ANALYTICA ACTA

Analytica Chimica Acta 312 (1995) 107-113

CHIMICA

Determination of nitrate in municipal waste water by UV spectroscopy

Mikael Karlsson a* *, Bo Karlberg b, Ralf J.O. Olsson a
a Department of Chemical Engineering and Technology, Royal Instirute of Technology, S-100 44 Stockholm, Sweden
b Department of Analytical Chemistry, Stockholm University S-106 91 Stockholm, Sweden
Received 11 November 1994; revised 3 March 1995; accepted 29 March 1995

Abstract
A method is proposed in which diode-array UV-visible spectroscopy and multivariate data analysis are applied to determine nitrate in municipal waste water. No filtering of the samples is required and no reagents are added. The working range for the nitrate determination is 0.5-13.7 mg/l (0.008-0.22 mM). The relative standard deviation was found to be 3.4%. Other constituents, notably total phosphorus, total nitrogen, ammonium nitrogen and iron can be determined simultaneously with the same method. The developed method can also be used to classify samples. The described method concept is well suited for in-line monitoring. Keywords: UV-Visible spectrophotometry; Nitrate; Waste water; Chemometrics;
Waters

1. Introduction
abundant form of inorganic is formed during the nitrification step when municipal waste water is aerated. Continuous monitoring of nitrate is desired during both the nitrification and the denitrification steps. Many methods have been proposed for nitrate measurement, for instance methods involving ion selective electrodes, direct UV spectroscopy, and wet chemistry methods, the latter methods being automated using either airsegmented flow or flow injection analysis principles. Most commercial nitrate selective electrodes are based on incorporating an ion exchange material in PVC membranes [l]. The dynamic range for the Nitrate is the most nitrogen. It

* Corresponding 0003-2670/95/$09.50

author. 0 1995 Elsevier Science B.V. All rights reserved

activity is typically 10-l to 10m5 M. However, several anions interfere, chloride ions in particular, which means that the electrode needs to be recalibrated frequently. The life-time of the membrane is limited due to the continuous leaching of the ion exchanger. The nitrate ion absorbs strongly in the UV range with a maximum absorbance at 205 nm and methods based on this property have been developed previously [2,3]. When such a sensor is used in municipal waste water samples, a filtering step has to be included before the UV measurement can be performed to account for turbidity. Furthermore, if the method is employed for continuous monitoring, the optical parts immersed in this type of sample must be regularly cleaned due to biofilm formation. To compensate for variations in sample turbidity, measurements are usually performed at two wavelengths, nitrate

SSDI 0003-2670(95)00179-4

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predominantly at 220 and 275 nm. The absorbance registered at the latter wavelength is subtracted from the absorbance value observed at 220 nm in accordance with the proposed manual method [4]. The commonly applied official method for determination of nitrate is based on reduction of nitrate to nitrite in a cadmium column followed by addition of sulphanilamide and N-1-naphthylethylenediamine hydrochloride (NED) [4-71. Again, the sample solutions must be filtered. Automated versions of this batch nitrate method exist either developed for airsegmented flow systems or for flow injection systems. The automated versions can be used for monitoring purpose but they involve use of toxic reagents and they require frequent recalibrations. In order to avoid cadmium, hydrazine has been suggested as a reducing agent for nitrate. This reduction is slow at room temperature which means that heating and temperature control are required in order to obtain desired sensitivity [7,8]. Thomas et al. [9,10] have developed a method for nitrate based on UV absorbance measurements at several wavelengths. The calculation of the nitrate concentration was made using a multilinear regression method. The method presented in this paper comprises steps of absorbance measurements in the spectral range 180-820 nm directly on unfiltered samples, comprehensive calibration using samples with known nitrate concentrations and chemometric data treatment (PLS, partial least squares).

preserved and had to be analyzed within 8 h of acquisition. Altogether 114 samples are included in this study and they were treated as follows. An accredited laboratory, Stockholm Vatten, performed determination of nitrate according to a reference method based on measurement of absorbance at 220 nm and at 275 nm [4]. The absorbance difference was calculated and used in a traditional calibration and evaluation procedure. Total phosphorus, total nitrogen, ammonium nitrogen, suspended solids, COD, BOD, alkalinity, and iron were determined according to their respective official methods [4-71 by the same accredited laboratory being a part of the authority approved control program for the waste water plants in Stockholm. For determination of ammonium nitrogen, nitrate, alkalinity and iron all samples were filtered through a 0.45 pm membrane filter; in all other tests unfiltered samples were used. In addition to these determinations, but simultaneously, an unfiltered portion of each sample was subjected to the proposed method: the sample portions were shaken and then directly transferred to a 10 mm quartz cuvette. A spectrum in the wavelength range 190-820 nm was recorded consisting of 316 absorbance values (one absorbance value at every second nm) for all 114 samples by using two different HP 8452A instruments in random order. The software used in this study was MATLAB@ Version 4.2 [ll].

3. Theory 2. Experimental Samples were taken daily in three different municipal waste water treatment plants in the Stockholm area during a period of six months to be able to cover seasonal and occasional variations. For each plant, three different locations were selected for sample acquisition, namely the incoming water site, the basin holding water that just had been processed in the chemical step, and, finally, the effluent water site; in all, nine sampling sites. Week samples consisted of aliquots collected daily during one week and these samples were preserved by adding concentrated sulphuric acid (1 ml acid/100 ml sample). Day samples were not 3.1. Selection of multivariate calibration technique Due to the shape of spectra obtained for unfiltered, municipal waste water samples containing nitrate, see Fig. 1, it is impossible to correlate absorbance values at just one wavelength to the concentration of nitrate. An interfering phenomenon such as light scattering due to turbidity is observed. Absorbance maxima occur in the region 200-220 nm as expected for samples containing nitrate. However, the presence of UV absorbing species other than nitrate cannot be excluded which adds to the complexity of calibration and evaluation of results. Consequently, a multivariate data analysis approach is required using as many absorbance values as possi-

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Fig. 1. Absorbance water samples.

spectra

of 114 unfiltered

municipal

waste

ble in a spectral scan. Several different approaches were initially tested. Classical multiple linear regression (MLR) using all wavelengths fails completely as a calibration technique. However, by using an iterative selection of five wavelengths for classical and inverse MLR useful results can be obtained, see Table 1. In the same table results from applying the partial least squares (PLSl) method are given. The MLR models were made with absorbance values at every single wavelength and the wavelength that resulted in the best model, i.e., the model that yielded the lowest RMSEP value, was selected. The selected wavelength was combined with all other wavelengths and the best combination was selected, this was performed until in all five wavelengths had been established. When comparing the MLR models with the PLSl model the latter gives the best predictions of nitrate and it offers the useful additional advantages of being self-diagnostic and being able to

detect outliers. Consequently, the PLSl model became the model of choice for all further work. The PLSl model is based on the NIPALS algorithm [12]. It is an obvious advantage if one common calibration model can be used for any sample in a waste water plant. A large concentration range of the analyte can then be covered since all samples are included in the model. The regression model will furthermore be more robust if a large concentration range is covered. The samples used for calibration purposes were collected over a period of at least six months and the concentration ranges for the various analytes were rarely exceeded during more than one years time judging from analytical data produced by the accredited laboratory (Stockholm Vatten). However, if a dramatic change of the conditions in the sewage plants would occur an extended calibration must be performed. When the PLSl method is used such a change would be detected with the present calibration model. 3.2. Selection of pretreatment method for the spectral data The assumptions leading to Beers law are not fulfilled when light scatter effects are present. These effects are caused by the physical properties of the sample, e.g., turbidity variations. Various spectral pretreatment methods and theories have been developed to improve the accuracy in the PLS regression among which the following three methods have been used in this paper: . the multiplicative scatter correction (MSC) method in which each spectrum is corrected in both offset and slope by comparison with the mean spectrum of the total dataset [13];

Table 1 Comparison

of different

multivariate PLSl using all wavelengths RMSEP

calibration

models for nitrate Classical MLR using 5 wavelengths Inverse MLR using 5 wavelengths RMSEP 0.4215 0.2676 0.1573 0.3880 Slope/bit. 0.99/0.01 o.so/o.ss O.SS/O.lO 0.99/0.01

Sample origin

Slope/Int. 0.99/0.05 0.86/0.40 0.98/0.01 0.99/0.01

RMSEP 1.9550 0.2988 0.2502 5.5322

Slope/Int. 4.67/ - 37.5 1.23/ - 0.61 0.59/0.30 0.70/ - 1.15

Basin 1 Basin 2 Basin 3 All samples

0.3041 0.2359 0.1518 0.3619

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the 2nd derivative method -the absorbance values forming a spectrum are derived twice which results in a transformed spectrum consisting only of the relative changes between absorbance values obtained at adjacent wavelengths. Derived peak intensities tend to change more linearly with analyte concentration than corresponding raw intensities [14]. The 2nd derivative was performed according to the Savitzky-Golay convolution derivation [ 151; the auto-scaling method (or z-transform) is a combination of mean-centering and normalization [ 161. The normalization was done by dividing each variable by its standard deviation (this gives each variable unit variance). The MSC was performed according to A,= R,-hi ii (1)

formed separately for nitrate but also, separately, for other sample constituents. In order to evaluate the influence of the different pretreatments the root mean squared error of prediction (RMSEP) [191 was calculated according to RMSEP = $(ei-ci) I= 1

(2)

where A, = transformed absorbance; R, = apparent absorbance; Lii= least squares estimation of the intercept parameter; Ai = least squares estimation of the slope parameter; i = the sample spectrum available (i = 1, 2, . . . , 114); k = the absorbance values at available wavelengths in a scan (k = 1, 2, . . . , 316). A factorial design model [17] was used to establish the optimal pretreatment or combination of pretreatments (MSC, 2nd derivative, auto-scaling) and thus minimizing the prediction errors arising from the modeling of the data. In the factorial design the different pretreatments have been modeled as variables at an on/off state [18]. The design is a full 23 design (see Table 2). This methodology was per-

where IZ= the number of samples (114); ti = modelled descriptor value; ci = the traditionally measured descriptor value; i = the sample number (i = 1, 2, . . ., 114). The RMSEP was then evaluated as a function of a number of latent variables kept in the PLSl model. The pretreatments yielding the smallest RMSEP for the different descriptors were used in the subsequent PLS modelling. All evaluations were made with leave-one-out cross validation modelling [20] instead of dividing the samples into a calibration set and a validation set.

4. Results and discussion The results from the evaluation of the different spectral data pretreatments can be seen in Table 2: A - sign in the Pretreatment column stands for a pretreatment that was not performed and a + sign for a performed pretreatment; the first column corresponds to MSC, the second is the 2nd derivative and the third column is auto-scaling. The largest difference in model quality is dependent upon the use of auto-scaling, see Table 2, alone

Table 2 Results of the pretreatment Pretreatment + + _ + _ + _ + + + +

optimization

for nitrate PCS Intercept (ppm) - 0.020 0.0441 - 0.005 -0.001 0.3852 0.3700 0.3480 0.3897 Slope 1.0001 0.9960 0.9991 1.0009 0.9969 1.0025 1.0051 0.9985 r2 RMSEP (ppm) 0.9955 0.9948 0.9960 0.9955 0.9951 0.9947 0.9936 0.9935 0.0843 0.0975 0.0743 0.0841 0.2297 0.2448 0.2557 0.2684

_ _ _ + + + +

15 15 13 1.5 14 12 7 8

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or in combination with any of the other two data pretreatment methods. There is a significant difference, a factor three reduction of precision when using auto-scaling. The best results in this study are obtained when the 2nd derivative data pretreatment is used alone. The explanation for this might be that this pretreatment results in a spectrum consisting of only the relative changes between absorbance values at adjacent wavelengths. In this situation light scattering effects due to particles in the sample are reduced since they are mathematically filtered away. The method also decreases spectral noise since the transformed points are the evaluation of a polynome which has been fitted to the adjacent spectral points. This means that the transformed spectra contain the main chemical information. The MSC method, which also adjusts for light scattering, does not improve the model. One probable reason for this is that spectral data differs for samples taken at various sites in the municipal waste water treatment plants. When applying the MSC method all spectral data are used simultaneously in the algorithm and problems may then arise if the spectra are too different. The method does not comprise a noise reduction step. Auto-scaling fails to improve the model since all variables are given the same mathematical weight in

Fig. 3. Histogram showing the residuals between predicted and reference values. The normal distribution curve (m = 0.0429; s = 0.3101) is denoted by the full line.

spite of the fact that the chemical information is not equally distributed over all variables. In Fig. 2 the predicted values of nitrate in 114 samples are plotted against the nitrate values obtained with the reference method. Only the 2nd derivative data pretreatment method was applied in this case. Significantly, the prediction error is smaller than the error reported for the reference analysis. It may seem peculiar that a second order analytical method has a smaller error than the reference method. However, it can be explained as follows.

-0

4 6 0 REFERENCe CONCWTRAITON,

10

I2

14 -0 6 7 6 9 10 $1 12 13 I4 15

ppn

1
16

Fig. 2. Predicted concentration vs. reference concentrations of nitrate (114 waste water samples). Slope = 0.9991; intercept = - 0.005; RMSEP = 0.2727; r 2 = 0.9960.

Fig. 4. Score plot (PC1 vs. PC2) showing taken at three different stages in the plants.

waste water samples

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Table 3 Results of modelling for total phosphorus, total nitrogen, ammonium nitrogen and iron Constituent Total phosphorus Total nitrogen Ammonium nitrogen Iron r2 0.6731 0.7643 0.8144 0.9124 Range (ppm) 0.03-5.20 15.0-39.0 0.70-30.20 0.30-21.20 1.0137 1.0073 0.9917 1.0090 Slope Intercept (ppm) 0.0264 -0.2320 0.1462 - 0.0370 RMSEP (ppm) 0.9162 2.8378 2.5742 2.1741

If: the predicted value, 2, equals the true value, Y, with a prediction error, 2, Eq. 3; the reference value equals the same true value with a reference analysis error, crep, Eq. 4 Y=Y+o Yref = Y+ %rer (3) (4)

The reference errors will be normally distributed and centered around zero if no systematical errors were introduced during the analysis, i.e.:
%f E Nmd (5)

However, the prediction errors are most probably not normally distributed since they are obtained from a regression model, i.e., the prediction model does not contain any random source; thus E,,f +LWr,o) (6)

Consequently, when examining the distribution of the residuals for the difference between p and Yef one can determine the one of .? or &,,which will dominate since Y- Yef = .Z- E,,f (7)

In Fig. 3 the residuals for the difference between Y and Yef for all 114 samples are shown in a histogram. The residuals have zero mean and are normally distributed. This indicates that the prediction error is smaller than the reference error, i.e.: Eref> > B (8)

improve the model for these parameters a comprehensive calibration is required entailing samples with larger matrix and concentration variations than those utilized in this study. Most likely, further parameters like, for instance, suspended solids, BOD and COD can be semiquantitatively predicted along with nitrate, total phosphorus, total nitrogen, ammonium nitrogen, and iron based on one single run of a sample spectrum. This possibility is certainly very challenging when considering unattended monitoring of waste water since no handling of reagents is needed. When the PLS algorithm is used to build a regression model it is also possible to use the intermediate scores and loadings vectors to obtain qualitative models, e.g., classifications. In Fig. 4 the score vector of the 1st and 2nd principal component plotted against each other and the samples forms three clusters. The clusters represent samples from the three different sites in the municipal waste water plant. Thus, it is possible in this case to identify from which site a sample is taken by the scores. The classification models can consist of n number of score vectors. It is also possible to identify samples that have been tampered with and abnormal samples containing unusual chemicals that can be harmful for the biological activity in the plant. Samples spiked with a UV absorbing compound such as nitrobenzene were identified as outliers by the model.

The same model was also used in an attempt to predict sample constituents other than nitrate. The model results for prediction of total phosphorus, total nitrogen, ammonium nitrogen and iron can be seen in Table 3, where the model quality parameters for the best model, as discussed above, are listed. To

5. Conclusions The developed concept has been shown to work well for collected, real samples and it should also have a large potential for in-line monitoring. Samples have been collected from three different waste

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water plants at three different sites during a time period of six months which means that seasonal and occasional variations are accounted for in the calibration model. By studying the laboratory reports over a period of more than one year the conclusion is that the total concentration ranges of most of the analytes were covered in the calibration. This does not mean, however, that the calibration model would be adequate for waste water samples collected in another geographic area with a different industrial structure than that of Stockholm. The selection of PLS as a multivariate calibration technique was based on the obvious advantages of the outlier detection and the inherent self-diagnostic abilities of this technique. Samples can furthermore be classified. The working range for the nitrate determination was 0.5-13.7 mg/l and the relative standard deviation was 3.4%. Simultaneously, a quantitative determination of analytes other than nitrate in the sample can be performed, e.g., total phosphorus, total nitrogen, ammonium nitrogen and iron. References [I] J.E.W.
Davies, G.J. Moody and J.D.R. Thomas, (1972) 87. Analyst, Analyst, 97 104

131 K.C. Thompson and M. Blankley, Analyst, 109 (1984) 1053. [4] Standard Methods for the Examination of Water and Wastewater, 17th edn., American Public Health Association, Washington, DC, 1989. IS] Analytical Methods Manual 1979, Inland Waters Directorate, Canada NAQUADAT, 1979. [6] American Society for Testing and Materials, ASTM Standards, Water, Philadelphia, PA, 1977. [7] Methods for Chemical Analysis of Water and Wastes, Environmental Protection Agency, Cincinnati, OH, 1971. [8] L. Kamhake, S. Hannah and J. Cohen, Water Res., 1 (19671 205. [9] 0. Thomas and S. Gallot, Fresenius J. Anal. Chem., 338 ( 1990) 234. [lo] 0. Thomas, S. Gallot and N. Mazas, Fresenius J. Anal. Chem., 338 (1990) 238. MATLAB, The Mathworks Inc., Natick, MA, 1977. 1111 WI H. Wold, in F. David @d.), Research Papers Statistics, 1966, pp. 411. [131P. Geladi, D. MacDougall and H. Martens, Appl. Spectrosc., 39 (1985) 491. [141T C. OHaver and T. Begley, Anal. Chem., 53 (1981) 1876. D51 A. Savitxky and M.J.E. GoIay, Anal. Chem., 36 (1964) 1627. [161R. Carlsson, in B.G.M. Vandeginste and O.M. Kvalheim (Eds.), Data Handling in Science and Technology, Vol. 8, Elsevier, Amsterdam, 1992, p. 1. [171G.E.P. Box, W.G. Hunter and J.S. Hunter, Statistics for Experimenters, Wiley, NY, 1978, p. 653. b31 R.J.O. Olsson, in K.I. Hildum, T. Isaksson, T. Noes and A. Tandberg (Eds.), Near Infra-Red Spectroscopy, Ellis Horwood, Chichester, 1992, p. 103. Wiley, D91 H. Martens and T. Nss, Multivariate Calibration, New York, 1989. DOI S. Wold, Technometrics, 20 (1978) 397.

[2] J.P. Rennie, A.M. Summer and F.B. Basketter, (1979) 837.