AN A LYTI C A L HANDBOOK FOR THE DETERMINATION O F
ARSENIC , C ADMIUM , COBA LT , COPPER, IRON,
LE AD , MANGANESE , MERCURY , NICKE L , SILVER AND Z INC
IN THE
MARINE AND ESTUARINE ENVIRONMENTS
by
Ralph G. Smith, Jr.
and
Herbert L. Windom
Skidaway Institute of Oceanography
55 West Bluff Road
Savannah, Georgia 3 1406
September 1 9 72
The Technical Report Ser ies of t he Marine Sc ience Progr am , Univers ity System
of Georgi a, embodies reports of a technic al nature c arried out in respons e to
spec ific requests by industry , loc al , regional , or state governme nt and the public
interest . Informat ion contained in these reports is in the public domain. Copies are
available to interested pers ons by writ ing to :
THE M ARINE RESOURCES C ENTER
55 We st Bluff Road
Savannah , Georgia 3 1406
If this prepublic at ion copy is c ited,
it
should be c ited
as
an unpublished manuscript .
TABLE OF CONTENTS
Page
Introduct ion
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Part I - Sea Water
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A.
Collection and Storage
B.
Metal An.alysis
1. Arsenic
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2. C admium
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3 . Cobalt , Copper, Nickel and Z inc . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 . Iron
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5 . Le ad
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6. Manganes e
7. Mercury
8. Silver
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Part II - B iological Samples
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A. Collection and Storage o f B iological Samples
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B.
Preparat ion of Biologic al Samples for Trace Metal Analys is
C.
Metal Analys is
1. .Arsenic
2. Mercury
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3 . Other Metals
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43
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TAB LE OF CONTEN TS
( Continued)
Part III
-
Sediment Samples
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A.
Total Digestion of Sediment Samples
B.
Preferent i al Le aching of Trace Metals from Sediments
Part IV - Anodic Stripping Volt ammetry
References
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51
58
1
INTRODUCTION
Due to the incre ased interest in the geochem istry and b iochemistry of
heavy metals in m arine and estuarine e nvironments more sens it i ve analyt ical
techniques amenable to rapid analysis of larger numbers of s amples have been
developed . Although m any of the new techniques requi re neutron activat ion c ap a­
bilit ies most have bee n des igned for the more commonly available colorimeter
or atomic abs orption spectrophotometer. This m anual descr ibed analyti c al tech­
niques for several he avy met als ut iliz ing the latter instrumentat ion. While some
of the techniques descr ibed have been in use by marine s c ient ists for s ome time ,
most have been only recent ly developed and all have been modified to some degree
as our experience has dictated.
In this manual a description of s ampling techniques is given along
w ith var ious methods for prepar at ion of the different types of s amples which
through exper ie nce we have found to be successful . All of the chemic al analyses
have been rout inely used in our lab and have bee n demonstrated to be successfully
applic able to both estuar ine and mar ine samples . In the c as e of water s amples
most of the techniques are s al inity i ndependent or have a correct ion for salinity
built-in. In the sect ions on chemic al analys is it is assumed that the reader has
a working knowledge of colorimetry and atomic abs orption s pe ctrophotometry .
In addit ion to sections des cribing useful technique for the analys is of
heavy metals in water, biologic al and sedime nt s amples a sect ion on anodic str ipping
analys is is presented . This sect ion was included to introduce a recent ly developed
voltametric technique that shows gre at potent ial for applic at ion to m arine and
estu ar ine problems .
2
PART I
SEA WAT ER
Of the types of s amples from the m arine environment t o be analyzed ,
water i s the most difficult analytically. Since the concentrat ions o f the metals are
extremely low requiring preconcentration, large volume s amples must be c ollected.
Some of the difficulty inherent with l arge s amples has been ove rcome by s ome labs
utilizing neutron activat ion where small s amples are sufficient (Schultz and Ture ­
kian, 1965 and Robertson, 1 970) . However , s ince neutron act ivation capab ility is
not commonly available to most m arine s c ient is t s , other techniques must be pur ­
sued. As a result of this , many techniques suitable for relatively inexpens ive
equipment have been devised. Notable among these are those techniques employing c olorimetry, atomic absorption s pectrometry, or polarigraphy such as anodic
stripp ing volt ammetry. Of these three techniques , this chapter de als with those
employing c olorimetric and atomic abs orption spectrophotometric techniques . The
applic ation of anodic stripping voltammetry will be cons idered in Part IV of this
manu al.
As an introduction to this chapter , Table 1 indicates , in gener al , the
method preferred for the analysis of the part icular metal along with the concentra­
t ion of the metals found in se awater. Also listed is the m aj or s pecies in which the
met al occurs in s e awater as determined by Mangel (1 9 7 1 ) using theoret ical c alcu­
lations s imilar to Sillen ( 1 96 5 ) .
A.
CO LLEC TION AN D STORAGE
Collection and storage of cont amination free sea water s amples is a part i­
cular problem in regard to trace metal analys is s ince the very metals that are of concern
T ABLE 1
Element
Concentration
in sea water
�-tg/1
Method
Species
Reference
Arsenic
Colorimetric
(Arsine generation - A A)
Cadmium
A PDC/MIBK- A A
Ion exchange -AA
ASV
0.01-0. 1
CdC� , Cd(Gly)2
Windom and Smith (1972)
Mullin and Riley (1956)
Cobalt
Ion exchange - A A
0. 01-0. 2
Co(Gly):3
Windom and Smith (1972)
Robertson (1970)
Copper
APDC/MIBK
ASV
AA
0.1 -1.0
Cu(OH)2 (Cit); 6
Cu(OH);-2, Cu (Gly);
Spencer and Brewer (1969)
Iron
Colorimetric
1.0 - 5.0
Fe(OH);
Menzel and Spaeth (1962)
Lead
Ion exchange - A A
ASV
0.01-0.1
Pb OH+a
Tatsumoto and Patterson (1963)
Manganese
Ion exchange - A A
0.3 - 5. 0
MnF
Mercury
Cold vapor A A
0.01- 0.05
Hg I-a
4
Windom (1972)
Nickel
Ion exchange - A A
0.1 - 0. 5
Ni( Asp)2
Windom and Smith (1972)
Spencer and Brewer (1969)
-
Portmann and Riley (1969)
2
2
-+3
Riley and Taylor (1968a)
TABLE 1 (cont'd)
Method
Element
Concentration
in sea water
�g/1
Species
Reference
Silver
Co-crystallization colorimetric
o. 0 1 - 0 . 1
�r12, Agr-;
Schutz and Turekian (1965 )
Zinc
Ion exchange - A A
APDC/MIBK- AA
0. 5
Zn(OH):
Windom and Smith (1972)
Spencer and Brewer ( 1969)
- 5. 0
APDC/MIBK - ammonium pyrolidine dicarbamate - methylisobutyl ketone extraction
A A - atomic absorption spectrophotometry
ASV-
anodic stripping voltammetry
Cit - citrate ion
Gly - glycine ion
5
are commonly used in such things as sampling bottles, wires and ships. The first
step in collecting "good" samples is the choice of sampler. The water sampling
device used should be large enough to collect a q�presentative uncontaminated
sample of sufficient size for the metal analysis. The sampler should also be as
metal free
criteria.
as
possible. The VanDorn and Niskin sampler appear to meet these
Matson (1968) has reported that VanDorn bottles adsorb certain trace
metals from sea water samples. Van Dorn bottles have also been found to contami­
nate samples with metals such as zinc from their rubber closers. Niskin bottles
on the other hand have no rubber closers and are made with an inert PVC material.
In order to evaluate possible contamination from Niskin bottles, double distilled
water was loaded into a Niskin bottle, filtered, and acidified in the same manner
as a sample is treated after collection and returned to the lab for analysis. Con­
centrations of the metals analyzed (Cd, Co, Cu, Zn, Hg) were below detectability.
Niskin bottles therefore appear to meet all the requirements for a good sampling
bottle.
In sampling, the bottle should be lowered at least ten meters below the
ship's keel to avoid contamination from the ship.
If
a "weight" is placed on the
hydrowire it should be at least five meters below the sampling bottle. Due to the
great amount of contamination associated with oceanographic vessels, it is our
opinion that uncontaminated surface samples cannot be obtained by any conventional
method.
Just subsequent to sampling and prior to storage, the sample should be
filtered through a 0 . 45 micron membrane filter. A nitrogen pressurized filtration
system such as that shown in Figure 1 using a standard Millipore R filter holder is
6
FIGURE 1
N2 Pressurized Filtration System
7
quite adequate aboard ship. After flushing one to two liters of sea water through
the system, the remainder is collected. This initial flushing is necessary to
clean and leach the filter and filtration system. The sample should be collected
in 1:1 HC1 cleaned polypropylene bottles. These have not been found to absorb
metals when the sample is acidified to a pH of 2 or less. It is advisable to age
the bottles with filtered sea water before they are used for sample collection
to equilibrate the container walls. Samples stored in aged, precleaned poly­
propylene bottles have been found to retain the same metal concentration after
several months.
8
B.
M E T AL AN ALYSIS
1. ARSENIC
Arsenic m ay be c once ntrated from s e a water by the co-cryst alli z at ion
with thionalide and determ ined photometric ally as described by Portmann and
Riley (1964). Since only a few s amples may conveniently be prepared at a t ime , the
method is too t ime consuming for a large number of s amples .
The arsine generat ion method using atomic absorption with an argon­
hydrogen entrained air flame was evalu ated. The procedure followed that des cribed
in Sect ion C. 1 of Part II for biologic al s amples with the exception that a l arger
s ample solution volume was used. Unfortunately , the larger volume of the system
makes the sens it iv ity much poorer than that reported for biologic al s amples . At ­
tempts to conc e ntrate the s ample by abs orbing the ars ine gas generated into a dilute
solution of s i.lver nitrate were u nsuccessful. Further work is needed in order to
develop this method.
The cocrystallization and photometr ic technique as described by Portman
and Riley ( 1964 ) is outlined below.
Reagents
1.
Ascorbic Acid 5% W/V
2.
Thionalide (thioglycollic-,8 -aminonaphthalide) 2 % W/V in acetone
3.
Sulphuric Ac id �5N)
4.
As corbic Acid 0. 1 m (prepare d aily)
5.
Pot as s ium Ant imonyl Tartrate 0 . 274%
6.
Ammonium Molybdate 4 . 8 %
9
7.
Mixed Reagent
-
50 m l of 5N H S04
a
+
15 ml of ammonium molybdate and 5 ml
pot as s ium ant imony t artrate and 30 ml of 0 . 1 m ascorbic ac id .
Dilute to 1 25 ml.
Re agent should be used within one hour of preparat ion.
Procedure
1.
To 1 L of filtered sea water in a 1 L Erle nmeyer flask, add 4 ml of 5% asc orbic
ac id.
2.
Cover w ith a watch glas s and he at t o boil ing .
3.
Add an additional 2 m l of 5% ascorbic ac id and cool to room temper ature .
4.
While stirr ing the s ample w ith a magnetic s t irre r , add 1 0 m l of 5N
� S0 4
and 7 ml of thionalide s olution.
5.
Stir the solution five minutes to coagulate the prec ipitate .
6.
Allow to st and 10 minutes , then heat to a gentle boil for 30 minutes .
7.
Cool and stir to coagulate the precipitate .
8.
Allow to st and overnight .
9.
Filter off the prec ip it ate w ith gentle suction us ing a 4 . 2 5 e m diameter Whatm an
No. 541 filter paper.
10.
Wash the prec ip it ate and filter with double distilled water.
11.
Place the filter and precipit ate in a 2 5 ml Erlenmeyer flask with glass stopper .
12.
Add 7. 5 ml of cone. HN03, close the flask and he at gently until the solut ion is
p ale yellow (24-36 hours ) .
13.
Evaporate the nitric ac id at low temperature .
14.
When a yellow viscous l iquid remains , p as s C 02 g as over surface while heating.
15.
Cont inue the gas until the dense white fumes subs ide .
present .
This destroys any
HN03
10
�S04•
16.
Cool the residue ( a pale yellow s olid) and add 1 ml o f IN
17.
Warm gently t o dissolve and transfer t o a 1 0 ml volu metric flask; add 2 ml of the
m ixed reagent and dilute to volume .
18.
Allow 30 m inutes t o elapse and measure the absorbance at 866 miJ us ing a 4 em
cell , and compare with standards and blanks made up in 1 L of double distilled
water and tre ated in th£ above m anner.
11
2.
C ADMIUM
C admium in sea water occurs at le ve ls too low for direct meas urement
by colorimetry or atomic absorpt ion spectrophotometry .
Atomic absorpt ion analyses
require s ample concentration in order to re ach concentrat ions above its limit of
detection.
Evaporation
of sea water is lengthy, cumbersome, and the
inevitable
concent r at ion of the salts pres e nt makes analys is difficult due to matrix i nterference.
The extraction technique of Brooks et al. (1967) using ammonium pyrolidine dicar ­
b am ate as a complexing agent m ay b e useful i f cadm ium conce ntrat ions are seve ral
parts per billion.
However , normal sea water levels are less than a part per
b illion, making this technique applic able to e nr iched waters such
as
polluted estu ar ies
or sedime nt pore waters .
Riley and T aylor (1968b) have described a method us ing a chelating ion
exchange resin for concentration and the subsequent determination by atomic ab­
s orpt ion .
The method i s also applic able to other trac e metals (Co, M n , N i , Zn) .
It involves chelating the trace metals on c helex 100 res in and eluting them with a
small volume of acid.
The procedure outlined below follows that described by Riley and Taylor
( 1968b) w ith several modific ations .
The eluate from the column after be ing evapor ated
to dryness is dissolved in HCl rather than acetone .
It was found that the addition of
acetone or any other misc ible organic solvent produces a pre c ip itate which clogs
the asp irat ion ass embly of the atomic absorption system.
Also , if larger than
1 L s amples are used, the ion exchange column should be b ackwashed occasionally
to avoid res in comp act ion.
St andards m ay be prepared by spiking s e a water whic h
h as been stripped of trace metals by pass age through a column o f the c helat ing res in.
12
A blank i s prepared by proces sing stripped s e a water in the absence of the spike.
The absorption from the blank is subtracted from the absorption of the s amples
and standards .
This w ill correct for absorpt ion due to m atrix e ffects of s amples
having s im ilar s alinity (within 1 0 °/oo).
°
Samples differing by more than 10 /oo
s al i nity should be treated individually.
The pH of the sea water s ample must be adjusted to 7. 8 :_ 0 . 1 prior to
analys is .
The ion-exchange resin is pH selective and the pH adj ustme nt is critic al
if 100 % recovery is to be obtained.
The s ample flow rate through the column must
not exceed 5 ml/minute since the res in has a very slow exchange rate . Replic ate
analyses of one large water s ample have indicated a prec ision of ::!:_7% us ing this
technique .
Reagents
1.
Chelex-100 ion exchange res in.
2.
HNq,
3.
HCl (2N) ( 1 6 7 ml/L)
5 0 - 100 mesh.
(2N) ( 1 2 7 ml cone. HNq/L).
Procedure
1.
Wash a suit able aliquot of the res in w ith excess 2 N HNO� three times .
( 1 ml/ml of res in) .
2.
Approximately 10 ml of res in are needed per column.
Wash the res in with double dist illed
�0
(1 em diameter) to a depth of 9 . 0 e m .
pour a few ml of double distilled
in a thick s lurry.
}\0
and pack an ion exchange column
T o avoid air bubbles in the res in,
into the c olumn, then pour the resin
The water level in the column should never be allowed to
13
drop below the resin bed level.
This is best accomplished by connecting a piece
of tygon tubing to the bottom of the column and loop ing it above the res in bed.
3.
Connect the ion exchange column to a reservoir and wash the res in with an
additional 50 ml of H:P·
4.
Allow the sea water s ample , p H
=
7 . 6 , t o flow through the column (5 ml/m i n) .
Check the flow rate occ as ionally with a stop watch and graduate cyl inder.
If
more than 1 L of s ample is used (we usually use 5 L) , the column s hould be bac k­
washed every two hours with dist illed deionized water.
5.
Wash the column with 250 m l H :P and discard the wash.
6.
Elute the met als w ith 30 m l of 2 N HN03 followed by 20 ml of 2 N HCl and finally
with 20 ml of double distilled w ater.
7.
Combine the eluants in a vycor flask.
Evaporate the solution to dryness at low temperature and dissolve the res idue
with 1 ml of 2N HN03•
8.
Dilute the solut ion to 5 ml.
necess ary for Ni and Zn.
If 5 L of s ample were used a 1/5 dilution may be
14
3.
COBALT , COPPER , NICKE L AND ZINC
Cobalt has bee n determined in sea water by a method proposed by Weiss
and Reed (1960 ) . The met al is co-crystallized w ith O:-nitroso-.8-napthol and deter­
mined calorimetrically w ith nit roso-R-s alt . However , the use of 50 to 100 L
s amples required by this technique limits its fe as ibil ity . Cobalt m ay also be
determined by atomic absorption after concentration by ion- exchange (Riley and
T aylor (1968) as described under c admium ) ; however , the normal level found in sea
water approaches the limit of detect ion of the method. The most feas ible and eas ily
accomplished analytic al technique for Co appe ars to be the APDC/MIB K extraction
of Brooks et al . (196 7 ) followed by atomic absorption spectrophotometry .
Copper has been determ ined by a dithizone-carbon tetrachloride extrac ­
t ion
as
described by Sandell ( 1 96 5 ) but the complex is not very specific. Le ngthy
separat ion steps are nece s s ary to isolate copper . Atomic absorpt ion analy s is after
s ample extract ion w ith APDC -MIBK offers greater sens itivity w ithout the necess ity
of s ep ar at ion (Brooks , et al . , 196 7) . C admium , cobalt , iron, nickel and z inc m ay
also be determined by the method if concentrations are above one part per billion.
The trace met als are chelated and extracted from a 750 ml volume of water into a
small volume of organic s olvent . standards are prepared by s piking water from
whic h the trace met als have bee n pre viously extracted. The sp iked s amples are
chelated and extracted following the s ame procedure
as
the s amples . Replic ate
analys is indicate a prec is ion of ,:t 1 2 % for copper .
Nickel m ay be determined i n sea water b y extract ion with dimet hylgloxime
(Kenter et al., 1 9 69 ) . The method has a l imit of detect ion of about 0 . 35 ug/1 and
is free from any s alt effect. Cobalt and copper form dimenthylgloxime complexes
15
only at concentrations much greater than that found in sea water.
Nickel m ay also
be determined by the atomic absorpt ion - APDC/MIBK method with a precision of
�12 % ( Brooks , et al. , 1 96 7 ) .
At concentrations below 2 . 0 ppb , the precis ion drops
substant i ally.
Zinc m ay be determ ined c olorimetrically by
a
dithizone extraction method
(Sandell , 1 96 5 ) , but the presence of other trace metals interfere, so that separat ion
prior to analys is is required. Riley and T aylor ( 19 6 8 a) c laim 100 % retent ion of z inc
on Chelex - 100 ion-exchange resin.
The z inc is eluted from the resin and deter­
mined by atomic absorpt ion. Replicate analysis i ndicates a pre c is ion of !10% (see
c admium technique) .
Brooks et al. ( 1 9 6 7 ) extract ion method m ay also be useful,
espe c i ally when other trace metals ( Co, Cu, Fe , Ni) are of interest .
is about the s ame as for the above method .
The precis ion
All glassware must be cle aned with
scrutiny and the use of rubber in any app aratus must be avoided.
In summary , the most univers ally applic able technique for the analys is
of Co, Cu, Ni and Zn is the APDC/MIBK-AA method.
m any labs and is the preferred method here .
It has found gener al use in
The following therefore describes
this technique in det ail.
Reagents
1.
Redistilled methy is obutyl ketone .
2.
APDC ( 1 % ) (prepared d aily ) .
Shake the 1 % APDC ( ammonium pyrolidine
dicarbamate) solution with an equal volume of MIBK ; separate and filter the
aqueous phase through Whatm an #5 filter paper.
solution.
This cle ans the APDC
16
Procedure
1.
C arefully adjust the pH of the s ample to between 4 and 5 .
2.
Measure and pour 750 ml of s ample into a polypropylene Erlenmeyer flask.
3.
Add 7 ml of 1 % APDC and 3 5 ml of MIBK.
4.
Equilibrate the sample for
30
minutes on a shaker, then pour into a lL.
separatory funnel .
5.
Separate the phas es and put the organic phase into a plastic be aker and cover
the beaker with par afilm.
6.
Add 2 0 m l of MIBK to the aqueous l ayer, shake for five minutes , separate and
disc ard the organic phase .
7.
Homogeniz e the aqueous phase of all s amples and measure the temperature of
the homogenized s ample .
8.
From a tempe rature -solubility chart (Table 2 ) , determine the % of s oluble MIBK
in the homogenized s ample .
9.
Separate the homogenized water into four 750 m l aliquots and spike with in­
crement al amounts of trace metals . Thes e serve as standards .
10.
Add 7 m l of 1 % APDC to e ach sp iked s ample.
11.
Add 35 ml of MIBK les s the volume found soluble from step 8 .
12.
Equilibrate the s amples on a shaker and extract as before .
13.
Determine abs orbance for st andard and s amples by AA.
14.
Prep are a c alibration curve from the standards and determine the s ample
concentrations .
17
TABLE 2
TEMPERATURE SOLUBILITY CHART
Temp. f C)
% Solubility
0
2 . 63
15. 27 ML
2
2 . 54
15 . 98
4
2 . 45
16.66
6
2 . 36
1 7 . 30
8
2 . 28
1 7 . 91
10
2 . 20
1 8 . 50
12
2 . 13
1 9 . 05
14
2 . 06
19 . 5 8
16
1 . 99
20 . 0 7
18
1 . 92
20 . 54
19
1 . 90
20 . 76
19 . 5
1 . 88
20. 87
20
1 . 87
20 . 9 7
20 . 5
1 . 86
21. 07
21
1 . 84
21. 18 '
21. 5
1 . 83
2 1 . 28
22
1. 82
21. 38
24
1. 77
2 1 . 75
26
1. 72
22 . 10
28
1. 68
22 . 41
30
1 . 64
2 2 . 70
Insoluble MIBK
18
4.
IRON
Atom ic absorptio n analys is (s im il ar to that of Co, Cu , Ni and Zn) of iron
after extraction with APDC/MIBK has bee n described by Brooks et al. ( 1 96 7) .
p re c is ion based on replicate analys is is .:!:12%.
The
A sens it ive colorimetric , and
generally more useful , method requiring only 100 ml of s ample us ing bathophenan­
throline has been described by Strickland and P arsons (196 8 ) .
mines the amount of biologic ally react ive iron.
The method deter­
Any pre c ipitated ferric hydroxide
in the s ample is dissolved with HCl and hydroxylam ine hydrochloride is added to
reduce Fe �3 to Fe +a.
An acetate buffer is added to buffer the solution to the pH at
which the b atho - iron complex is formed.
Bathophenanthroline is added and the
complex extracted into isobut anol for added sensit ivity.
The alcohol s olution is
diluted to a constant volume with acetone which also less ens c louding due to occluded
s e a water .
A c al ibration curve is prepared by spiking double distilled water with
known amounts of iron and complexing
as
above .
The absorbance of s amples and
standards is determined at 533 �· Replic ate analys is of sea water s amples
indicate a precision of .:!:20%.
The method as described below v;rr i es from Strickland and P arsons (1968)
slightly in that the hydroxylamine was not recrystallized prior to purific ation, and
isobut anol is used in the place of is amyl alcohol, due to the obnoxious odor of the
l att er.
Reagents
1.
Bathophenanthroline solution ( B atho-s olut ion) - Diss olve 0. 07 gm of bathophenan­
throline in 100 ml ethyl alcohol and then add 100 ml of d ist illed water.
is stable indefinitely if kept in polyethylene bottle .
Solution
19
2.
Is obutanol
3.
Hydroxylamine Hydrochloride - Dissolve 1 0 gm i n 100 ml dist illed water. Add
5 ml of Batho-solut ion and extr act with 10 ml of isobut anol. Separate the lower
aqueous layer and continue re -extr action until the extract is colorless . Store
in a polyethylene bottle .
4.
Sodium acetate buffer - d is s olve 75 g m of s odium acetate trihydrate i n 100 ml of
water. Add 2 ml of hydroxylamine soluti on and 5 ml of Batho-s olut ion and allow
mixture to stand 15 minutes . Extract the solution with 10 ml port ions of isobut anol
alcohol until the alcohol portions are colorles s . Add a further 5 ml of Batho­
solut ion and reextract . Store in a polyethylene bottle .
5.
Iron Extract ion Reagent: Dilute 20 . 0 ml of cone . HCl t o 500 ml (0 . 4 8 N) .
Procedure
1.
Transfer 100 ml of filtered sea water to a 2 50 ml separ atory funnel.
2.
Add 1 0 ml of i ron extraction re agent followed by 2 ml of hydroxylamine hydrochloride solution.
3.
Mix the solut ions and let stand for 5 m inutes .
4.
Add 2 . 0 m l of acetate buffer followed by 5 ml of Batho-solution.
5.
Mix and let st and 1 0 minutes .
6.
Measure from a dry gr aduated cyl inder 30 ml of isobut anol. Shake the funnel
vigorously one minute and let stand 5 minutes .
7.
Discard the lower aqueous layer and swirl the funnel to dislodge w ater droplets .
Separate all of the aqueous layer .
8.
Run the alcohol layer into a graduated cylinder and make t o a 30 m l volume with
acetone and mix.
20
9.
10.
Me asure the opt ical dens ity in a 4 e m cell (533 mJJ ) against a reage nt blank.
Subtr act absorption o f blank from that o f s ample .
21
5.
LEAD
Lead in sea water m ay be determined by the method descr ibed by Riley
and Taylor ( 1 96 8b) if concentrat ions are about 0 . 5 ug/ L. The sens it ivity m ay be
increased by us ing a l arge s ample volume {5 - 10 L) . Upon analys is by atomic ab­
s orption, the abs orbance obse rved is largely due to molecular absorption interferences. The absorb ance due to lead is only a fraction of the total observed
abs orb ance . Therefore , the prec is ion of the method unfortunately is very poor at
the concentrat ions normally found i n sea w ater. For this re ason the technique
has applic at ion only u nder unusual c ircumstances such as polluted are as . A more
suit able technique short of the is otope dilut ion technique of Tasumoto and Patter­
son (1 963) is ASV which is described in Part IV.
22
6.
MANGANESE
The permanganate method as descr ibed by Sandell ( 1 9 6 5 ) lacks the sens i-
t iv ity required for sea water s amples , s ince neither per iodate and persulfate have
been used as an oxidiz ing agent with acceptable results .
Strickland and Parsons '
( 1968) photometric method for manganese determinat ion i n s e a water involves the
cat alyt ic effect of manganese on the oxidat ion of leuco base .
The method is quite
sens it i ve but suffers the dis advantage of a s alt effect and is therefore of limited use fulness in estuarine areas .
The sens it ivity increases gre atly at low s al init ies ;
the refore , only s amples with nearly the s ame s alinities should be analyzed together .
Riley and T aylor ( 1 9 6 8 a) described a method for manganese analys is involving a chelat ing ion-exchange res in and the subsequent determ ination by atomic
absorption.
The method is s imilar to the ir e arlier work (Riley and T aylor , 1 96 8b),
the chief differe nce in the two methods be ing that i n the m anganese method the
chelat ing ion-exchange res i n is converted to the ammonium form and the sea water
sample is loaded on the column at a pH of 9. 0 .
The authors found that the ammonium
form of the res in is more s elective for m anganese.
The procedure outlined below follows that described by Riley and Taylor
, w as
( 19 6 8 a) with the exceptio n that the eluate , after being evaporat ed to dryness
dissolved in HCl rather than aceton e .
It was found that acetone causes irregul ar
m.
aspi ration into the flame of the atomi c absorption syste
Als o, st andards are
n quant ities of m anganese . The
pre pared by sp iking strip ped s e a water with know
ange colum n tre ating them as s amples .
sp iked s ampl es are run through the ion exch
str ipped s e a wate r as desc ribe d above
A blank should be prep ared by proc ess ing
23
in the absence of trace metals. Absorption from the prepared blank is subtracted
from the sample absorption. This will correct for matrix effects. Samples with
large variations in salinity
(> 10
°
/oo) should be run separately.
It is important that the pH of the water be adjusted to 9 . 0 � 0.1 just prior
to loading on the column since the resin selectivity is pH critical. Also, the sample
flow rate through the column must be maintained at 5 ml/minute.
Reagents
1. Chelex-100 Ion Exchange Resin
2. HN03 (2N) 127 ml cone. HNq/L.
3. NH40H (2N) 268 ml cone. NH40H/L.
Procedure
1.
Wash a suitable aliquot of the resin with excess 2N HN03 three times (1 ml
acid/ml resin).
2.
Wash the resin with redistilled water and convert to the ammonium form by
washing three times with 2N NH40H (1 ml/ml resin).
3.
Wash the resin three times with redistilled water (1 ml �0/ml resin).
4.
Pack a 1 em diameter ion exchange column to a depth of 9 em by pouring the
resin in a slurry.
5.
Allow 1 L of filtered sea water, pH 9 . 0, to flow through the ion exchange column
(5 ml/minute).
6.
Wash the column with 30 ml of redistilled water and elute manganese with
30 ml of 2N HN03• (Collect the eluate in vycore glassware.)
7.
Evaporate the solution to dryness at low temperature.
24
8.
Dissolve the residue with 2 N HC l and dilute to a 5 m l volume .
9.
Determine absorbance by atomic absorption spectrophotometry us ing Mn
hollow c athod lamp and compare with st andards and blanks .
25
7.
MERCURY
A colorimetr ic method for the determinat ion of mercury i nvolv ing a
dithizone extraction is described by Sandell ( 1 96 5 ) .
in both specificity and sens it ivity.
This method, howe ver , l acks
Atomic absorption with an air-acetylene flame
l acks the sens it ivity needed for sea water analys is even after prior conce ntration.
Rece ntly , flameless atomic absorpt ion has been established as a sens it ive method
for analysis of microgram and submicrogram amounts of mercury .
Kalb ( 1 9 70 ) describes a method whereby mercury is amalgamated on
s ilver foil and subsequently heated in an induction fur nac e , liberating the mercury
which is swept into the instrument's opt ical path.
Howe ver , matrix problems m ake
reproduc ible results difficult to obt ain. Liberat ing mercury from organic matter
by permanganate oxidat ion has been described by Hatch and Ott ( 1 96 8 ) .
The s ample
is subsequently reduced w ith a solut ion of sodium chloride/hydroxylam ine sulfate/
stannous sulfate.
This one step reduct ion suffers from irreproduc ibility.
Uthe ,
et al . , ( 1 9 70 ) have devised a s ophisticated re act ion chamber which they claim gives
more reproduc ible results than that of Hatch and Ott .
Chau and Saitoh ( 1 9 70 ) described a method for mercury analys is of lake
water which presently is in use in this lab for s e a water.
The method involves a
dithizone-chloroform extract ion and the subsequent b ack extraction into 5N HCl.
The ac idic s olution is reduced with stannous chloride and vaporized by a peristalt ic
pump .
Samples spiked with methyl and phenylmercury have been found to be quanti-
t atively extracted with the dithizone s olution.
1
Interferences from other metallic
ions are m inimized by the addit ion of E DT A and hydroxylamine hydrochlor ide to
t he s ample .
26
The absorpt ion tube (16
x
2 . 5 em) thr ough which the mercury v apor passes
is made of glass , has an inlet and outlet opening , and quartz windows on either e nd .
This tube m ay be placed i n the opt ic al path of any convent ional atomic abs orption
system . The re action chamber used is a 1 25 ml gas washing bottle. Air from a
per ist altic pump ( 1 L/m in) is forced through the glass fritted tube ins ide the gas
washing bottle . A drying tube containing magnes ium perchlorate is placed between
the re action flask and the absorption tube to prevent wate r vapor from entering the
abs orption tube . This tube should be replaced or refilled after 12-15 analysis to
avoid loss in sens it ivity . The mercury v apor is recycled in a clos ed system u nt il
the absorbance reaches a maximum . The m aximum absorbance is more e as ily
determined when a recroder is connected to the AA system .
Standards m ay b e prepared by spiking 40 ml o f 2 . 5 N HC1 w ith i ncrement al
amounts of mercury. To as cert ain qu ant it ive recovery s ever al previously extracted
s amples m ay be spiked and re -extracted. A s ample blank m ay be prepared simil arly.
Reagents
1.
Dithizone-chloroform. Dis solve 6 mg of dithizone in 1 L C HC!a. Remove any
mercury prese nt in this solut ion by extracting with an equ al volume of 5N HCl.
2.
Disodium EDT A solution - 0 . 10 M .
3.
Hydroxylamine hydrochloride ( 5 0 % W/V) .
4.
Stannous chloride - 20% ( W/V) i n 6 N HCl.
5.
HCL (5N) 4 1 7 ml cone . HC1/L.
27
Procedure
1.
T o 500 m l of filtered sea water (pH adjusted t o 2 us ing d ilute HCl), add 2 m l of
hydroxylamine hydrochloride and 1 ml of EDT A.
2.
Add 2 5 ml of dithizone -c hloroform s olut ion and shake 20 m inutes on a mechanic al
shaker .
3.
Allow the s ample t o stand 2 0 minutes , then separ ate the organic layer into a
60 ml separatory funnel.
4.
Add 2 0 m l o f 5 N H C l and shake 1 0 minutes
5.
Disc ard the organic layer and transfer the ac idic s olut ion into a s m all beaker .
Wash the funnel with 2 0 m l of redist illed water and add the wash t o the beaker.
6.
Pour the contents of the beaker into the re action flask.
7.
Add 1 m l of SnC � and close re act ion flask quickly .
Adjust pump speed to
1 . 0 L/m in.
8.
Allow the absorbance to re ach a maximum before opening the system .
28
8.
SILVER
Silver concentrat ion i n sea water is at the submicrogram per l iter level.
It must , therefore, be concentrated prior to analys is by convent ional me ans . Riley
and Taylor's \1968b) chelating ion exchange technique claims 1 0 0 % rete nt ion of s il­
ver ion on the chelat ing res in. Howe ver , only 10% of the s ilver can be eluted from
the res in. Attempts to digest the res in w ith HC104 and HNO� have been uns at is ­
factory .
Lai and We iss (1962) have inve st igated the use of thionalide for the co­
cryst allizat ion of 27 elements includi ng s ilve r . The c ryst als are diss olved with hot
nitric ac id and the organic matter destroyed. Silver is s ep arated from interfer ing
ions by extraction with tri-N-butyl thiophosphat e . The organics present must the n
b e destroyed with nitric and perchloric ac ids .
The subsequent determinat ion of s i lver m ay be determined by two methods .
Method I is b ased on the c at alyt ic act ion of s ilver on the persulfate oxidat ion of
m anganous ion to permanganate as described by Underwood, et al . ( 1 9 5 2 ) . This
method is recommended for the determinat ion of less than 0 . 25 J.Lg of s ilver. Method
II involves the photometr ic determ inat ion using p-dimethylaminobenz alrhodanine as
des cr ibed by Sandell and Newmayer (195 1 ) . This method is recomme nded for the
determination of greater than 0 . 5 u.g of s il ve r . Method I was c hosen in this work
due to its s ens it ivity. Standards may be prepared by running s piked s amples throug.r
the colorimetric procedure only. A check on the effic iency of the co-cryst allizat ion
m ay be accomplished by running st andards through the e ntire procedure .
29
Reagents
1.
Thionalide solution ( 1 % W/V in acetone ) .
2.
Acetic ac id (cone . )
3.
Tri -N-butyl thiophosphate ( 1 . 5 % W/V i n CC1 ) .
4
4.
Phosphoric ac id ( 1 :1 ) .
5.
Manganous sulfate ( 0 . 006 M)
6.
Potas s ium persulfate (granular )
Procedure
1.
Adjust the p H of 1 L sea water sample to 3 . 5 -4. 0 with acetic acid.
2.
Heat the s ample to 800 C , then add 20 ml of the thionalide solut ion.
3.
Allow the s olut ion to cool at room temperature , then place i n a refrigerator
(5 ° C ) overnight .
4.
Collect the crystals i n a s intered glass funnel .
5.
Wash the crystals w ith a s mall volume of redistilled water and discard the wash.
6.
Dissolve the cryst als with 7 5 ml of hot c one. HN03•
7.
Add 1 0 m l of cone . �804 and boil until carbonizat ion oc curs .
8.
Add s everal 2 m l portions of 1 : 1 HNO-HC10 to c lear the solution, the n c are ­
4
fully evaporate to dryne s s .
9.
Dis solve the white inorganic res idue with 20 ml of 6N HNq_ .
,
10 .
Extract this solut ion four times with 10 ml portions of thiophosphate s olut ion.
11.
Combine the extracts and wash with 40 ml redist illed water.
12.
Evaporate the CC1 at room temper ature , then wet ash the res idue with 1 0 m l of
4
HN03 and 3 ml of HC10 •
4
30
13.
After comp lete evapor at io n o f the ac id, the res idue is dissolved with 5 m l of
redist illed water.
14.
To a 15 ml conic al centrifuge tube cont ai ning 1 ml of phosphoric ac id s olut ion
and 1 ml of m anganous sulfate solut ion, add the 5 ml s ample .
15.
Adjust the total volume to 10 ml us ing redistilled water w ashing of the previous
s ample container and add 2. 0 gm of potass ium persulfate .
16.
Mix the solut ion and place all s amples and st andards s imultaneously in a boiling
water bath for 1 0 minutes .
17.
Simultaneously place s amples and standards in an ice bath and allow to stand
20 minute s with occ as ional s hak ing.
18.
Centrifuge the s amples and standards and place back i n the ice bath until re ady
to determine their optical density ( at 525 mu) .
19 .
Avoid bubbles in the cell by keeping the t ip of the transfer pipet below the
surface of the liquid during deli very.
20 .
Compare s amples with st andards .
31
PART II
BIOLOGICAL SAMPLES
Metal concentrations in m arine organs ims are much higher than that in
sea water therefore generally eliminat ing the necess ity of preconcentrat i on prior
to analys is . However , other d ifficulties are encountered with b iological s amples
that are not as severe in water s amples .
The gre atest problem in the chemical analysis of mar ine organisms is
t he l ack of uniformity from s ample to s ample . As a result almost e ach species
presents a u nique matrix problem . The following discuss ion of metal analysis of
b ilogical s amples is therfore devoted to atomic absorption spectrophotometric
techniques emphas izing the is olat ion of the metal u nder cons iderat ion by e ither
separation (arsenic and mercury ) or matrix correction (other met als) .
A.
COLL E C TION AND STORAGE 0 F BIOLOGICAL SAMPLES
As is the c ase with water s amples , biologic al samples may also be con­
t am inated during collection if cert ain precautions are not t aken. Since most
oce anographic biologic al s ampling ge ar have met al materials , metal cont amination
if pos s ible . In collecting large organisms by such methods as fish netting, rod
and reel fishing or trawling, cont amination may not be severe s i nce tissues of
the larger organisms can be t ake n so that the surface tissues are excluded. Col­
lecting of s m aller organisms such as plankton , however, where the whole organism
is used.for analys is presents greater difficult ies . Plankton s amples in part icular
suffer from cont am ination during c ollectio n result ing from the debris associated
with oceanographic vessels . During plankton towing, paint chips and other ship's
debris are usually collected along w ith the s ample . Once included with the s ample ,
32
it is very difficult to isolate these materials from the biologic al material .
Therefore,
i n plankton s ampling, great c are must be t aken to avoid trailing plankton nets in the
wake of the ship or in a w ay so that debris from the s hip c an cont am inate the s ample.
In general, when collect ing any biologic al s amples , metal material on s ampling ge ar
such
as
that in the cod e nd of plankton nets should be m inim ized and rep laced, where
poss ible , with plast ic or other nonmet alic m ate ri als.
Immediately upo n collection, samples should not be allowed to come in
contact w ith the deck of the ship if at all poss ible .
Also , immediately upon colle c ­
tion, or as s oon thereafter as pos s ible , s amples should be labelled, placed i n plastic
bags or cont ainers and frozen.
In the case of large fishes , dissection of t issue
s amples may be des ired aboard ship .
During the collection of v arious t issues e ithe r
aboard ship o r back in the lab, great care should b e exerc ised i n order to not cross
cont aminate one tissue with another s i nce concentrations of met als w ill vary several
orders of m agnitude from t is sue to t issue .
For example , it is best in collect i ng
muscle t issue to open the organism first and take muscles from the ins ide out r ather
than penetrating the skin from the exterior to get at the mus cle.
33
B.
P REPARATIO N O F BIOLO GI CAL SAMPLES FOR TRACE META L ANA LYSIS
Wet ashing of biologic al s amples prov ide s an adequate way of bre aking
down organic matter with little c hance of loss of metal due to volat il ization, except
mercury, which will be cons idered in a following sect ion.
Middleton and Stuckey
(1954) have des cribed a wet ashing procedure using nitric ac id at low temperature.
In our laboratorie s we use a modific at ion of this technique .
After drying at 8 00 C
s amples are digested us ing fuming nitric acid directly.
Care must be exercised
when us ing fum ing nitric to avoid ignition upon heat i ng.
If this becomes troubles ome
it is best to add the fuming nitric and allow the s ample to st and thirty minutes before
applying he at .
If the s ample should ignite at any point , it s hould be extinguished
with redist illed water immediately to avoid volat ization of trace met als . Also ,
hotplate temperature must be below 300 ° C to avoid trace metal volat ization.
Samples should be covered with a w atch glass during the digestion to
avoid the rapid evaporat ion of the nitric acid.
Volat ilized fats m ay deposit a film
on the watch glass and m ay be removed with Kimwipes .
After the res idue becorre s dry , fum ing nitric acid s hould be added to
moisten the res idue ( approximately 1 . 0 ml) .
This process is repeated unt il a white
res idue rem ains . At this point the res idue is dissolved with HCl and diluted to
volume .
This res idue may not be completely s oluble in HCl due to the presence
of metaphosphates .
This small amount of insoluble matter may be removed by
filtering the s ample through Whatman #4 filter paper prior to br inging it to volume.
Vycor be akers axe used to avoid pos s ible le aching or adsorpt ion of trace
metals.
Fused quartz be akers are preferred for Pb analysis .
Beakers having less
than 250 ml capac ity should be avo ided s ince the s m aller surface are a will lengthen
digest ion time cons iderably .
34
Blanks are prepared by heat ing fum ing nitric acid in a cle an vycor be aker.
Upon e vaporat ion the res idue is dissol ved with HCl and brought to volume .
Blanks
for all trace met als cons idered here should be below detect ability by atomic absorp­
t ion spectrophotometry .
This wet ashing technique has many advant ages over dry ashing and other
wet ashing techniques .
For example , the loss of t r ace metals (mercury not included)
t hrough volat ization is eliminated s ince the temperature never exceeds 300° C , and
s ample digestion time is s ignific antly less and requires very l ittle personal atte ntion.
P rocedure
1.
To 0 -5 gm of dried s ample in a 250 ml vycor be ake r , add 5 . 0 ml of fuming
nitr ic acid and cover w ith a watch glas s .
2.
If there is danger of ignition, allow s ample to stand 30 minutes .
3.
He at on a hotplate at 3000 C .
4.
\.Vhen the residue becomes dry , add 1 . 0 m l of fuming nitric .
Repe at as many
t imes as necess ary to produce a white. res idue.
5.
Dissolve the residue with 1 : 1 HCl.
If the res idue is not complete ly soluble ,
filter through Whatman #4 and d ilute to the des ired volume .
35
C.
META L ANALYSIS
1.
ARSEN IC
Color imetr ic methods for the determ inat ion of arsenic in biological
material lack sens it ivity and spec ific ity.
Portm ann and R iley ( 1964) descr ibe a
method involving a co-crystallization w ith thionalide and the subsequent format ion
of arsenomolybdenum blue complex after digestion.
The method , although be ing
spec ific and sens iti ve enough, is terribly laborious and time consuming.
Atomic abs orption spectrophotometry has unt il recently been poor at
best for the determ ination of arsenic .
The reas on for this is that arsenic resonance
line l ies in the ultraviolet region be ing difficult for some older spectrophotometers
t o resolve .
Also, the most commonly used flame in atomic absorption work ( air­
acetyle ne ) absorbs some 65-70% of the trans m itted UV light .
By us ing an argon­
hydrogen-entrained air flame absorption is cut to 1 5-20% of the transm itted light
at the 193
mu
resonance line.
However , the low temperature of the argon-hydrogen
flame e ncounters interferences in real s amples due to incomplete s alt d issociation
and molecular absorption.
This necessit ates either matching the s ample matrix
very c losely or chemic al s ep arat ion.
The gener at ion of arsine gas has the advant age of concentrat ing and
separating the arsenic from the original s ample matrix.
The ars ine is generated
by the addition of zinc to an ac idic s ample solut ion in a re action flas k.
The zinc
l iberates hydroge n from the acid which reacts with As -h3 to liberate ars ine
(As� ) .
Holak (1969) collected the ars ine in a liquid nitrogen cold trap, subsequently w arming
and sweep ing it into an argon-hydrogen fl ame with nitrogen.
Madsen ( 1 9 7 1 ) found
that arsine is rapidly absorbed by a dilute s ilver nitrate solut ion.
The s ilver nitrate
solution c an subsequently be introduc ed into the flame ; however, the lower limit of
36
s ensitivity of this procedure is approximately 1 . 0 IJg. Dalton { 1 9 7 1 ) reported that
the ars ine m ay be c arried directly into the flame by the excess hydrogen generated.
For this purpose Fernandez ( 1 9 7 1 ) described a collection balloon attached to the
react ion flask to collect the ars ine and excess hydrogen that is gene r ate d. Repro­
duc ibil ity is difficult to obt ain due to loss of e last ic ity from ac id att ack and the
balloon often deve lops pinholes .
Improved sens it ivity m ay be obtained by purging the re action flask w ith
argon as suggested by the Jarell Ash Corpor at ion ( 197 1 ) . The ars ine gas generated
is then pas sed through a hydrogen sulfide absorber pr ior to introduction into the
flame . This m ay be acc omplished by pas s ing the gas through c otton impregnated
with le ad acet ate.
Biological s amples should be wet ashed at low temperature with fum ing
nitric acid in order to avoid loss of AB -lo. The final solut ion should contain no
nitric acid so that the AB
+s
m ay be reduced to AB
+3
with K1. and SnC � . This m ay
be accomplished by heating the s amples while blowing C02 over the s ample until
dens e white fumes cease . A s ample aliquot cont aining between 0 . 25 and 1 . 00 JJ. g
of arsenic is diluted to a 2 5 ml volume with a 20% HCl
-
5 % � S04 diluent .
Sensit ivity and Reproducib ility
At 5X s c ale expans ion, st andards in the range of 0 . 25 to 1 . 25 JJ. g arsenic
give an average abs orpt ion of 15% and 75% of the s c ale respective ly .
. Replicate analyses of orchard le aves prepared by the National Bureau of
Standards (#1 5 7 1 ) were digested and analyzed as described above . The result ing
value obt ained w as 12 . 8 ,=t. 1 . 5 ,u g/gm. The NBS reported value is 1 4 JJ.g/gm .
37
Equipment
Beckman model 495 atomic abs orpt ion spectrophotometer equipped with
a 10 inch line ar recorder or other equivalent equipment .
Optimum pressure for
the argon and hydrogen are 15 and 3 PSI respect ively .
Spec ial Appa ratus
1.
5 0 m l pear shaped, two neck flask 1! 14/20 .
2.
Adapter with aerator assembly (Sc ient ific Glas s J A 7970 ) .
3.
Adapter, reducing 1' 14/20
4.
Polyethylene drying tube .
5.
Flowmeter
6.
3 -way stopcock
7.
2 ml auto-dispens er
-
'$' 24/40 .
Reagents
1.
Pot ass ium Iodide 2 0 % W/V solut ion
2.
Stannous chloride 2 0 % ( W/V) i n cone . HCl
3.
Zinc powder suspens ion ( 10 gm z inc dust per 2 0 m l redistilled water).
4.
Le ad acet ate 10 % ( W/V) s olution.
5.
Dilue nt 5 %
6.
Arsenic st andard 1000 ppm solution
�S04
20 % H C L solut ion.
-
0 . 4165 gm Na2H AS 04
•
7 }\0/100 ml
of diluent . All subsequent dilutions must be made with the d iluent .
38
Apparatus Set -Up
The inlet of the aerator assembly is connected to an auxili ary argon supply
w ith tygon tubing ( Figure 2 ) .
This connect ion should be intercepted with a flow
The three way stopcock allows the argon
meter and a 3-way stopcock respect ively .
to bypas s the system or purge through the system.
The flow meter enables main­
t ainence of a constant flow rate (70 -80 ml/min) through the system .
The outlet
s ide of the aerator is connected to the drying tube cont aining cotton impregnated
with le ad acetate.
The tygon tubing leading from the
�S
absorber is connected to
the burner by way of a hypodermic needle connected to c apillary tubing.
Method
1.
After remo v al of HN0:3 us i ng C0 , a 1 . 0 ml aliquot of the s ample is diluted to
2
a 25 ml volume with the dilue nt .
2.
Add 1 . 0 ml of the KI solution and 0 . 5 ml of SnC1 , mix and let stand at le ast
2
15 minutes .
3.
P l ace the s ample in the react ion flask
•
.
Adjust the stopcock s o that the auxilliary
argon purges the system for 30 seconds . Readjust so that the argon now bypas ses
the system .
4.
Add 2 . 0 m l o f the Zn s uspens ion with the auto-dispenser and quickly stopper the
flask.
5.
When the recorder shows a shift in the absorbance s ignal, turn the stopcock in
ord<;lr to purge the system with argon.
6.
The absorbance peaks very quickly and returns to the b aseline . After the absor­
b ance has pe aked, turn the stopcock s o the argon will byp as s the system.
39
3 WAY
STOPC O C K
FLOW
METER
Reagent
Por t
�
R E A CT I ON
F LA S K
S U L F I DE
ABSOR BER
.._ A
To AA
s p 1 r a fo r
.
FI GURE
2
A rs i n e G e n e r a t o r
40
2.
MERCURY
Kothny (1969), Miller and Swanberg (1958) described colorimetric methods
for biological s amples which require lengthy extract ion and separation steps.
More
recently, flameless atomic absorption has been accepted as a fas t , accurate and
amenable method for mercury determination using conventional AA equipment.
In
the conventional flame method of atomic absorption only a very s m all portion of the
Hg atoms are in a state capable of absorbing light at the mercury resonance line.
With flameless AA a much larger percent age of the mercury atoms vaporized into
the optic path are in the proper state.
In this method, an absorption tube maclr · of
glass with inlet and outlet openings and quartz windows on either end is placed in
the optical path of any atomic absorption system.
Air from a peristaltic pump is
forced through a glass fritted tube to vaporize chemically reduced mercury.
Most analytical methods for the determination of total mercury in bio­
logical material depend upon the complete destruction of organic matter without loss
of mercury from the system.
Dry ashing should therefore be avoided s ince mercury
volatilizes at relatively low temperature.
Hatch and Ott (1968) have described a wet oxidation method in which the
s ample is digested with sulphuric acid and oxidized with permanganate .
After a
one step reduction, the mercury is vaporized and swept into the optical path of the
atomic absorption system.
.,
A recorder connected to the AA system plots the s ample
absorbance which has a linear relat ionship to concentration
•
A modification of the Hatch and Ott (1968) procedure has been used in
this lab with good reproduc ibility.
This procedure calls for s ample digestion with
a 2 : 1 sulphuric-nitric acid s olution on a water bath at 5� C overnight .
It was found
41
that shorter digestion periods are insuffic ient for the complete destruct ion of organic
m atter. Samples are t he n trans formed quant it at ively to BO D bottles and further
oxid ized w ith a minimum of perm anganate and persulfate . The excess oxidizing
agents are reduced w ith a solut ion of sodium chloride -hydroxylamine sulfate . fhe
result i ng cle ar solut ion is reduced w ith st annous sulfate and immediately c onnected
to an aer at ion as sembly . A peristalt ic pump s weeps t he mercury vapor into the
opt ic al path of the atomic absorpt ion of mercury analyzer (such as the Perkin
Elmer MAS 50) system . A drying tube filled w ith magnes ium perchlorate should
be placed between the s ample and the cell to prevent moisture from enter ing the
cell . St andards m ay be prepared by spiking redist illed w ater and adding all of
the reagents used.
Contam inat ion of BO D bottles becomes a m aj or problem if great c are is
not taken in cle aning. The suggested cle aning procedure is as follows : wash BO D
bottles w ith hot , s oapy water immediately after use. Hot water should be used ex­
clus ively for washing and rins ing. The bottles should then be filled w ith concen ­
trated nitric acid several hours before reuse . After emptying the ac id, the bottles
should be rins ed w ith t ap water followed immediately with dist illed w ater. The
glass fritted aeration tube s hould be soaked in soapy water when in use to
prevent hiJh blank values .
Reagents
IJi'04
1.
CoQ.centrated
2.
Concentrated HN�
3.
KMn04 5 % W/V solut ion
4.
Ka Sa 08 5 % W/ V s olut ion
42
5.
Sodium chloride-hydroxylamine sulfat e .
( 1 2 gm NaCl & 1 2 gm hydroxylamine
sulfate per 100 ml redist illed water ) .
6.
� S04 ) .
Stannous sulfate (25 gm/250 m l of 0 . 5 N
Procedure
1.
We igh 0 . 5 gm of frozen t issue into a 100 ml be aker.
2.
Add 4 . 0 ml of cone . H2 S04 and 2 . 5 ml of cone . HN03 •
3.
Cover beaker with a watch glass and place i n a water bath at 5 80 C overnight .
4.
Carefully transfer the s ample s olution t o a BOD bottle , w ashing the original
s ample be aker with redistilled water .
5.
Dilute s ample s olut ion t o 100 ml and add 1 ml of KMn04 solut ion.
6.
Shake and add addit ional port ions of KMn04 unt il the purple color pers ists at
le ast 15 minutes .
7.
Add 2 m l of K S2 O solution and allow to st and 30 minutes .
2
e
8.
Add NaCl-hydroxysulfate i n 2 m l increments until a clear solution is observed.
9.
Add 5 ml of stannous sulfate and immediately attach t o aer ation assembly .
10 .
From recorder pe ak he ight , find the
pg
Hg from a c al ibration curve.
43
3.
O THER MET AI..S
For the analys is of other met als in biologic al s amples the init ial digest ion
is the s ame as that described in Sect ion B above . O nce the proper dilut ion of the
s ample is m ade s o that the metal concentrat ions are in the optimum working range
but prior to analys is , the matr ix of the s ample solution must be established. In the
c ase of arse nic and mercury, the problem w ith matrix interferences is corrected
essent ially be a separ at ion technique. For other metals , separ ation is t ime con­
suming, difficult and not necessary if proper steps are taken.
Atomic absorption spectrophotometry is relat ively free of interfere nces
in comparison to other spectral methods . However , interfere nces do exist , the
most serious for heavy met als being molecular absorpt ion. This interference is
due to blocking or absorbing of some of the light pas s ing through the flame . C alc ium
is reported to be the most serious interfer ing e lement ( Angina and Billings , 1967 ) ,
however , other specific element interferences i n trace met al analys is have been
observed and are listed in Table 3 for the metals of concern here . Biologi c al s am ­
ples are p art icularly suscept ible to this type of interference bec ause of the high
concentrat ions of alkali and alkaline e arth met als present . Eliminat ion of this
type of interference by standard-add ition is not effective s ince the s ame amou nt
of interference s ignal is present in s ample and s piked s ample .
The most effect ive means of dealing with the interference is by s imply
determ ining the amount of interfering ele ments present in the s ample . By pre ­
paring a trace blank and stadards w ith the s ame concentrat ion of interfering elements
found i n the s ample , the interfac ing s ignal is subtracted from the tot al s ignal .
44
T AB LE 3
SP E C I FIC E LEMENT INTERFERENCES
Met al
Calc ium
Magnes ium
Sodium
Pot as s ium
Iron
C admium
X
X
X
X
X
Cobalt
X
X
X
X
X
Copper
X
X
X
X
Iron
X
Lead
X
X
X
Manganese
X
X
X
Nickel
X
X
X
X
Silver
Zinc
X
X
X
X
X
45
Matching s ample and s t andard m atrices of every s ample is virtu ally
impos s ible and unneces s ary. At concentrat ions of the interfering element where
its rat io to the concentrat ion of the element of concern is less than 500 : 1 , the
interference is almost undetectible . At concentrations exceeding this the interfere nce incre as es continuously with increas ing concent rat ion of the interfering
e lement . It is only important however that s ample and st andard matrices do not
differ by more than about 500 ppm in the concentrat ion of the interfering element
for acceptable pre c is ion. This therefore allows for grouping of s amples w ith
s im ilar mat r ices .
Of all the met als considered in this manu al, atom ic absorption is inade­
quate only for the analys is of lead at the concentrations commonly occurring in
m arine organisms . The sensit ivity of the le ad analysis and the common interfer­
e nces encou ntered with m arine s amples m ake atomic abs orpt ion of little value
for many of the larger organisms such as finfis h. Samples containing higher con­
centrat ions of lead , such as mollusc , however, c an be adequately analyzed by
this method. Anodic stripping may provide a more amenable solution to this prob­
lem for l aboratories not owning m as s spectrometers .
46
P ART Ill
SEDIMEN T SAMP LES
Atomic absorpt ion spectrophotometr ic techniques are quite adequ ate for
met al analyses at the r ange encountered in organic rich, clay rich mar ine and
estuar ine sediments .
Procedures for analys is of m arine sediments by atom ic ab­
sorpt ion spectrophotometry are essent i ally analogous to those des cribed for
biological mater i al us ing standards made up to duplic ate the m atrix of the s amples
(See P art II, Section C 3 ) .
The m ain difference i n the analyt ic al techniques used
for these two types of s amples is in the digestion procedures .
Several different
d igest ion procedures are available which allow the analys is of different fr actions
of a given sediment .
m anu al .
Essentially three diges ion techniques are cons idered in this
These are : ( a) total digestion, whe n the tot al concentrat ion of the met al
in the sediment s ample is des ired,
(2) preferential le ach, when an est imation of ·
the concentration of authigenic met als and those absorbed on clay part ic les or
assoc iated w ith c arbonate mater ial, is des ired, and (3) preferent i al digestion of
the organic fraction of the sediment , when an estimate of the amou nt of metals
assoc iated with organic m atter is desired .
The first two d igestion techniques w ill be described below.
technique is essent i ally the s ame
as
The third
that used for the digestion of biologic al materials
for mercury analys is as given above (Sect ion C2 of Part II) .
This technique pre ­
ferenti ally destroys organic m atter; however , it will also attack authige nic and
abs orbed phases as well.
Its only advant age over the "prefere ntial leach" is that
it destroys organic material th at the preferent ial leach will not .
47
A.
TO TAL DIGESTIO N O F SEDIME NT SAMP LES
Sed iment s amples m ay be tot ally digested w ith perchlor ic ac id afte r the
removal of s il ic a.
This m ay be accomplished with hydrofluoric ac id .
heating, the s il ic a is volatilized as s ilicon tetrafluoride .
Upon gentle
After the complete des ­
truction of organic matter with perchloric and nitric acids , the res idue may be brought
into solut ion with 1 : 1 hydrochloric ac id .
This analytical procedure follows that
des cr ibed by Hendr ick ( 1 9 6 8 ) .
Digestions should b e c arr ied out in teflon beakers having high ac id and
he at res istanc e .
The beakers and all glassware used should b e c le aned before use
by submerging in hot nitric acid and rinsed with redist illed water.
Reage nts
1.
Hydrofluoric ac id (48%) .
2.
Nitric ac id (cone . )
3.
Perchlor ic acid ( 70%) .
4.
Hydrochloric acid (cone . ) .
Procedure
1.
Weigh 0 . 5 0 gm s ample onto weighing paper.
2.
Cover the bottom of a teflon be aker w ith redist illed water.
Dump the we ighed
s ample into the water .
3.
Add 1 5 ml of H F and 10 ml cone . HNq� to e ach s ample .
4.
Cover with a teflon beaker cover and allow t o st and two hours .
5.
Add 2 ml of HC10 and heat on hot plate at low temperature (s ett ing of 3) until
4
dense fumes of perchloric ac id sub s ide .
48
6.
Allow to cool and wash t he s ides of the be aker with a minimum of redistilled
water.
7.
Evaporate again to drynes s .
8.
Diss olve the res idue with 4 m l of hot 1 : 1 HCl and dilute t o a 10 . 0 m l volume .
9.
Determine m atrix and prepare s ample accordingly.
49
B.
PRE F E RENTIAL LEACHING O F TRACE METALS FROM SEDIMEN TS
Chester and Hughes ( 19 6 7 ) have investigated several chem ic al techniques
for the separ ation of part it ioned trace met als from pe lagic sediment s .
Among t he
techniques investigated are : the use of EDTA (Goldberg and Arrhenius , 195 8 ) ,
1
M - hydrochlor ic ac id ( Arrhenius and Korkish , 1 95 9 ) , dilute acet ic ac id (Hirst
and N ic holls , 1 9 5 8 ) , and reducing agents ( Arrhenius and Korkish, 1959) .
The effect
of EDT A was found to be very slow and the destruct ion of EDTA was found to be diffi­
cult .
Ray , et al . ( 1 9 5 7 ) have s hown that 1 M - hydrochloric ac id will attack the lattice
structures of certain clay minerals .
The effect of dilute acetic ac id on the ferrous
and m anganese oxide phases of ferro-manganese nodules was determined by thes e
authors as wel l .
The ir findings indicate that dilute acet ic acid i s not sufficie nt t o
completely dissolve the iron oxide present in the nodules .
Reducing agents were in­
vest igated and the use of 1 M hydroxylamine hydrochloride was found to dissolve
50% of the iron oxide present .
A combined acid-reduc ing age nt solution was invest i ­
gated by Chester and Hughes ( 1967) and fou nd to dissol ve 90 % o f the tot al iron and
9 6 % of the total manganes e from nodules .
They also investigated the effect of the
comb ined reagent on other maj or pelagic sediment s .
Their results indicate that the
trace met als leached from clay m iner als is c omp atible with that expected to be found
in non-lattice pos itions .
Reagents
1.
Ac�tic acid 35% (v/v) .
2.
Hydroxylam ine hydrochlor ide 2 5 % ( v/v) solut ion.
3.
M ixed acid-reduc ing re agent .
and 350 ml of 35% acet ic ac id) .
( Mix 150 m l o f 25% hydroxylam ine hydrochloride
50
4.
N it ric ac id (cone . ) .
Procedure
1.
Place 0 . 1 gm of powdered air dried sediment in a 50 ml Erlenmeyer flas k.
2.
Add 1 0 ml of t he mixed re agent and stopper the flask.
( Do not use a rubber
stopper ) .
3.
*4.
Shake the flask o n a mechanic al shake r for four hours .
Filter the s olut ion through a Whatman #40 filter paper and wash the res idue
and filter paper with a s m all volume of redistilled water .
5.
Add 1 ml of cone . HN03 t o the filtrate .
6.
Evaporate the solut ion at low temperature to near dryness unt il strong fu mes
of HN�_, are e volved.
7.
Cool the filtrate and d ilute to 5 . 0 ml i n a volumetric flask w ith redis t illed
water.
* Samples s hould be c arried through this step in one working day.
51
PART I V
ANODIC STRIPPING VOLT AMMETRY
Sea water is an ideal medium for anodic str ipp ing voltammetry s ince it
provides an excelle nt electrolyte s olution.
The technique achieves great sens itivity
by electrolyt ic ally reducing trace m et als on a mercury electrode for a given length
of time .
The met als are then reoxidized (stripped) from the electrode by line arly
varying the pote nt i al in the pos it i ve d irect ion over a short t ime .
By monitoring the
anodic curre nt dur ing reoxidation of the metal , the pe ak current response corre ­
ponds to the qu ant ity of met al in solution.
Theoretic ally , the technique is applic able
to any met al which c an undergo a reduction react ion at -1 . 5 volts versus a silver­
s ilver chloride electrode.
The b as ic components of a convent ionally used ASV
system is shown in Figure 3.
In our laboratory , McKee -Pederson electrometric system ( MP-100 0 ) is
used as polarograph in conjunction w ith a Hewlett Packard X-Y recorder ( 7034A) .
The instrument al set up includes a m illivolt source, an integrator for produc ing a
voltage ramp , an ope r at ional amplifier , and a chopper stab ilizer module for accurate
potent i al me asurements .
Matson (19 6 8) and F itzgerald ( 1 9 70 ) have determined c admium, copper,
lead and zinc routine ly us ing a compos ite mercury gr aphite electrode ( C MGE) . The
C MG E cons ists of a graphite rod impregnated with paraffin under vacuum at high
temper ature .
The are a to be used for plating (4 . 0 cm2 ) is cle aned of wax and
polished with a very fine grade of emergy cloth, followed by filter paper .
film of mercury (2
x
A thin
l 0 -7 moles/cm2 ) is plated onto the polished ends of the electrode
at - 1 . 1 volts versus a s i lver-s ilver chlor ide electrode for 45 -60 minutes .
The
52
A SV
S yst e m
A g /Ag CI
e l ec t rode
P o l a r o­
graph
R e c o r d er
CMGE
·�
Pt e l e c t r o d e
FIGU R E
3
53
mercury is added as the chloride t o a 0 . 5 N NaCl solution for electroplat ing. The
mercury film should be completely removed and replated after 75 -100 analyses or
at least every three days . An increas ing b as e l ine m ay be evidence of a deteriorating
electrode . Often electrodes may be rej u venated by repe ating the polishing process
before replat ing the mercury film. A diagram of an ASV cell using the compos ite
mercury graphite electrode is shown in Figure 4 .
Quartz cells , 3 5 ml volume , are used for plat ing, howe ver , l arger ce lls
m ay be used with a longer plating tim e . The electrodes are c ontained in a machined
teflon he ad which provides a cover for the c e lls .
The silver-s ilver chloride refe rence electrode may be prepared by ano­
diz ing a 0 . 0 4 inch diameter silver w ire in a 0 . 1 N NaCl solution. The reference
and platinum counter electrodes are four inches in length and are isolated from the
s ample s olution by means of 4 mm teflon tubes fi.lled with 0 . 5 N NaCl and s e aled at
the bottom with leached vycor plugs .
In order to rid the s ample of diss olved oxygen and provide a me ans of
st irring, nitrogen is bubbled through the s olut ion at a rate of 130- 160 ml/min.
before and during the plat ing proces s . Teflon tubing stiffened with platinum wire
and terminating just below the CMGE s erves as the bubbler .
The plat i ng t ime for a 2 5 ml s ample is 1 000 sec . , w ith a potent i al of - 1 . 1
volts vs . Ag/ AgCI . Just prior to the complet ion of the plat ing step the nitroge n
bubbling should be completely stopped. The optimum s c an rate for a volt age ramp
of - 1 . 1 to -0 . 2 is 16 . 7 mv/sec .
54
P t c ou n t e r e I
e
c t rode
\
f
C M GE
/
�
Ag / A g C I e l e c t r o d e
�
T e f l on
N2 Tu be
,.
,. n-Jo�,___- T e f l o n H e a d
,.
,.
,.
,.
/
,....
l.._
_
_
Q u a rtz V i a l
Lea ched
Vy c o r
F I GU R[
4
A SV
Ce l l .
Plugs
55
The method o f st andard addit ion i s used t o determine metal concentrat ions
from peak heights . Difficulties m ay arise in providing water suit able for prepar i ng
st andards { i . e . , f:ree of met al contaminant s ) . This m ay be elim inated by the use of
a reagent cle aning system which cons ists of a large flat bottom flask, 2 -3 pounds of
mercury met al , a s ilver/s ilver chloride electrode , and plat inum wire ( Fig . 5 ) .
Platinum wire sealed i n pyrex tubing, isolat i ng the wire from the syste m , is placed
in a mercury pool at the bottom of the flask. A negative potent i al with respect to a
Ag/AgCl e lectrode is m aintained between the mercury pool and a platinum counter
e lectrode near the surface of the solution to be purified. The Ag/AgC l and platinum
counter e lectrodes are isolated from the system by me ans of teflon tubing sealed at
the bottom with vycor plugs and containing a reservoir of 0 . 5 N NaCl. Met als c apable
of u ndergoing a reduct ion re action at
-1. 5
volts vs . Ag/AgC l form an amalgam with
the mercury. N itrogen bubbling through the system removes diss olved oxygen and
provides a high purity stirring mechanism .
Fitzger ald (1 9 70 ) has demonstrated the usefulness of anodic stripping
volt am metry in studying trace met al spe c i at ion. Upon ac idific at ion of tlie s ample
up to pH = 3 , the copper response incre as es . Further ac idification increas es the
potent ial at which hydrogen is generated, therefore , limiting the quant ity of ac id
which may be added. According to Fitzger ald ( 1 9 70 ) , ac idifi c at ion rele ases se­
questered metals from we akly ac idic complexing agents and only very stable complexes
exist at a pH of 3 . In order to· determine t ot al metal concentration, Fitzgerald
:1 9 70 ) suggests photo -oxidat ion with an ultra-violet l amp. This is favored over
other methods bec ause of its s implic ity and due to cont am inat ion c ons iderations .
57
Armstrong, e t al . (1966 ) used a 1 200 W lamp t o completely oxidize organic compounds
in sea water . With this type of syste m , howev er, cooling of the s ample during irra­
diat ion is requ ired in order to elimi nate boiling of the s ample.
58
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