Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 42 SEPARATION OF SELECTED PESTICIDES BY AN HPLC TECHNIQUE; PERFORMANCE PARAMETERS AND VALIDATION *Camelia POPA1 1 Faculty of Food Engineering, tefan cel Mare University of Suceava, Romania camelia.popa@fia.usv.ro *Corresponding author Received 25 August 2012, accepted 14 September 2012 Abstract: The aim of this work is to get the performance parameters investigated by hight performance liquid chromatography (HPLC), for the separation method of 2,4-Dichlorophenoxiacetic acid, 3,6-Dichloro-2-methoxybenzoic acid, an organic mixture with herbicide action. The chromatographic separation with better peak shape was achieved. The retention times (tR), peak resolutions (RS), separation factors ( ), column efficiency (Neff), height of theoretical plates (HETP), indicate that the mobile phases in gradient, containing acetonitrile and water with 1% acetic acid are the best for the separation of investigated components on cromatographic column C18. Also it has been shown that in data conditions the methode is sensitive, precise and reproductible. Keywords: HPLC, environment, performance, validation 1. Introduction Pesticides are chemical protection tools for plants. They are obtained from one ore more biological compounds efficient.With few exception like growing regulators, the biological active ingredients are toxic. Due to this toxicity are dictate good practice in dose, distribution and in use of pesticides. Also pesticides pass from ground water to vegetables [1], plants and foods and finally they are accumulate in animal fat. Pesticides affect the structure and immune efficiency and reduce the immunity at infection. The farmers that use the pesticides must take into acount the parameters, as follows: the configuration of marketing, the technology of conditioning, the technology to apply, the maximum limit of waste.The mixture of these two components with herbicide action are used in disproof of weed from beating cereals. 2,4- Dichlorophenol is the toxic component born in the of 2,4- dichloro-phenoxiacetic acid manufacturing process and retrieved in the end of the mix in acceptable limit.The performance parameters of this separation method have a great importance, they reflect the correct and the exactely dosage of the components in the mixture and on ground. The presence of pesticides in environment induces the modification of the quality environment components- ground, underground and surface water, the optimization of these quntities meaning an important factor in environmental quality protection. 2. Experimental Chemicals and reagents The components of mobile phases: acetonitrile and water (LABOSI), HPLC grade. Acetic acid, glacial degree, 2,4- diclorphenoxiacetic acid 99,9 % purity (named 2,4D acid), 3,6-dichloro-2- metoxibenzoic acid 99,9% purity (named dicamba), 2,4 dichlorophenol 99% purity (named DCF) from Merck. mailto:camelia.popa:@fia.usv.ro Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 43 Instrumentation and conditions The cromatographic investigations was carry out on a VARIAN PROSTAR liquid cromatograph system equipped with: quaternary pump (model 9100), autosampler (model 9010), UV detector (model 9065). The data were aquired via Prostar data aquisition workstation. Mobile phase consists of water and acetonitrile HPLC grade, injection volume: 20 µl, flow rate : 1ml/minute, : 280nm. Reversed phase analysis was performed at 220C using an Bondesil C18 column, 5µ (25cm, 4mm ID) [2]. Table 1 shows the gradient elution used. Table 1 Gradient of mobile phase Time(minutes) B (%) C (%) 0 95 5 9 95 5 17 50 50 30 50 50 B = 1% acetic acide in HPLC water; C = 1% acetic acid in HPLC acetonitrile. Elution order: dicamba, 2,4D acid, 2,4DCF Standard preparation To get the separation parameters was used synthetic standard solution named stock solution: 0.07 g 2,4D acid, 0.025g Dicamba, 5ml DCF standard solution, completed to 25 ml with (alkaline) HPLC grade water. Syntetic standard solution keeps the same report between the components like in the mixture with herbicide action. DCF standard solution was prepared from 0,1gDCF diluted to 25ml with (alkaline) HPLC water. Sample preparation A representative quantity of sample is weighed and the active ingredients are extracted with selective solvents. Follow the evaporation of solvent and than active ingredients are solved and diluted to 25 ml with HPLC grade water. Calculations Capacity factor (K’) [3] was calculated using equation (1): ot ' Rt ot otRt'K (1) where: tR is the retention time of the solute t0 is the time for an unretained solute; t’R is the adjusted retention time of the solute The condition of strong separation from technical book of Varian instrument is K’ 1 [3]. Column selectivity ( ). The separation factor ( ) [3] was calculated using equation (2): ' 1Rt ' 2Rt (2) where: ' 2Rt and ' 1Rt are adjusted retention times for two adjacent peaks. The selectivity condition is 1 . Peak resolution (RS). The peak resolution (RS) [4] was calculating using the equation (3): R S 1 2 1.18× t R = w +w (3) where: tR is the difference in retention times between the two peaks; w1 and w2 are widths of the two peaks at half of their height. The condition of separation is: RS = 1 means 98% separation; RS = 1.5 means 99.7% separation. Column efficiency (Neff ). The column efficiency [4] was calculated as number of theoretical plates using equation (4): 2 w ' Rt54.5effN (4) Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 44 From technical book of Varian instrument the efficiency condition is Neff > 400. Height of theoretical plates (HETP) [4] was calculated using equation (5): effN L HEPT (5) where: L is the length of the column (cm); Neff is the effective number of theoretical plates. Also, from technical book the accepted value is HETP = 0.001 ÷ 0.002 mm. Standard deviation (Sr) [5, 6] was calculated using equation (6): 1n n 1k 2 xkx rS (6) Repeatability limit (r) [9] was calculated using equation (7): rS2tr (7) where t = 1.96, student coefficient for 95% confidence interval. 3. Results and discussions For cromatographic separation of 2,4D acid, Dicamba and DCF on C18 stationary phase (4,6mm, 5µm) with varying column lengths from 150 to 250 was attempted. Different mobile phase composition containing water and acetonitrile with 1% acetic acid were tried. The column 250mm x 4,6mm, 5 µm showed higher elution times and good resolutions for the components of interest, respectively 21,89 seconds for dicamba, 24,60 seconds for 2,4D acid and 26,20 seconds for DCF. System suitability is shown in Table 2. Using equations from ,,calculations” capter and the chromatogram obtained, was calculated the performance parameters that shows the efficiency of separation in the conditios of the method. Performance parameters are shown in Table 3. We can see strong values for performance parameters in the conditions of the method: peak resolution, column efficiency and height of theoretical plates. The results show very good performance parameters of this separation methode.This HPLC separation method of the organic mixture with herbicide action is selective, fact demonstrated by the selectivity (specificity) of the instrument/equipment and the separation conditions on chromatographic column, C18. Table 2 System suitability Component dicamba 24D acid DCF tR (minutes) 21,89 24,60 26,20 Table 3 Efficiency of separation Performance parameter Accepted value [4] Obtained value Capacity factor (K’) 1 K’Dicamba = 16.6 K’2,4DAcid = 17.6 K’DCF = 18.3 Column selectivity ) 1 Dicamba = 1.04 2,4Dacid = 1.06 DCF = 1.05 Peak rezolution RS 1 for 98% separation 1.5 for 99.7% separation RDicamba = 3.8 R2,4DAcid = 6.5 R DCF = 4.6 Column efficiency Neff (number of theoretical plates) 400 NeffDicamba = 258475 Neff2,4Dacid = 171600 NeffDCF = 307000 Height of theoretical plate (HETP) 0.001÷0.002mm HETPDicamba = 0.001mm HETP2,4Dacid = 0.002mm HETPDCF = 0.001mm Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 45 Method validation The proposed method was validated with respect to linearity, accuracy, precision, specificity, following the HP Guide for HPLC, CE and UV-Vis spectroscopy [7]. Linearity (sensitivity) and range Linearity test solutions were prepared by diluting stock solution at five concentration levels of analytes concentration. The solutions were injected in triplicate and following regresion equations were found by plotting peak area versus concentration. The response is linear on area of concentration chosen if the results dont’t have a signifiant deviation from linearity, this means, an corelation coefficient bigger than 0,997 for all components. The obtained equations for regression lines are: YDicamba = 9770,32x + 15685,5; Y2,4Dacid = 79022x - 77878,5; YDCF = 4441,75x-8114,5. The coefficient of determination (R2) obtained for regression line demonstrates the excellent relationship between peak area and components concentrations. The results are shown in Table 4. Precision [7] in retention times and peak area (or height) are major criterion of separation systems. The precision of the chromatographic method reported as percent of relative standard deviation (Sr) was estimated by measuring repeatability on five replicate cromatograms [8]. Table.4. Linearity results for LC method Compo- nent Concen- tration Equation for regression line R2 Dicamba 0,25 - 2,25 mg/mL Y = 9770,32 x + + 15685,5 0,998 2,4D acid 0,7-3,5 mg/mL Y = 79022 x – – 77878,5 0,999 DCF 5-25 µg/mL Y = 4441,75 x – – 8114,5 0,999 The relative standard deviation values (Sr) and the repeatability limit (r) for retention times and areas are shown in Table 5. Xk – Xk-1 is the difference between two individual results that must be smaller than repeability limit.The condition Xk – Xk-1 r, is accomplished. Precision in analisys and accuracy Accuracy was estimated by spinking the sample matrix of interest with a known concentration of reference material: C2,4DAcid = 28.5%; CDicamba = 9.5%; C2,4DCF = 0.1% the same as the concentration of formulated herbicides. It was compared the response obtained after the extraction of analyte from the sample and injection in the column with the response of the reference material added to the pure solvent (Table 6). Table 5. Repeatability for retention times and areas Component Dicamba 2,4D acid DCF Parameter/RUN Area (counts) rt (minutes) Area (counts) rt (minutes) Area (counts) rt (minutes) Run 1 424827 21.89 2017948 24.56 1477 76 26.18 Run 2 392906 22.01 1864533 24.49 1334 77 26.02 Run 3 418781 22 1934687 24.53 1418 34 26.06 Run 4 403003 22.05 1893372 24.6 1562 84 26.18 Run 5 383215 21.97 1854117 24.56 1325 26.24 Food and Environment Safety - Journal of Faculty of Food Engineering, tefan cel MareUniversity - Suceava Volume XI, Issue 3 – 2012 46 40 Sr 17369 0.06 66494 0.05 9985 0.18 r 48003 0.16 183762 0.13 2759 5 0.49 Xk – Xk-1 r OK OK OK OK OK OK Table 6 Precision and accuracy of measurement RUN 2,4D acid (%) Dicamba (%) DCF (%) 1 28.50 9.50 0.093 2 28.00 9.60 0.100 3 28.51 9.56 0.075 4 28.36 9.90 0.103 5 28.78 9.47 0.09 Accuracy- Student variable ( 1) 0.148 0.086 0.016 Precision as Sr, (%) 0.28 0.17 0.03 4. Conclusions The resuls show that HPLC separation method of the organic mixture with herbicide action is selective, fact demonstrated by the selectivity of the instrument/equipment and the separation conditions on chromatographic column, C18. To note, the performance parameters with strong values in the conditions of the methode: the resolution of separation, column efficiency and height of theoretical plate. Also, it has been shown that in data conditions the methode is sensitive, precise and reproductible. 5. References [1]. JONHSON, A.C. et al., Penetration of Herbicides to Groundwater in an Unconfined Chalk Aqiufer Following Normal Soil Applications, J. Contam. Hidrol. 53., 100-120, (2001) [2]. POPA, C. et al., HPLC separation methode for the content of 2,4- diclhorphenol, diclhorphenoxiacetic acids and 3,6-dichloro-2 metoxibenzoic acid from an mixture with herbicide action B.I.115002B1, OSIM (1994) [3]. Nomenclature for Cromatography (IUPAC reccomendations 1993). Pure Appl Chem 65. 820-870, (1993) [4]. Operation book for HPLC VARIAN PROSTAR instrument, (1994) [5]. SR ENV 13005:2003. Guide to the expression of uncertainty in measurement. Standardization Association of Romania, Bucharest, Romania [6]. CHAIRMAN W. et al, EURACHEM/CITAC Guide CG4-The Fitness for Purpose of Analitycal Methods. A Laboratory Guide to Method validation and Related Topics, 2000. [7]. HUBER, L. Good Laboratory Practice, HP publication number 12-5091-6259E, Marcel Dekker Inc. 31-62, (1993) [8]. Validation of Compendial Methodes, US Pharmacopeia XXII, , National Formulary, XII, The US Pharmacopeial Convention Inc, Rockville, MD, 1990, General Chapter (1225), 1710-1612 [9]. ISO 3534-1:2009, Statistics.Vocabulary and symbols. Part 1: General statistical terms and terms used in probability, Standardization Association of Romania, Bucharest, Romania, (2009)