STUDIES ON ORGANIC FOULANTS IN THE SEAWATER
FEED OF REVERSE OSMOSIS
PLANTS OF SWCC1
Abdul Ghani I. Dalvi, Radwan A. Al-Rasheed
And Mohammad Abdul Javeed
Research & Development Center,
Saline Water Conversion Corporation
P.O.Box # 8328, Al-Jubail 31951, Kingdom of Saudi Arabia
SUMMARY
The inherent seawater characteristic is one of the main hurdles in success of any desalination
process. An inorganic constituent of seawater, which accounts for more than 99% of the
total ionic composition is a prime hindrance in multistage flash (MSF) desalination
technique.
The organic substances though negligible cause teething trouble in reverse
osmosis (RO) technique due to its fouling on the membranes. Ninety per cent of organic
constituents in seawater are humic substances. Method of isolation and purification of humic
substances which are about 2 ppm in seawater of Gulf region has been standardized and
gram quantities could be isolated using XAD –2 and XAD-7 resins from the selected sites of
Al-Jubail, Al-Khobar and Jeddah. Isolated humic substances have been characterized using
elemental, UV-visible and infrared spectroscopy and fluorescence techniques. Methods for
the estimation of humic substance also have been developed using UV-visible and
fluorescence spectropic techniques.
Humic substances contained about 80% of fulvic acid
and 20% of humic acid. Total acidity of Jeddah sample is 5.0 meq/gm whereas in Al-Jubail
and Al-Khobar samples are 3.36 and 3.23 meq/gm respectively. Various functional groups
like carboxyl, phenolic and alcoholic OH group, derivatives of benzene and group containing
sulphur could be detected and identified due to their characteristic absorption frequencies.
From the present study it is evident that nature of humic substances from Al-Jubail and AlKhobar are similar but on the contrary humic substance from Jeddah showed remarkable
compositional deviation specially in respect of sulphur content which is 10 % whereas it is
absent in Al-Khobar and Al-Jubail.
Samples from Jeddah indicated a favorable condition
for high bacterial activity in comparison with Eastern Province sites of Al-Jubail and AlKhobar.
1. Issued as Technical Report No. TR 3803/APP 95010 in November, 1999.
2247
Two methods have been developed for the estimation of humic substances.
UV-visible
absorption spectrometry method for the estimation needs isolation and preconcentration of
humic substance prior to their estimation.
Amount of humic substances estimated at Al-
Jubail, Al-Khobar and Jeddah by absorption spectrometry technique are 1.99, 1.93 and 1.2
ppm, respectively.
spectrometry.
Another method developed in this study involved fluorescence
The concentration of humic substance estimated by the technique after
preconcentration for Al-Jubail, Al-Khobar and Jeddah samples are 2.095, 2.06 and 1.39
ppm, respectively. Concentration estimated by two techniques is fairly in good agreement.
Estimation of humic substance, however, could be carried out by fluorometry directly in
seawater without isolation and preconcentration provided standards are run along with
samples to nullify the interference due to seawater matrix and its constituents.
Direct
estimation in seawater by fluorescence spectrometry is estimated at about 10-12% higher
concentration compared to isolated and preconcentrated samples.
1.
INTRODUCTION
Desalination in the broad sense is a process through which water of low salinity is produced
to an extent that it becomes potable. Among the known desalination processes, multistage
flash (MSF) distillation and reverse osmosis (RO) membrane filtration are most popular and
widely used techniques. Major chemical constituents (40000-50000 ppm) of seawater are of
inorganic origin and the minor (2-4 ppm) are of organic origin.
Though organics are
negligible in concentration as compared to inorganic constituents, they pose more acute
problems in reverse osmosis desalination process. It is well known that fouling in RO
membranes causes serious problems including (i) a gradual decline of membrane flux thereby
decrease in permeate production, (ii) an increase in ∆P thereby increasing requirement of high
pressure pump rating and (iii) degradation of membrane itself.
All these factors reflect on
the cost of water production. Hence, nowadays attempts are being made to deplete the
concentration of organic and some of the inorganic constituents from the feed to RO to
overcome these problems by various pretreatment methods. Other than conventional methods
such as coagulation [1], filtration and separately passing through activated carbon or clays for
decreasing the organic load from the feed of RO, some of the more recent techniques [2,3] are
2248
also emerging such as nanofiltation and ultrafiltration which are felt to quite promising
pretreatment method to overcome the said problems and to reduce the overall cost of
production. Moreover, these organic contaminants have been found to be the precursors for
the formation of organic derivatives, some of which are carcinogenic. The dissolved organic
matter is not a single substance but a mixture of many ill-defined aliphatic and aromatic
compounds. However, among the total dissolved organic substance (DOS) in seawater 8090% is represented by the humic materials or substances. Much attention has, therefore, been
paid to isolate them and study their chemistry. Humic substances account for significant and
variable proportions of organic matter in soils, sediments, and they are also found as soluble
organic matter in fresh and seawaters [6-8]. Humic substances comprise a general class of
ubiquitous substances in terrestrial and aquatic environments. Studies on humic substance in
the earlier days [ 9,10], were confined only to soil scientists. The term 'humus' (Latin
equivalent to soil) was initially introduced to describe the dark colored organic matter in soil,
and later, the term was modified as Humus Acid or Humic Acid. These substances are
known to play a role in the food chain, affect the aesthetic quality of water by imparting color
and act as a complexing agent for inorganic ions. Thus there has been a growing interest to
determine the contribution by humic substances to biological, physical and geochemical
processes in natural water systems. Chloroform was found to be ubiquitous in chlorinated
drinking water, hence the US Environmental Protection Agency (USEPA) issued a regulation,
limiting the concentration of chloroform and its sister trihalomethanes (THMs) to 100 ppb
[11, 12]. Various authors have supported the hypothesis that it is the natural organic matter
(Humic substance), which are the most common reaction precursor to trihalomethane
formation [13-15]. Aquatic humic substances are organic acids that are derived from soil
humus and aquatic plants. Generally more than half of the dissolved organic carbon in water
is due to humic substances. As there is growing concern over the role of humic substance in
the various aspect of water chemistry, more and more efforts are now concentrated to
understand the role, structure and chemistry of humic acid in aqueous system mainly because
of their complexation with metal ions, mobilization of toxic trace metal [16], formation of
chlorinated methanes in water treatment, role in bacterial growth after chlorination of feed
and interaction with organics like solublization of pesticides and hydrocarbon [17]. To asses
the potential for bacterial growth and THMs formation potential due to humic substance in
sea water, it is essential to isolate and characterize the humic substances. Furthermore, there
is a need to develop methods to estimate the humic substances in the seawater and other
2249
aqueous systems. The isolation and estimation of humic substances are two difficult tasks to
be achieved but they are very important, as various aspects are related to humic substances.
Low concentration of humic substances in seawater or aqueous system and non-availability of
efficient sorbent to obtain gram quantities makes the isolation and characterization, a tedious,
lengthy and difficult task. The methods once developed will enable to map out the areas of
high humic substances content where preventive measures could be taken to avoid biofouling
on RO membrane. Humic molecules as such are refractory in nature, not easily assimilable by
microorganisms. However, during the chlorination of feed water, humic substances are
broken down into smaller fragments thereby loosing their inert nature and become primary
source of bacterial nutrient promoting in membranes biofouling. Smaller fragments formed
during the chlorination process are also major source for the formation of carcinogenic
organic compound such as trihalomethanes, haloacetic acids and other disinfection byproducts. Both these problems are of utmost importance in RO process. Thus much attention
is being paid recently to better understanding the situation. In this direction research work
leading to abatement of organic fouling is actively being pursued.
2.
OBJECTIVES
Present study was carried out with a view to standardize the procedure for extraction of
dissolved organic substances from seawater, their isolation and characterization, and to study
the concentration and distribution of humic substances, in the aquatic systems near the RO
desalination plants located in the Kingdom. The data obtained will be used in the RO
desalination process to understand the biofouling of membranes. Apart from chemical and
biological water quality of aquatic system in relation to humic substances, environmental and
ecological nature of the aquatic environment would also be ascertained.
Following were the major objectives of the project:
(i)
To standardize extraction and purification procedure of humic substances in
seawater to get at least gram quantities.
(ii)
To isolate humic substances from sea near the locations of SWCC plant in (a)
Eastern Province (Arabian Gulf) and (b) Western Province (Red Sea).
2250
(iii) Characterization and estimation of pure isolated humic substances by physicochemical techniques, viz., UV, IR, TOC and CHNS analyzer.
(iv) Development of a method for the determination of concentration of humic
substances in aqueous system by direct or indirect methods using techniques like
absorption spectrometry or flourometry.
3.
EXPERIMENTAL
The project was aimed at the extraction, isolation, characterization and estimation of humic
substances from seawater. There are several methods and techniques such as precipitation
[18], solvent extraction [19], Ultrafiltration [20], for isolation and extraction of humic
substances. However, all these methods are tedious, time consuming and involve complicated
procedures to isolate and concentrate humic substances from large volumes of water. After
the development of macroporous resins for chromatography [21-23], these resins were fully
exploited for removing trace organic solutes from water [24-27]. Humic acid concentration is
less then 2 ppm whereas the total dissolved organic substance (DOS) is itself only about 2-3
ppm. To get gram quantities of humic substances, from such low concentration present in
seawater generally sorption and ion exchange technique is used. Large amount of seawater
under specific conditions is passed through exchange column of specific resins having high
efficiency absorption or adsorption for humic substance and then eluted under different
conditions to obtain concentrated matter. However, before extraction, resin would require
treatments such as cleaning or purification by extraction and washing. The resin then placed
in column which needs to properly packed and conditioned.
3.1
Isolation of humic substances
3.1.1
Resin Cleaning
Amberlite XAD-2 and/or XAD-7 resins (40-60 mesh) were chosen for purpose of isolating
[24-27] humic substances from seawater. Before using these resins they were cleaned to
remove organics which may be present during the synthesis of these resins as contaminants.
The following standard procedure was adopted for purification. Initially, the resin was rinsed
2251
with distilled water and extracted with 0.1N NaOH in a beaker. Stirring in a beaker with
0.1N NaOH, the resin was allowed to settle, and the supernatant was decanted. Stirring and
decantation process were continued for 5 successive days. Next, the resin was put in soxhlet
extractor and extracted sequentially for minimum of 2 days each successively with methanol,
diethyl ether, acetonitrile and methanol. Finally it was stored in methanol until used.
3.1.2
Column Preparation
Glass column of 55 cm length and 5 cm diameter fitted with high porosity disc and stopcock
at the bottom and with B24/40 female socket at the top end was used. The column was
thoroughly cleaned to remove any organic or grease and finally rinsed with distilled water.
Resin was packed in column as methanol water slurry without any void or gas bubbles in the
resin column. Glass wool was put at the top of resin to avoid floating of resin and to retain
any suspended particles. Distilled water was passed through the column filled with resin to
remove methanol. Packed column (up to a height of 40 cm) was then rinsed alternatively
with 0.1 N NaOH and 0.1 N HCl to remove any impurities.
Top end of the column, which is fitted with B24/40 female socket, has on the top male
B24/40 cone, which can be fitted on the top of column. Other end of B24/40 cone is
connected with 18 mm tygone tube, which is part of master, flex pump with microprocessor
pump drive. Other end of the Tygone tube is immersed in a drum of 200 liters capacity,
which is filled with seawater to be passed through the column. In this set up, flow can be
adjusted with master flex pump to obtained the desired rate.
3.1.3 Procedure of isolation
Schematic diagram of experimental set up used for isolation of humic substance from
seawater is given in Figure 1. Unchlorinated seawater from intake was pumped into a
cleaned 200 liters drum through bolting silk cloth (75 µ mesh size) to remove suspended
particles. After filtration, the pH of seawater was adjusted to 1.9 to 2.00 using sulphuric acid
and pumped through column with help of master flex pump at the rate of 110-120 ml/min.
Thus about 160 to 170 liters of seawater was passed through the column per day. After
passage of 2500 to 3000 liters of seawater in about 20 days through the column, it was
2252
washed with distilled water, the pH of which was adjusted to 1.9 to 2.0. Washing of column
with distilled water of pH 2.0 was carried out till the effluent was free of chloride. This
indicated that all salts have been removed from column. After thorough washing of column
with distilled water of pH = 2.0, humic acid which was adsorbed on the column was eluted
with a mixture of 5M NH4OH and methanol in equal proportion. Elution was kept very slow
at the rate of 5 to 10 ml /Hr. In about 2 to 3.0 liter of eluting solution all adsorbed humic
substances were eluted which was further concentrated using a Rotavapour assembly
consisting of buchi water bath and water condenser coupled with refrigerated constant
temperature circulator. Evaporation was carried out under vacuum at a temperature of (4045oC). When the solution containing concentrated humic substances reduced from 2-3 liters
down to about 30-40 ml, it was transferred to a dish which was kept in desicator in vacuum.
Here, the solution was further concentrated by slow evaporation under vacuum and finally
obtaining a solid, dark brown colored residue after about 8-10 days. This solid residue was
ground into fine powder and stored in a glass bottle in desicator for further studies.
3.1.4 Humic Substances Isolation in Al-Khobar Plant (Eastern Province)
In Al-Khobar plant, isolation experimental set up was arranged on the Jetty. Submergible
pump was used to pump unchlorinated seawater into a 200-liter drum. Submergible pump
was situated at about 8-10 feet below the surface level of seawater and located away from the
discharges of Al-Khobar plants. After properly conditioning seawater in 200 liter drum, it
was passed through conditioned column and washed with distilled water of pH =2. Humic
acid was eluted with methanol ammonia mixture, concentrated by evaporation under vacuum
at 40 oC as per procedure discussed above in section “3.1.3”. After passing approximately
2600 liters of seawater it was possible to isolate 2.1 grams of humic substance in the solid
form.
3.1.5 Humic Substances Isolation in Al-Jubail Plant (Eastern Province)
An experimental set up was arranged in the pilot plant of the R&D Center, Al-Jubail.
Unchlorinated seawater was collected in a 200-liter drum from an intake, which is used for
reverse osmosis plant. Isolation of humic substances from Al-Jubail seawater was carried out
2253
by passing the conditioned seawater in 200 liters drum through the column packed with
XAD-2 resins. Approximately 2400 liters of seawater yielded 1.8 gms of humic acid.
3.1.6 Humic Substances Isolation in Jeddah Plant (Western Province)
At Jeddah plant experimental set up was installed in the laboratory for isolation of humic
substances. Unchlorinated seawater was collected in a 200 liters drum-using pump which
was located near the intake bay. The drum containing seawater was transported to laboratory
and transferred to another 200 liters drum situated near the isolation system. During the
transfer of seawater to the second 200 liters drum, it was made to pass through the bolting silk
cloth to remove the suspended particulates. The pH of seawater was adjusted to 2 and then
about 3100 liters of seawater was passed through the conditioned column to isolate humic
substances on the resin column. After properly washing of columns with distilled water (at
pH = 2) to remove the salts, humic substances were eluted with methanol ammonia mixture.
The solution containing humic acid was transported to Research Center, Al-Jubail to
concentrate and recover humic substances by evaporation as mentioned in the procedure
(Section 3.1.3).
3.2
UV-Visible and Infra Red Spectrometer
To obtain UV-visible spectrum of humic substance, Shimadzu UV-2100S Spectrometer was
used. This dual beam spectrometer utilizes 1 cm path length cell in the wavelength range of
190 to 800 nm.
To record the infra red spectra of humic substances, KBr pallets (disc) were used.
Spectroscopic grade KBr was used to prepare disc. 5 mg of the humic substance was
thoroughly mixed with 0.3 gm of KBr and pressed using pelletizer resulting in transparent
disc. Disc was mounted on window holder and IR spectra were recorded using Hitachi 27050 spectrometer in the range of 250-4000 cm-1.
2254
3.3 Spectrofluorometer
To obtain the excitation and fluorescence spectrum of humic acid Shimadzu RF-1501 was
used. This spectrometer is capable of varying the wavelength of both excitation and emission
(fluorescence) from 220 to 900 nm.
3.4
Elemental Analyer
Carlo Erba EA. 1108 was used for elemental analysis of Carbon (C) Hydrogen (H), Nitrogen
(N) and Sulphur (S). Elemental standards of known composition were used for calibration
purposes.
3.5
Total Organic Carbon
To estimate total organic carbon (TOC) Shimadzu TOC 500 model was used.
3.6
Quantification of Humic Substances
For quantification of humic substance a small glass column about 30 cm in length and 1.2 cm
diameter filled with XAD-2 resins was used. Two liters of unchlorinated seawater from sites
of estimation were taken. The pH value of seawater was adjusted to 2 and passed through the
column which was already conditioned at pH=2.
Seawater was passed at the rate of 6-8
drops per minutes. After loading 2 liters of seawater, column was washed with distilled water
whose pH is adjusted to 2 in order to remove all salts. Then humic acid adsorbed on the
column was eluted with eluting mixture of equal proportion of 5 M NH4OH and methanol of
equal proportion. Elution rate was adjusted to 3-4 drops per minute. About 10 times column
volume (~ 300 ml) eluting solution was used for complete removal of humic substances
absorbed on the column. This effluent was concentrated at 40oC to 125-130 ml to remove
ammonia. Later the eluted concentrate solution was transferred to a 250 ml volumetric flask
and made up to mark. Portion of these solution were used for quantification of humic
substance by UV-visible and fluorescence techniques.
2255
4. RESULTS & DISCUSSION
4.1 Estimation and characterization of isolated humic substances
It is practically difficult to characterize and estimate any material by means of single
technique. In order to get more reliable and dependable result, it is always recommended to
use as many techniques as possible to characterize or estimate any material or substance.
Hence, various techniques were adopted to characterize and estimate humic substances
isolated from seawater at three different locations of Red Sea and Arabian Gulf coasts. The
techniques used to characterize and estimate were:
(a)
Elemental analysis
(b)
Total organic carbon (TOC)
(c)
Ultraviolet and visible absorption spectroscopy
(d)
Infrared Spectroscopy
(e)
Fluorescence Spectroscopy
To isolate the humic substances from seawater, a large amount of seawater was passed
through the resin to get very small quantities of humic substances, as concentration of humic
substance is very low in seawater. Table 1 indicates that a huge amount of seawater was
used to obtain humic substance in gram quantities.
Last column of the Table 1 gives
approximate concentrations of humic substances in seawater based on amount recovered from
the total volume of seawater used.
The humic substances usually consist of humic and fulvic acids in addition to other
components. Humic acid components are soluble at higher pH but insoluble at low pH while
fulvic acid component is soluble in both bases and acids [28]. Humic substances isolated
from three locations viz. Al-Jubail, Al-Khobar and Jeddah were subjected to analysis, it was
found that 80.4, 79.5 and 84% of substances contained fulvic acid component respectively in
above said sites. It can be seen from Table 2 that humic acid component in isolated humic
substance contained only 19.5% in Al-Jubail, 20.5% in Al-Khobar and 16% in Jeddah. High
percentage of fulvic acid in Jeddah sample ( ≈ 84%) means more aliphatic components in
humic substance and this was also confirmed by high hydrogen to carbon ratio compared to
other samples. In general, aliphatic components are higher than aromatic component in all
the samples from three sites.
Furthermore, total acidity was high in Jeddah sample, which
was 5.01 meq/gm, compared to 3.36 and 3.23 meq/gm for Al-Jubail and Al-Khobar samples
respectively.
2256
4.2
Elemental Analysis
Elemental analysis is probably the most common technique used in characterization of humic
substances [29]. In fact, chemical analysis is the cornerstone of all chemical inquiry. Since
most humic substances are mixtures of many components elemental analysis does not provide
an absolute or an accurate molecular formula but it does provide general compositional
information and could set limits as to possible or probable molecular composition. Elemental
analysis could also be useful in establishing the purity of humic substances preparations
methods. Elemental composition, along with elemental ratios such as C/H, O/C, N/C, are
fundamental approaches used in describing and understanding the geochemistry of the
isolated substances and often are single most useful indicator of unique nature of a given
humic substance. Having stated the importance of elemental data for the interpretation, it is
necessary to obtain reliable and accurate results. However, there are limitations in obtaining
accurate data for various reasons. Foremost limitation is the availability of very small
samples with high purity.
The result of elemental analysis for C, H, N, S for all three samples collected from Al-Jubail,
Al-Khobar and Jeddah are tabulated in Table 3. However, oxygen values are computed
after taking into account the ash content of the sample. A standard of a known composition
was also run along with the above samples to ascertain the analysis. It can be seen that C %
in Al-Khobar and Al-Jubail were around 42% while in Jeddah it was only 27%. However,
hydrogen in Jeddah was 5.46% compared to 6.23% in Al-Jubail and Al-Khobar samples. In
other words, percentage ratio of H/C in Jeddah was very high, thereby indicating that
aliphatic components were more in Jeddah sample compared to samples from Al-Khobar and
Al-Jubail. Nevertheless, H/C ratio in Al-Khobar and Al-Jubail plant samples were also high
(~1.77) which indicated that aliphatic components in humic substances from these sites were
also high but not as high as was in Jeddah sample (which was highest among the samples
from all three locations). In addition, ratio of O: C was also above 0.7 in all three samples
indicated high proportion of fulvic acid in humic substances. This fact was supported by
solubility test in acid as shown in Table 2 where it was indicated that all the three samples
from Al-Jubail, Al-Khobar and Jeddah contained 80.4%, 79.5% and 84% of fulvic acid,
respectively compared to 19.6, 20.5 and 16% of humic acid component respectively in these
2257
plants. Hence, it can be stated that aromatic components in all three humic substances were
less compared to aliphatic components. However Jeddah sample contains higher aliphatic
component compared to other samples. The decrease in aromacity of Jeddah humic substance
indicated less refractory or inert nature and thus more viable or assimilable nature of carbon
as a nutrients for microorganisms. This could be one of the important factors for high SDI
and biofouling in Jeddah. The difference in nature of humic substance isolated from Jeddah
compared to Al-Jubail and Al-Khobar can also be noted from N/C ratios. This indicates a lot
of bacterial activity in Jeddah in comparison with other two sites. Secondly relatively high
N/C in Jeddah samples indicated that large proportions of humic substances produced were
from aquagenic refractory material. Sulphur content in Jeddah sample was around 10%
whereas it was absent in Al-Jubail and Al-Khobar samples. High nitrogen and sulphur
content may provide an ideal breeding ground for microorganism. However the source and
origin of sulphur need further investigation.
Based on elemental analysis the most likely empirical formula for humic substances in AlJubail and Al-Khobar area is C8H14NO whereas for Jeddah sample it is C4H8NOS0.5. Visual
observation of samples also revealed that Al-Jubail and Al-Khobar were similar of having
dark brown color fine powder which formed lumps while Jeddah sample was very fine
crystalline powder with light yellowish brown color. It did not form lump.
4.3
Molecular Weight
Humic substances molecular weight determination using gel permeation chromatography for
Al-Jubail, Al-Khobar and Jeddah lied in the range of 3000-3700. Average molecular weight
of three site samples are 3440, 3620, and 3718 for Al-Jubail, Al-Khobar and Jeddah,
respectively.
4.4
Temperature Dependence of Weight Loss
Weight loss determination of humic substances have been carried out on three samples from
different sites of Al-Jubail, Al-Khobar and Jeddah and results are shown in Table 4.
All
three samples indicated gradual increase of weight loss as temperature increased up to 100oC.
This is due to loss of moisture. It has been reported [30] that loss of weight at 55-60 oC
2258
corresponds to water determined by Karl Fisher water content in humic substances. Here in
this case also loss of weight at 55 oC corresponds to water content in these three samples.
Loss in weight beyond the temperature of 80oC is considered as loss due to decomposition of
humic substances. Samples dried at different temperatures up to 80 oC regained the weight
when again exposed to air whereas the samples heated at temperature 80 oC or above, did not
regain or regained negligible weight [30] was the basis for the conclusion that the humic
substances heated beyond the temperature of 80 oC start decomposing. A present sample was
heated up to 750 oC to determine the ash content and results of ash content are also shown in
Table 4. Ashes content were 7.5 to 8.0% in Al-Jubail and Jeddah sample whereas AlKhobar sample contained only 4%.
4.5
Total Organic Carbon (TOC)
Total organic carbon of the isolated humic substances from all three sites were determined by
dissolving known amount of dry humic substance in water (free of carbon). The results are
shown in Table 5. TOC of humic substances obtained from Al-Jubail and Al-Khobar were
higher compared to Jeddah. In fact, Al-Jubail and Al-Khobar humic substances as function of
TOC can be represented by equation (1) while equation (2) represents the case for Jeddah,
Al-Khobar and Al-Jubail:
Y = 0.386 X - 0.0894
(1)
Jeddah:
Y = 0.228 X - 0.0636
(2)
where Y = TOC & X = humic substance both in ppm
Figure 2 represents the graphic relation of humic substance with TOC. Both the graph or
the equations show that values of TOC obtained were in reasonable agreement with the
values of carbon obtained by elemental analysis for these samples as shown in Table 3. For
Al-Jubail and Al-Khobar (eastern province) samples carbon values by TOC were about 38%
whereas for Jeddah it was 22%. However values of carbon obtained by elemental analysis
were 42% and 27% for eastern province location and Jeddah, respectively. As it can be seen
2259
in Figure 2 that two different equations and graphs followed by humic substances obtained
from eastern and western provinces indicate that the composition and perhaps nature and
origin are different for humic substances isolated from these sites.
4.6
IR Spectroscopic Studies
Spectroscopic method like other investigation techniques are severely limited when applied to
humic substances. This is because humic substances comprised of very complicated illdefined mixtures of various long chained organic compounds and macromolecules. Thus the
spectra of humic substances represent the summation of the responses of many different
species and functional groups present in these substances. In some cases only small fraction
of total number of molecules or groups contribute to total spectra. Occasionally, spectral
contribution due to some groups may be masked partially or totally, thereby causing
hindrance, hence no clear understanding which in certain cases could lead to totally different
inferences or interpretations.
Infrared spectrum in the region of 4000 to 250 cm-1 obtained by KBr pallet are shown in
Figures 3 to 5. It can be seen that all IR spectra obtained for samples from three sites viz.
Al-Jubail (Figure 3), Al-Khobar (Figure 4) and Jeddah (Figure 5) had in general, similar
features and bands. The samples of Al-Khobar and Al-Jubail showed the closer resemblance
whereas Jeddah sample IR showed distinct difference due to the presence of some additional
peaks. Prominent bands which were observed in all samples are 3150-3500 cm-1, 2800-3050
cm-1, 1550-1725 cm-1, 1375-1450 cm-1, 1220 cm-1, and 1050-1150 cm-1 region. However, in
the case of Jeddah additional sharp intense band at 600 cm-1 was observed, in addition to the
absence of a band around 1220, which could be clearly observed in samples from other two
sites. Bands around 1230 cm-1 due to C-O and OH deformation were more clearly seen in AlKhobar and Al-Jubail samples which was absent or perhaps merged into the band around
1100 cm-1 in Jeddah sample. The band around 1100 cm-1 is very intense in the case of Jeddah
sample. In addition, very sharp absorption was noticed at 600 cm-1 with shoulder peak at 460
cm-1 in Jeddah sample. However peaks of 600 and 480 cm-1 also observed in Al-Jubail and
Al-Khobar samples but spectra were diffused and peaks were broad and less intense.
2260
The broad peaks in the region 3150 to 3500 cm-1 which was observed in all the three samples
corresponds to absorption where OH stretching occurs, [31] and its broadness is generally
attributed to hydrogen bonding [32,33]. When Hydrogen is bonded to the more
electronegative atoms such as oxygen or nitrogen the bond is polarized, leaving the hydrogen
atom with partial positive charge. This partially positive charge hydrogen atom can then
interact electrostatically with other atoms, which are oppositely charged. This interaction can
be intramolecular (occur within the same molecule) or intermolecular (between functional
group of the different molecules). Thus hydrogen-bonding results in increased separation
between the hydrogen atom and the atom to which it is co-valently attached or bonded. This
brings the change in frequency of absorption and broadening result due to the statistical
distribution in the extent of hydrogen bonding in the molecule. All the three samples of humic
substances indicated the band around 3400 cm-1 due to OH stretching broadened because of
hydrogen bonding. A shoulder peak around 2920-2950 cm-1 was also evident in the spectra
of all the three humic substances.
These bands were attributed to the asymmetric and
symmetric stretching vibration of aliphatic C-H bonds in methyl and/or methylene units in the
compound [32]. The assignment of this band was confirmed by some worker in this field by
methylation of humic substance and corresponding observation of increased absorbance in the
spectra [31-34].
Absorbance band observed around 1700-1710 cm-1 is generally attributed to the C=O
stretching vibration mainly due to carbonyl group [31,32]. This band was very sharp and
distinct in the spectra of the Jeddah sample compared to Al-Jubail and Al-Khobar samples.
This probably points out to the fact that fulvic acid component is high in concentration in case
of Jeddah sample compared to Al-Jubail and Al-Khobar samples.
The band at 1650 cm-1 is assigned to aromatic C = C stretching vibration, i.e., carbon –
carbon double bonds conjugated C = O or COO [35]. Generally C = C bond in benzene is
infrared inactive, however benzene derivatives which decrease the symmetry of the molecule,
bond becomes infrared active. This band was more pronounced in Al-Jubail and Al-Khobar
samples compared to Jeddah again support the aromatic component in Jeddah is relatively
less compared to aliphatic component.
2261
Band at 1450 cm-1 (as shoulder) has been attributed to the bending vibration of aliphatic C-H
group [33] and band at 1400 cm-1 is due to O-H bending vibrations of alcohol or carboxylic
acids as occurs with simple model compounds. These two bands were superimposed on each
other and were not resolved completely. These bands were present in all three samples.
The band at 1200-1230 cm-1 has been assigned to C-O stretching vibration and OH bonding
deformation mainly due to carbonyl groups. This band disappears on producing salts [33].
This band 1050-1100 cm-1 was present in all three sites samples but more intense in Jeddah
sample. This band is attributed to the C-H bond (single bond) stretching in a branched CH3
unit [36]. More intensity of this band in Jeddah compared to other samples again support the
idea that the Jeddah sample is characterized to be more aliphatic in nature.
Presence of intense and sharp band at 600 cm-1 in Jeddah sample makes the Jeddah sample
IR spectra look very different from those of Al-Jubail and Al-Khobar IR spectrum.
This
band was attributed to C-S bond [36]. As it can be seen from (Table 3) that elemental
analysis indicates the presence of 10.23% S in the Jeddah samples whereas Al-Khobar and
Al-Jubail did not contain any sulphur. Thus the presence of intense band at 600 cm-1 only in
Jeddah sample was as expected. Further it was reported that treated and untreated sewage is
discharged into the Red Sea and this could be one of the source of high sulphur content in
seawater and its humic substances.
4.7
UV-Visible Spectroscopic
UV-visible spectra of samples are generally appear as broad band, thus fine structural
information is lost due to this broadening. Olsen [37] demonstrated that how one can get
misleading information due to impurities present in the samples. Considering the ill-defined
multicomponent nature of humic substances, UV-visible spectra results from overlap of
absorbances of various chromophores or functional groups[38] are very broad and diffused.
The UV-visible absorptivities of humic substances do vary as a function of pH [39] and these
are due to the ionization of carboxylic and phenolic functional groups. Though, UV-visible
spectroscopy is a valuable tool in identification of chromophoric functional groups in discrete
organic molecules but there are limitations. In spite of the limitation of UV-visible
2262
spectroscopy, it has been exploited and used for estimation of degree of humification by
Chen et al., 1977 [40].
Figure 6 shows the UV-visible absorption spectrum obtained with
isolated humic substance from three sites.
Irrespective of site of sample, spectrum with a
broad band having absorption peaks around 300 nm was observed. This absorption band
could be utilized for estimation of humic substances in seawater. After isolation of humic
substance from seawater by procedure, which is described in experimental part, was subjected
to estimation by UV-visible spectral studies. Absorbance of isolated solution containing the
humic substance was used to compute the concentration of humic substance. It can be seen
from Table 5 that concentration of humic substances estimated using UV-visible
spectroscopy technique after separation from 2 liters of seawater of all three sites, viz. AlJubail, Al-Khobar and Jeddah were 1.99, 1.93 and 1.2 ppm respectively. The percentage
relative standard deviation of the method is 3.8%. Calibration curve used for estimation of
humic substances was obtained by dissolving known quantities of humic substances and
taking spectrum as shown in Figure 7.
This figure shows a linearity up to 20 ppm and
expressed by the following equation:
Y = 0.0125 X + 0.0184
(3)
Hence from the above equation and the absorbance (Y) of the unknown humic substance
solution, it would be possible to compute concentration (X) of humic substances in this
unknown solution.
4.8
Fluorescence Spectroscopy Studies
Absorption of radiation by an atom or a molecule raises the molecule from electronic ground
state to higher level. Most of the absorbed energy is dissipated in the form of heat and come
back to ground state by radiationless transitions depending upon the excitation. However, in
some molecules major portion of absorbed energy is emitted as electromagnetic radiation at
higher wavelength (low energy as part of it is dissipated in radiationless transition) than the
excitation radiation wavelength which depends on allowed electronic transitions according to
spectroscopic rules. Relatively few organic compounds exhibit fluorescence mainly in UVvisible region of electromagnetic radiation spectrum. Absorption is a pre-requisite of
fluorescence. Some functional groups if present in certain molecules (or compound) may
enhance the fluorescence intensity while other functional group may quench it. Humic
substances are known to fluoresce [41-44]. Fluorescence Spectroscopic studies suffer from all
2263
the limitations of UV-visible spectroscopy in regard to obtaining information about
functionality in humic substances. It is known that only small fraction of the absorbed
radiation is emitted as fluorescence and since humic substances are comprised of
multicomponent mixture, it is likely that fluorescence spectra may represent even small
portion or part of the total molecule rather than those represented by UV-visible spectra.
These limitation have been highlighted in number of the review [43, 44, 45]. In short, it can
be concluded that fluorescence spectroscopy can not be used for direct determination of
functional groups in humic substances. However, fluorescence technique could be utilized
for estimation of humic substance like UV-visible spectroscopy. Initially, it is necessary to
determine the absorption maximum wavelength for excitation of molecules to get
fluorescence at the optimum intensity.
This is obtained by taking excitation spectrum i.e.
observing the fluorescence at a fixed wavelength and varying excitation wavelength. The
excitation spectra for humic substances in water are shown in Figure 8. It can be seen from
the figure that there is broad bands absorption which peaks at 256 nm and 661 nm in addition
to direct emission from excitation source at 421 nm and 842 nm which are sharp in nature.
Broad bands at 256 nm and 661 nm are due to molecular absorption levels which are diffused
and overlap with molecular orbital levels of longer life time compared to atomic levels which
are sharp and have short life time [46]. Excitation spectra of any molecule, in fact reveals
their absorption bands or level. Figure 8 shows the fluorescence spectrum of isolated humic
substance. It was evident from the fluorescence spectrum that there is a broadband emission
at 462 nm. This fluorescence emission at 462 nm is a characteristic of humic substance and
could be used for quantification. Pure humic substance isolated from seawater was used for
obtaining fluorescence calibration curve. Calibration was found to be linear up to 20 ppm and
can be seen in Figure 9. Using this calibration curve, the concentration of humic substance
could be obtained directly either in seawater by obtaining fluorescence intensity at excitation
wavelength of 256 nm or from eluted humic substance solution obtained after separating it
from 2 liters of seawater by XAD-2/XAD-7 resin as discussed in detail in experimental
section. The results obtained by fluorescence spectroscopic technique using two types of
samples i.e., seawater as such or isolated humic solution from seawater are shown in Table
6.
It can be seen that humic substance concentration estimated after isolation were 2.09,
2.06 and 1.39 ppm whereas amount estimated directly in seawater prior to separation were
2.7, 2.34 & 1.4 ppm in Al-Jubail, Al-Khobar and Jeddah, respectively. The percentage
relative standard deviation of the method is 1.75% for isolated samples. Amount estimated
2264
prior to separation is higher compared to isolated samples. This is due to interference of
impurities in seawater. Perhaps these impurities or other species present in seawater also
fluoresce at the same wavelength as humic substance thereby enhancing the estimation.
5.
CONCLUSIONS
(1)
Procedure for the separation of humic substances in gram quantities has been
standardized.
(2)
XAD-2 and XAD-7 resins could be used for isolation of humic substances from
seawater. This procedure of preconcentration of humic acid in conjunction with other
techniques could also be used for their quantitative estimation.
(3)
Elemental analysis of isolated humic substances from all three sites indicated that
humic matter isolated from eastern and western provinces are of two different nature
and compositions. Their empirical formula (C8H14NO and C4H8NOS0.5) and degree
of aromacity were different.
(4)
Total acidity in Jeddah sample is 5.0 meq/gm whereas in Al-Jubail and Al-Khobar
contain 3.36 and 3.23 meq/gm, respectively. Further about 80% content of humic
substance is fulvic acid and 20% is humic acid in all three sites.
(5)
Jeddah samples indicated more favorable conditions for bacterial activity in
comparison to eastern province sites sample, viz. Al-Jubail and Al-Khobar.
(6)
Al-Jubail and Al-Khobar samples contained respectively 6.6 and 6.1% water content
compared to 2.6% in Jeddah samples.
(7)
Infrared spectroscopic studies revealed presence of various functional groups like
carboxyl, phenolic, OH groups, derivatives of benzene and sulphur group, etc.
(8)
A method has been developed for the estimation of humic substances in seawater by
UV-visible spectrometry technique after preconcentration.
By this method,
concentration of humic substances estimated in the samples of Al-Jubail, Al-Khobar
and Jeddah are 1.99, 1.93 and 1.2 ppm, respectively. The percentage relative standard
deviation of the method is 3.8%.
(9)
A fluorescence spectrometric method was also developed for the estimation of humic
substances in seawater after their preconcentration from seawater by XAD resin.
Concentration of humic substances in the three sites of Al-Jubail, Al-Khobar and
2265
Jeddah are 2.095, 2.06 and 1.39 ppm, respectively. The percentage relative standard
deviation of the method is 1.75%.
(10)
Such fluorescence technique could also be used for the estimation of humic
substances without separation provided a standard is run along with it and correction
factor could be applied as seawater impurities could interfere in the estimation.
6.
RECOMMENDATIONS
In future, following studies could be carried out:
1.
To map out the humic substances along the coasts of Red Sea and Arabian Gulf, the
method developed has to be applied to various locations specially where RO plants are
situated. Seasonal variations also need to be established.
2.
To study the effectiveness of pretreatment of RO system in removal of humic
substances, each RO plant feed and after pretreatment samples has to be evaluated in
detail for their humic substances behavior.
3.
To study the degradation of humic substances into smaller components and its
identification by GC/MS to arrive at probable structure or composition of humic
substances.
4.
Identification of carcinogenic compound on chlorination of humic substances and
their carry over to MSF distillate and SWRO permeate.
2266
Table 1. Volume of seawater used for isolation of humic substance and their
concentration
Station
Volume of
Seawater
(liters)
Amount isolated by Concentration of humic substances in
resin (dry form)
seawater (ppm)
(gram)
Based on 100% Based on 50%
retention on resin retention on resin
Al-Jubail
2400
1.8
0.75
1.5
Al-Khobar
2600
2.1
0.80
1.6
Jeddah
3100
1.77
0.57
1.14
Table 2. Percentage of Fulvic and Humic Acid of Humic Substances
Components
Stations
Al-Jubail
Al-Khobar
Jeddah
Fulvic acid %
80.4
79.5
84
Humic acid %
19.6
20.5
16
Total acid meq/gm
3.36
3.229
5.01
2267
Table 3. Elemental analysis
Element %
Sample
Identity
C
H
N
S
Atom %
O
Ash
C
H
Atom Ratio
N
S
O
H/C
C/H
O/C
N/C
Khobar HA
42.25
6.23
7.19
0
40.43
3.9
3.52
6.23
0.514
0
2.53
1.77
0.565
0.719
0.146
Jubail HA
42.19
6.23
6.38
0
37.5
7.7
3.516
6.23
0.456
0
2.34
1.772
0.564
0.666
0.130
Jeddah HA
26.95
5.46
10.28
10.23
38.98
8.1
2.246
5.46
0.734
0.319
2.44
2.131
0.41
1.08
0.327
Standard
30.14
3.8
8.23
9.9
27.85
-
2.512
3.8
0.588
0.309
1.74
1.513
0.661
0.693
0.237
Actual
Value
(31.35) (4.0)
(8.13)
(9.3)
(27.85)
-
2.612
4.0
0.581
0.290
1.74
1.531
0.653
0.666
0.222
Values in brackets are actual value of standard
2268
Table 4. Weight Loss Analysis and Ash Content
Station
% Weight loss @ Temp.
55oC
% Ash
100 oC
750 oC
Content
Al-Jubail
6.6
9.1
92.2
7.7
Al-Khobar
6.1
8.2
96.1
3.9
Jeddah
2.6
4.0
91.9
8.1
Table 5. Total Organics Carbon of Isolated Humic Substances
S. No.
Humic Substances
(ppm)
TOC (ppm)
Al-Jubail
Al-Khobar
Jeddah
1
1
0.32
0.36
0.12
2
5
1.8
2.44
1.1
3
10
3.8
3.7
2.2
4
20
7.4
7.9
4.5
2269
Table 6. Quantification of Humic Substance by Different Techniques
S.No.
Technique
Al-Jubail
(ppm)
1.99
Al-Khobar
(ppm)
1.93
Jeddah
(ppm)
1.21
2.095
2.06
1.39
(b) Seawater (unisolated)
2.7
2.34
1.4
(c) Standard
10.3
10.37
19.88
(10)
(10)
(20)
1
UV-visible spectrometry
(isolated samples)
2
Fluorescence spectrometry
(a) Isolated sample
(Actual value)
All values mentioned are average of six samples with duplicate determination
2270
Glass wool
XAD Resin
SW Container
Glass Frit
SEA
Master
flex
Submersible
pump
Figure 1. Schematic Diagram For the Extraction of Humic Substances from the Seawater
2271
Jeddah
Y= 0.386 x - 0.0894
Al-Jubail &
Al-Khobar
y = 0.227 x – 0.0636
Humic Substances ppm
Figure 2. Variation of total organic carbon (TOC) as function of
humic substances
2272
100
90
80
%
T
70
60
50
40
30
20
10
4000
3500
3000
2500
2000
1800
1600
1400
1200
1000
800
600
Wave Number cm-1
Figure 3. Infra Red Spectrum of Humic Substances Isolated from Al-Jubail Site.
2273
400
250
100
90
80
%
T
70
60
50
40
30
2
10
4000
3500
3000
2500
2000
1800
1600
1400
1200
1000
800
Wave Number cm-1
Figure 4. Infra Red Spectrum of Humic Substances isolated from Al-Khobar Site
2274
600
480
250
100
90
80
70
%
T
60
50
40
30
20
10
4000
3500
3000
2500
2000
1800
1600
1400
1200
1000
800
600
Wave Number cm-1
Figure 5. Infra Red Spectrum of Humic Substances Isolated from Jeddah Site
2275
400
250
(c) Jeddah
(b) Al-Khobar
(a) Al-Jubail
Figure 6. UV-Visible Spectra of Humic Substances from (a) Al-Jubail
(b) Al-Khobar (c) Jeddah
2276
Humic Substance Concentration ppm
Figure 7. Calibration curve of humic substances (UV-Visible
2277
500
0.0
900
220
Wavelength (nm)
Excitation Spectrum
500
0.0
220
900
Wavelength (nm)
Fluorescence Spectrum
Excitation spectrum
(1)
(2)
(3)
(4)
Fluorescence Spectrum
Peak (nm)
Peak (nm)
256 (broad)
421 (sharp)
661 (broad)
843 (sharp)
256 (sharp)
462 (broad
514 (sharp)
706 (broad)
Figure 8. Excitation and fluorescence spectrum
2278
Fluorescence Intensity vs Humic Substances
Y = 16.701 x + 10.412
Humic Substances Concentration ppm
Figure 9. Fluorescence calibration curve for humic substances
2279
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28
29.
Croue, J.P., Lefebore, F, Martin, B., Legube, B., (1993), Water Science, Techno. 27,
143-152.
Alborzfar, M., Jonson, G. and Grouch, (1998), Water Research, 32, 2983-2994.
Kati, R., Vaisanen, P., Metasa M. S., Kutovaara, M. and Nystrom, M (1998),
Characterization and removal of humic substances in Ultra and nanofiltration, 118, 273283.
Belzile, N., Joly, H. A., and Li, H. C. (1997), J. of Chem. , 75, 14-27.
Permonova, I.V., Frimmel, F.H. K., (1998), Water Research, 32, 872.
Gareels R. M., Mackenzie F. T., Hunt C., “Chemical Cycles and Global Environment”
William Kantmann Inc., Los Altos, C. A. (1973)
Steelink C., J. Chemical Education (1977), 54, 599-003.
Black A. P., Christman R. F., J. American Water Works Association (1963), 55 897912.
Waksman S. A., “Humus, Origin, Chemical Composition and importance in Nature”
2nd Edition, William & Wilkins, Baltimore M. D. 526 pp (1938).
Kononova M. M., “Soil Organic Matter” Pergamon, Elmsford. N. Y., 544 pp (1966).
Sympons J. M., Bellar T. A., Carswell J. K., DeMenco, J, Jrop K. L., Robeck G. G.,
Seegar D. R., Solcum C. J. Smith B. L., Steven A. A., J. Am. Water Work Asso.
(1975), 67, 634-647.
National Interim Primary Drinking Water Regulation Fed. Regist. (1979), 44, 682468707.
Steven A. A., Slocum C. J., Seegar D. R, Robeck G. G., J. Am. Water Work Asso.
(1976) 68, 615.
Bebcock, D. B., Binger P. C., J. Am. Water Work Asso. (1979), 71, 149.
Muttanara, S., Sales, E.I., Gazali, Z. (1995), Water supply, 13, 105-117.
Mierle, G., Ingram, R. (1991), Role of humic substances in the mobilization of Hg from
watersheds, Water, Air, Soil Pollution, 56, 349.
Boehm P D., and Quinn J. G. Solubilization of hydrocarbons by the dissolved organic
matter in seawater, Geochim,, Cosmochim Acta, (1973), 37, 2459-2477.
Martin F. D., Fierce R. H., J. Environ. Letter (1971), 1, 49.
Eberle, S. H. Sheweer, K. H., Wom Wasser (1974), 41, 287.
Gjessing E. T., Environ, Sci. Tech, (1970), 4, 437.
Riley J. P., Taylor D., Anal Chim. Acta (1969) 46, 307.
Montoura R. F. C., Riley J. P., Analy Chim. Acta, (1975) 76, 97.
Leenher J. A., Huffman E. W. D., J. Res US. Geol. Surv. (1976) 4, 737.
Gustafson R. L., Albright R. L., Heister J., Lino J. A., Reid O. T., Ind Eng. Chem. Prod.
Res. Dev. (1968), 7, 107.
Simpson R. “The separation of organic chemicals from water” Rohm and Haas,
Philadelphia, P A., 1972.
Thurman, E. M., Malcolm R. L., Aiken G. R., Anal, Chem. (1978) 50, 775.
Aiken G. R., Thurman E. M., Malcolm R. L., Anal. Chem. (1979) 51, 1799.
Malcolm, R.L. and Mac Carthy, P. (1982), Envin. Sci Techno. 16, 20.
Rice, J.A., Maccarthy, P. (1991), Organic Geochemistry, 17, 635-648.
2280
29. Rice, J.A., Maccarthy, P. (1991), Organic Geochemistry, 17, 635-648.
30. Aiken, G.Rl, Macknight, D.M., Wershaw, R.L. and Maccarthy, P. (1985), Humic
substances in soil, sediment and water, Wohn Willing & Sons.
31. Wagner, G.H. and Steven, F.J. (1965), Structural arrangement of functional groups in
soil humic acid as revealed by analysis, Soil Sci. Soc. Am. Proce., 29, 43-48.
32. Theng, B.K.G., Wake, J.R.H. and Pasner, A.M. (1966), The infrared spectrum of humic
acid, Soil Sci., 102, 70-72.
33. Juo, A.S.R. and Barber, S.A. (1969), Reaction of stronium with humic acid, Soil Sci.,
108, 89-94.
34. Warshaw, R.L., Mikita, M.a. and Steelink, C. (1981), Direct 13C NMR evidence for
carbohydrate moieties in fulvic acid, Envion. Sci. Techno., 15, 1461-1463.
35. Schnitzer, M. and Skinner, S.I.M. (1968), Alkali versus acid extraction of soil organic
matter, Soil Sci., 105, 392-396.
36. Van der Mass, J.H. (1972), Basic Infrared Spectroscopy, 2nd edi., Heyden & Sons Ltd,
London.
37. Olsen, E.D. (1975), Modern optical methods of analysis, McGraw Hill, New York.]
38. Stevenson, F.J. (1982), Humus Chemistry, Genesis, Composition, Reaction, Wiley
Interscience, New York.
39. Mac Carthy. P. and O’Cinneide, S. (1974), Fulvic acid I partial fractionation, J. Socil.
Sci., 25, 420-428.
40. Chen, Y., Senesi, N. and Schnitzer, M (1977), Information provided on humic
substances by E4/E6 ratio., Soil Sci. Soc. Am. J., 41, 352-358.
41. Miano, T.M., Sposito, G, Martin, J.P. (1988), Soil Science Soc. Of American Journal,
52, 1016-1019.
42. Seal, B.K. Roy, K.R. and Mukherjee, S.K. (1964), Fluorescence emission spectra &
extraction of hum and fulvic acid, J. Ind. Chem. Soc., 41, 212-214.
43. Schnitzer, M. (1971), Characterization of humic constituents by spectroscopy in soil
biochemistry, 2, edi. By A.M.MacLarn & J. Skujins, Marcel Dekker, New Yark.
44. Stevenson, F.J. (1982), Humus Chemistry, genesis, composition, reaction, Weiley Inter
Science, New York, 443.
45. Schnitzer, M. and Khan S. U. (1972), Humic substances in environment, Marcel
Dekker, New York.
46. Flippora, E.M., Fadeov., V.V., Chubarov, V.V. (1995), Origin & Structure of
Fluorescence and from Aquatic Humic Substances, 5th Internal Conf. On Laser
Application in Life Science, Moscow, Runia, Sponsored by International Society for
Optimal Engineering, Bellingham, USA.
2281