INTRODUCTION

1. Determination of nitrate in different matrices using capillary electrophoresis Theo P.E.M. Verheggen, Jetse C. Reijenga*, Fred Huf and Frans M. Everaerts Laboratory of Instrumental Analysis, University of Technology, Eindhoven, the Netherlands (*corresponding author, tgtejr@chem.tue.nl) INTRODUCTION Most teams were rather optimistic about the confidence interval. By observation it appeared that several mistakes were made. The most important one was that the standard deviation of the determination was supposed to the one originating from the use of the linear regression of the calibration graph, without taking into account contributions from sample preparation and dilution. As the correlation coefficients were generally excellent, the standard deviation from the fit would be small. The standard deviation of the extraction volume alone could easily have been 5% or more. Previous experiments in our laboratory indicate that the amount of sample (50 g) and the extraction volume (250 ml) should remain constant from run to run. Deviating from these guidelines even resulted in systematic differences in nitrate content found. Some student teams used a very small sample amount (<5g) in order to ovoid the dilution step. Most teams realized that they had been a bit optimistic on the confidence interval, but it must be pointed out it was the statistics of the total results (Fig.5.) that convinced them, probably not an increased insight into the contribution from the sample preparation (Fig.6.). Almost all of them recognized the few outliers, either by common sence, or by applying one of the established outlier tests. All teams agreed that the difference between the two potatoe brands was significant (results from team j and l would not support this). Systematic errors in the overall averaged results cannot be excluded. In our view the student's results, summarized in Figures 5, 6 and 7 are quite interesting from the didactical point of view. We intend to use the data sets in our present analytical chemistry course to illustrate aspects of accuracy and precision. Non-fertilized lettuce contained significantly less nitrate (2000 vs 2500 mg/kg) at only slightly smaller plant mass. The cadmium electrolyte system allows the simultaneous determination of nitrate and nitrite in the presence of excess chloride, such as in fysiological samples. Significant differences in nitrate content of different potatoe brands were found (80 and 270 mg/kg respectively). The sample preparation of the vegetables was the main cause of the total confidence interval and it would seem that the sample preparation method for the potatoes requires some adjustement so that an optimised, constant mass of sample is mixed with a constant extraction volume 1. Jandik P and Jones W R, J Chromatogr 546 (1991) 431-443 2. Kaniansky D, Zelensky I, Hybenova A and Onuska F I, Anal Chem 66 (1994) 4258-4264 3. Janini G M, Muschik G M and Issaq H J, J Cap Elec 1 (1994) 116-120 4. Guan F Y, Wu H F and Luo Y, J Chromatogr A 719 (1996) 427-433 5. Hargadon K A and McCord B R, J Chromatogr 602 (1992) 241-247 6. Gebauer P, Deml M, Bocek P and Janak J, J Chromatogr 267 (1983) 455-457 7. Kawamura Y, Takahashi M, Arimura G, Isayama T, Irifune K, Goshima N and Morikawa H, Plant and Cell Physiol 37 (1996) 878-880 8. Bondoux G, Jandik P and Jones W R, J Chromatogr 602 (1992) 79-88 9. Jimidar M, Hartmann C, Cousement N and Massart D L, J Chromatogr A 706 (1995) 479-492 10. Verheggen, Th.P.E.M. and Everaerts, F.M., J.Chromatogr., 638 (1993) 147-153 Potatoe analyses were carried out by 2nd year students of the Department of Chemical Engineering at the Eindhoven University of Technology in May 1998. The students processed their own data. under these conditions could be constructed, and nitrate, nitrite and chloride could be quantified simulaneously if necessary. Electropherogram of the determination of nitrate in a typical undiluted urine sample. 1=nitrate, 2=chloride, 3=sulfate, 4=nitrite (spiked), 5, 6 and 7 are unknowns. The analysis of nitrate and other inorganic anions has been a significant field of application of CE for years, and was reviewed in 1994 [1]. The optimization of the separation and trace level detection has been the subject of a number of papers [2-4]. Applications to the analysis to food related samples were also reported [5-9]. The present paper reports the application of CE to analyse nitrate in different matrices. Separation and detection Detection of nitrate in capillary electrophoresis can be carried out in the direct and the indirect mode. We chose for the former. Larges excess of chloride, such as in the case of biological fluids, requires additional measures regarding resolution. Because a chloride excess locally induces a lower fieldstrength this leads to a distorted peak of the co-migrating nitrate, which normally had about the same effective mobility. For this reason a background electrolyte was chosen that selectively retards chloride and sulfate by complexation with cadmium. The buffering co-anion in that case was acetate at pH 4.5. equipment Lettuce and urine were analysed in a home-made closed CE system, described previously [10]. The injection was 5 seconds at 2500 Pa pressure. The capillary was 75 m inner diameter fused silica. Potatoe analyses were performed using P/ACE 2000 and P/ACE 5500 (Beckman) electrophoresis systems, using 75 m capillaries of different lengths, detection was at 214 nm. Calibration graphs were made by changing the injection time. chemicals Background electrolytes consisted of a 0.01 M solution of hydrochloric acid, adjusted to pH 3.0 with -alanine (for the students analyses of potatoes), or to pH 8.1 with TRIS (for the lettuce analyses). The background electrolyte for the urine analysis was 0.005 Mol/l cadmium acetate, buffered to pH 5.0 with acetic acid. EOF was suppressed [10] by the addition to the BGE of 0.05% polyvinylalcohol (Mowiol 88-8, Hoechst, Frankfurt, FRG) and 5.10-5 M cetyltrimethylammonium bromide. The latter was anion-exchanged to remove the bromide. procedures calibration IS, sample pretreatment Lettuce samples were prepared by taking either the whole plant (excluding roots), or taking half or a quarter by vertically cutting the plant in slices and thus mixing several plants. An approximately constant weighed amount was put in an ordinary kitchen blender together with 250 ml of deionized water and blended for 5 minutes. The resulting mixture was filtered through a white band filter in a funnel and and 10 ml of the supernatant was pipetted into a 100 ml calibrated volumetric flask, 1 ml of a 2.5*10-2 M internal standard solution of potassium bromide was added and made up with deionized water. This was then filtered through a 0.45 m syringe type filter and injected. The potatoe analyses procedure for the students was given as follows: "Make a caplibration graph by injecting a 2.10-4 Mol/l standard solution of sodium nitrate with injection times between 1 and 20 seconds. Extract nitrate from a weighed amount of a roughly sliced (peeled) potatoe with a measured amount of deionized water in a kitchen blender for 5 minutes. The blender works best when using between 100 and 300 ml water. Make an estimate of the amount of sample to extract on the basis of an order of magnitude content of 500 mg/kg. Dilute the filtered extract if necessary." Lettuce analyses The lettuce analysis were carried out by constructing an internal standard calibration graph and injecting equal volumes of different nitrate standards. The individual peak areas had a relative standard deviation of 0.9% (Nitrate) and 1.5% (Bromide). The peak areas ratios had a relative standard deviation of 1.2%, so that the use of the internal standard was not necessary after all. A calibration graph without internal standard over one decade was excellent. Fig.1. nitrate calibration graph for lettuce analysis. Urine analyses Nitrate was determined in urine also with direct UV detection. Although chloride does not adsorb even at 200 nm, a large excess comigrating with chloride will lead to unacceptable peak distortion and poor detectability, caused by the locally lower fieldstrength. More selectivity between chloride and nitrate was required and accomplished by using complex formation [6], in this case using Cadmium to retard chloride. Although mobility matching is fair, a good calibration graph of chloride Potatoe analyses The student teams generally made calibration graphs consisting of 6-12 points and analysed the samples in duplicate or triplicate. The one obvious outlier must be due to a calculation error. Intervals indicate 95% confidence interval. Note the logarithmic concentration axis. EXPERIMENTAL Individual results of the two different potatoe samples were apparently correlated (Fig.6.) which can only be explained by individually different systematic bias in the sample preparation procedure. Finally all student teams processed all collected data in order to give a final verdict as to the nitrate content. The results are depicted in Fig.7. CONCLUSIONS The non-fertilized plants ended up with a slightly reduced mass at the end of an exponential growth curve. Fig.2. Growth curve of the lettuce plants. REFERENCES It would seem that in spite of the initial nitrate amount, the concentration dropped gradually after 8 weeks. When harvested after 12 weeks, concentrations were slightly less than 2000 mg/kg for the non-fertilized lettuce and less than 2500 for the fertilized crop. The standard deviations indicated in Fig.3. include the total determination, where aspects of sample preparation predominated. ACKNOWLEDGEMENT