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Determination of charge density and adsorption behaviour of DMAEA-Q-based cationic polymers by fluorimetric analysis
water research 43 (2009) 1905–1912
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Determination of charge density and adsorption behaviour of DMAEA-Q-based cationic polymers by fluorimetric analysis Hans Saveyn*, Daan Curvers, Ruben Dupont, Paul Van der Meeren Ghent University, Particle and Interfacial Technology Group, Coupure Links 653, 9000 Gent, Belgium
article info
abstract
Article history:
In wastewater and sludge treatment, cationic polymers are applied at large scale. A correct
Received 9 December 2008
determination of the charge density and adsorption efficiency is of high importance for an
Received in revised form
economic and ecologically sound operation. Although several analytical techniques exist
28 January 2009
for charge density and polymer concentration determination, they often suffer from
Accepted 3 February 2009
laborious sample pretreatment, complex instrumentation or interference from background
Published online 10 February 2009
components present in sludge. In this work, an alternative method has been studied to determine the charge density of an important series of cationic polymers used in water and
Keywords:
sludge treatment, viz. copolymers containing quaternised dimethylaminoethylacrylate
Polyelectrolytes
(DMAEA-Q). The method is based on the basic hydrolysis of the cationic moiety, resulting
DMAEA-Q
in choline chloride, which is measured by a fluorimetric technique based on the enzymatic
Conditioning
conversion of choline. It was demonstrated that the new technique ensures a highly reli-
Amplex red
able determination of the charge density of these polymers, based on a comparison with
Choline oxidase
the traditional charge titration technique and the data supplied by the manufacturer. Moreover, the specificity of the enzymatic conversion method also allows the determination of non-adsorbed polymer in conditioned sludge samples, without interference from other components. As a consequence, it enables the determination of the optimal polymer dose in practical conditioning and dewatering operations. ª 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
During wastewater treatment, large amounts of sludge are produced, which have to be disposed of. Prior to disposal, the sludge is concentrated by a series of steps including thickening, mechanical dewatering and thermal drying. One of the steps generally involved in thickening and dewatering is the flocculation of sludge with cationic polymers, called conditioning. This process results in an improved solid liquid separation through particle size increase and deswelling of the water holding sludge matrix (Legrand et al., 1998). Throughout the years, there has been a shift from mainly using inorganic conditioners, such as aluminium or ferric salts, to synthetic organic polymers (Bolto and Gregory, 2007).
A good overview of the different synthetic cationic polymers, also called polyelectrolytes, used in practice is given in Dentel (2001). The synthetic polyelectrolyte that takes up the vast majority of the world market for municipal wastewater treatment, about 75%, is a copolymer of acrylamide and quaternised dimethylaminoethylacrylate (DMAEA-Q) (Ciba, 2007). One of the main challenges in conditioning is finding the polymer dose leading to optimum results. Underdosing will lead to unsatisfactory dewatering results, overdosing might result in economic loss and potential ecological risks due to polymer residues in the effluent. The optimum dose depends, amongst other parameters, on the charge density of the polymer. The experimental determination of the polymer charge density mostly relies on colloid titration, in which the
* Corresponding author. Tel.: þ32 9 264 60 25; fax: þ32 9 264 62 42. E-mail address: [email protected] (H. Saveyn). 0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.02.001
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cationic polymer is titrated against an anionic polymer. The endpoint can be detected by a variety of methods, going from visual detection over spectrophotometric to streaming current detector methods. An overview of possible detection techniques has been discussed by Kam and Gregory (1999), who investigated different charge titration techniques in detail. Furthermore, we recently published a study that deals with charge loss for DMAEA-Q polymers, thereby affecting the accuracy of charge determination by colloid titration (Saveyn et al., 2008). The latter work describes the high susceptibility of DMAEA-Q polymers to basic hydrolysis at the ester bond, whereby choline chloride is released into solution, even at mildly acidic pH values down to pH 5 when the polymer concentration is sufficiently low. This choline chloride release may be a drawback in many cases: it can lead to efficiency loss during conditioning, it can be a precursor to odour nuisance (Chang et al., 2005) or it may affect the measurement accuracy during charge titration. Yet, this hydrolytic choline chloride release may also open new ways of polymer characterisation. By quantifying the released choline chloride content using a suitable analytical method, it may become possible to determine the polymer’s charge density, provided that complete hydrolysis is ensured. Analytical methods to determine choline vary from HPLC with electrochemical detection (Potter et al., 1983), biosensors based on amperometric or potentiometric detection (Garguilo et al., 1993), colorimetric detection (Hayashi et al., 1962), and fluorimetric detection (He et al., 2002). Many of these methods require specialized equipment and training, which make them less useful for routine analysis. Colorimetric and fluorimetric methods, based on the enzymatic conversion of choline chloride to a reaction dependent marker molecule, offer the advantage of being relatively simple to perform and requiring less complex instrumentation. Hereby, fluorimetric detection benefits from an improved specificity and sensitivity, compared to colorimetric detection. In addition to measuring polymer charge density, the specificity of the fluorimetric measurement can be exploited for the measurement of choline in a wastewater matrix, and hence to determine residual polymer levels in a conditioned sludge or filtrate sample, following basic hydrolysis. As such, it becomes possible to study the efficiency of cationic polymer adsorption upon sludge conditioning. Very few analytical techniques allow the correct determination of polymer concentrations in wastewater or sludge, due to interference from a variety of components. It is obvious that techniques sensitive to pollution, such as biosensor detection or colorimetric determination will fail for this purpose. Charge titration of the sludge solution after conditioning is the method of choice in practice. However, both under- and overestimation are likely to occur with charge titration of sludge centrate or filtrate. Overestimation of the polymer concentration happens when the anionic titrant interacts with non-polymeric components that carry positive surface charges, like hematite particles or certain proteins. Underestimation of the polymer content occurs when the polymer is attached to suspended solids present in the centrate or filtrate. The latter was demonstrated by Chang et al. (2002), who proposed a method based on nuclear magnetic resonance (NMR) and compared it with charge titration and polymer determination from
viscosity measurements. In their study, they noticed the highest polymer recoveries for the NMR method. Although the NMR approach yielded very good measurement results, the authors already pointed to the fact that it is very expensive, laborious and complex. It is the aim of this study to investigate the possibilities of fluorimetric choline analysis as a tool for reliable and practical determination of DMAEA-Q polymer charge density and residual dissolved polymer concentrations in conditioned sewage sludge samples.
2.
Materials and methods
2.1.
Materials
Thickened activated sludge was sampled from the Ossemeersen Waste Water Treatment Plant (Aquafin, Ghent, Belgium). After sampling, sludge was stored at 4 C for maximum 3 days, to reduce the effect of biochemical composition change. Before testing, a 600 ml sludge sample was acclimatized for 30 min in a water bath at 20 C. Polyelectrolyte chemicals were a gift from Ciba Specialty Chemicals Belgium, and were delivered as beads or liquid dispersion formulation. Except for the LT 22 type, which belonged to the Magnafloc series, all polyelectrolytes were from the Zetag product range (Zetag 7650, 7867FS40, 7899, 7555, DP7-7442, 7878FS40, 7651 and 7878FS25). All products are copolymers of polyacrylamide and quaternised dimethylaminoethylacrylate (CAS Number 69418-26-4) (Dentel, 2001). Aqueous polyelectrolyte solutions were prepared at a 2 g/l active polyelectrolyte concentration (0.2%) in double demineralised water, according to instructions given by the manufacturer (Ciba, 2000). Polyelectrolyte solutions were prepared at least 24 h prior to application, in order to allow the polyelectrolyte chains to completely unfold for optimized contact efficiency. Poly diallyl dimethyl ammonium chloride (polyDADMAC) was used as an alternative flocculant without choline moieties (Aldrich, 20% weight solution in water, MW 400 000–500 000). Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217, Amplex Red Acetylcholine/AChE Assay Kit) was purchased from Invitrogen Molecular Probes (Merelbeke, Belgium). This kit contained Amplex Red Reagent (N-acetyl3,7-dihydroxyphenoxazine), dimethylsulfoxide (DMSO), horseradish peroxidase (200 U), hydrogen peroxide, 250 mM Tris–HCl buffer and choline oxidase (12 U) from Alcaligenes sp. The kit was frozen upon receipt at 18 C, according to instructions by the manufacturer. Choline chloride for making standard solutions was purchased from Acros (>99% purity).
2.2.
Methods
2.2.1.
Sludge conditioning
For sludge conditioning, a Stuart Scientific Jar Test device was used, operating at 270 rpm during 1 min for intense mixing of the polyelectrolyte into the sludge, followed by a 10 min slow stirring period at 35 rpm to promote floc growth. Half a litre of sludge was mixed with 120 ml of a polyelectrolyte solution and tap water mixture. Following conditioning, 50 ml samples
water research 43 (2009) 1905–1912
of conditioned sludge were centrifuged during 10 min at 2850 G (Centrifuge Heraeus Sepatech Labofuge GL, supplied by Goffin Meyvis, Belgium). About 25 ml of the supernatant was then transferred to a polypropylene recipient, capped and stored in a freezer (18 C) for later measurements. The capillary suction time (CST) of conditioned sludge was measured with a Triton Electronics 304 M CST meter (Triton Electronics, Dunmow, Essex, England) using Triton CST paper (7 9 cm).
2.2.2.
Polyelectrolyte characterisation by charge titration
Polyelectrolyte charge density values were kindly supplied by Ciba Specialty Chemicals. The polymer charge titration was performed with a Charge Analyser II (Rank Brothers, UK) using a Metrohm 765 Dosimat titrator and a streaming current detector. Hereto, the method described by Kam and Gregory (1999) was used with the modification proposed in a previous paper (Saveyn et al., 2008). Solutions of 2 g polyelectrolyte/l were made and for every measurement 1 g of this solution was put into 650 ml of a 2 mM sodium acetate solution at pH 4.5 and titrated under continuous magnetic stirring. As anionic titrant, a 206.2 mg/l sodium polystyrene sulfonate (PSS, Acros Organics, dried overnight at 105 C) solution was prepared. This solution was standardized against a 1 meq/l cetyltrimethyl ammonium bromide solution (CTAB, Sigma) by spectrophotometric titration. Based on the absorbance ratio at 630 nm and 565 nm, a PSS charge density of 4.05 meq/g was measured.
2.2.3.
1907
high viscosity of the samples, which made a one step dilution less accurate and irreproducible. Subsequently, 5 ml of the diluted sample was taken and 0.5 ml of a 1 M NaOH (AnalaR NORMAPUR, VWR Prolabo, 99%) solution was added. The sample was then shaken in a capped glass vial and stored overnight (14 h), in order for the choline chloride moieties to completely hydrolyse from the polymer backbone. Choline chloride standard samples (0–5 mg/l) underwent a pretreatment identical to the polymer containing samples. Polymer containing samples of frozen supernatant obtained after centrifugation of conditioned sludge samples were thawed prior to measurement and prepared in the same way. In order to assure that the choline concentration in the supernatant would fall within the measurement range of choline (0–5 mg/ l), a series of dilutions were made. This series consisted of a non-diluted sample, a 10-fold diluted sample and a 100-fold diluted sample, and was prepared in duplicate, together with a choline concentration series from 0 to 5 mg/l (also prepared in duplicate). The measurement was performed with a Spectra Max Gemini XS fluorimeter (Molecular Devices). Black microplates (Greiner Bio-one, 96 wells, V-shaped) were filled with 25 ml of pretreated sample, 75 ml of 50 mM Tris–HCl buffer and 100 ml of the final reagent. Measurements were performed following incubation at 37 C, with an excitation wavelength of 571 nm and an emission wavelength of 585 nm according to instructions from the manufacturer. Every plate contained at least two choline standard series.
Fluorimetric analysis of choline chloride
The fluorimetric analysis is based on the reaction depicted in Fig. 1 (He et al., 2002). When using polymers as a starting material, basic hydrolysis of the cationic moieties of the DMAEA-Q copolymers generates anionic polymers and choline chloride molecules. Either this choline chloride, or pure choline chloride when using a standard calibration series, is subsequently transformed into betaine chloride and hydrogen peroxide (H2O2) through the enzymatic conversion by choline oxidase. Finally, in the presence of horseradish peroxidase, the generated hydrogen peroxide transforms the non-fluorescent Amplex Red molecule into highly fluorescent Resorufin. First of all, 1 mg of Amplex Red reagent was dissolved in 200 ml DMSO in a small plastic vial covered with aluminium foil to protect it from degradation by light. The 250 mM Tris– HCl buffer was diluted 5 times with deionized water, resulting in a 50 mM solution. The individually packed horseradish peroxidase was dissolved in 1 ml of the 50 mM Tris–HCl buffer and the choline oxidase was dissolved in 600 ml of the 50 mM Tris–HCl buffer. The final reagent was prepared by mixing 200 ml of the Amplex Red in DMSO solution, 100 ml horseradish peroxidase solution, 100 ml choline oxidase solution and 10 ml 50 mM Tris–HCl buffer. The reagent was kept in a plastic container wrapped in aluminium foil to protect it from light. Secondly, pure polymer samples were prepared for measurement. Based on approximate charge density information supplied by the manufacturer, 2 g polymer/l solutions were diluted so that the estimated released choline chloride concentration was around 3 mg/l. The dilution from the 2 g polymer/l solution was performed in two steps, due to the
3.
Results and discussion
3.1. Fluorimetric measurements on choline standard series In Fig. 2, the fluorescence evolution as a function of time after addition of the final reagent is displayed for a standard series of choline chloride. It is noticed that the fluorescence intensity increases to a maximum after about 50–75 min, suggesting first-order reaction kinetics at a first glance. However, after reaching their maximum fluorescence intensity, a slight decline in fluorescence is noticed for some of the samples, especially the highly concentrated ones. The reason for this may be twofold. First of all, a competing reaction between horseradish peroxidase and resorufin that results in the formation of a non-fluorescent complex was reported by Zhou et al. (1997). Furthermore, the fluorescent resorufin molecule may be transformed into an unknown non-fluorescent molecule in the pH range 6.2–7.7 (Towne et al., 2004). Therefore, fitting first-order kinetics to the measured fluorescence pattern, in order to use the kinetic parameters for calibration purposes, may yield erroneous results. He et al. (2002) used a single time point from the kinetic data (at 20 min) to set up a calibration curve. Although this approach worked well for many samples, in some cases the fluorescence intensity showed some outliers at certain time points. Therefore, it was chosen to use the slope of the fluorescence intensity curve on the linear part of the curves, between 15 and 40 min, which yielded good calibration curves with a determination coefficient R2 larger than 0.95 in all cases.
1908
water research 43 (2009) 1905–1912
R CH2
CH O
CH
O
O
C
O
O CH2
Hydrolysis
+
Cl
HO
CH2
CH2
N
C
CH3 CH2
N
+
Cl
-
CH3
CH3
-
Betaine chloride
CH3 OH-/H 2O
N
O
+
CH2 H 3C
C
OH
Anionic polymer
C
NH2
O
NH2
CH2
C
C
C
R CH2
CH2
+
Cl
+
-
Choline oxidase H2O2
CH3
CH3 Horseradish
Choline chloride
peroxidase
-
CH3
CH3
HO
Cationic DMAEA-Q copolymer
CH C
O C
HC
C CH
CH C C
N
CH CH
HO
CH C
O C
HC
C CH
CH C C
N
O C CH
CH
Resorufin
C O
OH C
CH3
(Fluorescent)
Amplex Red (Non-fluorescent)
Fig. 1 – Schematic representation of the reactions involved for the fluorimetric analysis of choline chloride (whether or not derived from polymer).
3.2. Fluorimetric determination of polymer charge density Determination of polymer concentrations by charge titration techniques generally relies on a prior determination of the charge density of this polymer. Whereas the latter is usually also performed by charge titration, this study proposes an alternative method based on quantitative choline determination.
3.2.1.
Method development
A prerequisite for allowing charge determination by choline analysis is the complete hydrolysis of choline from the polymer within a reasonable time frame. In fact, it was shown in
Relative fluorescence units (-)
30000
Choline chloride concentration (mg/l)
25000 20000
4.76 3.81 2.86 1.90 0.95 0.00
15000 10000 5000 0 0
25
50
75
100
125
150
Measurement time (minutes)
Fig. 2 – Fluorescence evolution as a function of time after addition of the final reagent, for a standard series of choline chloride.
a previous study that DMAEA-Q polymers are sensitive to hydrolysis and that the percentage of hydrolysis reaches a more or less stable value, determined by the solution pH, after about 5–12 h (Saveyn et al., 2008). In the latter work, charge titration results showed that a pH value of 12 yields nearly complete hydrolysis upon overnight storage of the sample, so it was decided to let the samples in the current study hydrolyse at pH 12 for one night. However, after the basic hydrolysis step, the pH of the samples had to be readjusted. According to the instructions supplied by the manufacturer of the enzyme kit, the reaction with Amplex Red should be performed within a quite narrow pH interval of pH 7–8 for two main reasons. Some reagents are unstable at pH values above 8.5, whereas at lower pH values the fluorescence quantum yield of resorufin becomes markedly lower. Although the pH of the samples could have been adjusted by neutralising the base with acid, followed by addition of the 50 mM Tris–HCl reaction buffer, extensive acid/base addition could have led to high salt concentrations affecting the good functioning of the enzymes needed for the fluorescence reaction. Therefore, it was tested whether the Tris–HCl buffer was able to bring down the pH below 8 after basic hydrolysis of the sample. Samples of 5 ml of an aqueous polymer solution, with a similar concentration as encountered in the liquid phase of conditioned sludge (around 200 mg/l), were treated with 0.5 ml of a 1 M NaOH solution. The same was done for a random sample of sludge supernatant after conditioning with polymer and centrifugation and a sample of 5 mg/l choline in water. It should be mentioned that dosing higher amounts of NaOH could have led to choline being transformed into trimethylamine, a volatile odorous compound (Chang
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et al., 2005). After addition of the NaOH solution, the pH of the different samples was 12.36 (polymer in water), 12.30 (sludge supernatant) and 12.07 (choline in water). These values showed that the pH of the different samples was high enough to allow complete hydrolysis upon overnight storage. After overnight storage, the samples were treated with 50 mM Tris– HCl buffer solution in a 1:7 ratio as was the case for the fluorimetric measurements. After this, the pH was measured again. The pH values now were 7.78 (polymer in water), 7.85 (sludge supernatant) and 7.80 (choline in water), which were all in the interval of 7–8, required for optimum reaction with Amplex Red. For the sake of completeness, it has to be mentioned that the buffer capacity of the 50 mM Tris–HCl buffer was not sufficient to reach a pH below 8 if 1 ml or more of 1 M NaOH was added to the sample.
3.2.2.
Method evaluation: comparison to charge titration
Nine different polymers, as used for sludge conditioning and dewatering experiments in a previous study (Saveyn et al., 2005), were investigated for their charge properties. The results of the charge determination by the choline method are depicted in Fig. 3, together with the results from charge titration and the charge density values supplied by the manufacturer. The latter data are based on the reaction stoichiometry, i.e. on the relative amounts of charged and uncharged monomers used in the process. The confidence intervals for the values supplied by the manufacturer are based on the product classification in 4 different categories: being 20, 40, 60 or 80% cationic. A 100% cationic polymer of this composition would only consist of polymerised DMAEA-Q with a molecular weight of 193.5 for the repeating unit, and hence 1 equivalent of charge per 193.5 g, i.e. a charge density of 5.17 meq/g. As a consequence of the broadness of the different charge density categories supplied by the manufacturer, the estimated uncertainty on every category is 10% cationicity, equalling 0.1 5.17 meq/g ¼ 0.517 meq/g. This uncertainty is depicted as the error bar on the manufacturer’s data in Fig. 3.
It is seen from Fig. 3 that the charge titration technique and the choline based determination of polymers yield very similar results. Except for polymers DP7-7442 and 7899, there is no statistical difference between the two methods (at 95% confidence level). This is confirmed by plotting the values of the choline based charge determination versus the charge titration data. The linear trendline through the obtained scatter plot has a slope that is not statistically different from 1 (95% confidence interval of [0.89; 1.17]) and an intercept that is not statistically different from 0 (95% confidence interval of [0.25; 0.44]). The determination coefficient R2 equals 0.98. The charge densities determined by both methods agree relatively well with the values supplied by the manufacturer. For the choline based determination, only polymer 7555 yields a significantly different value from the data given by the manufacturer. On the other hand, for the charge titration technique, 4 polymers yield a significantly different value from the manufacturer’s data (DP7-7442, 7555, 7651 and 7878FS25). With the exception of polymer 7651, which has a rather broad confidence interval for the choline based measurements, the charge titration technique and the choline based determination exhibited a similar reproducibility. One of the reasons why the charge titration technique may yield lower charge densities than the choline based technique or the data supplied by the manufacturer in some cases, could be related to the structure of the polymers. Polymers DP77442, 7867FS40, 7878FS40 and 7878FS25 are known to be crosslinked polymers. Therefore, some of their charges may not be readily accessible for the anionic polymer used in the titration, due to diffusion limitations. Although this problem is sometimes partially solved by shearing of the cationic polymer prior to titration, it remains uncertain whether all charges become accessible for titration in this way. This may also explain why the choline based measurement results, allowing a release of all charged sites from the polymer, may be closer to the manufacturer’s information.
3.3.
Charge density (meq/g)
5.0
1909
Residual polymer in sludge filtrate
Given that the choline based measurement delivered reliable results for the charge density of the different polymers, it was tested whether the same fluorimetric method could be used to quantify non-adsorbed polymers in conditioned sewage sludge samples. In this way, it would be possible to assess the optimal polymer dose and to avoid overdosage.
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
3.3.1. Sensitivity of fluorimetric method to wastewater matrix impurities and low temperature sample conservation
0.5
51
S2 5 78 78 F
40
76
42
78 FS 78
D
P
774
75 55
99
40
78
67 FS
50 78
76
LT
22
0.0
Polymer name Charge titration
Choline determination
Manufacturer's information
Fig. 3 – Charge density determination for different DMAEA-Q type cationic polymers according to two techniques (error bars represent 95% confidence intervals, based on at least 3 repetitions). For comparison, values as supplied by the manufacturer are also given; error bars indicate a 10% cationicity (0.517 meq/g) uncertainty in this case.
Prior to performing the sludge sample analyses, it had to be ensured that the proposed technique would be applicable on previously collected centrifuged supernatant samples that had been stored by freezing. One of the first issues to be dealt with was the possible background interference from naturally occurring choline or hydrogen peroxide in the bulk solution of the sludge sample. To assess this potential problem, activated sludge was conditioned with polyDADMAC, a flocculant that did not contain any precursors of choline. This sample was then centrifuged to remove all suspended material. Finally, a standard series of choline chloride were made in the
water research 43 (2009) 1905–1912
3.3.2.
Polyelectrolyte adsorption isotherms
Fig. 4 shows the adsorption curves of a series of polymers, applied on the same sludge sample, as a function of the added dose. Five polymers had been selected. Polymers LT22, 7555 and 7878FS25 have similar molecular weights, but clearly differ in measured charge density. Polymers 7899, 7555 and DP7-7442 have similar measured charge densities, but markedly differ in molecular weight. According to a previous classification (Saveyn et al., 2005), polymer DP7-7442 has a low molecular weight, polymers LT22, 7555 and 7878FS25 have a high molecular weight and polymer 7899 has an ultra-high molecular weight. Fig. 4 shows that the various polymers exhibited a clearly different adsorption behaviour, related to their charge density and molecular weight. For the polymer series LT22, 7555 and 7878FS25 with similar molecular weights but different charge densities, the data show that the lower the charge density, the higher the adsorption efficiency at higher doses. This can be explained by the fact that polymers with a high charge density will cause charge neutralisation of the sludge surface at low doses. Increasing the dose will then result in repulsion between the positive surface charge of the sludge and the positively charged polymers. For the polymer series 7899, 7555 and DP7-7442 with similar charge density but different molecular weight, it is noticed that a higher molecular weight results in lower adsorption efficiencies at high doses. Due to their bridging abilities, higher molecular weight polymers are assumed to form flocs without the need to completely neutralize the surface charge of the individual particles. As
14
Amount of polymer adsorbed (mg/kg DM)
supernatant from the latter, as well as in distilled water. These samples were then measured in duplicate according to the regular procedure. The calibration curves for choline chloride in distilled water yielded a slope of 50.3 RFU mg min1 l1 with a 95% confidence interval of [43.5; 57.2] RFU mg min1 l1 and a determination coefficient of 0.964. The calibration series in polyDADMAC treated sludge centrate yielded a slope of 48.1 RFU mg min1 l1 with a 95% confidence interval of [40.9; 55.3] RFU mg min1 l1 and a determination coefficient of 0.957. This shows that the choline measurement is not significantly affected by the multitude of components in a complex matrix such as sludge. A second concern was the influence of freezing on the samples. In this study, the supernatant samples had been taken immediately after centrifugation of the conditioned sludge samples and were then frozen. Therefore, it was necessary to assess the impact of freezing and subsequent thawing processes on the determination of choline from hydrolysed polymer. Hereto a solution of polymer 7878FS40 was made with an approximate concentration of 2 mg choline chloride/l. Part of the solution was kept in a fridge (4 C) and the remaining part was frozen overnight and thawed the next day. The choline concentrations were then measured in 6-fold for both samples. The choline concentrations measured were 2.36 0.10 mg/l and 2.23 0.26 mg/l for the cooled and frozen samples, respectively (95% confidence intervals). This demonstrates that the freeze/thaw process did not significantly affect the choline yield of the polymer sample. Therefore it was decided that the newly proposed method could be applied on the frozen sludge supernatant samples.
12
Polymer type 10
LT 22 (High MW) 7899 (Ultra-High MW) 7555 (High MW) DP7-7442 (Low MW) 7878FS25 (High MW)
8 6 4 2 2
4
6
8
10
12
14
Amount of polymer added (mg/kg DM)
Fig. 4 – Adsorption of DMAEA-Q type cationic polymers onto sludge. Data are derived from choline based measurement of supernatant obtained after centrifugation of conditioned sludge. The diagonal line represents complete adsorption, data below the diagonal line indicate less than 100% adsorption.
a consequence, flocculated entities are formed with a positive surface charge, which will prevent additional polymer adsorption due to electrostatic repulsion. The choline based adsorption data are confirmed by both electrophoretic mobility data of the sludge supernatant (Fig. 5) and by the capillary suction time (CST) values of the different conditioned sludge samples (Fig. 6). The electrophoretic mobility (m) of the supernatant indicates whether negatively charged particles are still present, or whether all particles have become neutralized and free polymer is in solution leading to a positive m value. CST, although not the perfect indicator for dewaterability, is a frequently used tool for determination of the optimum polymer dose. At lower than optimum dose, CST will be high due to inferior floc formation, at optimum polymer dosing CST shows a minimum value, whereas at overdosing the CST increases again because of a viscosity increase of the solution due to non-adsorbed polymer (Christensen et al., 1993). When comparing Fig. 4 to Figs. 5 and 6, it is noticed that polymers 7899 and 7878FS25
Electrophoretic mobility of conditioned sludge (1e-8m-2/s/V)
1910
3.5 3 2.5
Polymer type
2
LT 22 (High MW) 7899 (Ultra-High MW) 7555 (High MW) DP7-7442 (Low MW) 7878FS25 (High MW)
1.5 1 0.5 0 -0.5 -1 -1.5 2
4
6
8
10
12
14
Amount of polymer added (mg/kg DM)
Fig. 5 – Electrophoretic mobility data for sludge conditioned with different DMAEA-Q type cationic polymers. The data reflect samples identical to the ones depicted in Fig. 4.
water research 43 (2009) 1905–1912
CST of conditioned sludge (s)
50
4.
45
1911
Conclusions
40
Polymer type
35
LT 22 (High MW) 7899 (Ultra-High MW) 7555 (High MW) DP7-7442 (Low MW) 7878FS25 (High MW)
30 25 20 15 10 5 0 2
4
6
8
10
12
14
Amount of polymer added (mg/kg DM)
Fig. 6 – Capillary suction time (CST) data for sludge conditioned with different DMAEA-Q type cationic polymers. The data reflect samples identical to the ones depicted in Fig. 4.
(symbolised by triangles and circles, respectively), which show the lowest adsorption efficiencies at medium to high doses, also exhibit clear positive electrophoretic mobility values and high CST values at these same doses. Polymers like the LT22 (diamonds), which still show a high adsorption efficiency at high doses, also exhibit negative electrophoretic mobilities and low CST values at these doses. This clearly indicates that the optimum polymer dose, i.e. before the CST notably starts to increase, coincides with the point at which polymer adsorption is still nearly complete. A possible issue with the adsorption data concerns the reliability of choline chloride as an indicator molecule for dissolved polymer concentration. It may be argued that some of the choline molecules in solution are not derived from dissolved polymer, but rather have been hydrolysed from polymers that are still adsorbed onto the sludge particles. In the latter case, the choline measurement would yield an overestimation of the polymer concentration in solution. Yet, there are several arguments against this hypothesis. First of all, it is noticed in Fig. 4 that for the lower polymer concentrations, the adsorption is nearly 100%. The latter implies that negligible amounts of choline were measured in the supernatant, even though a considerable amount of polymer was present in the flocculated sludge system. Secondly, a combination of short sampling time, relatively high polymer concentration and mild pH ensured that the degree of polymer hydrolysis in the sludge sample was restricted to a minimum. Indeed, Aksberg and Wagberg (1989) demonstrated that at polymer concentrations of 400 mg/l, the time needed to hydrolyse half of the cationic charges of two DMAEA-Q type polymers was 24 h and 62 h at pH 7. In this study, the used polymer concentrations were of the same order of magnitude, with around 100–400 mg polymer/l conditioned sludge. Furthermore, the sludge pH was 7.0 on average, and situated in a narrow interval of 6.7–7.4. The time between the start of conditioning the sludge samples and freezing the isolated supernatant after centrifugation was 40 min at maximum. All these factors resulted in a low probability of hydrolysis of choline moieties from polymer chains adsorbed to the sludge particles.
Overnight basic hydrolysis at pH 12 of the cationic moieties of DMAEA-Q type cationic polymers resulted in the release of all choline chloride groups into solution. Choline chloride was determined analytically by fluorimetric analysis based on the enzymatic conversion of choline to H2O2, which allowed the transformation of Amplex Red into the fluorescent Resorufin molecule. It has been demonstrated that this technique enables a more reliable determination of the true charge density of a series of DMAEA-Q type polymers, in comparison to the traditional charge titration technique. Moreover, the specificity and sensitivity of the enzymatic conversion method also allowed for the determination of residual non-adsorbed polymer in conditioned sludge samples, without interference from other components and with limited sample pretreatment. As a consequence, it can be used to determine the adsorption behaviour of DMAEA-Q type polymers on sludge in practical conditioning and dewatering operations.
references
Aksberg, R., Wagberg, L., 1989. Hydrolysis of cationic polyacrylamides. Journal of Applied Polymer Science 38 (2), 297–304. Bolto, B., Gregory, J., 2007. Organic polyelectrolytes in water treatment. Water Research 41 (11), 2301–2324. Ciba, 2000. Ciba Specialty Chemicals, Publication No. TPD 2000 8990. Ciba Specialty Chemicals, Bradford, UK. Ciba, 2007. Personal communication. Chang, J.S., Abu-Orf, M., Dentel, S.K., 2005. Alkylamine odors from degradation of flocculant polymers in sludges. Water Research 39 (14), 3369–3375. Chang, L.L., Bruch, M.D., Griskowitz, N.J., Dentel, S.K., 2002. NMR spectroscopy for determination of cationic polymer concentrations. Water Research 36 (9), 2255–2264. Christensen, J.R., Sorensen, P.B., Christensen, G.L., Hansen, J.A., 1993. Mechanisms for overdosing in sludge conditioning. Journal of Environmental Engineering – ASCE 119 (1), 159–171. Dentel, S.K., 2001. Conditioning. In: Spinosa, L., Vesilind, P.A. (Eds.), Sludge into Biosolids. IWA Publishing, London, pp. 278–314. Garguilo, M.G., Huynh, N., Proctor, A., Michael, A.C., 1993. Amperometric sensors for peroxide, choline, and acetylcholine based on electron-transfer between horseradish-peroxidase and a redox polymer. Analytical Chemistry 65 (5), 523–528. Hayashi, M., Unemoto, T., Miyaki, K., 1962. Improvement on colorimetric determination of choline with iodine. Chemical and Pharmaceutical Bulletin 10 (6), 533–535. He, X.X., Chen, F., McGovern, M.M., Schuchman, E.H., 2002. A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann–Pick disease and other disorders of sphingomyelin metabolism. Analytical Biochemistry 306 (1), 115–123. Kam, S.K., Gregory, J., 1999. Charge determination of synthetic cationic polyelectrolytes by colloid titration. Colloids and Surfaces A: Physicochemical and Engineering Aspects 159 (1), 165–179. Legrand, V., Hourdet, D., Audebert, R., Snidaro, D., 1998. Deswelling and flocculation of gel networks: application to sludge dewatering. Water Research 32 (12), 3662–3672.
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Potter, P.E., Meek, J.L., Neef, N.H., 1983. Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. Journal of Neurochemistry 41 (1), 188–194. Saveyn, H., Meersseman, S., Thas, O., Van der Meeren, P., 2005. Influence of polyelectrolyte characteristics on pressure-driven activated sludge dewatering. Colloids and Surfaces A: Physicochemical and Engineering Aspects 262 (1–3), 40–51. Saveyn, H., Hendrickx, P.M.S., Dentel, S.K., Martins, J.C., Van der Meeren, P., 2008. Quantification of hydrolytic charge loss of DMAEA-Q-based polyelectrolytes by proton NMR spectroscopy and implications for colloid titration. Water Research 42 (10– 11), 2718–2728.
Towne, V., Will, M., Oswald, B., Zhao, Q., 2004. Complexities in horseradish peroxidase-catalyzed oxidation of dihydroxyphenoxazine derivatives: appropriate ranges for pH values and hydrogen peroxide concentrations in quantitative analysis. Analytical Biochemistry 334 (2), 290–296. Zhou, M.J., Diwu, Z.J., Panchuk-Voloshina, N., Haugland, R.P., 1997. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Analytical Biochemistry 253 (2), 162–168.
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Determination of charge density and adsorption behaviour of DMAEA-Q-based cationic polymers by fluorimetric analysis Hans Saveyn*, Daan Curvers, Ruben Dupont, Paul Van der Meeren Ghent University, Particle and Interfacial Technology Group, Coupure Links 653, 9000 Gent, Belgium
article info
abstract
Article history:
In wastewater and sludge treatment, cationic polymers are applied at large scale. A correct
Received 9 December 2008
determination of the charge density and adsorption efficiency is of high importance for an
Received in revised form
economic and ecologically sound operation. Although several analytical techniques exist
28 January 2009
for charge density and polymer concentration determination, they often suffer from
Accepted 3 February 2009
laborious sample pretreatment, complex instrumentation or interference from background
Published online 10 February 2009
components present in sludge. In this work, an alternative method has been studied to determine the charge density of an important series of cationic polymers used in water and
Keywords:
sludge treatment, viz. copolymers containing quaternised dimethylaminoethylacrylate
Polyelectrolytes
(DMAEA-Q). The method is based on the basic hydrolysis of the cationic moiety, resulting
DMAEA-Q
in choline chloride, which is measured by a fluorimetric technique based on the enzymatic
Conditioning
conversion of choline. It was demonstrated that the new technique ensures a highly reli-
Amplex red
able determination of the charge density of these polymers, based on a comparison with
Choline oxidase
the traditional charge titration technique and the data supplied by the manufacturer. Moreover, the specificity of the enzymatic conversion method also allows the determination of non-adsorbed polymer in conditioned sludge samples, without interference from other components. As a consequence, it enables the determination of the optimal polymer dose in practical conditioning and dewatering operations. ª 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
During wastewater treatment, large amounts of sludge are produced, which have to be disposed of. Prior to disposal, the sludge is concentrated by a series of steps including thickening, mechanical dewatering and thermal drying. One of the steps generally involved in thickening and dewatering is the flocculation of sludge with cationic polymers, called conditioning. This process results in an improved solid liquid separation through particle size increase and deswelling of the water holding sludge matrix (Legrand et al., 1998). Throughout the years, there has been a shift from mainly using inorganic conditioners, such as aluminium or ferric salts, to synthetic organic polymers (Bolto and Gregory, 2007).
A good overview of the different synthetic cationic polymers, also called polyelectrolytes, used in practice is given in Dentel (2001). The synthetic polyelectrolyte that takes up the vast majority of the world market for municipal wastewater treatment, about 75%, is a copolymer of acrylamide and quaternised dimethylaminoethylacrylate (DMAEA-Q) (Ciba, 2007). One of the main challenges in conditioning is finding the polymer dose leading to optimum results. Underdosing will lead to unsatisfactory dewatering results, overdosing might result in economic loss and potential ecological risks due to polymer residues in the effluent. The optimum dose depends, amongst other parameters, on the charge density of the polymer. The experimental determination of the polymer charge density mostly relies on colloid titration, in which the
* Corresponding author. Tel.: þ32 9 264 60 25; fax: þ32 9 264 62 42. E-mail address: [email protected] (H. Saveyn). 0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.02.001
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cationic polymer is titrated against an anionic polymer. The endpoint can be detected by a variety of methods, going from visual detection over spectrophotometric to streaming current detector methods. An overview of possible detection techniques has been discussed by Kam and Gregory (1999), who investigated different charge titration techniques in detail. Furthermore, we recently published a study that deals with charge loss for DMAEA-Q polymers, thereby affecting the accuracy of charge determination by colloid titration (Saveyn et al., 2008). The latter work describes the high susceptibility of DMAEA-Q polymers to basic hydrolysis at the ester bond, whereby choline chloride is released into solution, even at mildly acidic pH values down to pH 5 when the polymer concentration is sufficiently low. This choline chloride release may be a drawback in many cases: it can lead to efficiency loss during conditioning, it can be a precursor to odour nuisance (Chang et al., 2005) or it may affect the measurement accuracy during charge titration. Yet, this hydrolytic choline chloride release may also open new ways of polymer characterisation. By quantifying the released choline chloride content using a suitable analytical method, it may become possible to determine the polymer’s charge density, provided that complete hydrolysis is ensured. Analytical methods to determine choline vary from HPLC with electrochemical detection (Potter et al., 1983), biosensors based on amperometric or potentiometric detection (Garguilo et al., 1993), colorimetric detection (Hayashi et al., 1962), and fluorimetric detection (He et al., 2002). Many of these methods require specialized equipment and training, which make them less useful for routine analysis. Colorimetric and fluorimetric methods, based on the enzymatic conversion of choline chloride to a reaction dependent marker molecule, offer the advantage of being relatively simple to perform and requiring less complex instrumentation. Hereby, fluorimetric detection benefits from an improved specificity and sensitivity, compared to colorimetric detection. In addition to measuring polymer charge density, the specificity of the fluorimetric measurement can be exploited for the measurement of choline in a wastewater matrix, and hence to determine residual polymer levels in a conditioned sludge or filtrate sample, following basic hydrolysis. As such, it becomes possible to study the efficiency of cationic polymer adsorption upon sludge conditioning. Very few analytical techniques allow the correct determination of polymer concentrations in wastewater or sludge, due to interference from a variety of components. It is obvious that techniques sensitive to pollution, such as biosensor detection or colorimetric determination will fail for this purpose. Charge titration of the sludge solution after conditioning is the method of choice in practice. However, both under- and overestimation are likely to occur with charge titration of sludge centrate or filtrate. Overestimation of the polymer concentration happens when the anionic titrant interacts with non-polymeric components that carry positive surface charges, like hematite particles or certain proteins. Underestimation of the polymer content occurs when the polymer is attached to suspended solids present in the centrate or filtrate. The latter was demonstrated by Chang et al. (2002), who proposed a method based on nuclear magnetic resonance (NMR) and compared it with charge titration and polymer determination from
viscosity measurements. In their study, they noticed the highest polymer recoveries for the NMR method. Although the NMR approach yielded very good measurement results, the authors already pointed to the fact that it is very expensive, laborious and complex. It is the aim of this study to investigate the possibilities of fluorimetric choline analysis as a tool for reliable and practical determination of DMAEA-Q polymer charge density and residual dissolved polymer concentrations in conditioned sewage sludge samples.
2.
Materials and methods
2.1.
Materials
Thickened activated sludge was sampled from the Ossemeersen Waste Water Treatment Plant (Aquafin, Ghent, Belgium). After sampling, sludge was stored at 4 C for maximum 3 days, to reduce the effect of biochemical composition change. Before testing, a 600 ml sludge sample was acclimatized for 30 min in a water bath at 20 C. Polyelectrolyte chemicals were a gift from Ciba Specialty Chemicals Belgium, and were delivered as beads or liquid dispersion formulation. Except for the LT 22 type, which belonged to the Magnafloc series, all polyelectrolytes were from the Zetag product range (Zetag 7650, 7867FS40, 7899, 7555, DP7-7442, 7878FS40, 7651 and 7878FS25). All products are copolymers of polyacrylamide and quaternised dimethylaminoethylacrylate (CAS Number 69418-26-4) (Dentel, 2001). Aqueous polyelectrolyte solutions were prepared at a 2 g/l active polyelectrolyte concentration (0.2%) in double demineralised water, according to instructions given by the manufacturer (Ciba, 2000). Polyelectrolyte solutions were prepared at least 24 h prior to application, in order to allow the polyelectrolyte chains to completely unfold for optimized contact efficiency. Poly diallyl dimethyl ammonium chloride (polyDADMAC) was used as an alternative flocculant without choline moieties (Aldrich, 20% weight solution in water, MW 400 000–500 000). Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217, Amplex Red Acetylcholine/AChE Assay Kit) was purchased from Invitrogen Molecular Probes (Merelbeke, Belgium). This kit contained Amplex Red Reagent (N-acetyl3,7-dihydroxyphenoxazine), dimethylsulfoxide (DMSO), horseradish peroxidase (200 U), hydrogen peroxide, 250 mM Tris–HCl buffer and choline oxidase (12 U) from Alcaligenes sp. The kit was frozen upon receipt at 18 C, according to instructions by the manufacturer. Choline chloride for making standard solutions was purchased from Acros (>99% purity).
2.2.
Methods
2.2.1.
Sludge conditioning
For sludge conditioning, a Stuart Scientific Jar Test device was used, operating at 270 rpm during 1 min for intense mixing of the polyelectrolyte into the sludge, followed by a 10 min slow stirring period at 35 rpm to promote floc growth. Half a litre of sludge was mixed with 120 ml of a polyelectrolyte solution and tap water mixture. Following conditioning, 50 ml samples
water research 43 (2009) 1905–1912
of conditioned sludge were centrifuged during 10 min at 2850 G (Centrifuge Heraeus Sepatech Labofuge GL, supplied by Goffin Meyvis, Belgium). About 25 ml of the supernatant was then transferred to a polypropylene recipient, capped and stored in a freezer (18 C) for later measurements. The capillary suction time (CST) of conditioned sludge was measured with a Triton Electronics 304 M CST meter (Triton Electronics, Dunmow, Essex, England) using Triton CST paper (7 9 cm).
2.2.2.
Polyelectrolyte characterisation by charge titration
Polyelectrolyte charge density values were kindly supplied by Ciba Specialty Chemicals. The polymer charge titration was performed with a Charge Analyser II (Rank Brothers, UK) using a Metrohm 765 Dosimat titrator and a streaming current detector. Hereto, the method described by Kam and Gregory (1999) was used with the modification proposed in a previous paper (Saveyn et al., 2008). Solutions of 2 g polyelectrolyte/l were made and for every measurement 1 g of this solution was put into 650 ml of a 2 mM sodium acetate solution at pH 4.5 and titrated under continuous magnetic stirring. As anionic titrant, a 206.2 mg/l sodium polystyrene sulfonate (PSS, Acros Organics, dried overnight at 105 C) solution was prepared. This solution was standardized against a 1 meq/l cetyltrimethyl ammonium bromide solution (CTAB, Sigma) by spectrophotometric titration. Based on the absorbance ratio at 630 nm and 565 nm, a PSS charge density of 4.05 meq/g was measured.
2.2.3.
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high viscosity of the samples, which made a one step dilution less accurate and irreproducible. Subsequently, 5 ml of the diluted sample was taken and 0.5 ml of a 1 M NaOH (AnalaR NORMAPUR, VWR Prolabo, 99%) solution was added. The sample was then shaken in a capped glass vial and stored overnight (14 h), in order for the choline chloride moieties to completely hydrolyse from the polymer backbone. Choline chloride standard samples (0–5 mg/l) underwent a pretreatment identical to the polymer containing samples. Polymer containing samples of frozen supernatant obtained after centrifugation of conditioned sludge samples were thawed prior to measurement and prepared in the same way. In order to assure that the choline concentration in the supernatant would fall within the measurement range of choline (0–5 mg/ l), a series of dilutions were made. This series consisted of a non-diluted sample, a 10-fold diluted sample and a 100-fold diluted sample, and was prepared in duplicate, together with a choline concentration series from 0 to 5 mg/l (also prepared in duplicate). The measurement was performed with a Spectra Max Gemini XS fluorimeter (Molecular Devices). Black microplates (Greiner Bio-one, 96 wells, V-shaped) were filled with 25 ml of pretreated sample, 75 ml of 50 mM Tris–HCl buffer and 100 ml of the final reagent. Measurements were performed following incubation at 37 C, with an excitation wavelength of 571 nm and an emission wavelength of 585 nm according to instructions from the manufacturer. Every plate contained at least two choline standard series.
Fluorimetric analysis of choline chloride
The fluorimetric analysis is based on the reaction depicted in Fig. 1 (He et al., 2002). When using polymers as a starting material, basic hydrolysis of the cationic moieties of the DMAEA-Q copolymers generates anionic polymers and choline chloride molecules. Either this choline chloride, or pure choline chloride when using a standard calibration series, is subsequently transformed into betaine chloride and hydrogen peroxide (H2O2) through the enzymatic conversion by choline oxidase. Finally, in the presence of horseradish peroxidase, the generated hydrogen peroxide transforms the non-fluorescent Amplex Red molecule into highly fluorescent Resorufin. First of all, 1 mg of Amplex Red reagent was dissolved in 200 ml DMSO in a small plastic vial covered with aluminium foil to protect it from degradation by light. The 250 mM Tris– HCl buffer was diluted 5 times with deionized water, resulting in a 50 mM solution. The individually packed horseradish peroxidase was dissolved in 1 ml of the 50 mM Tris–HCl buffer and the choline oxidase was dissolved in 600 ml of the 50 mM Tris–HCl buffer. The final reagent was prepared by mixing 200 ml of the Amplex Red in DMSO solution, 100 ml horseradish peroxidase solution, 100 ml choline oxidase solution and 10 ml 50 mM Tris–HCl buffer. The reagent was kept in a plastic container wrapped in aluminium foil to protect it from light. Secondly, pure polymer samples were prepared for measurement. Based on approximate charge density information supplied by the manufacturer, 2 g polymer/l solutions were diluted so that the estimated released choline chloride concentration was around 3 mg/l. The dilution from the 2 g polymer/l solution was performed in two steps, due to the
3.
Results and discussion
3.1. Fluorimetric measurements on choline standard series In Fig. 2, the fluorescence evolution as a function of time after addition of the final reagent is displayed for a standard series of choline chloride. It is noticed that the fluorescence intensity increases to a maximum after about 50–75 min, suggesting first-order reaction kinetics at a first glance. However, after reaching their maximum fluorescence intensity, a slight decline in fluorescence is noticed for some of the samples, especially the highly concentrated ones. The reason for this may be twofold. First of all, a competing reaction between horseradish peroxidase and resorufin that results in the formation of a non-fluorescent complex was reported by Zhou et al. (1997). Furthermore, the fluorescent resorufin molecule may be transformed into an unknown non-fluorescent molecule in the pH range 6.2–7.7 (Towne et al., 2004). Therefore, fitting first-order kinetics to the measured fluorescence pattern, in order to use the kinetic parameters for calibration purposes, may yield erroneous results. He et al. (2002) used a single time point from the kinetic data (at 20 min) to set up a calibration curve. Although this approach worked well for many samples, in some cases the fluorescence intensity showed some outliers at certain time points. Therefore, it was chosen to use the slope of the fluorescence intensity curve on the linear part of the curves, between 15 and 40 min, which yielded good calibration curves with a determination coefficient R2 larger than 0.95 in all cases.
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R CH2
CH O
CH
O
O
C
O
O CH2
Hydrolysis
+
Cl
HO
CH2
CH2
N
C
CH3 CH2
N
+
Cl
-
CH3
CH3
-
Betaine chloride
CH3 OH-/H 2O
N
O
+
CH2 H 3C
C
OH
Anionic polymer
C
NH2
O
NH2
CH2
C
C
C
R CH2
CH2
+
Cl
+
-
Choline oxidase H2O2
CH3
CH3 Horseradish
Choline chloride
peroxidase
-
CH3
CH3
HO
Cationic DMAEA-Q copolymer
CH C
O C
HC
C CH
CH C C
N
CH CH
HO
CH C
O C
HC
C CH
CH C C
N
O C CH
CH
Resorufin
C O
OH C
CH3
(Fluorescent)
Amplex Red (Non-fluorescent)
Fig. 1 – Schematic representation of the reactions involved for the fluorimetric analysis of choline chloride (whether or not derived from polymer).
3.2. Fluorimetric determination of polymer charge density Determination of polymer concentrations by charge titration techniques generally relies on a prior determination of the charge density of this polymer. Whereas the latter is usually also performed by charge titration, this study proposes an alternative method based on quantitative choline determination.
3.2.1.
Method development
A prerequisite for allowing charge determination by choline analysis is the complete hydrolysis of choline from the polymer within a reasonable time frame. In fact, it was shown in
Relative fluorescence units (-)
30000
Choline chloride concentration (mg/l)
25000 20000
4.76 3.81 2.86 1.90 0.95 0.00
15000 10000 5000 0 0
25
50
75
100
125
150
Measurement time (minutes)
Fig. 2 – Fluorescence evolution as a function of time after addition of the final reagent, for a standard series of choline chloride.
a previous study that DMAEA-Q polymers are sensitive to hydrolysis and that the percentage of hydrolysis reaches a more or less stable value, determined by the solution pH, after about 5–12 h (Saveyn et al., 2008). In the latter work, charge titration results showed that a pH value of 12 yields nearly complete hydrolysis upon overnight storage of the sample, so it was decided to let the samples in the current study hydrolyse at pH 12 for one night. However, after the basic hydrolysis step, the pH of the samples had to be readjusted. According to the instructions supplied by the manufacturer of the enzyme kit, the reaction with Amplex Red should be performed within a quite narrow pH interval of pH 7–8 for two main reasons. Some reagents are unstable at pH values above 8.5, whereas at lower pH values the fluorescence quantum yield of resorufin becomes markedly lower. Although the pH of the samples could have been adjusted by neutralising the base with acid, followed by addition of the 50 mM Tris–HCl reaction buffer, extensive acid/base addition could have led to high salt concentrations affecting the good functioning of the enzymes needed for the fluorescence reaction. Therefore, it was tested whether the Tris–HCl buffer was able to bring down the pH below 8 after basic hydrolysis of the sample. Samples of 5 ml of an aqueous polymer solution, with a similar concentration as encountered in the liquid phase of conditioned sludge (around 200 mg/l), were treated with 0.5 ml of a 1 M NaOH solution. The same was done for a random sample of sludge supernatant after conditioning with polymer and centrifugation and a sample of 5 mg/l choline in water. It should be mentioned that dosing higher amounts of NaOH could have led to choline being transformed into trimethylamine, a volatile odorous compound (Chang
water research 43 (2009) 1905–1912
et al., 2005). After addition of the NaOH solution, the pH of the different samples was 12.36 (polymer in water), 12.30 (sludge supernatant) and 12.07 (choline in water). These values showed that the pH of the different samples was high enough to allow complete hydrolysis upon overnight storage. After overnight storage, the samples were treated with 50 mM Tris– HCl buffer solution in a 1:7 ratio as was the case for the fluorimetric measurements. After this, the pH was measured again. The pH values now were 7.78 (polymer in water), 7.85 (sludge supernatant) and 7.80 (choline in water), which were all in the interval of 7–8, required for optimum reaction with Amplex Red. For the sake of completeness, it has to be mentioned that the buffer capacity of the 50 mM Tris–HCl buffer was not sufficient to reach a pH below 8 if 1 ml or more of 1 M NaOH was added to the sample.
3.2.2.
Method evaluation: comparison to charge titration
Nine different polymers, as used for sludge conditioning and dewatering experiments in a previous study (Saveyn et al., 2005), were investigated for their charge properties. The results of the charge determination by the choline method are depicted in Fig. 3, together with the results from charge titration and the charge density values supplied by the manufacturer. The latter data are based on the reaction stoichiometry, i.e. on the relative amounts of charged and uncharged monomers used in the process. The confidence intervals for the values supplied by the manufacturer are based on the product classification in 4 different categories: being 20, 40, 60 or 80% cationic. A 100% cationic polymer of this composition would only consist of polymerised DMAEA-Q with a molecular weight of 193.5 for the repeating unit, and hence 1 equivalent of charge per 193.5 g, i.e. a charge density of 5.17 meq/g. As a consequence of the broadness of the different charge density categories supplied by the manufacturer, the estimated uncertainty on every category is 10% cationicity, equalling 0.1 5.17 meq/g ¼ 0.517 meq/g. This uncertainty is depicted as the error bar on the manufacturer’s data in Fig. 3.
It is seen from Fig. 3 that the charge titration technique and the choline based determination of polymers yield very similar results. Except for polymers DP7-7442 and 7899, there is no statistical difference between the two methods (at 95% confidence level). This is confirmed by plotting the values of the choline based charge determination versus the charge titration data. The linear trendline through the obtained scatter plot has a slope that is not statistically different from 1 (95% confidence interval of [0.89; 1.17]) and an intercept that is not statistically different from 0 (95% confidence interval of [0.25; 0.44]). The determination coefficient R2 equals 0.98. The charge densities determined by both methods agree relatively well with the values supplied by the manufacturer. For the choline based determination, only polymer 7555 yields a significantly different value from the data given by the manufacturer. On the other hand, for the charge titration technique, 4 polymers yield a significantly different value from the manufacturer’s data (DP7-7442, 7555, 7651 and 7878FS25). With the exception of polymer 7651, which has a rather broad confidence interval for the choline based measurements, the charge titration technique and the choline based determination exhibited a similar reproducibility. One of the reasons why the charge titration technique may yield lower charge densities than the choline based technique or the data supplied by the manufacturer in some cases, could be related to the structure of the polymers. Polymers DP77442, 7867FS40, 7878FS40 and 7878FS25 are known to be crosslinked polymers. Therefore, some of their charges may not be readily accessible for the anionic polymer used in the titration, due to diffusion limitations. Although this problem is sometimes partially solved by shearing of the cationic polymer prior to titration, it remains uncertain whether all charges become accessible for titration in this way. This may also explain why the choline based measurement results, allowing a release of all charged sites from the polymer, may be closer to the manufacturer’s information.
3.3.
Charge density (meq/g)
5.0
1909
Residual polymer in sludge filtrate
Given that the choline based measurement delivered reliable results for the charge density of the different polymers, it was tested whether the same fluorimetric method could be used to quantify non-adsorbed polymers in conditioned sewage sludge samples. In this way, it would be possible to assess the optimal polymer dose and to avoid overdosage.
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
3.3.1. Sensitivity of fluorimetric method to wastewater matrix impurities and low temperature sample conservation
0.5
51
S2 5 78 78 F
40
76
42
78 FS 78
D
P
774
75 55
99
40
78
67 FS
50 78
76
LT
22
0.0
Polymer name Charge titration
Choline determination
Manufacturer's information
Fig. 3 – Charge density determination for different DMAEA-Q type cationic polymers according to two techniques (error bars represent 95% confidence intervals, based on at least 3 repetitions). For comparison, values as supplied by the manufacturer are also given; error bars indicate a 10% cationicity (0.517 meq/g) uncertainty in this case.
Prior to performing the sludge sample analyses, it had to be ensured that the proposed technique would be applicable on previously collected centrifuged supernatant samples that had been stored by freezing. One of the first issues to be dealt with was the possible background interference from naturally occurring choline or hydrogen peroxide in the bulk solution of the sludge sample. To assess this potential problem, activated sludge was conditioned with polyDADMAC, a flocculant that did not contain any precursors of choline. This sample was then centrifuged to remove all suspended material. Finally, a standard series of choline chloride were made in the
water research 43 (2009) 1905–1912
3.3.2.
Polyelectrolyte adsorption isotherms
Fig. 4 shows the adsorption curves of a series of polymers, applied on the same sludge sample, as a function of the added dose. Five polymers had been selected. Polymers LT22, 7555 and 7878FS25 have similar molecular weights, but clearly differ in measured charge density. Polymers 7899, 7555 and DP7-7442 have similar measured charge densities, but markedly differ in molecular weight. According to a previous classification (Saveyn et al., 2005), polymer DP7-7442 has a low molecular weight, polymers LT22, 7555 and 7878FS25 have a high molecular weight and polymer 7899 has an ultra-high molecular weight. Fig. 4 shows that the various polymers exhibited a clearly different adsorption behaviour, related to their charge density and molecular weight. For the polymer series LT22, 7555 and 7878FS25 with similar molecular weights but different charge densities, the data show that the lower the charge density, the higher the adsorption efficiency at higher doses. This can be explained by the fact that polymers with a high charge density will cause charge neutralisation of the sludge surface at low doses. Increasing the dose will then result in repulsion between the positive surface charge of the sludge and the positively charged polymers. For the polymer series 7899, 7555 and DP7-7442 with similar charge density but different molecular weight, it is noticed that a higher molecular weight results in lower adsorption efficiencies at high doses. Due to their bridging abilities, higher molecular weight polymers are assumed to form flocs without the need to completely neutralize the surface charge of the individual particles. As
14
Amount of polymer adsorbed (mg/kg DM)
supernatant from the latter, as well as in distilled water. These samples were then measured in duplicate according to the regular procedure. The calibration curves for choline chloride in distilled water yielded a slope of 50.3 RFU mg min1 l1 with a 95% confidence interval of [43.5; 57.2] RFU mg min1 l1 and a determination coefficient of 0.964. The calibration series in polyDADMAC treated sludge centrate yielded a slope of 48.1 RFU mg min1 l1 with a 95% confidence interval of [40.9; 55.3] RFU mg min1 l1 and a determination coefficient of 0.957. This shows that the choline measurement is not significantly affected by the multitude of components in a complex matrix such as sludge. A second concern was the influence of freezing on the samples. In this study, the supernatant samples had been taken immediately after centrifugation of the conditioned sludge samples and were then frozen. Therefore, it was necessary to assess the impact of freezing and subsequent thawing processes on the determination of choline from hydrolysed polymer. Hereto a solution of polymer 7878FS40 was made with an approximate concentration of 2 mg choline chloride/l. Part of the solution was kept in a fridge (4 C) and the remaining part was frozen overnight and thawed the next day. The choline concentrations were then measured in 6-fold for both samples. The choline concentrations measured were 2.36 0.10 mg/l and 2.23 0.26 mg/l for the cooled and frozen samples, respectively (95% confidence intervals). This demonstrates that the freeze/thaw process did not significantly affect the choline yield of the polymer sample. Therefore it was decided that the newly proposed method could be applied on the frozen sludge supernatant samples.
12
Polymer type 10
LT 22 (High MW) 7899 (Ultra-High MW) 7555 (High MW) DP7-7442 (Low MW) 7878FS25 (High MW)
8 6 4 2 2
4
6
8
10
12
14
Amount of polymer added (mg/kg DM)
Fig. 4 – Adsorption of DMAEA-Q type cationic polymers onto sludge. Data are derived from choline based measurement of supernatant obtained after centrifugation of conditioned sludge. The diagonal line represents complete adsorption, data below the diagonal line indicate less than 100% adsorption.
a consequence, flocculated entities are formed with a positive surface charge, which will prevent additional polymer adsorption due to electrostatic repulsion. The choline based adsorption data are confirmed by both electrophoretic mobility data of the sludge supernatant (Fig. 5) and by the capillary suction time (CST) values of the different conditioned sludge samples (Fig. 6). The electrophoretic mobility (m) of the supernatant indicates whether negatively charged particles are still present, or whether all particles have become neutralized and free polymer is in solution leading to a positive m value. CST, although not the perfect indicator for dewaterability, is a frequently used tool for determination of the optimum polymer dose. At lower than optimum dose, CST will be high due to inferior floc formation, at optimum polymer dosing CST shows a minimum value, whereas at overdosing the CST increases again because of a viscosity increase of the solution due to non-adsorbed polymer (Christensen et al., 1993). When comparing Fig. 4 to Figs. 5 and 6, it is noticed that polymers 7899 and 7878FS25
Electrophoretic mobility of conditioned sludge (1e-8m-2/s/V)
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Fig. 5 – Electrophoretic mobility data for sludge conditioned with different DMAEA-Q type cationic polymers. The data reflect samples identical to the ones depicted in Fig. 4.
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CST of conditioned sludge (s)
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4.
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30 25 20 15 10 5 0 2
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Fig. 6 – Capillary suction time (CST) data for sludge conditioned with different DMAEA-Q type cationic polymers. The data reflect samples identical to the ones depicted in Fig. 4.
(symbolised by triangles and circles, respectively), which show the lowest adsorption efficiencies at medium to high doses, also exhibit clear positive electrophoretic mobility values and high CST values at these same doses. Polymers like the LT22 (diamonds), which still show a high adsorption efficiency at high doses, also exhibit negative electrophoretic mobilities and low CST values at these doses. This clearly indicates that the optimum polymer dose, i.e. before the CST notably starts to increase, coincides with the point at which polymer adsorption is still nearly complete. A possible issue with the adsorption data concerns the reliability of choline chloride as an indicator molecule for dissolved polymer concentration. It may be argued that some of the choline molecules in solution are not derived from dissolved polymer, but rather have been hydrolysed from polymers that are still adsorbed onto the sludge particles. In the latter case, the choline measurement would yield an overestimation of the polymer concentration in solution. Yet, there are several arguments against this hypothesis. First of all, it is noticed in Fig. 4 that for the lower polymer concentrations, the adsorption is nearly 100%. The latter implies that negligible amounts of choline were measured in the supernatant, even though a considerable amount of polymer was present in the flocculated sludge system. Secondly, a combination of short sampling time, relatively high polymer concentration and mild pH ensured that the degree of polymer hydrolysis in the sludge sample was restricted to a minimum. Indeed, Aksberg and Wagberg (1989) demonstrated that at polymer concentrations of 400 mg/l, the time needed to hydrolyse half of the cationic charges of two DMAEA-Q type polymers was 24 h and 62 h at pH 7. In this study, the used polymer concentrations were of the same order of magnitude, with around 100–400 mg polymer/l conditioned sludge. Furthermore, the sludge pH was 7.0 on average, and situated in a narrow interval of 6.7–7.4. The time between the start of conditioning the sludge samples and freezing the isolated supernatant after centrifugation was 40 min at maximum. All these factors resulted in a low probability of hydrolysis of choline moieties from polymer chains adsorbed to the sludge particles.
Overnight basic hydrolysis at pH 12 of the cationic moieties of DMAEA-Q type cationic polymers resulted in the release of all choline chloride groups into solution. Choline chloride was determined analytically by fluorimetric analysis based on the enzymatic conversion of choline to H2O2, which allowed the transformation of Amplex Red into the fluorescent Resorufin molecule. It has been demonstrated that this technique enables a more reliable determination of the true charge density of a series of DMAEA-Q type polymers, in comparison to the traditional charge titration technique. Moreover, the specificity and sensitivity of the enzymatic conversion method also allowed for the determination of residual non-adsorbed polymer in conditioned sludge samples, without interference from other components and with limited sample pretreatment. As a consequence, it can be used to determine the adsorption behaviour of DMAEA-Q type polymers on sludge in practical conditioning and dewatering operations.
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