurs.
4 . 3 -3.
_________________
Potassium a n t i m o n x________________
l t a r t r a t e s o l u t i o-L_---------_----_
n 0.1 moles/litre
Dissolve 3.25 g of t h e s a l t K(SbO)C4H406 i n distilled
water and dilute to 100 ml. T h e s o l u t i o n should be renewed i f
a precipitation occurs.
- molybdate
reagent
__________
___-_------
4 . 3 - 4 - Acid
200 m l of the sulphuric a c i d ( 4 . 3 . 1 ) i s mixed under
continuous stirring with 45 ml o f t h e molybdate s o l u t i o n (4.3.2).
F i n a l l y , 5 rnl of the tartrate s o l u t i o n ( 4 . 3 . 3 ) i s a d d e d . I f s t o r e d
cold i n a d a r k bottle the reagent i s stable for s e v e r a l m o n t h s .
7 . 0 g of ascorbic a c i d , C H 0
i s dissolved i n distilled
water and diluted to 100 ml. If s g o F e 8 ’ c o l d i n a dark b o t t l e , t h e
reagent i s stable f o r some weeks.
F o r preservation, 0.05 g of EDTA ( e t h y l e n e diamine t e t r a a c e t i c
acid) and 1 m l formic a c i d , H C O O H , may be a d d e d .
4.3.6.
18
-
Phosphate stock solution
P o t a s s i u m d i h y d r o g e n p h o s p h a t e i s dried o v e r concentrated
s u l p h u r i c a c i d o r a t 100°C. 0.3403 g o f KH2P04 p.a. is dissolved
i n distilled w a t e r a n d d i l u t e d t o 1 000 ml in a v o l u m e t r i c
f l a s k . S o m e d r o p s o f chloroform a r e a d d e d for preservation. This
s o l u t i o n c o n t a i n s 2.5 p m o l e s / m l PO4-P. The solution is s t a b l e
for many m o n t h s i f kept i n a r e f r i g e r a t o r .
10.0 ml o f the s t o c k s o l u t i o n i s diluted with distilled water
t o 1 000 ml in a v o l u m e t r i c flask. 1 ml contains 0 . 0 2 5 h m o l e
POLI-P. T h i s s o l u t i o n should not be s t o r e d for f u t u r e use.
4.4.
APPARATUS AND EQUIPMENT
4 . 4 . 1 . S p e c t r o p h o t o m e t e r o r f i l t e r photometer w i t h filter at o r close
t o 8 8 2 nm. C e l l s o f 5 cm a n d / o r 10 cm l e n g t h .
4 . 4 . 2 . G l a s s s t o p p e r e d t e s t t u b e s , 3 0 - 5 0 ml v o l u m e , o r o t h e r
suitable containers.
4 . 4 . 3 . Kipp pipette ( F i g u r e 2 ) o f 35 ml o r 2 5 m l volume for
d i s p e n s i n g of s a m p l e s . T h i s h a n d y pipette i s marketed under a
v a r i e t y of n a m e s e . g . p o u r - o u t d i s p e n s e r , TIPET, a u t o m a t i c transfer
p i p e t t e , r e p e a t e r pipette.
-
Automatic s y r i n g e p i p e t t e s o f 1 ml or 2 ml v o l u m e for reagent
additions.
T h e s e last t w o t y p e s o f pipettes are not n e c e s s a r y , but they
m a k e the r o u t i n e h a n d l i n g o f a great number of s a m p l e s m u c h easier.
4.5.
SAMPLING AND PRETREATMENT
The a n a l y s i s should be c o m m e n c e d as soon a s possible after
s a m p l i n g , p r e f e r a b l y within h a l f an h o u r , and definitely before
two hours. S a m p l e s s h o u l d be k e p t i n a cool dark place a n d not
w a r m e d to room t e m p e r a t u r e until the t i m e of analysis. I f the analysis
has t o be delayed for m o r e t h a n a b o u t one hour, s t o r e the samples
i n the r e f r i g e r a t o r . O n s h o r t t r i p s it m a y be possible t o a d d the
r e a g e n t s t o the s a m p l e s a n d perform the measurements a s h o r e . There
i s c o n f l i c t i n g e v i d e n c e c o n c e r n i n g the effect of different
p r e s e r v a t i o n m e t h o d s and the f a t e o f phosphate m o l e c u l e s in glass and
p l a s t i c b o t t l e s . No e n t i r e l y s a t i s f a c t o r y preservation method is
k n o w n (for s a m p l e s c o l l e c t e d for nutrient analysis). H o w e v e r , quick
f r e e z i n g i n a dry i c e / a c e t o n e bath has o f t e n been used when there is
no alternative to storing samples.
-
Figure 2
Kipp Pipette
19
-
I!.
6.
20
-
ANALYTICAL PROCEDURES
Calibration
--_--------
4.6.1.
A s e r i e s o f w o r k i n g s t a n d a r d s a r e prepared from t h e phosphate
w o r k i n g s o l u t i o n by d i l u t i o n w i t h d i s t i l l e d water. If 50 m l volumetric
f l a s k s are u s e d , T a b l e IV c a n be u s e d .
I
I
I
II
0.40
It
0.80
It
11
I1
It
II
It
II
2.00
II
11
II
4.00
I1
II
As described i n ( 4 . 6 . 2 ) b e l o w , 3 5 ml ( o r 25 m l ) portions o f
t h e s e s o l u t i o n s a r e t r a n s f e r r e d t o t e s t tubes and reagents are added.
F o r t h e c a l i b r a t i o n no t u r b i d i t y b l a n k s (samples w i t h only the acid
m o l y b d a t e r e a g e n t ) a r e n e e d e d . A r e a g e n t blank i s prepared from the
s a m e v o l u m e s o f distilled water a n d reagents.
-
After m e a s u r e m e n t t h e a b s o r b a n c e s - corrected for the reagent
a r e plotted v e r s u s c o n c e n t r a t i o n on millimetre paper. This
blank
c a l i b r a t i o n s h o u l d give a s t r a i g h t l i n e up to at least 10 ,umoles/litre.
o f *P04-P.
-
4.6.2.
Analysis
All g l a s s w a r e t o be used must be absolutely clean and s h o u l d
n o t be used for o t h e r purposes. P o w d e r e d synthetic detergents contain
p h o s p h a t e (large a m o u n t s ) i n m o s t c a s e s , and s u c h products should
n o t be used. A s o l u t i o n of distilled water containing the detergent
u s e d for c l e a n i n g mu.st be tested f o r phosphate c o n t e n t . T h i s is the
o n l y s a f e way t o avoid u n n e c e s s a r y complications.
S e a water for t h e d i f f e r e n t a n a l y s e s is preferably poured
d i r e c t l y from t h e water s a m p l e r s i n t o stoppered g l a s s bottles of
s u i t a b l e v o l u m e . F o r r o u t i n e a n a l y s i s o f a great number o f samples
i t i s v e r y convenient t o measure t h e different s u b s a m p l e s with a n
a u t o m a t i c d i s p e n s i n g p i p e t t e ( K i p p p i p e t t e ) with a ground glass joint
f i t t i n g t o t h e g l a s s s a m p l e bottle.
-
21
-
From each s a m p l e t w o 35-ml p o r t i o n s a r e t r a n s f e r r e d t o test
tubes. One o f the portions is r e g a r d e d a s the s a m p l e a n d t h e o t h e r
is the turbidity blank. To each o f the p o r t i o n s 1 m l o f t h e a c i d
molybdate solution (4.3.4) i s a d d e d a n d t o t h e s a m p l e a l s o 1 m l o f
the ascorbic acid s o l u t i o n (4.3.5). Mix w e l l b e t w e e n the a d d i t i o n s .
In case 25 m l o f sample is a n a l y z e d use 0.7ml portions of the reagents.
-
After five m i n u t e s each s a m p l e i s measured a g a i n s t i t s
turbidity blank in the photometer at 882 nm. I f t h i s w a v e - l e n g t h
i s not p r o v i d e d , it i s possible t o m e a s u r e a t a shorteu. w a v e - l e n g t h
but this r e s u l t s in decreased s e n s i t i v i t y .
T h e colour o f t h e s a m p l e s i s s t a b l e for a t least 24 h o u r s .
H o w e v e r , t h e reduction of t h e a r s e n a t e m o l y b d a t e c o m p l e x i s c o m p l e t e
a f t e r one h o u r (see a l s o p a r a g r a p h 4.6.3.2.).
--_NOTE
: The u s e of a t u r b i d i t y blank may be o m i t t e d i n case o f h i g h
c o n c e n t r a t i o n s of phosphate and - a t t h e s a m e t i m e
insignificant
turbidity. H o w e v e r , t o test the s i g n i f i c a n c e o f t h e t u r b i d i t y , i t
i s recommended to a n a l y s e at l e a s t s o m e s a m p l e s w i t h and w i t h o u t
a turbidity blank.
-
I f 5-cm cells a r e used i n s t e a d o f 10 c m , the s a m p l e v o l u m e s
may be r e d u c e d to 25 ml a n d the r e a g e n t v o l u m e s t o 0.7 ml.
4.6.3.
Disturbances
_____-_-_---
4.6.3.1.
Disturbance from s i l i c a t e
It i s a well k n o w n fact t h a t t h e blue p h o s p h o m o l y b d e n u m
c o m p l e x formec! with a s c o r b i c acid is s t a b l e f o r h o u r s . I f g r e a t
amounts o f silicate are present, especially in some types o f river
and l a k e w a t e r s and s t a g n a n t s e a w a t e r t h a t c o n t a i n s hydrogen s u l p h i d e
a blue silicomolybdate c o m p l e x is g r a d u a l l y f o r m e d . With the s t a t e d
c o n c e n t r a t i o n s of s u l p h u r i c acid a n d m o l y b d a t e , a n d by m e a s u r i n g
t h e a b s o r p t i o n after f i v e m i n u t e s , L ~ Jt o 10 m g o f S i 0 4 - S i p e r l i t r e
can be present without d i s t u r b a n c e .
4.6.3.2.
Disturbance f r o m a r s e n a t e
T h e arsenate i o n gives w i t h s u l p h u r i c a c i d , m o l y b d a t e a n d
ascorbic a c i d , a blue c o m p l e x . H o w e v e r , t h e r e a c t i o n i s r a t h e r
s l o w , being complete a f t e r one h o u r . By m e a s u r i n g t h e a b s o r p t i o n
a f t e r five m i n u t e s the d i s t u r b a n c e from a r s e n a t e i s a v o i d e d . I t m a y
be mentioned that with s t a n n o u s c h l o r i d e a s t h e r e d u c t a n t t h e blue
a r s e n a t e c o m p l e x is formed i m m e d i a t e l y .
-
4.6.3.3.
22
-
I n f l u e n c e from t h e c o n c e n t r a t i o n of s u l p h u r i c a c i d
T o a c h i e v e a rapid c o l o u r development a n d to depress the
i n t e r f e r e n c e from s i l i c a t e , i t i s o f fundamental i m p o r t a n c e that the
f i n a l r e a c t i o n t a k e s place a t a pH l o w e r than 1 a n d that t h e ratio
b e t w e e n s u l p h u r i c a c i d (in m o l e s / l i t e r ) and molybate (in per c e n t )
i s kept between 2.0
2 . 5 . W i t h the r e a g e n t s described the concent r a t i o n o f s u l p h u r i c a c i d i n t h e s a m p l e i s about 0 . 1 mole/liter.
I t h a s been s h o w n e x p e r i m e n t a l l y that w i t h the c o n c e n t r a t i o n o f
a n t i m o n y i o n s used h e r e ( 0 . 0 0 0 6 8 per c e n t ) the a c i d concentration
m a y v a r y between 0.06 and 0 . 1 5 moles/liter whereas a t 0.18
moles/liter t h e a b s o r b a n c e d e c r e a s e s by about 30 per cent.
-
4.7.
CALCULATION AND REPORTING OF RESULTS
T h e c o r r e c t e d a b s o r b a n c e i s used for evaluation of t h e r e s u l t ,
u s i n g the c a l i b r a t i o n g r a p h described i n paragraph 4.6.1).
F O routine
~
analyses a calibration factor is calculated.
S e l e c t from the g r a p h a n a b s o r b a n c e v a l u e (e.g. 0.170) and find i t s
c o r r e s p o n d i n g c o n c e n t r a t i o n figure ( e . g . 1.65 umoles/litre 1 and
calculate.
F5cm
= ----1.65
0.170
:
which g i v e s
F5cm
9.71
Then t h e c o n c e n t r a t i o n o f a s a m p l e , i n moles/litre, i s obtained
by m u l t i p l y i n g i t s a b s o r b a n c e with t h e factor F.
4.8:
ESTIMATION OF PRECISION AND ACCURACY
From a n u t r i e n t i n t e r c a l i b r a t i o n reported by Koroleff and
P a l m o r k (see G r a s s h o f f 1976) a c c u r a c y was given a s + 15 per cent
a t low l e v e l ( 0 . 2 umole/litre ), +5 per cent at a n i n t e r m e d i a t e - l e v e l
(0.9 umo1e:Liter) a n d +2 per cent' at a h i g h level (2.8 umoles/litre,).
-
-23-
5.
DETERMINATION OF TOTAL PHOSPHORUS
A great number of sensitive analytical procedures for determination
of total phosphorus have been published during recent years (see e.g. Koroleff
1976). All of these methods involve the use of peroxodisulfate as an oxidant
to transform the various phosphorus fractions to dissolved orthophosphate.
However, in most marine chemistry programmes not only total phosphorus
but also total nitrogen are determined. In 1977 Koroleff presented a
practical method for the simultaneous determination of these parameters
from the same sample and in 1981 Valderrama published a slightly modified
variant of the same method. Therefore, nr, separate procedure for the
determination of total phosphorus is presented here, but the analyst is
guided to the technique described in section 10 "Simultaneous persulfate
oxidation of phosphorus and nitrogen compounds in seawater".
6.
DETERMINATION OF
REACTIVE SILICATE
6.1 SCOPE A N D FIELD OF APPLICATION
Depending on pH the yellow silicomolybdate complex that is formed
in this analysis exists in two isomeric forms, which differ only in their
hydration. The
-silicomolybdic acid which is formed below a pH value
of 2.5 is rather unstable, but has a higher molar absorbance than the -acid.
In early work silicate was determined by visual or photometric estimation
of the
-complex. The literature has been reviewed by Mullin and Riley
(1955), and in 1968 a careful study of the optimum conditions for this
technique was performed by a research group at the Sagami Chemical Center
in Japan. In their modified method the colour of the complex is stable
from 5 to 20 minutes at room temperature, in both distilled water and
sea water; the salt effect is diminished to 3 percent.
Grasshoff (1984) has developed a method based on the formation
of the
-isomer at a pH of 3.7 to 4.0, and states that the colour in
sea water is stable (after two hours) for days.
6.2 PRINCIPLE
The determination of dissolved silicon compounds in natural waters
is based on the formation of a yellow silicomolybdic acid, when a more or
less acidic sample is treated with a molybdate reagent. Only silicic
acid and its dimer react with molybdate at any appreciable speed and the
methods therefore give only the amount of "reactive" silicate, which is
probably a reasonable measure of the silicate available to growing
phytoplankton.
-24-
Since both of the yellow silicomolybdic acid isomers are rather weak
in colour, several methods have been developed in which they are reduced
to intensely coloured, blue complexes. A wide range of inorganic and organic
reducing agents have been used. A mixture of metol and sulphite is commonly
used. In the method described here ascorbic acid acts as the reductant,
The molar absorption of the coloured complex
This implies that if the concentrations are given
1.000 is equal to 45 pmoles/litre Si with a 1 cm
With this cell length up to about 80 poles/litre
(absorbance about 1.7). With a 10 cm cell, 0.045
determined. As this corresponds to an absorbance
as the limit for direct determination.
6.3
is about 22 200 litres/mole/cm.
as poles/litre, the absorbance
cell used for the measurement.
of Si can be determined
pmoles/litre of Si can ke
of 0.010, it can be regarded
REAGENTS
All solutions should be prepared from reagent grade (pro analysi)
chemicals, using good quality distilled water. The solutions should be stored
in plastic bottles rather than glass bottles.
6.3.1 Ammonium heptamolybdate solution 0.16 mole/litre
Disssolve 49.5 g (NH4)6M07024.4H20 and dilute to 250 ml..
salt dissolves slowly, moderate heating may be applied.
6.3.2
As the
Sulphuric acid solution about 3,7 moles/litre
198 ml H2204 (concentrated sp.gr. 1.82) is carefully poured, under
constant cooling and mixing, into distilled water and finally diluted to
1000 ml.
6.3.3 Molybdate reagent
Add a measured volume of the molybdate solution (6.3.1) to an equal
volume of the sulphuric acid solution (6.3.2) and mix.
6.3.4 Oxalic acid solution 0.7 mole/litre
Dissolve 63 g C204H2 and dilute to 1000 ml.
6.3.5 Ascorbic acid solution 0.1 mole/litre
Dissolve 4.4 g C6H806 and dilute to 250 ml.
bottle, the reagent is stable for weeks.
Stored cold in a brown
6.3.6 Silicate stock solution
Sodium silicofluoride, Na SiF6 p.a. is dried over concentrated
sulphuric acid to constant weigzt. Weigh in 0.4701 g of the salt and
dissolve in distilled water in aplastic beaker. Transfer to a 500-ml
volumetric flask and dilute to the specified volume with d-istilledwater.
-25-
Finally, transfer to a plastic bottle. 1 ml contains 5.0 umoles Si.
The solution is stable for several months.
6.3.7
Silicate working solutions
50 ml of the stock solution (6.3.6) is diluted with distilled
water or synthetic sea water of appropriate salinity (see below) to a final
volume of 500 ml in a volumetric flask made of plastic materials. 1 ml
contains 0.50 pole/litre Si. This solution should be used at once.
6.3.8
Synthetic sea water
25 g sodium chloride, NaCl p.a., and 8 g magnesium sulphate
heptahydrate, MgS04.7H20 p.a. are dissolved in distilled water for every
litre of solution. This water must be stored in a plastic bottle. For
analytical purposes it is equivalent to a salinity of 28.10-3. For
calibration work it may be diluted to the desired salinity. the silicate
concentration should be below 1-2 ,moles Si/litre.
6.4
APPARATUS AND EQUIPMENT
6.4.1
Test tubes. Stoppered tubes of glass may be used but plastic
ones are preferred (inexpensive tubes made from polystyrene are commercially
available). Small plastic bottles (25-50 ml) may also be used.. If glass
tubes are used they must initially be carried through the analytical
procedure at least five times, otherwise they will yield too low results.
6.4.2
Automatic syringe pipettes (3 pcs.), capacity 2 ml.
6.4.3
Spectrophotometer, or filter photometer with the filter at or
close to 810 nm. If this is not provided, use a filter having a maximum
transmission above 700 nm; in this case the sensitivity is reduced and a
slight deviation from the Lambert-Beer's law may occur. Cells of 1,5 and
10 cm length as required.
6.5
SAMPLING AND PRETREATMENT
Samples of sea water for silicate determination should not be
stored in glass bottles €or more than a short while prior to analysis
and it is best, therefore, to transfer samples directly into containers
of polyethylene or waxed glass. To minimize the effects of diatom
multiplication, store samples in the dark and for nc longer than a day
prior to analysis. Conflicting opinions and evidence exist as to whether
samples can be stored €or long periods or not, and if stored, whether they
should be deep frozen or not. If deep frozen, the silicate concentration
can be expected to be acceptable only for samples containing less than
50 poles/litre. When stored frozen, silicate tends to polymerize.
Therefore, samples must be allowed to stand for at least three hours at
room temperature after thawing. Thus it is recommended to perform the
-26-
analysis as soon as possible after sampling. If the sampler have to he
stored, the tolerable storage time ought to be investigated and the possible
size of the error estimated.
6.6
ANALYTICAL PROCEDURES
6.6.1
Calibration
From the working solution (6.3.7) a series of working standards
are prepared by diluting with distilled water or synthetic sea water
(6.3.8) in volumetric flasks. Table V below may be used.
For routine analysis in sea water it is normally sufficient to
prepare the calibration solutions in synthetic sea water of a salinity
representing a mean value of the salinities in the working area. However,
for the greatest accuracy, it should be more convenient to calibrate
in distilled water and then correct for the salt error. This is of
particular importance in areas where the silicate concentrations in surface water may become very low as a result of phytoplankton primary
production.
TABLE
V
Preparation of working standards for silicate
ml of working solution/100 ml
(dilute with synthetic sea water
or distilled water)
0.0
0.5
1.0
2.0
5.0
10.0
20.0
25.0
30.0
poles/litre si
0.0
2.5
5.0
LO. 0
25.0
50.0
100.0
125.0
150.0
I
Some of the standards are chosen depending on the expected
concentration range in the samples. Samples are pipetted from the
working standards to the test tubes. The distilled water or synthetic
sea water of the chosen salinity respectively, should be used as the
blank. The reagent blank must be prepared together with every calibration.
-27-
The blank sample compensates for the silicate content in the reagents,
as well as in the distilled water (or synthetic sea water) used for the
dilution of the standards. However, the same blank value cannot be used for
the analysis of the natural water samples. In this latter case a reagent
blank has to be used. Preparation of reagent blank and analysis of samples
is described in the section below.
The absorbance is plotted versus concentration on
millimetre
paper. The result should be a straight line over the entire range of concentration.
6.6.2
Analysis
To 35 ml of the sample in a plastic test tube 1.0 ml of the molybdate
reagent (6.3.3) is added. After 10 to 20 minutes (for distilled water)
or 5 to 10 minutes (for sea water), 1.0 ml of the oxalic acid solution
(6.3.4) is added followed immediately by 1.0 ml of the reductant ascorbic
Swirl gently during the reagent additions. After at least
acid (6.3.5).
30 minutes the absorbance of the sample is measured against distilled
water in a cell of suitable length at a wave-length of 810 nm. Correct
the measured absorbancy of the sea water samples by substracting that of
the reagent blank. The reference sample is used for correcting the absorbances
of the calibration samples.
The reagent blank is prepared by carrying out the above procedure
using 35 ml of distilled water. The absorbance is denoted Al. Repeat the
determination, but with only 0.5 mL of the molybdate reagent. In this case
measure the absorbance after one hour (A2).
Absorbance of reagent blank =
2(A1
- A2)
The absorbance caused by the silicate content of the reagents only
is thereby obtained. This reagent blank should be performed every time
any of the reagent solutions are prepared. In addition, it is advisable
to check the reagent blank from time to time.
For the most precise estimate of low amounts of silicate using a
10 cm cell, a reference absorbance should be measured for every sample to
compensate for its natural turbidity. The reference solution is prepared
by adding 3 ml of 0.25 mole/litre sulphuric acid solution (e.g., prepared
by diluting 7 ml of the 3.7 moles/litre solution to 100 ml with distilled
water) to 35 ml of sample. Thus the absorbance of the sample is:
A
6.7
sample
= A
measured
- Areference
- Areagent blank
CALCULATIONS AND REPORTING OF RESULTS
For the routine analyses a calibration factor is calculated. Select
from the graph (described in section 6.6.1) an absorption value, corrected
as described in section 6.6.2 (e.g., 0.730) and find its corresponding
concentration figure (e.g., 37,8 umoles/litre) and calculate:
F1 cm
--
37,8
0,730
which gives F
1 cm
=
58,8
-28-
Then the concentration of a sample, in poles/litre, is obtained
However, for the most
by multiplying its absorbance with the factor F.
accurate work in marine waters a correction must be applied for the salt
error. It is known that the dissolved salts of sea water reduce the
final colour intensity to some extent. It has been found that the
variation of the salt error can be expressed as a linear function of the
sea water salinity:
Atrue ,
distilled water
=
A
observed
. (1 +
0.0045
. sample salinity)
Thus, if the calibration factor F determined for distilled water
is F
dist:
F
sample
Fdist.
(1 f 0.0045
. sample
salinity)
Note that these figures cannot be guaranteed for general use.
Because of the varying quality of reagents, etc., every laboratory should
construct a correction formula of its own, by calibrating at several
salinities and then calculating from the observed variations of the absorbances.
6.8
ESTIMATION OF PWCISION AND ACCURACY
From a nutrient intercalibration reported by Koroleff and Palmork
(see Grasshoff 1976) accuracy was given at &4 per cent at a low level
(4.5 poles/litre), 2 2 , 5 per cent at an intermediate level (45 pmoles/litre)
and 26 per cent at a high level (150 poles/litre).
6.9
REMARKS
6.9.1
Effect of the oxalic acid.
Oxalic acid is added for two reasons:
(a) To avoid the reduction of any excess molybdate reagent. The
stability of the silicomolybdic acid in the presence of oxalic
acid is limited, and the reductant is therefore added
immediately after the acid;
(b) to eliminate the influence of any phosphate present. Trials
have been made with up to 10 umoles/litre of P and no increase
in colour intensity was observed.
6.9.2
Time for colour development and colour stability
For up to about 30 poles/litre of Si, the colour reaches its
maximum after about 30 minutes and is then - provided any evaporation
stable for several days. However, for practical purposes
is prevented
it may be advisable to regard thecoloured complex as being stable for
several hours. For higher concentrations, up to an absorbance of
1.7 (1 cm cell) , the intensity increases extremely slowly after 30 minutes,
only about 0.1 percent per hour.
-
-29-
7.
DIRECT DETERMINATION OF AMMONIA WITH THE INDOPHENOL BLUE METHOD
7.1
SCOPE AND FIELD OF APPLICATION
This method, which is specific for ammonia, is applicable to
all kinds of natural waters and is the recommended method for sea water.
It is four times as fast as the Nessler reaction. "Ammonia" here refers
to the sum of ammonia and ammonium iens, because the original proporitons
of them in a water sample are pH dependent.
7.2
PRINCIPLE
In a weakly alkaline solution ammonia reacts with hypochlorite
to form monochloramine, which in the presence of phenol, catalytic amounts
of nitroprusside ions and an excess of hypochlorite yields indophenol blue.
By increasing the concentrations of the reagents used in this method,
labile organic nitrogen compounds may also react, while the reaction time
is decreased. The reaction mechanism leading to the coloured complex is
complicated and not yet fully understood. Probably, a quinone chlorimide
is formed in an intermediate step. The intensity of the coloured complex
is measured in a photometer.
7.3
REAGENTS
7.3.1
"moria-free" water
There is not a standard procedure for the preparation of water
with a very low ammonia content, as the results obtained depend on the
equipment and working facilities as well as the quality of the water used.
Each laboratory has therefore to find the method that gives the most
satisfactory result.
Deionized water may sometimes be used, with or without subsequent
distillation, but it must be remembered that some ion exchange resins may
leach out ammonia containing organic substances.
Ordinary distilled water may be used after a second distillation.
In this second step, 2 ml concentrated sulphuric acid and 1 q potassium
peroxodisulphate (K2S298) are added per litre. Freshly distilled, such
water should contain less than 0.3 p o l e of nitrogen per litre.
Another alternative is to use surface water samples in an open sea
area, preferably shortly after a plankton bloom. This water usually
contains very little ammonia and it should be stored in tightly closed
plastic containers with thick walls.
7.3.2
Reagent A, phenol
- nitroprusside solution
Dissolve 35 grams of analytical grade phenol, C6H50H, (a slightly
pink quality may be accepted) and 400 mg of sodium nitsoprusside dihydrate
(Na2Fe(CN)5N0.2H20) in ammonia free distilled water and dilute to 1 litre.
If the solution is stored in a dark bottle in arefriqerator it is
-30-
stable for several months. When the reagent becomes greenish it should
be discarded. (It is usually a good idea to store also the pure phenol
in the refrigerator).
7.3.3
Reagent €3, alkaline solution with free chlorine, variant I
Prepare a solution of hypochlorite containing 0.14 per cent
of free chlorine in 0.5 pole/litre sodium hydroxide in the following
way.
Commercial bleaching substance or concentrated sodium hypochlorite,
NaC10, can be used as stock solution. the hypochlorite content is tested
as follows. Dissolve 0.5 g potassium iodide (KI) in 50 ml distilled
water and acidify with 5 ml sulphuric acid, 9 poles/litre (as used in the
determination of oxygen) and titrate the liberated iodine with thiosulphate
solution, 0.1 pole/litre, using starch as indicator. 1 ml of the thiosulphate
solution is equivalent to 3.54 mg active chlorine.
Prepare the reagent B by dilutirg the required volume with sodium
hydroxide, 0.5 )unoLe/litre (20 g NaOH dissolved in 50 ml ammonia-free
distilled water and then made up to l litre with the same water to yield
a solution with 140 mg chlorine per litre. Store the reagent in the
refrigerator in a dark bottle. It is stable for weeks.
7.3.4
Reagent B, variant I1
The reagent can be conveniently prepared from a solid substance
instead of the hypochlorite solution. Trion is the commercial name for
dichloroisocyanuric acid (dichloro-s-triazine-2,4,6 (lH, 3H, SH)-trione).
Trion is available in at least technical grade. The product has to be
tested for its actual content of available chlorine using the procedure
described above. A typical figure may be that 1 g dissolved in the
sodium hydroxide, l)xnole/litre, produces a soluton containing 0.6 per cent
chlorine.
Thus, roughly 2 g Trion should be dissolved per litre of the
sodium hydroxide solution (prepared as in variant I) to yield a solution
with 140 mg chlorine per litre. The reagent should be stored in a dark
bottle in the refrigerator and it is stable for weeks.
The advantage with variant is that the solution is easy to prepare
without having to analyze the Trion at every occasion.
However, the
reactions in the analysis will proceed somewhat slower with this reagent.
7.3.5
Buffer solution
Dissolve 66.7 g trisodium citrate dihydrate, Na3C6H507.2H20, 34 g
boric acid, H3BO3, and 19.4 g citric acid dihydrate, C6H807.H20 in m o n i a
free distillea water and dilute to 1 litre.
7.3.6
Ammonia stock solution
As standard substance, either ammonium sulphate or ammonium
chloride of p.a. quality can be used. Before weighing, the salts must
be dried to constant weight at 100°C and then preferably stored in a
dessicator.
-31-
Dissolve 0.1320 g of (NH4)2S04 or 0.0535 g NH 4c1 in ammonia-free
water and dilute to 100 ml. Add a drop of chloroform as preservant. The
solutions should be stored in glass bottles. They are stable if protected
from direct sunlight and evaporation. Each solution contains 10.0 p o l e s
of NH3-N per ml.
7.4
APPARATUS AND EQUIPMENT
7.4.1
Test tubes with ground glass (or plastic) stoppers and a volume of
30 ml or more
7.4.2
Automatic syringe pipettes of 2 m l capacity (3 pcs.)
7.4.3
Spectrophotometer or filter photometer with a filter having maximum
transmission at (or close to) 630 nm. Photometric cells of 1, 5
or 10 cm length as required.
7.5
SAMPLING AND PRETREATMENT
The samples may not be preserved with sublimate (HgC1 ). Acid
preservation is possible, but in that case every sample must 2 e carefully
neutralized with sodium hydroxide before the analysis is started, and
corrections made €or the ammonia content in acid and hydroxide. This makes
the analysis uncertain and the acid preservation should be avoided if
at all possible. Preferably, the samples should be analyzed within a
short time after the sampling, and any necessary storage should be in
a dark, cold place.
Polluted waters are often turbid and may be filtered through
washed glass wool filters. However, such waters frequently contain high
concentrations of ammonia and may therefore be diluted before the analysis.
Any residual turbidity can be compensated with a similarly diluted sample
in the reference cell. Thus, filtration should be avoided, if possible.
7.6
7.6.1
ANALYTICAL PROCEDURES
a
Calibration
All work with ammonium analysis must be carried out where smoking
is not permitted,in order to avoid disturbances from ammonium. Before
the test tubes can be used for calibration or analysis, they must be
carefully cleaned; it is not sufficient just to wash them, but the
following subsequent cleaning procedure must be carried out. To every tube
about 25 ml distilled water (not necessarily ammonia-free water) and
reagents are added as described in paragraph 7.6.2.
Let the reaction
proceed for at least two hours and shake the tubes from time to time
during the reaction period. A11 ammonia contained in the tubes (dissolved
in the water or adhered to the glass wall) will react. then the tubes
are rinsed with ammonia-free water and kept stoppered when not in use. The
tubes should not be washed between the different sets of calibrations or
analyses, but just rinsed with ammonia-free water.
-32-
The analysis of ammonia is under the influence of disturbances from
variations in pH and salinity of the samples. To account for this, the
calibration can be carried out in either of two ways. For work in true
oceanic areas, where the salinity variations are small, the ammonia working
solution and the working standards should be diluted with ammonia-free sea
surface water instead of distilled water
For work in e.q., estuaries where
the brackish water displays large salinity variations, a calibration in
distilled water, followed by corrections for the salinity of each sample,
is preferred.
With the guidance of Table VI, the given volumes of ammonia working
solution are pipetted into 100 ml volumetric flasks and diluted to the mark
with distilled water, or sea surface water, that is free from ammonia.
TABLE
I
VI
Preparation of working standards for ammonium
ml of working solution
to be diluted to 1OC ml
0.0
0.5
1.0
2.0
6.0
10.0
20.0
resulting concentration
moles/litre
0.00
0,25
0.5
1.0
3.0
5.0
10.0
1
Note that the concentration pole/litre refers to NH3-N, which
means nitrogen coming from ammonia (and not the concentration of ammonia
itself).
From each of the standard concentration solutions above, 26 ml
(preferably three portions of 25 ml) transferred to the test tubes. These
samples are then analyzed as described in paragraph 7.6.2.
The following is an example oE a calibration series (measured
in a 1 cm cell) where the absorbances have not been corrected for the
blank value:
-33-
poles/li tre
0
0.022
A
7.14
17.85
0.171
0.397
35.7
0.772
53.6
1.105
The blank sample here corrects for the absorbance caused by the
ammonia in the reagents and also the residual ammonia in the ammonia-free
water. After subtraction of the blank absorbance, a calibration graph
is constructed, showing corrected absorbance versus concentration.
The calibration graph is reproducible within the entire range
for direct determination and it is linear up to a concentration of about
36 poles/litre.
For concentrations up to 14 pmoles/litre, a 5 cm cell
can be used, and with this cell, concentrations down to 0.04 pole/litre
can be determined.
A new calibration is required every time any of the reagents is
exchanged for a new solution.
The reagents , especially the buff er solution (7.3.5) , contain
ammonium ions and hence the abosrbance caused by the ammonium in the
reagents - the reagent blank - must be determined. To 22 ml of ammoniafree water (preferably three portions of 22 ml), the normal reagent
volumes are added
1 ml of each. To a second set of 22 ml portions,
double the reagent volumes - 2 ml of each
are added. After two hours,
3 m l of ammonia-free water is added to each sample in the first set, and
the absorbances of all samples are measured immediately. The difference
between the mean values of the two sets is the reagent blank. This
difference is caused only by the ammonia content of single volumes of the
reagents.
-
-
For example: the mean of the set with 2 ml of
A = 0.040 and the mean of the second set is A = 0.022.
the reagent blank
is then 0.018 (for 1 cm cell).
-
each reagent is
The difference -
If this is compared with the blank value given in the calibration
series it can be noticed that the nitrogen content in the water used for
dilution of the calibration samples gives an absorbance of 0,022-0.018
ED
0.004, which corresponds to a concentration of about 0.2 pnole/litre.
7.6.2
Analysis
Before the analysis and between analysis of different sets of
samples the test tubes must be cleaned as described in the beginning
of section 7.6.1.
For each sample a volume of 25 ml is measured and
transferred to a test tube. Add 1 ml of the buffer solution (7.3.5)
Mix
and 1 ml each of the reagents A (7.3.2) and B (7.3.3 or 7.3.4).
properly, e.g., by swirling the test tube, after each addition. Stopper
the tubes and keep them in a dark place duxing the reaction period
(2 to 6 hours; see also below in paragraph 7.9.2).
-34Measure the abosorbances of the samples in the photometer using
cells of suitable length depending on the colour intensity of the samples.
Use distilled water as reference and take the measurements at the wavelength 630 nm.
Depending on how the calibration was done, the result of the analysis
has to he calculated according to one of the procedures in the following
section (7.7).
7.7
CALCULATION AND REPORTING
For
routine
OF RESULTS
analysis a calibration factor is calculated.
Select
from the graph an absorption value (e.g., 0.240) and find its corresponding
concentration figure (e.g., 2.85 pmoles/litre and calculate:
5cm
=
2.85
0.240
which gives
F
--
5cm
11.88
How this calibration factor should be used in the calculations
is shown below.
The absorbance should be corrected with the absorbance of the
reagent blank that is described in paragraph 7.6.1. If the calibsation
has been made, using sea surface water of about the same salinity as the
sample has, the calculation is made using the calibration factor F, thus:
NH4-N
poles/litre
Areagent blank 1
F .
If, however, the calibration was made with solutions of distilled
water it is necessary to correct for the actual salinity of each sample.
Choose for each sample the salinity correction factor, Fs, from the table
in paragraph 7.9.1. Thus we have:
NH -N
4
7.8
poles/litre
(Asamp1e
-
Areagent blank)
FS
ESTIMATION OF PRECISION AND ACCURACY
High quality analysis of ammonia with good precision and accuracy
is highly dependent on how successful one can avoid contamination of samples by
tobacco smoke and other airborne ammonia as well as contamination of reagents
and glassware.
An intercalibration exercise described by Dah1 (see Grasshoff 1976)
yielded a precision of f' 4.8 per cent and an accuracy of 25.5 per cent.
-35-
7.9
INFLUENCING FACTORS AND DISTURBING SUBSTANCES
7.9.1
Salinity and pH
-
It can be noted from the calibration factors found for distilled
water and sea water solutions that in sea water the blue colour produced
will be less intense for any given concentration of ammonia. This depends on
the fact that in the final sample solution the pH will be a function of
the sample salinity. The reaction is complete and quantitative ,in the
pH-range between 10.4 and 11.3. Thus, for each Sample a correction has
to be made with respect to its salinity and the resulting pH. The
correction factor is selected from Table VII.
TABLE VI1
Salinity correction factors for ammonia and analysis
Salinity
3
( .10 1
PH
I
0-8
11
14
17
20
23
27
30
33
36
11.4-10.8
10.6
10.5
10.4
10.3
10.2
10.0
9.95
9.90
9.80
1.00
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
Fs
7.9.2
Reaction time
At room temperature the reaction is complete after two hours
in distilled water and sea water with salinity below 15 if the reagent B
used contains hypochlorite instead of Trion.
In ocean water the reaction requires four hours.
If the reagent
B with Trion is used the reaction will proceed slower and another two hours
reaction time should be added. However, as the blue colour produced is
stable for many hours it is convenient in routine analysis to let the
samples react over night.
The reaction time may be shortened by using a higher concentration
of hypochlorite. This cannot be recommended as some organic substances
containing nitrogen may decompose and form monochloramine, thus giving too
high results.
I
-367.9.3
Disturbing substances
With the recommended concentration of hypochlorite the method
is specific for ammonia and other nitrogen compounds do not react. Among
inorganic substances, Hg-ions are capable of impeding the reaction.
Consequently, samples should never be preserved with sublimate (HqCl2).
Likewise it has been observed that iron, in concentrations higher than
2 mg/l causes disturbance.
Stagnant water ofen contains hydrogen sulphide and if its
concentration exceeds 2 mg/l a disturbing effect is obtained. In such
waters the concentration of armnonia is normally rather high, and thus
the disturbance may be eliminated by diluting the samples.
8.
DETERMINATION OF NITRITE
8.1
SCOPE AND FIELD OF APPLICATION
This method is applicable for the determination of nitrite
nitrogen (N02-N) in most types of waters (including sea water and waste
water). It constitutes a modification of the well known Bendschneider
and Robinson method (Koroleff, 1973)
.
The colorimetric reaction is almost specific for nitrite ions.
Interferences may be caused by aromatic amines, copper (more than 0.5 mg/l),
iodide ion (more than 0.1 mg/l). Suspended matter and strong colour
in the sample also interfere.
This method is not appreciably affected by salinity, small
changes in reagent concentration 01: volume, or by temperature. Of the
interfering compounds, none are normally present in significant amounts
in the ocean, inshore or estuarine waters unless excessive pollution from
land drainage is encountered.
8.2
PRINCIPLE
The determination of nitrite is based on the classical Griess'
reaction, in which the nitrite ion at pH 1.5-2.0 is diazotized with
sulphanilamide, resulting in a diazo compound, which in turn is coupled
with N-(1-naphthyl)-ethylenediamine to form a highly coloured azo dye
with an absorption maxima at 545 nm whichis measured colorimetrically.
8.3
REAGENTS
8.3.1
Masnesium sulphate solution, about 2 moles/litre
Dissolve 50 g MgS04
. 7H20
in distilled water and dilute
to 100 ml.
8.3.2
Sodium hydroxide solution 4 moles/litre
Dissolve 16 g NaOH in distilled water and dilute to 100 ml.
-37-
8.3.3
Sulphanilamide reagent
Dissolve 8 g sulphanilamide, NH2C6H4S02NH2, in a mixture of
80 ml concentrated hydrochloric acid (HC1, 37 percent sp. qr. 1.19) and
400 ml water. Dilute to 500 ml. The reagent is stable for several months.
8.3.4
Diamine solution
N- (1-naphthy1)-et& lenediamine dihydrochloride.
Dissolve
0.8 q of C10H7NHCH2CH2~H .2HC1 in distilled water and dilute to 500 ml.
Store the solution in a iark bottle. Renew the solution about once
a month, or as soon as it develops a strong brown colour.
8.3.5
Nitrite stock solution
0
Anhydrous sodium nitrite, NaN02 p.a. is dried at 110 C for
several hours. Dissolve 0.3449 g of the dry salt in distilled water and
dilute to 1,000 ml. Store the solution in a dark bottle with 1 ml chloroform as preservant. The solution is stable for at least 1-2 months.
1 ml contains 5 m o l e s as N02-N.
8.3.6
Nitrite working solution
10 ml of the stock solution is transferred to a volumetric
flask and diluted to 1,000 ml with distilled H20. The solution must be
used the same day. 1 ml contains 0.05 umole NO -N.
2
8.4.
APPARATUS AND EQUIPMENT
8.4.1
Test tubes with glass or plastic stoppers and with a capacity
of 25 ml or more.
8.4.2
Automatic syringe pipettes or piston pipettes for dispensing
of reagents (not necessary, but very handy equipment).
8.4.3
Colorimetric equipment,
(i)
one of the following is required:
Spectrophotometer for use at 545 nm, with 1
longer as required;
cm cells and
(ii) Filter photometer, equipped with a yellow-green filter having
maximum transmittance near 545 nm. 1 cm cells or longer
as required.
8.5
SAMPLING AND PRETREATMENT
Nitrite is an intermediate compound in the simplified redox
chain ammonia - nitrite - nitrate. Samples for analysis of nitrite cannot
be properly preserved. Storage in a dark and cold place can be tolerated
for up to about five hours. If possible, samples should be analyzed without
delay.
-38-
Avoid filtering the turbid samples, and use coprecipitation as
pretreatment in extreme cases: Add 0.2 ml magnesium sulphate solution and
0.2 ml 4 moles/litre sodium hydroxide solution per 100 ml sample; agitate,
and after 30 minutes, take the decanted part of the sample for analysis.
Samples that are slightly turbid and contain no other disturbing
substances, as sea water from near-shore areas, may be analyzed together
with turbidity blanks instead of being pretreated.
8.6
ANALYTICAL PROCEDURES
8.6.1
Calibration
As there is no salinity effect in the formation of the azo dye,
the calibration can be done in solutions made with distilled water.
Using the nitrite working solution (1 ml = 0.05 pole) a series
of standard solutions is prepared by diluting with distilled water. If
100-ml volumetric flasks are used Table VI11 below can be followed:
TABLE VI11
r-
Preparation of working standards for nitrite
0.10 ml working solution/100 ml
0.05 pmole/litre N02-N
0.20
I1
II
0.10
0.50
I1
11
0.25
1.00
11
11
0.50
I1
2.00
I1
I1
a. 00
I1
I1
I'
I1
From the flasks, 25 ml samples are transferred to stoppered
test tubes. Distilled water is used as the blank. The reagents are added
and the analysis is performed as described below. The absorbances
(corrected for the blank) are plotted versus concentration on a millimetre
paper. In a 10 cm cell, the concentration 1 pmole/litre NO2-N gives an
absorbance of about 0.500.
-39-
8.6.2
Analysis
For precise analysis of low concentrations of nitrite in sea
water, any turbidity in the sample must either be removed (as described
in section 8.5) or compensated for by a turbidity blank.
25 ml of the possibly pretreated sample is transferred to a
stoppered test tube. If a turbidity blank is to be analyzed, another
25 ml is transferred to a second tube. Add 0.5 ml of the sulphanilamide
solution (8.3.3) to the sample and turbidity blank, then mix well. After
not less than three minutes, but not longer than eight minutes, 0.5 ml of the
diamine solution (8.3.4) is added to the sample but not to the turbidity blank.
Mix the sample once again.
-
After ten minutes, the absorbances of the sample and the
turbidity blank are measured in a photometer at 545
in a cell length
as required. The colour intensity is constant for two hours. The
concentration of the sample is evaluated from the calibration graph, when
the abosrbance of the turbidity blank has been subtracted from that of
the sample.
As the addition of acid to a water sample usually changes its
turbidity, it is important that the acidic sulphanilmi.de solution is added
not only to the sample, but to the turbidity blank as well.
a. 7
CALCULATION AND REPORTING OF RESULTS
For routine analysis a calibration factor is calculated. Select
from the graph an absorption value (e.g., 0.230) and find its corresponding
concentration figure (.e.g., 0.904 pole/litre) and calculate:
which gives F
5 m
=
.9
Then the concentration of a sample, in pnoles/litre, is found
by multiplying its absorbance, corrected for absorbance caused by turbidity
and the reagent blank, by the factor F.
8.8
ESTIMATION OF PRECISION AND ACCURACY
Turbidity of the sample may introduce a considerable systematic
error, particularly at low concentration levels. For highest accuracy and
turbidity should, therefore, be removed as-described in section 8.5 or
compensated for as described in section 8.6.2.
about
In routine analytical work the precision can be expected to be
pole/litre.
2 0.02
-40-
9.
DETERMINATION OF NITRATE
9.1
SCOPE AND FIELD OF APPLICATION
This method based on a previous publication (Koroleff, 1973)
is applicable for the determination of the sum of nitrate and nitrite,
in natural waters.
If the sample contains considerable amounts of nitrite, a separate
determination of nitrite must be conducted and the result subtracted from
that obtained with this method.
Nitrate concentrations up to about 40 poles/litre of nitrogen
(NO -N) can be determined without prior dilution of the samples.
3
The colorimetric reaction is almost specific for nitrite ions.
Interferences may occur, caused by aromatic amines, copper (more than
0.5 mg/l), and iodide ion (more than 0.1 mg/litre). Suspended matter
and a strong colour in the sample also interfere. Instead of filtering
the sample, use the precipitation technique. However, moderate amounts
of suspended matter can be tolerated as it will be trapped in the reduction
column.
At concentrations higher than about 15 )unoles/litre of NO3-N
in sea water, the dissolved salts cause a deviation from the linear
relationship between concentration and the intensity of the coloured
nitrite complex.
9.2
P RINC IPLE
Nitrate is reduced to nitrite almost quantitatively (about
90-95 percent) by amalgamated cadmium. The nitrite is then determined
according to the classical Griess' reaction as described in the method
for nitrite analysis (section 8).
The reduction is carried out at a pH of about 8.5. An ammonium
chloride buffer is added to the sample to control the pH and to complex
the liberated cadmium ions.
9.3
REAGENTS
Generally, the ordinary distilled or de-ionized water of the
laboratory should be suitable for the analysis of nitrite as well as
nitrate. However, if problems are encountered with the quality of
the water, "nitrogen-free" water has to be prepared.
Distilled (or de-ionized) water is distilled in all-glass
equipment. For this distillation 2 ml concentrated sulphuric acid and
1 g potassium peroxodisulphate (K s 0 ) are added per litre.
2 2 8
All chemicals should be of reagent grade (E
analysi).
-41-
9.3.1
Hydrochloric acid, about 0.5 moles/litre
Dilute
10 ml concentrated HC1 (density l.l9/ml) to 250 ml with water.
9.3.2
Mercuric chloride solution, 1 percent.
distilled water.
9.3.3
Hydrochloric acid, 2 moles/litre. Dilute 166 ml concentrated HC1
(density 1.19 g/ml) to 1 litre with distilled water.
9.3.4
Concentrated buffer solution
Dissolve 1 g HgC12 in LOO ml
Dissolve 250 g ammonium chloride, NH C1, in distilled water, add
25 ml concentrated ammonium hydroxide, NH40H (%ensity 0.91 g/ml) and dilute
to 1 litre.
9.3.5
water.
9.3.6
Dilute buffer solution
Dilute 20 ml of the concentrated buffer to 1 litre with distilled
Renew daily.
Acid washing solution
Dissolve 4 g NH CJ. in distilled water, add 15 ml hydrochloric
4
acid, 2 moles/litre, and dilute to 1 litre with water.
9.3.7
Sulphanilamide reagent
(same reagent as for nitrite determinations)
Dissolve 8 g sulphanilamide, NH2C6H4S02NH2, in a mixture of 80 ml
concentrated hydrochloric acid (sp. gr. 1.19) and 400 ml water. Dilute
to 500 ml with water. The reagent is stable for several months.
9.3.8
N- (1-naphthyl)-ethylenediamine dihydrochloride solution
(same reagent as for nitrite determinations)
Dissolve 0.8 g C10H7NHCH2CH2NH2.2HC1 in water and dilute to 500 ml.
Store the solution in a dark bottle. Renew the solution about once a month,
or immediately if it develops a strong brown colour.
9.3.9
Magnesium sulphate solution, about 2 moles/litre
Dissolve 50 g M SO4.7 H20 in water and dilute
9
9.3.10
Sodium hydroxide solution 4 moles/litre
Dissolve 160 g NaOH in water
9.3.11
to 100 ml.
and dilute to 1 litre.
Nitrate stock solution
Dissolve 1.0111 g potassium nitrate, KN03, in distilled water
and dilute to 1,000 ml. Add 1 m l chloroform as preservant. The solution
is stable for several months if evaporation is prevented. 1 ml = 10.0 ugat
(140 ug) NO3-N.
-42-
9.3.12
Diluted nitrate solution
10 ml of the stock solution is diluted with distilled water
0.1 Pgat (1.4~9)NO3-N.
to 1,000 ml. 1 ml
=
9.3.13
Synthetic sea water
Dissolve 310 g sodium chloride, NaC1, IO0 g magnesium sulphate,
MgSO4,7H 0 and 0.50 g sodium hydrogen carbonate, NaHC03.H20 in 10 litres
of distifled water. The salinity of this water is about 33.7 percent.
9.3.14
Amalgamated cadmium
WARNING: Cadmium is a poisonous metal. For reasons of occupational
health and safety it should be handled with care. Perform all operations on
the dry metal, particularly the granules, in a well ventilated area e.g., a
fume cupboard. Never inhale the dust. If granulated cadmium (for instance,
Merck Cadmium grob gepulvert, zur Fiillunq von Reduktoren) is not available,
one has to prepare it in the following way:
File sticks of pure cadmium metal (reagent grade) with a coarse metal
hand file (about second cut) and collect the fraction which passes a sieve
with 1-mm openings and is retained on a sieve with 0.5-mm openingsl).
Calculate the required amount of cadmium about 35 g per reductor column
(see Figure 3). Rinse the filings quickly with 0.5 mole/litre hydrochloric acid (9.3.1) and then with distilled water until the rinse water
does not give the chloride reaction with silver nitrate.
Transfer about 35 g of the washed metal into a 100 ml glass bottle and
fill the bottle with the mercuric chloride solution (9.3.2) so that all
air is excluded from the bottle. Seal the bottle with a glass stopper.
After this step avoid all contact between air and the metal. Rotate the
bottle for 90 minutes in a horizontal position with suitable equipment.
Open the bottle and rinse out the turbid sublimate solution with distilled
water which is introduced through a glass tube a' the bottom of the bottle.
Collect the sublimate solution.
If a rotary evaporator
carried out. Transfer
or 250 ml round bottom
is allowed to turn for
is available the treatment can be conveniently
the cadmium and the sublimate solution to a 100 ml
flask that is aligned on the evaporator where it
the required time.
Do not pour the used mercuric chloride (sublimate) solution into the
sewer! When a suitable vDlume is coliected, add 25 ml concentrated
HC1 per litre and precipitate with hydrogen sulphide (H2S) or sodium
sulphide (Na2S). Filter the liquid. Store the precipitate and discard
the clear filtrate.
1)
There are different sieves available commercially.
the suitable sieves.
TABLE
Table IX gives
IX
Comparison of sieve dimensions according to different systems
Opening :
upper sieve
Lower sieve
mm
1
0.5
DIN
6
12
Mesh/cm
7
13
Mesh/ inch
17.2
32
Sieve No.
18
35
-43-
30
75
fl
$2
a
180
b
Figure 3.
Reduction column for analysis of nitrate and total nitrogen
(all measures given in millimetres)
(a) glass wool
(b) Cd-amalgam
(c) copper wire
-44-
9.4
APPARATUS AND EOUIP?ENT
9.4.1
Reduction columns (see Figure 3)
9.4.2
Volumetric flasks or test tubes with a capacity of 25 ml.
The tubes should
9.4.3
9.5
either be graduated or marked at the 25 ml volume.
Colorimetric equipment; one of the
following is required:
(a)
spectrophotometer, for use at 545 run and provided with
cells of 1 cm (and 5 cm) length, or
(b)
filter photometer, equippped with a filter having maximum
transmittance at (or near) 545 nm, with cells of 1 cm (and
5 an) length.
SAMPLING AND PRETREATllENT
The analysis should preferably be undertaken immediately after
the sampling. If this is not possible, the samples may be preserved by the
addition of 1 ml sulphuric acid 4 moles/litre per litre. The sample has
to be neutralized again before the analysis of this preservation is applied.
Suspended matter and a strong colouration in the sample are removed
by using a precipitation technique: Add 0.2 ml of magnesium sulphate solution
and 0.2 ml of sodium hydroxide solution per 100 ml sample. Agitate, and
after 30 minutes, use the decanted solution for analysis.
9.6
ANALYTICAL PROCEDURES
9.6.1
Preparation of the reduction columns
Place a small ball of thin copper wire at the bottom of the
reduction column and fill it entirely with water. Open the bottle containing
the amalgamated cadmium fillings and put a finger (or a rubber plate) on the
opening. turn the bottle obliquely downward and place the opening under the
water surface in the reductor column. The metal should flow down into
the reductor column without coming into contact with air. Make sure that
no cavities are formed in the reductor. Fill the column with the metal
to about 1 cm below the reservoir and plug the column with glass wool. This
operation should be carried out so that all liquid that flows from the column
etc. can be collected and precipitated as described above.
Flush the column several times with the acid washing solution
(9.3.6) and with the dilute buffer solution (9.3.5) until the effluent shows
a weakly alkaline reaction (for instance, blue colouration with bromothymol blue).
The mlumnshould never be left with the acid washing solution. Flush the
column weekly when it is used regularly or more often if the flow decreases
through the column. A newly prepared column reduces nitrate almost
immediately with an efficiency of 95-100 percent (see section 9.7 for further
details). If the reduction efficiency decreases below 85 percent, empty the
column, wash the filings quickly with 2 moles/litre HC1 and rinse very thoroughly
with distilled water. Dry the filings, sieve again, and reamalgamate as
described above.
-45-
If a column does not give a proper reduction when it is newly
prepared it may be "started" by running through a nitrate standard solution,
e.g., 10 pmoles/litre, or common tap water. In most cases, less than one
litre will suffice.
9.6.2
Calibration
A series of standard concentrations is prepared from the nitrate
stock solution (9.3.11) and the diluted nitrate solution (9.3.12)' by
diluting with distilled water or synthetic sea water. As there is no salt
error at concentrations below about 15 poles/litre the synthetic sea water
will probably seldom be required.
As several columns are likely to be calibrated at the same time,
the standard solutions are prepared in at least 1 litre quantities according
to Table x.
TABLE
-
X
-.
Preparation of working standaxds €or n i t r a s
m l diluted
- nitrate solution/l,OOO
ml
1.00
5.00
10.00
50.00
ml nitrate stock solution/1,000 ml
1.00
2.00
3.00
4.00
!
pmoles/litre
0.10
0.50
1.00
5.00
pmoles/litre
10.0
20.0
30.0
40.0
I
The standard solutions are analyzed as described below.
of each solution must be analyzed.
Duplicates
The absorbances are plotted versus concentration for each of the
reduction columns. If the results are very uniform from column to column,
one calibration graph may be used €or the subsequent evaluation of the samples.
However, most probably one graph will be required for every column.
If samples have been treated according to the precipitation
procedure in paragraph 9.5, then the calibration solutions and blank samples
should be treated in the same way.
-46-
9.6.3
Analysis
9.6.3.1
Reduction of nitrate
Neutralize acid preserved samples with 0.1 or 0.5 rnole/litre
NaOH after addition of two drops of bromothymol blue indicator. (Calculate
prospective dilution) .
(1) Fill the resexvoir of the reduction column with the dilute buffer
solution (9.3.5) and let it flow through. Discard the solution.
(2)
Fill the reservoir again with 50 ml distilled water and 1 ml concentrated
buffer solution (9.3.4). Discard the first 20 ml which flows through
the column and collect 25 ml in a volumetric flask or test tube. Proceed
in the same manner with all the columns in order to obtain a blank value.
These blank samples are then analyzed for nitrite as described in (9)
and onward. If samples have been treated according to the precipitation
procedure in paragraph 9.5, then blank samples and the calibration
solutions should be treated in the same way.
(3)
The reduction efficiency of each column must be controlled for every
analytical batch. The standard concentration used for this control
depends on the types of concentration range of the samples. Analyze
duplicates of this control standard as described in (4) to (10).
Take a 50 ml sample or less containing up to 15 pmoles/litre NO -N)
3
and adjust the volume to 50 ml.
Add 1 ml of concentrated buffer solution (9.3.4) and mix; use an
automatic pipette if possible.
Draw off the excess solution in the reductor by using a capillary-tipped
glass tube connected to a suction pump and proceed immediately with (7).
Transfer the entire sample (4) to the reservoir of the reductor. Discard
the first 10 ml flowing through the column and use 10 mL to rinse a 25 m l
volumetric flask (or test tube). Then collect 25 ml in the flask or tube.
50 ml should flow through the column in ten to twelve minutes. Proceed
as described in (9). (The determination of nitrite should be carried
out within one hour.)
Draw off the remaining sample from the reductor and begin directly
with the next sample.
The reductor column must be washed immediately after every analytical
batch. F 11 the reservoirs twice with the dilute buffer solution (9.3.5).
Cover the reductors and make sure that they do not dry.
9.6.3.2
Determination of nitrite
(3) To 25 ml of the reduced sample, coming
from the reductor, add to
samples, standard solutions and blank samples 0.5 ml sulphanilamide
solution (9.3.7) and mix. After not less than 3 minutes, and not
longer than 8 miwtes, add 0.5 m l of N-(1-naphthyl)-ethylenediamine
solution (9.3.8) and mix again.
-47-
(10)
9.7
The absorbance of the coloured sample i.- measured after l? minuter,
but within two hours, against the reference sample at 545 nm. Use
a cell of suitable length depending on the intensity of the colour.
(The absorbance oE the blank sample against water should not
exceed 0.010, measured in a 1 cm cell.)
CALCULATION AND REPORTING OF RESULTS
Subtr.rlct the absc-rbznceof the blank sample from the absorbances
of the sa11p1.esa2d evaluate their Concentrations from the czlihration gra7h.
For routine analysis a calibration factor is calculated. Select
from the graph an absorption value (e.g., 0.715) and find its corresponding
concentration figure (e.g., 13.9 poles/litre) and calculate:
F
Ian
--
which gives F
13.9
0,715
lcm
--
19.44
Then the concentration of a sample, in poles/litre, is found
by multiplying its absorbance, corrected for the absorbance caused by the
blank, by the factor F.
NOTE :
If the nitrate determination is part of the determination of
total nitrogen the calculation is carried out as described in
section 10.7.
The result obtained is the sum of the concentrations of nitrite
and nitrate nitrogen in each sample. If a sample contains a significant
concentration of nitrite, this must be compensated for by subtracting the
result found from a separate determination for nitrite according to the
previous method (Section 8).
(If the sample was dilutedprior to the reduction this must be
taken into account in the calculation:
poles/litre N03-N
=
A x B
V
where :
=
v =
A
E
=
nitrate concentration, pnoles/litre, as obtained from the
calibration graph.
ml of original sample before dilution
final volume, after dilution.
For example: A 25 ml sample was diluted to 100 ml and of this
50 ml was analyzed. The result ob ained from the calibration graph was
26 umoles.
Then
26
x
25
100
=
104 poles/litre N 0 3 - ~
To check the reduction efficiency of the reductor (4) a nitrate
standard solution of suitable concentration is analyzed. For instance,
a solution of 1 pole/litre is analyzed and an absorbance of 0.241 is
obtained when using a 5 cm cell. An analysis of the nitrite solution with
the same concentration gives the absorbance 0.258 under the same conditions.
The reduction efficiency is then: 0.241 x 100
93 percent
0.258
9.8
ESTIMATION OF PRECISION AND ACCURACY
Deviation between duplicate samples analyzed with the aid of
one and the same reductor is c O . 1 pole/litre in the range 0 - 5 poles/litre,
-c0.2 umole/litre in the range 5 - 10 pmoles/litre and 0.5 pmole/litre at
higher concentrations. Deviation of results from different columns depend
on the reduction efficiency (yield factors) of the columns.
When systematic errors are avoided an accuracy o f i 3 per cent can
be assumed in the range between 0 - 10 poles/litre.
10.
SIMULTANEOUS PERSULPHATE OXIDATION OF PHOSPHORUS AND NITROGEN
CONTAINING COMPOUNDS IN WATER
10.1
SCOPE AND FIELD OF APPLICATION
Commonly used names of the parameters determined with this method
are total phosphorus and total nitrogen. Earlier methods published for their
determination have been designed for either of these parameters. In 1977
Koroleff presented a method for a simultaneous determination of total
phosphorus and total nitrogen in the same sample. The method was published
in 1981 by Valderrama in a slightly modified version. The method exhibits
improved reliability and precision and it allows for the storage of samples
to be analyzed later.
The yields obtained by oxidation of various nitrogen compounds
depend on the form of nitrogen linkage. Thus, €or example, nitrate, nitrite,
ammonia, urea, some aliphatic amino acids and some proteins give yields
over 92%. Pronouncedly poor yields have been obtained from compounds
containing nitrogen-to-nitrogen bonds, while a double bond between nitrogen
atoms seems to prevent their oxidation to nitrate entirely (Nydahl, 1978).
The digestion time, a function of the autodecomposition of peroxodisulphate,
needs to be no longer than 30 minutes, provided that the temperature
attained in the pressure cooker is 110-115°C.
10.2
PRINCIPLE
To allow nitrogen compounds to become oxidized, it is necessary
to use an alkaline medium, otherwise. nitrate is not produced in quantifiable
amounts. Conversely, the Oxidation of phosphorus compounds must be performed
on an acidified sample. In the simultaneous oxidation the reaction starts
at pH 9.7 and ends at pH 5-6. These conditions are obtained by a boric
acid-sodium hydroxide system. In seawater samples no precipitate is formed
when the oxidation reagent is added. At elevated temperature (100-120°C)
a precipitate is formed, which, however, almost dissolves as oxidation
proceeds. After the oxidation the remaining small amount dissolves upon
swirling. The free chlorine, which is formed in seawater samples, is
reduced by adding ascorbic acid before the molybdate reagent (Koroleff, 1977).
-49-
After oxidation the nitrogen compounds are determined as nitrate
according to the procedure in Section 9 and the phosphorus compounds are
determined as inorganic phosphate according to the procedure in Section 4.
10.3
REAGENTS
In addition to the reagents needed for determintion of dissolved
inorganic phosphate (Section 4) and nitrate (Section 9) the following items
are needed.
10.3.1
Dilute
hydrochloric acid, about 0.2 umoles/litre
Dilute 17 ml concentrated hydrochloric acid, HC1 (density 1.19 g/ml)
with distilled water to 1 litre.
10.3.2
Sodium hydroxide, 1 umole/litre
Dissolve 40 g sodium hydroxide, NaOH, in about 50-60 ml distilled
water and then dilute to 1 litre.
10.3.3
Oxidation reagent
Dissolve 50 g sodium peroxodisulphate, K2S208, and 30 g boric acid,
H3B03, in 350 ml of sodium hydroxide (1 mole/litre) and make up to 1 litre
with distilled water.
The peroxodisulphate has to have a very low content of nitrogen
compounds. The product with No. 5092 from Merck is usually of very satisfactory
quality.
If the oxidation reagent is stored in dark glass bottle and protected
from direct light in room remperature it is stable for at least six to eight
months.
10.3.4
Concentrated standard solution
Dissolve 0.4505 g dried glycine, C2H502N (reagent grade) and 0.1361 g
of potassium dihydrogen phosphate , KH2P04 (reagent grade), previously dried
at 1lOOC and kept in a desiccator, with distilled water and make up to a
final volume of 100 ml. 1 ml contains 10.0 ,poles of P and 60.0 p o l e s of N.
10.3.5
Dilute standard solution
Pipette 1.00 m l of the concentrated standard solution (10.3.4) to
a 100 ml volumetric flask and make up to the mark with distilled water. 1 ml
contains 0.10 p o l e s of P and 0.60 p o l e s of N.
10.4
APPARATUS AND EQUIPMENT
In addition to the equipment needed €or the determination of dissolved
inorganic phosphate (Section 4) and nitrate (Section 9) the following items
are needed.
-50-
Oxidation bottles of alkali resistent glass with screw caps
of non-nitrogen containing material and about 50 ml volume.
(Suitable bottles are Sovirel graduated bottles with screw caps. However,
as these caps, contain nitrogen compounds they have to be replaced. The
replacement must not be of polyethylene as they will become brittle and
crack as a result of the free chlorine that is developed in the oxidation.
Suitable replacements are polypropylene caps from Nalgene Labware, Nalge
Corporation).
10.4.1
10.4.2
Stainless steel pressure cooker of any ordinary kitchen type
that will maintain an internal temperature of 110-1L5°c.
10.5
SAMPLING AND PRETFLEATPG3NT
Soon after the sampling, e.g., in connection with the analysis
of the other nutrient parameters, 30 ml portions of the samples are
transferred to oxidation bottles. TO each portion 4 ml of the oxidation
reagent (10.3.3) are added with an automatic syringe pipette and the bottles
tightly stoppered.
Boil the samples for 30 minutes at 110-115°C in suitable batches
(e.g., 30-40 bottles) to fill the pressure cooker. Together with each batch
boil two oxidation reagent blank samples consisting of 4 ml reagent and no
extra distilled water.
After boiling, the samples should be gently swirled to promote
dissolution of the precipitate thay may have formed. Then the bottles
are allowed to cool down to room temperature. The bottles should not be
opened until the analysis of phosphate and nitrate is started.
It has been shown (Valderrama, 1981) that it is preferential
to add the oxidation reagent and boil the samples soon after sampling and
then store the samples in oxidized form. If samples are stored before oxidation
instead the analyses will receive a higher coefficient of variation. The
storage has been successfully tested €or at least three months.
10.6
ANALYTICAL PROCEDURES
10.6.1
Calibration
Using the dilute standard solution, prepare a series of standards
with the concentrations given in Table XI, below:
TABU
XI
Preparation of working standards for total phosphorus and total nitrogen
ml dilute standard/200 ml
14.0
10.0
6.0
2.0
phosphorus
poles/ Litre
nitrogen
p o les/ 1itre
7.0
5.0
3.0
1.0
42.0
30.0
18.0
6.0
-51-
Analyze 30 ml portions in duplicate from each standard according
to the description in section 10.5 (oxidation) and then section 10.6.2
(procedure).
Note that for the calibration the blank samples must consist
of 30 ml of the distilled water used with addition of 4 ml of oxidation
reagent (10.3.3) to each blank. This will compensate for any phosphorus
and nitrogen present in the distilled water and the oxidation reagent.
Correct the two absorbance figures obtained for each solution
with the corresponding blank absorbance and construct calibration graphs
as described previously in paragraph 13.6 for nitrate and paragraph 8.5
for phosphate. The calibration factors, F, can also be calculated as
described.
As the original sample taken for analysis (30 ml) is diluted to
35 ml in the procedure, the calibration factors will be about 1.167
times (35/30) higher than those €or nitrate and phosphate. Thus a factor,
FN, of about 24 for nitrogen and Fp, 11 for phosphorus can be considered
as representative.
Note that although the oxidized nitrogen sample is diluted from
10 ml to 50 ml in the analysis this is compensated for by using a 5 cm
cell for the photometric reading as compared with the 1 cm cell that is
(usually) used for nitrate. Thus the difference in the calibration
factors becomes rather small.
10.6.2
Analysis
Open the bottles containing the oxidized, cool, samples. As
each sample is to be used for two determinations it has to be divided
into two separate parts. Transfer each sample to a measuring cylinder,
preferably a stoppered one, and adjust the volume to 35 ml and mix well.
Continue as described below for each analysis.
After each batch of samples that have been oxidized it is convenient
to fill the oxidation bottles with the diluted hydrochloric acid (10.3.1)
and let them stand for a couple of hours before rinsing them with
distilled water.
10.6.2.1 Procedure for nitrogen compounds (total nitrogen)
Prepare the reduction columns for nitrate analysis as described
in steps (1) and (2) of section 9.6.3 Nitrate analysis.
Pipette 10 m l of the sample, adjusted to volume as described
above in section 14.7, and continue the analysis as for a nitrate sample
according to the steps (4) through (10) in section 9.6.3 Nitrate analysis.
In most cases a 5 cm cell is suitable for the photometric determination
as indicated above concerning calibration.
-5210.6.2.2
Procedure for phosphorus compounds (total phosphurus)
The remaining 25 ml from the sample in section 10.6.2 is
used for the analysis of phosphorus compounds. Transfer the sample to a
test tube and add 0.7 mL of the ascorbic acid solution (4.3.5).
Mix well
and let the sample rest 1-2 minutes so any remaining free chlorine, that
was liberated during the oxidation, is completely destroyed. Finally,
add 0.7 ml of the acid-molybdate reagent (4.3.4) and mix the sample well
again. (The preparation of these reagents were described in Section 4.3).
After five minutes read
the absorbance of the sample in a
5 cm cell at 882 nm using distilled water as a reference. See also
paragraphs 4.6.2 and 4.6.3 concerning the stability of the coloured
complex and possible disturbances.
10.7
CALCULATION AND REPORTING OF RESULTS
The absorbance measured for each sample, As has to be corrected
for the absorbance of the blank that contains the oxidation reagent, Aox.
The corrected absorbance is then used for evaluation of the result from
the calibration graph.
If a calibration factor, F ,is used for the calculation use
the formulas:
FN
Total nitrogen, poles/litre
=
Total phosphorus, poles/litre
10.8
- Aox.N 1
(A
1
s,P Aox,P.
(As,N
Px
F
ESTIMATION OF PRECISION AND ACCURACY
The actual capability of the method is summarized in Table XI1
below (from Valderrama 1981).
TABLE XI1
Accuracy and precision
__
.~
Total phosphorus
Total nitrogen
True concentration
values (pmolI-'
)
1.5
_~
-
.
30.0
-
0.25
1.00
3.0
8
8
8
No. measurements
8
6.0
8
Measured concentrations
M e a n values in (prnoiI-'
1.8
6.7
31.2
0.21
1.02
.1.94
1.7-1.9
6.4-6.9
29.9-3 2.7
0.25 4 . 2 9
0.98-1.0 4
4.52-4.95
0.063
0.217
0.988
0.014
0.020
0.056
3.5
3.2
3.2
11.7
4.0
2.0
2.0
1.1
20.0
5.2
8.0
Range
Standard deviation
Variation coefficient (%)
Relative error (%)
8
-1.2
-53-
REFERENCES
Bates, R.G., 1963: Determination of pH; Theory and Practice.
John Wiley and Sons, Inc., New York, 435 pp.
Grasshoff, K. (editor); 1976 : Methods of sea water analysis, Verlag
Chemie, Weinheim, 317 pp.
FAO, 1975: Manual of methods in aquatic environment research, Part 1.
Methods for detection, measurement and monitoring of water
pollution. FAO Fish.Tech.Pap., (137): 238 pp.
Gieskes, J.M., 1969: Intercalibration of pH measurements.
Unesco Techn.Paper.Mar.Sci. 9, 908.
Koroleff, F., 1976a: Determination of phosphorus. In Grasshoff,
K. (editor), Methods of sea water anazsis, Verlag Chemie,
Weinheim.
Koroleff, F., 1976b: Determination of ammonia. Ibidem.
Koroleff, F., 1977: Simultaneous persulphate oxidation of phosphorus
and nitrogen compounds in water. In: Grasshoff, K.,
Report of the Baltic Intercalibration Workshop, Annex.
Interim Commission for the Protection of the Environment
of the Baltic Sea.
Nydahl, F., 1978: On the peroxodisulphate oxidation of total nitrogen
in waters to nitrate. Water Research 12; 1123-30.
Valderrama, J.C., 1981: The simultaneous analysis of total nitrogen and
total phosphorus in natural waters. Marine Chemistry,
10 p p 109-122.