411
Ailalyticu Chimica A~'ta, 78 (1975) 411-422 ,~-S~Elsevier Scientific Publishing C o m p a n y . A m s t e r d a m - Printed in The N e t h e r l a n d s
A FLUORESCENCE METHOD FOR STUDIES OF SPENT L I Q U O R A N D H U M I C S U B S T A N C E S IN SEA W A T E R
SULFITE
T O M A L M G R E N . B J O R N J O S E F S S O N ~lnd G U N N A R N Y Q U I S T
Depup'tt~letlt o f Alutl)'tical Chemi.s'tr)'. Utlit,ersity of Gothenl~to'.q. Fack. S.402 2(I Giitehorq 5 (Swedetl ) (Received 26th F e b r u a r y 1975)
Spent liquors f r o m sulfite p u l p mills are d i s c h a r g e d into the Baltic Sea at different locations. T h i s a d d i t i o n o f c o n s i d e r a b l e a m o u n t s of o r g a n i c material is o f special interest for t w o m a i n r e a s o n s : (i) tracing the water flow p a t t e r n using the c o n c e n t r a t i o n d i s t r i b u t i o n of the o r g a n i c m a t e r i a l s ; a n d (ii) p o l l u t i o n , w h i c h m a y c a u s e d a m a g e to fish resources. C o m m o n m e t h o d s for the d e t e r m i n a t i o n of sulfite p u l p w a s t e s are u.v. s p e c t r o p h o t o m e t r y l'z, the P e a r I - B e n s o n c o l o r i m e t r i c n i t r o s o p r o c e d u r e 3"4 a n d s p e c t r o f l u o r i m e t r y s-s. S e r i o u s s h o r t c o m i n g s of the u.v. a b s o r p t i o n m e t h o d s a n d the n i t r o s o m e t h o d ~tre t h a t they are n o t specific for spent suifite l i q u o r b e c a u s e h u m i c s u b s t a n c e s a n d o t h e r m a t e r i a l s interfere. T h e present i n v e s t i g a t i o n was carried out in o r d e r to e s t a b l i s h a p r o c e d u r e by w h i c h the c o n c e n t r a t i o n o f s p e n t sulfite l i q u o r in different w a t e r s m i g h t be d e t e r m i n e d w i t h r e p r o d u c i b i l i t y , specificity a n d rapidity. T h e m a i n c o n s t i t u e n t s in s p e n t suifite liquors a r e w a t e r - s o l u b l e lignin sulfonates (60-65'X,) a n d sugars ( 2 5 /I )./ ) . Since the lignin s u l f o n a t e s are the m o r e conservative material, different m e t h o d s have been p r o p o s e d to f o r m specific c o m p o u n d s f r o m t h e m such as vanillin. N i t r o b e n z e n e o x i d a t i o n of lignin sulfonates, however, is n o t a specific test for iignin sulfonates, since lignin a n d h u m i c s u b s t a n c e s also give vanillin a c c o r d i n g to H o r n k e a n d H e i n i s c h 9 a n d M o r r i s o n ~°. T r e a t m e n t of lignin sulfonates with h o t alkali to f o r m vanillin is a better m e t h o d , since u n s u l f o n a t e d lignins form vanillin o n l y in p o o r yield. T h e e x t r a c t e d vanillin c a n t h e n be identified by p a p e r or gas c h r o m a t o g r a p h y 11 T h i s m e t h o d is, h o w e v e r , very t i m e - c o n s u m i n g a n d difficult to c a r r y o u t o n b o a r d ship. Lignin s u l f o n a t e s o l u t i o n s are highly fluorescent a n d c a n be d e t e c t e d at very low c o n c e n t r a t i o n s . F l u o r e s c e n c e s p e c t r o m e t r y also offers the a d v a n t a g e s of sensitivity, specificity a n d s p e e d o f analysis. T h e d e t e c t i o n limit in n a t u r a l w a t e r s is c o m plicated by the presence o f h u m i c substances, which also b e l o n g to the g r o u p o f p o l y p h e n o l s , a n d possess similar fluorescence characteristics, as has b e e n s h o w n by T h r u s t o n ° a n d A l m g r e n a n d J o s e f s s o n s. In earlier work, a s t a n d a r d a d d i t i o n m e t h o d was p r o p o s e d w h e r e the i n t e n s i t y o f the b a c k g r o u n d h u m i c s u b s t a n c e s was first d e t e r m i n e d in river w a t e r s a m p l e s collected a b o v e the d i s c h a r g e site o f t h e s p e n t sulfite liquors. In t h e sea it is difficult to establish the b a c k g r o u n d o f h u m i c substances. In the S c a n d i n a v i a n area, the c o n c e n t r a t i o n is m o s t l y d e p e n d e n t o n the o u t f l o w from rivers w h i c h are rich in h u m i c substances. B e c a u s e o f d i s p e r s i o n effects, the c o n c e n t r a t i o n o f h u m i c substances is to s o m e e x t e n t inversely p r o p o r -
412
T. ALMGREN. I]. JOSI!F'SSON. G. NYQUIST
tional to the salinity t-' j'~ Tile p r e s e n t r e s e a r c h w a s d i r e c t e d to establislaing .'l s u i t a b l e s t a n d a r d for w a t e r - s o l u b l e h u m i c s t t b s t a n c e s r e p r e s e n t a t i v e o f the w a t e r s s t u d i e d . An attenapt w a s m a d e to d i f f e r e n t i a t e b e t w e e n the tttnount ot" spent sulfite l i q u o r as lignin sulfonatt2s a n d the c o n c e n t r a t i o n o1' httnaic s u b s t a n c e s . I-X P l" R I ~1 t: N "I'AL
.'llJPtlV~lltts All ~]LlOl'eSCUlltde e x c i t a t i o n ttlld e m i s s i o n spectra w e r e tllettstlred Oll tin Aminco--Izlownmn S P F s p e c t r o f l u o r i n a e t e r . T h e excitation s o u r c e w a s e i t h e r a highi n t e n s i t y x e n o n 1,inlp i ~ r o d u c i n g a COlltinl.lOtiS spectruna with pettks tit 4 0 0 nm a n d tit 900 11111 ( b e y o n d the r a n g e o f the i n s t r u m e n t ) , o r ;i high=intensity x e n o n - - n i e r c u r y lanlp producing- high i n t e n s i t y at the v a r i o u s m e r c u r y lines b y s u p e r i m p o s i t i o n of" tile x e n o n c o n t i n u u m . T h e i n s t r u m e n t w a s s u p p l i e d with a i ~ h o t o m u l t i p l i e r t u b e ( A m i n c o - B o w m t m R I O 6 ) with :m o p t i m a l w o r k i n g rang-e o f 3 0 0 - 5 0 0 nm. By using- a r a t i o system, w h e r e the b e a m is split into reference a n d s:tmple be,'tms, varitltions in iaml~ intensity a r e a u t o n l a t i c a l l y nullified. T h e sensitivity is d e c r e a s e d b y tipp t ' o x i m a t e l y 1(1",, w h e n this item is used, b u t the increase in s t a b i l i t y w a s c o n s i d e r e d m o r e i m p o r t t t n t h~l" q u a n t i t : l t i v e tyleztstiremt~nls. T h e i n s t r t t m e n t wits a l s o u s e d for p h o s p h o r e s c e n c e analysis. A rotating- shutter ( A m i n c o - - B o w m a n ) w a s a t t a 6 h e d to the cell h o l d e r , a n d p e r m i t t e d septiration o f different e m i s s i o n lifetimes b y varying- the specct o f r o t a t i o n . F o r till fluorescence n l e a s u r e m e n ' t s , l - c m q u a r t z cells w e r e used. P h o s p h o r e s c ~ t l c e n l e a s u r e m e n t s were c a r r i e d o u t tit 7 7 K in tt q t m r t z ctq~illary t u b e ttnd a D e w a r b o t t l e with the b o t t o m tip c o n s t r u c t e d o f qtttit'tz. Ultravioh~t a b s o r p t i o n d a t a tit discrete wavelen,.uths w e r e o b t a i n e d on an U n i c a n l S P 5 0 0 s p e c t r o p l a o t o m e t e r " u.v. t i b s o r p t i o n s p e c t r a of lignin s t d l b n a t e s a n d h e r o i c s u b s t a n c e s w e r e r e c o r d e d o n a B e c k m a n Acta irl spectrol'~hotonaeter. Q u a r t z cells with I- a n d 10-cm p a t h l e n g t h s were used for the a b s o r p t i o n m e a s u r e m e n t s .
S(onplinq pi'oc'edure All s,'tmples ot" n u t u r a l w a t e r w e r e t a k e n with a 1-1 R u t t n e r plastic sampler. T h e s a l n p l e s were g-enerally s t o r e d in 0.5-1 d a r k glass bottles, b u t for c o m p t w i s o n s o m e d u p l i c a t e s a m p l e s w e r e s t o r e d in clem" glass bottles. Tile t t n a l y s e s w e r e c a r r i e d o u t as s o o n ;.is p o s s i b l e w i t h i n 3 d a y s : in the m e a n t i m e , tile b o t t l e s w e r e s t o r e d at t t b o u t 4 C. T h e s a m p l e s wtzre filtered t h r o u g h a 0.45-i~m M i l l i p o r e filter to r e d u c e particle scattering. S(llllp/lll,¢J (ll'(.'(l.'; T h e n l e t h o d w a s t e s t e d d t l r i l l g the p e r i o d f r o m O c t o b e r 1972 t o J,'lllkl;.tl'y 1974. T h e s e tests w e r e m a d e in t w o different types of w m e r . T h e first area s u i t a b l e Ibr this p u r p o s e w a s l-lan/5 B a y (Fig., 1). w h i c h is a shtdlow b a y w i t h m o d e r a t e w a t e r e x c h a n g e in the s o u t h - w e s t p a r t o f the Baltic Sea. T h e r e is a high n u t u r a l c o n t e n t ot" h e r o i c s u b s t a n c e s togt2ther w i t h lig.nin sultbn:ttes from the w a s t e w a t e r o f tt pai~er mill. T h e naouth o f tile o u t l e t p i p e is tit s t a t i o n 15. T h e o u t p u t o f Iignin s u l f o n a t e s is 1 5 . 1 0 " kg y e a r - t , w h i c h is nearly 2000 kg- I1 -~ T h e s e c o n d test area w a s the i n n e r p a r t o f the tirchipelttgo o f the S w e d i s h w e s t coast. H t t v s t e n s l ] o r d ,'ind ByWord
SPENT SUI+I.TI'F+ LIQUOI,P, IN SEA W.,Vrl)I+,
413
tire situtitcd inside tile isl;lnds O r u s t ;ind Tj~3rn. A snl;ill river ('4 nl 3 s - i ) rl.lns into the Bvl]ord. The w'titcr ~xtcll;.lnl~ l')¢lw~il the fiords i.intt thu sou (Sk;iti~l'r,'ik) is i'csti'ictud. "rhc salinity ()1" the w;itcr tlbo)vo the pycno~:iinu (;it ;.ibout 12 nl) is ;Ibotlt 24 i ' nl,linly ~)OCtltl.,.;g t)r d i l u t i o n of tile N o r t h S~tl W L i t o r w i t h B;iiti¢ Sea W;.lll21", Tllci'c tlrl,' lit) p;lpur inills in thu vicinity i.int| tilu (dt)ncolltr;liion oP Iignin sulfon;itus s h o u l d thcretbre b~ low.
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H I I g l I1~I VIll I
!
h
F'i t. 1. T h e h i c u t k ) n ~1' I h u I hu m ; I p t)l' S(.:;llldhl;I ~,i;I.
.~l;.ilit~ils
14 1~ in ihu
I
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It~l)'. I n . s u r l c d :
IS O0 The
loc~ilkin
tit" I hlliL~
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Chemictds All chcmic;lls w~l'e of ;inal)'tical grade. Trir)ly distilled w a t e r was tlsuct ;ind gl'~;lt (~I.i!'0 I;ikl~ll to avoid tdonttlmin;itiO)ll from pt,)iyetilyl¢ll(.' tl.lbing. Pr¢,p(u'ed .~'l(llld(ird.~ S o d i u m liL~nin sulfonatc was prcl'xu'cd by the F i n n i s h P:q~cr mid Pulp Ruscarch Institute and is cornmcrci~dly av~dlahlc. It was c×tr;ictcd Ii"om :l tccllnicul ucidic ~alciurn hydro~cnsull'itc c o o k by ion-pair amine e×tr~tction. T h e a b s o r p t i v i t y at 280 n m w;ts 13.7 I g - i c m - ~ (Fig. 2). T h e lignin s u l f o n a t ¢ w u s d i s s o l v e d in triply distilled wutcr. The h u n l i c subst;inccs were isol;itect fi'onl b r o w n i s h river w a t e r lute in the aLItLIllln. "l'hi2 w a t u r was first punil3ed t h r o u g h a sl.inct filter pa(~kud w i t h gruvel ;it the top aild fine sc;.l sand tit the [')oitonl; the dissolved h u n l i c subsi;lll(d~s ( o r ftllviu ;lcids) w~rc then unrichcd on ;l chelating resin ;is described below. Thu technique is sinlilar to thai (.tuscribcd b)' Siegel ;lnd D c l u n s l'~ for ;inlino ucids, b u t ihu resin was lo;idcd w i t h h ' o n ( l l l ) instc,id ot" COpl~er. h'oll ;.iprl~tii's to I,)t~ i.i vi2i.y ut'ficiunt Iigtind nlet;il tbr ftilviu tlcids i~'
414
T. ALMGREN. B. .IOSEFSSON. (3. NYQUIST
].e Z.S 10 IS
',..
I0
... ..........
OS
• ~00
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I ~SO
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WAVELENGYH
"'l'l-~- "' ''1~' ' ' ' ' ' ' ~ ' ' - ' ' ' ' ' llO
.....
400
i ........
i', 4
SO
(rim)
Fig. 2. Absorption spectra for 100 p.p.m. Ilumic substances ( . . . . . ) and 100 p.p.m, lignin sulft3natcs ( - - - ) with a cell path length or I cm. A c o l u m n (50 x 2 0 0 m m ) was l o a d e d with 50-100 mesh Chelex I00 in the i r o n ( I l l ) form a n d a t t a c h e d behind the sand filter. T h e river w a t e r was p u m p e d at a rate of 5 I h - ~ . After 100 I o f w a t e r had passed t h r o u g h , the c o l u m n was washed with distilled w a t e r and b r o u g h t back to the l a b o r a t o r y . A 5 x 20 m m Chelex 100 c o l u m n in the NH,~ form was a t t a c h e d in series after the iron c o l u m n ; this c o l u m n t a k e s up iron a n d free metal ions from' the eluant. T h e r e t a i n e d h u m i c acids were eluted with 500 ml of 3 M a m m o n i a solution. T h e ammoni~t was e v a p o r a t e d in a r o t a r y e v a p o r a t o r leaving the h u m i c substances in a dry form. By this m e t h o d . 200-300 m g o f h u m i c material was isolated from the river w~tter. T h e material was,,, easily redissolved in distilled water a n d had the same fltlorescence characteristics as the river w a t e r itself. S t a , d a r d achlitio, t e c h , i q u e This m e t h o d is a n extension o f the p r o c e d u r e described by Ahngren a n d Josefsson a. T h e w a t e r samples were excited at 315 n m a n d emission spectra were r e c o r d e d f r o m 350 to 500 nm. The optimal c o n d i t i o n s w e r e selected by varying the slits, the a m p l i f i c a t i o n and the excitation w a v e l e n g t h . W h e n the only fluorescent sttbstances present were h u m i c substtmces, a straight line with a negative slope was o b t a i n e d o n plotting the emission intensity ves'sus salinity owing to the dilution effect: the c o n c e n t r a t i o n of h u m i e substances is low in the N o r t h Sea water. When, however, an effluent o f non-saline w a t e r c o n t a i n i n g large a m o u n t s o f lignin sulfonates was i n t r o d u c e d a positive deviation from the line o c c u r r e d (Fig. 3). F u r t h e r m o r e , on close inspection o f the s p e c t r u m , a shift o f the intensity m a x i m u m to shorter wavelengths, c a u s e d by lignin sulfonate emission, was observed (Fig. 4). Since the salinity range is n a r r o w in Hanti Bay, a s a m p l e o f average salinity (station 18), indicating no lignin sulfonate c o n t r i b u t i o n , was chosen as a s t a n d a r d . Different a m o u n t s o f lignin sulfonates were a d d e d to this s t a n d a r d . A plot o f the increase in intensity t.ersus the a m o u n t of lignin sulfonates a d d e d , p r o d u c e d a calibration curve which cot, ld be used for evaluating the c o n c e n t r a t i o n of lignin sulfonates in the samples.
SPENT
SULF'ITE
LIQUOR
415
IN S E A W A T E R
0 0 i
to
0
I
I
l.~O
I
I
r. JO
I
I
t4o SALINITY
I
I
~ "~o
:.tO
(O/oo)
Fig. 3. Relative Iluores~en¢e enfission intensity versus s~flinity Ik~r sam,pies from I la,~O Bay. Open ¢irulus represent S;llllpl~s with a Illqrked dccret~se in wavelength of m:l×inltam emission. The .ex~zilalion wavelength was 31.~ nm.
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I
]SO
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I
4 t0 WAVELENGTH
I
IJ0
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t~0
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I
4 l0
(nm)
Fig.. 4. Fluores~zenc;u e m i s s i o n spetzsra Ik~r 2.5 p . p . m , h u n l i ¢ st, b s t a n ~ e ' s ( . . . . . . ). ().5 p . p . m , lignin sull'ontlle ( ........ ) a n d a m i x t u r e o f 2.5 p . p . m , hun'fi~z substtln~zes a n d 0.5 p . p . m , l i g n i n sulfon,'lle ( .). T h e ux¢iltttion w a v u l e n ~ t h ,,vtls 315 n m . T h e a p p a r ~ t t t l s s ~ l t i n g s w e r e t h e s n m u in all t h r e e cases.
D u , I w , v e l e , g t h tc,clmiqtle T h e fluorescence peaks o f h u m i c substances a n d lignin sulfonates have intensity m a x i m a at different w a v e l e n g t h s (Fig. 4). T h e m a x i m u m for hunaic substances a p p e a r e d at 420--430 n m d e p e n d i n g to some extent on the excitation wavelength. Lignin sulfonates s h o w e d an intensity m a x i m u m at 390-400 rim. A m i x t u r e o f hurnic substances and lignin sulfonates gave an intensity maximuna s o m e w h e r e between 390.4400 nm and 420--.430 nm d e p e n d i n g on the c o n c e n t r a t i o n of the different c o m p o n e n t s ( F i g s . 4 a n d 5). T h e fluorescence intensity of lignin sulfonates was a b o u t 3 times stronger than the intensity of' h u m i c substances in the range
416
"r. A L M G R E N .
B. J O S E F S S O N .
C;. N Y Q U I S T
0.01--10 mg 1-I. T h u s a low c o n c e n t r a t i o n o f lignin s u l f o n a t e s c o u l d be detected in the presence of a rehltively high b a c k g r o u n d o f h u m i c substances.
,',0 ,~0 ,~. . . . . . . . . . . . . . . . . . . . . . . WAVELENGTH
',, ".
Into)
l:'ig. 5. I : l u t ) r c s c c n c c e m i s s i o n s p e c t r a li~i" d i f f e r e n t m i x t t t r c s o f lignill s u l f o n a t c s a n d h t l m i c s t d ~ s t a n c c s . 2.(1 itn(.I 1.0 ( ). 1.0 lind 2.0 ( . . . . . . ) l i n d 0.5 lilld 4.0 ( - ) r).p.ln, l i g n i n st, l l b n i l t e l i n d h u n l i c s u b s t a n c e s r e s p e c t i v e l y , w e r e used. Tht: e x c i t a t i o n w a v e l e n g t h wlts 3 15 n m .
If the ;lbsorption in the s.'lnaple is low ( A < 0.02). the flttoresccnce intensity is directly p r o p o r t i o n a l to the c o n c e n t r a t i o n o f the sttbstances giving rise to the fluoresccnce. T h e e q u a t i o n for the fltiorcscence intensity is then given by: /i = k i C , . ~ + k z C .
(1)
w h e r e I,.. is tile fluorescence intensity. C.,~ a n d C,~ are the c o n c e n t r a t i o n s of the two s u b s t a n c e s A a n d B. a n d kl and I
Ailalyticu Chimica A~'ta, 78 (1975) 411-422 ,~-S~Elsevier Scientific Publishing C o m p a n y . A m s t e r d a m - Printed in The N e t h e r l a n d s
A FLUORESCENCE METHOD FOR STUDIES OF SPENT L I Q U O R A N D H U M I C S U B S T A N C E S IN SEA W A T E R
SULFITE
T O M A L M G R E N . B J O R N J O S E F S S O N ~lnd G U N N A R N Y Q U I S T
Depup'tt~letlt o f Alutl)'tical Chemi.s'tr)'. Utlit,ersity of Gothenl~to'.q. Fack. S.402 2(I Giitehorq 5 (Swedetl ) (Received 26th F e b r u a r y 1975)
Spent liquors f r o m sulfite p u l p mills are d i s c h a r g e d into the Baltic Sea at different locations. T h i s a d d i t i o n o f c o n s i d e r a b l e a m o u n t s of o r g a n i c material is o f special interest for t w o m a i n r e a s o n s : (i) tracing the water flow p a t t e r n using the c o n c e n t r a t i o n d i s t r i b u t i o n of the o r g a n i c m a t e r i a l s ; a n d (ii) p o l l u t i o n , w h i c h m a y c a u s e d a m a g e to fish resources. C o m m o n m e t h o d s for the d e t e r m i n a t i o n of sulfite p u l p w a s t e s are u.v. s p e c t r o p h o t o m e t r y l'z, the P e a r I - B e n s o n c o l o r i m e t r i c n i t r o s o p r o c e d u r e 3"4 a n d s p e c t r o f l u o r i m e t r y s-s. S e r i o u s s h o r t c o m i n g s of the u.v. a b s o r p t i o n m e t h o d s a n d the n i t r o s o m e t h o d ~tre t h a t they are n o t specific for spent suifite l i q u o r b e c a u s e h u m i c s u b s t a n c e s a n d o t h e r m a t e r i a l s interfere. T h e present i n v e s t i g a t i o n was carried out in o r d e r to e s t a b l i s h a p r o c e d u r e by w h i c h the c o n c e n t r a t i o n o f s p e n t sulfite l i q u o r in different w a t e r s m i g h t be d e t e r m i n e d w i t h r e p r o d u c i b i l i t y , specificity a n d rapidity. T h e m a i n c o n s t i t u e n t s in s p e n t suifite liquors a r e w a t e r - s o l u b l e lignin sulfonates (60-65'X,) a n d sugars ( 2 5 /I )./ ) . Since the lignin s u l f o n a t e s are the m o r e conservative material, different m e t h o d s have been p r o p o s e d to f o r m specific c o m p o u n d s f r o m t h e m such as vanillin. N i t r o b e n z e n e o x i d a t i o n of lignin sulfonates, however, is n o t a specific test for iignin sulfonates, since lignin a n d h u m i c s u b s t a n c e s also give vanillin a c c o r d i n g to H o r n k e a n d H e i n i s c h 9 a n d M o r r i s o n ~°. T r e a t m e n t of lignin sulfonates with h o t alkali to f o r m vanillin is a better m e t h o d , since u n s u l f o n a t e d lignins form vanillin o n l y in p o o r yield. T h e e x t r a c t e d vanillin c a n t h e n be identified by p a p e r or gas c h r o m a t o g r a p h y 11 T h i s m e t h o d is, h o w e v e r , very t i m e - c o n s u m i n g a n d difficult to c a r r y o u t o n b o a r d ship. Lignin s u l f o n a t e s o l u t i o n s are highly fluorescent a n d c a n be d e t e c t e d at very low c o n c e n t r a t i o n s . F l u o r e s c e n c e s p e c t r o m e t r y also offers the a d v a n t a g e s of sensitivity, specificity a n d s p e e d o f analysis. T h e d e t e c t i o n limit in n a t u r a l w a t e r s is c o m plicated by the presence o f h u m i c substances, which also b e l o n g to the g r o u p o f p o l y p h e n o l s , a n d possess similar fluorescence characteristics, as has b e e n s h o w n by T h r u s t o n ° a n d A l m g r e n a n d J o s e f s s o n s. In earlier work, a s t a n d a r d a d d i t i o n m e t h o d was p r o p o s e d w h e r e the i n t e n s i t y o f the b a c k g r o u n d h u m i c s u b s t a n c e s was first d e t e r m i n e d in river w a t e r s a m p l e s collected a b o v e the d i s c h a r g e site o f t h e s p e n t sulfite liquors. In t h e sea it is difficult to establish the b a c k g r o u n d o f h u m i c substances. In the S c a n d i n a v i a n area, the c o n c e n t r a t i o n is m o s t l y d e p e n d e n t o n the o u t f l o w from rivers w h i c h are rich in h u m i c substances. B e c a u s e o f d i s p e r s i o n effects, the c o n c e n t r a t i o n o f h u m i c substances is to s o m e e x t e n t inversely p r o p o r -
412
T. ALMGREN. I]. JOSI!F'SSON. G. NYQUIST
tional to the salinity t-' j'~ Tile p r e s e n t r e s e a r c h w a s d i r e c t e d to establislaing .'l s u i t a b l e s t a n d a r d for w a t e r - s o l u b l e h u m i c s t t b s t a n c e s r e p r e s e n t a t i v e o f the w a t e r s s t u d i e d . An attenapt w a s m a d e to d i f f e r e n t i a t e b e t w e e n the tttnount ot" spent sulfite l i q u o r as lignin sulfonatt2s a n d the c o n c e n t r a t i o n o1' httnaic s u b s t a n c e s . I-X P l" R I ~1 t: N "I'AL
.'llJPtlV~lltts All ~]LlOl'eSCUlltde e x c i t a t i o n ttlld e m i s s i o n spectra w e r e tllettstlred Oll tin Aminco--Izlownmn S P F s p e c t r o f l u o r i n a e t e r . T h e excitation s o u r c e w a s e i t h e r a highi n t e n s i t y x e n o n 1,inlp i ~ r o d u c i n g a COlltinl.lOtiS spectruna with pettks tit 4 0 0 nm a n d tit 900 11111 ( b e y o n d the r a n g e o f the i n s t r u m e n t ) , o r ;i high=intensity x e n o n - - n i e r c u r y lanlp producing- high i n t e n s i t y at the v a r i o u s m e r c u r y lines b y s u p e r i m p o s i t i o n of" tile x e n o n c o n t i n u u m . T h e i n s t r u m e n t w a s s u p p l i e d with a i ~ h o t o m u l t i p l i e r t u b e ( A m i n c o - B o w m t m R I O 6 ) with :m o p t i m a l w o r k i n g rang-e o f 3 0 0 - 5 0 0 nm. By using- a r a t i o system, w h e r e the b e a m is split into reference a n d s:tmple be,'tms, varitltions in iaml~ intensity a r e a u t o n l a t i c a l l y nullified. T h e sensitivity is d e c r e a s e d b y tipp t ' o x i m a t e l y 1(1",, w h e n this item is used, b u t the increase in s t a b i l i t y w a s c o n s i d e r e d m o r e i m p o r t t t n t h~l" q u a n t i t : l t i v e tyleztstiremt~nls. T h e i n s t r t t m e n t wits a l s o u s e d for p h o s p h o r e s c e n c e analysis. A rotating- shutter ( A m i n c o - - B o w m a n ) w a s a t t a 6 h e d to the cell h o l d e r , a n d p e r m i t t e d septiration o f different e m i s s i o n lifetimes b y varying- the specct o f r o t a t i o n . F o r till fluorescence n l e a s u r e m e n ' t s , l - c m q u a r t z cells w e r e used. P h o s p h o r e s c ~ t l c e n l e a s u r e m e n t s were c a r r i e d o u t tit 7 7 K in tt q t m r t z ctq~illary t u b e ttnd a D e w a r b o t t l e with the b o t t o m tip c o n s t r u c t e d o f qtttit'tz. Ultravioh~t a b s o r p t i o n d a t a tit discrete wavelen,.uths w e r e o b t a i n e d on an U n i c a n l S P 5 0 0 s p e c t r o p l a o t o m e t e r " u.v. t i b s o r p t i o n s p e c t r a of lignin s t d l b n a t e s a n d h e r o i c s u b s t a n c e s w e r e r e c o r d e d o n a B e c k m a n Acta irl spectrol'~hotonaeter. Q u a r t z cells with I- a n d 10-cm p a t h l e n g t h s were used for the a b s o r p t i o n m e a s u r e m e n t s .
S(onplinq pi'oc'edure All s,'tmples ot" n u t u r a l w a t e r w e r e t a k e n with a 1-1 R u t t n e r plastic sampler. T h e s a l n p l e s were g-enerally s t o r e d in 0.5-1 d a r k glass bottles, b u t for c o m p t w i s o n s o m e d u p l i c a t e s a m p l e s w e r e s t o r e d in clem" glass bottles. Tile t t n a l y s e s w e r e c a r r i e d o u t as s o o n ;.is p o s s i b l e w i t h i n 3 d a y s : in the m e a n t i m e , tile b o t t l e s w e r e s t o r e d at t t b o u t 4 C. T h e s a m p l e s wtzre filtered t h r o u g h a 0.45-i~m M i l l i p o r e filter to r e d u c e particle scattering. S(llllp/lll,¢J (ll'(.'(l.'; T h e n l e t h o d w a s t e s t e d d t l r i l l g the p e r i o d f r o m O c t o b e r 1972 t o J,'lllkl;.tl'y 1974. T h e s e tests w e r e m a d e in t w o different types of w m e r . T h e first area s u i t a b l e Ibr this p u r p o s e w a s l-lan/5 B a y (Fig., 1). w h i c h is a shtdlow b a y w i t h m o d e r a t e w a t e r e x c h a n g e in the s o u t h - w e s t p a r t o f the Baltic Sea. T h e r e is a high n u t u r a l c o n t e n t ot" h e r o i c s u b s t a n c e s togt2ther w i t h lig.nin sultbn:ttes from the w a s t e w a t e r o f tt pai~er mill. T h e naouth o f tile o u t l e t p i p e is tit s t a t i o n 15. T h e o u t p u t o f Iignin s u l f o n a t e s is 1 5 . 1 0 " kg y e a r - t , w h i c h is nearly 2000 kg- I1 -~ T h e s e c o n d test area w a s the i n n e r p a r t o f the tirchipelttgo o f the S w e d i s h w e s t coast. H t t v s t e n s l ] o r d ,'ind ByWord
SPENT SUI+I.TI'F+ LIQUOI,P, IN SEA W.,Vrl)I+,
413
tire situtitcd inside tile isl;lnds O r u s t ;ind Tj~3rn. A snl;ill river ('4 nl 3 s - i ) rl.lns into the Bvl]ord. The w'titcr ~xtcll;.lnl~ l')¢lw~il the fiords i.intt thu sou (Sk;iti~l'r,'ik) is i'csti'ictud. "rhc salinity ()1" the w;itcr tlbo)vo the pycno~:iinu (;it ;.ibout 12 nl) is ;Ibotlt 24 i ' nl,linly ~)OCtltl.,.;g t)r d i l u t i o n of tile N o r t h S~tl W L i t o r w i t h B;iiti¢ Sea W;.lll21", Tllci'c tlrl,' lit) p;lpur inills in thu vicinity i.int| tilu (dt)ncolltr;liion oP Iignin sulfon;itus s h o u l d thcretbre b~ low.
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Chemictds All chcmic;lls w~l'e of ;inal)'tical grade. Trir)ly distilled w a t e r was tlsuct ;ind gl'~;lt (~I.i!'0 I;ikl~ll to avoid tdonttlmin;itiO)ll from pt,)iyetilyl¢ll(.' tl.lbing. Pr¢,p(u'ed .~'l(llld(ird.~ S o d i u m liL~nin sulfonatc was prcl'xu'cd by the F i n n i s h P:q~cr mid Pulp Ruscarch Institute and is cornmcrci~dly av~dlahlc. It was c×tr;ictcd Ii"om :l tccllnicul ucidic ~alciurn hydro~cnsull'itc c o o k by ion-pair amine e×tr~tction. T h e a b s o r p t i v i t y at 280 n m w;ts 13.7 I g - i c m - ~ (Fig. 2). T h e lignin s u l f o n a t ¢ w u s d i s s o l v e d in triply distilled wutcr. The h u n l i c subst;inccs were isol;itect fi'onl b r o w n i s h river w a t e r lute in the aLItLIllln. "l'hi2 w a t u r was first punil3ed t h r o u g h a sl.inct filter pa(~kud w i t h gruvel ;it the top aild fine sc;.l sand tit the [')oitonl; the dissolved h u n l i c subsi;lll(d~s ( o r ftllviu ;lcids) w~rc then unrichcd on ;l chelating resin ;is described below. Thu technique is sinlilar to thai (.tuscribcd b)' Siegel ;lnd D c l u n s l'~ for ;inlino ucids, b u t ihu resin was lo;idcd w i t h h ' o n ( l l l ) instc,id ot" COpl~er. h'oll ;.iprl~tii's to I,)t~ i.i vi2i.y ut'ficiunt Iigtind nlet;il tbr ftilviu tlcids i~'
414
T. ALMGREN. B. .IOSEFSSON. (3. NYQUIST
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Fig. 2. Absorption spectra for 100 p.p.m. Ilumic substances ( . . . . . ) and 100 p.p.m, lignin sulft3natcs ( - - - ) with a cell path length or I cm. A c o l u m n (50 x 2 0 0 m m ) was l o a d e d with 50-100 mesh Chelex I00 in the i r o n ( I l l ) form a n d a t t a c h e d behind the sand filter. T h e river w a t e r was p u m p e d at a rate of 5 I h - ~ . After 100 I o f w a t e r had passed t h r o u g h , the c o l u m n was washed with distilled w a t e r and b r o u g h t back to the l a b o r a t o r y . A 5 x 20 m m Chelex 100 c o l u m n in the NH,~ form was a t t a c h e d in series after the iron c o l u m n ; this c o l u m n t a k e s up iron a n d free metal ions from' the eluant. T h e r e t a i n e d h u m i c acids were eluted with 500 ml of 3 M a m m o n i a solution. T h e ammoni~t was e v a p o r a t e d in a r o t a r y e v a p o r a t o r leaving the h u m i c substances in a dry form. By this m e t h o d . 200-300 m g o f h u m i c material was isolated from the river w~tter. T h e material was,,, easily redissolved in distilled water a n d had the same fltlorescence characteristics as the river w a t e r itself. S t a , d a r d achlitio, t e c h , i q u e This m e t h o d is a n extension o f the p r o c e d u r e described by Ahngren a n d Josefsson a. T h e w a t e r samples were excited at 315 n m a n d emission spectra were r e c o r d e d f r o m 350 to 500 nm. The optimal c o n d i t i o n s w e r e selected by varying the slits, the a m p l i f i c a t i o n and the excitation w a v e l e n g t h . W h e n the only fluorescent sttbstances present were h u m i c substtmces, a straight line with a negative slope was o b t a i n e d o n plotting the emission intensity ves'sus salinity owing to the dilution effect: the c o n c e n t r a t i o n of h u m i e substances is low in the N o r t h Sea water. When, however, an effluent o f non-saline w a t e r c o n t a i n i n g large a m o u n t s o f lignin sulfonates was i n t r o d u c e d a positive deviation from the line o c c u r r e d (Fig. 3). F u r t h e r m o r e , on close inspection o f the s p e c t r u m , a shift o f the intensity m a x i m u m to shorter wavelengths, c a u s e d by lignin sulfonate emission, was observed (Fig. 4). Since the salinity range is n a r r o w in Hanti Bay, a s a m p l e o f average salinity (station 18), indicating no lignin sulfonate c o n t r i b u t i o n , was chosen as a s t a n d a r d . Different a m o u n t s o f lignin sulfonates were a d d e d to this s t a n d a r d . A plot o f the increase in intensity t.ersus the a m o u n t of lignin sulfonates a d d e d , p r o d u c e d a calibration curve which cot, ld be used for evaluating the c o n c e n t r a t i o n of lignin sulfonates in the samples.
SPENT
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Fig. 3. Relative Iluores~en¢e enfission intensity versus s~flinity Ik~r sam,pies from I la,~O Bay. Open ¢irulus represent S;llllpl~s with a Illqrked dccret~se in wavelength of m:l×inltam emission. The .ex~zilalion wavelength was 31.~ nm.
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Fig.. 4. Fluores~zenc;u e m i s s i o n spetzsra Ik~r 2.5 p . p . m , h u n l i ¢ st, b s t a n ~ e ' s ( . . . . . . ). ().5 p . p . m , lignin sull'ontlle ( ........ ) a n d a m i x t u r e o f 2.5 p . p . m , hun'fi~z substtln~zes a n d 0.5 p . p . m , l i g n i n sulfon,'lle ( .). T h e ux¢iltttion w a v u l e n ~ t h ,,vtls 315 n m . T h e a p p a r ~ t t t l s s ~ l t i n g s w e r e t h e s n m u in all t h r e e cases.
D u , I w , v e l e , g t h tc,clmiqtle T h e fluorescence peaks o f h u m i c substances a n d lignin sulfonates have intensity m a x i m a at different w a v e l e n g t h s (Fig. 4). T h e m a x i m u m for hunaic substances a p p e a r e d at 420--430 n m d e p e n d i n g to some extent on the excitation wavelength. Lignin sulfonates s h o w e d an intensity m a x i m u m at 390-400 rim. A m i x t u r e o f hurnic substances and lignin sulfonates gave an intensity maximuna s o m e w h e r e between 390.4400 nm and 420--.430 nm d e p e n d i n g on the c o n c e n t r a t i o n of the different c o m p o n e n t s ( F i g s . 4 a n d 5). T h e fluorescence intensity of lignin sulfonates was a b o u t 3 times stronger than the intensity of' h u m i c substances in the range
416
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B. J O S E F S S O N .
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0.01--10 mg 1-I. T h u s a low c o n c e n t r a t i o n o f lignin s u l f o n a t e s c o u l d be detected in the presence of a rehltively high b a c k g r o u n d o f h u m i c substances.
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l:'ig. 5. I : l u t ) r c s c c n c c e m i s s i o n s p e c t r a li~i" d i f f e r e n t m i x t t t r c s o f lignill s u l f o n a t c s a n d h t l m i c s t d ~ s t a n c c s . 2.(1 itn(.I 1.0 ( ). 1.0 lind 2.0 ( . . . . . . ) l i n d 0.5 lilld 4.0 ( - ) r).p.ln, l i g n i n st, l l b n i l t e l i n d h u n l i c s u b s t a n c e s r e s p e c t i v e l y , w e r e used. Tht: e x c i t a t i o n w a v e l e n g t h wlts 3 15 n m .
If the ;lbsorption in the s.'lnaple is low ( A < 0.02). the flttoresccnce intensity is directly p r o p o r t i o n a l to the c o n c e n t r a t i o n o f the sttbstances giving rise to the fluoresccnce. T h e e q u a t i o n for the fltiorcscence intensity is then given by: /i = k i C , . ~ + k z C .
(1)
w h e r e I,.. is tile fluorescence intensity. C.,~ a n d C,~ are the c o n c e n t r a t i o n s of the two s u b s t a n c e s A a n d B. a n d kl and I
