MICROWAVE SOLVENT EXTRACTION TECHNIQUES FOR VARIOUS REGULATORY COMPOUNDS

Transcription

1 MICROWAVE SOLVENT EXTRACTION TECHNIQUES FOR VARIOUS REGULATORY COMPOUNDS J.R. Fish, FAS Analytical Services, Monroe, NC M.D. Miller, CEM Corp., Matthews, NC Presented at the 1996 Pittsburgh Conference Chicago, IL March 3-8, 1996

2 The 1970 s and 80 s represented a period of rapid growth in the separation sciences, with gas and liquid chromatography as well as the hyphenated techniques becoming commonplace. Now in the 1990 s there is a need to streamline the extraction techniques preceding these chromatographic analyses. Time and solvent usage have become critical factors. In response to this need several new sample preparation technologies have emerged. As a result analysts are faced with the challenge of adapting current methods or developing entirely new ones depending on the extraction technology they choose to employ. (Slide 1) Microwave solvent extraction is one of those techniques which can utilize current solvent regimes with minor modifications and with significant solvent reduction. However certain factors need to be taken into consideration such as the thermal stability of the compounds of interest and their polarity. The dielectric constants of the solvents used are also important as this determines how much heating occurs in the presence of microwave energy. Water in the sample can either interfere with or aid in the extraction process. One should also take into consideration any co-extractives that may be reduced by solvent choice. (Slide 2) For this study, we utilized a MES-1000 microwave extraction unit made by the CEM Corp. that utilizes microwave energy to heat polar solvents in a closed vessel. The resulting pressure allows these solvents to reach temperatures often 100 C above their normal boiling points while being monitored and controlled by a fiber optic temperature probe. (Slide 3) Earlier studies on the recovery of chlorinated pesticides from a soil certified reference material established that the 1:1 acetone/hexane solvent system of the standard Soxhlet method could be adapted for use in a microwave extraction system. However, optimum recoveries were obtained by using a 3:2 acetone/hexane combination that represents the actual azeotrophic solvent ratio present in a Soxhlet extraction. (Slide 4) From this earlier work it was also determined that an optimum minimum extraction temperature needed to be established. It can be seen here how recoveries of the spiked chlorinated pesticides in dry soil increased as the temperature of the extraction increased to an optimum temperature of 120 C. (Slide 5) Out of this study arose questions concerning the ability to run multiple samples simultaneously with varying moisture contents. High moisture samples would operate at higher temperatures due to the water s microwave absorbing properties but it was not known how much that temperature could vary or how it would affect recoveries. (Slide 6) To determine the extent of temperature variations, we prepared vessels with oven-dried soils to which water was added to represent 0, 7.5%, 15% and 22.5% moisture. 35 ml of 1:1 acetone /hexane was added and the vessels were sealed and loaded into the carousel for extraction.

3 The unit s fiber optic temperature probe that controls the temperature of the run monitored the sample containing no water. The temperature of the other vessels were monitored by individual fiber optic temperature probes connected to an external Luxtron unit and a computer. The vessels were then heated to 120 C and held for 5 minutes. (Slide 7) The controlling vessel, with no added water, reached the set point temperature in about 1.5 minutes while those containing moisture continued to heat to about 6 % above the set point. Since the magnetron of the unit only operates when the control vessel drops below the set temperature only a limited amount of heating of the other vessels is possible. (Slide 8) We then looked at another solvent system: 1:1 acetone/ dichloromethane. The same procedure was followed only this time the moisture range was 0%, 12.5%, 25% and 37.5% water. (Slide 9) The temperatures in this instance varied by about 12 %. This variation in temperature indicates that the operator should make an effort to see that the controlling vessel contains a matrix with the least amount of water if minimum temperatures are critical and to take into consideration the increased pressure in the vessels containing more moisture. (Slide 10) To determine if this variation could have an effect on the recovery of chlorinated pesticides, this test was then repeated with the addition of Matrix Spike Mix according to EPA Soil Spike protocol. (Slide 11) There was very little variation in recovery for each compound at the different moisture levels. The exception being Aldrin which showed low recoveries at moisture levels of 25% and 37.5%. Water was removed from the extract by stirring about 5 grams of anhydrous sodium sulfate into the vessel before filtering. This could possibly account for the low aldrin figures. (Slide 12) We then considered very high moisture samples such as vegetable material. Although many chlorinated pesticides for example are soluble in non-polar solvents, traditional methods of extraction involve the use of water miscible polar solvents such as acetone to aid in breaking down the sample matrix to allow the residues to solublize. These polar solvents also tend to remove compounds that necessitate cleanup procedures before further analysis and many are not compatible with element specific GC detectors. We proposed that the large amounts of water within the cell structure could be used to break down the matrix by directly absorbing microwave energy, rapidly heating to above the boiling point of water with an accompanying increase in pressure inside the cell. This action could release the compounds of interest directly into a non-polar solvent. To determine if pesticides could be recoverable with only hexane present, we prepared samples of potatoes and green beans in a food chopper and weighed 12 grams of each into a Teflon liner. Each sample was then spiked with Matrix Spike Mix as for the soil spike

4 The unit s fiber optic temperature probe that controls the temperature of the run monitored the sample containing no water. The temperature of the other vessels were monitored by individual fiber optic temperature probes connected to an external Luxtron unit and a computer. The vessels were then heated to 120 C and held for 5 minutes. (Slide 7) The controlling vessel, with no added water, reached the set point temperature in about 1.5 minutes while those containing moisture continued to heat to about 6 % above the set point. Since the magnetron of the unit only operates when the control vessel drops below the set temperature only a limited amount of heating of the other vessels is possible. (Slide 8) We then looked at another solvent system: 1:1 acetone/ dichloromethane. The same procedure was followed only this time the moisture range was 0%, 12.5%, 25% and 37.5% water. (Slide 9) The temperatures in this instance varied by about 12 %. This variation in temperature indicates that the operator should make an effort to see that the controlling vessel contains a matrix with the least amount of water if minimum temperatures are critical and to take into consideration the increased pressure in the vessels containing more moisture. (Slide 10) To determine if this variation could have an effect on the recovery of chlorinated pesticides, this test was then repeated with the addition of Matrix Spike Mix according to EPA Soil Spike protocol. (Slide 11) There was very little variation in recovery for each compound at the different moisture levels. The exception being Aldrin which showed low recoveries at moisture levels of 25% and 37.5%. Water was removed from the extract by stirring about 5 grams of anhydrous sodium sulfate into the vessel before filtering. This could possibly account for the low aldrin figures. (Slide 12) We then considered very high moisture samples such as vegetable material. Although many chlorinated pesticides for example are soluble in non-polar solvents, traditional methods of extraction involve the use of water miscible polar solvents such as acetone to aid in breaking down the sample matrix to allow the residues to solublize. These polar solvents also tend to remove compounds that necessitate cleanup procedures before further analysis and many are not compatible with element specific GC detectors. We proposed that the large amounts of water within the cell structure could be used to break down the matrix by directly absorbing microwave energy, rapidly heating to above the boiling point of water with an accompanying increase in pressure inside the cell. This action could release the compounds of interest directly into a non-polar solvent. To determine if pesticides could be recoverable with only hexane present, we prepared samples of potatoes and green beans in a food chopper and weighed 12 grams of each into a Teflon liner. Each sample was then spiked with Matrix Spike Mix as for the soil spike

5 tests. After the addition of 15 ml hexane, each vessel was sealed and all were heated together to 110 C for 10 minutes. The control vessel contained a potato sample with moisture levels of about 80% compared to the green beans 90%. After cooling, an internal standard was added to each vessel to calculate recoveries. The hexane layer was decanted through a filter funnel containing anhydrous sodium sulfate and rinsed with about 5 ml hexane. (Slide 13) Recoveries were 85% and above in all cases. Again Aldrin had low recoveries, especially in the potatoes. However acceptable spike data appears, it is not necessarily representative of the efficiency of the extraction of field incurred (naturally incorporated) pesticides. Since it is difficult to find field incurred samples, we decided to grow our own. (Slide 14) For our target compounds, we selected Pentachloronitrobenzene or PCNB (also known as quintozene)and hexachlorobenzene or HCB. Because these are soil fungicides it was a simple matter to control application by just working them into the soil. They are also stable, non-polar chlorinated compounds that are easily resolved by electron capture detection with short run times. Most importantly, they are known to be taken up by radishes during growth. Radishes grow quickly and easily in a small space, and we were able to have samples ready in a matter of weeks. Seeds of the radish variety Early Scarlett Globe were sown in 12 X 36 plastic planter boxes filled with sterile soil-less potting mix. Before planting, about 0.75 grams each of PCNB and HCB was combined with about 1/2 cup of fine sand to facilitate even distribution and then uniformly worked into the potting mix. This amount was approximately equivalent to the proportion of active ingredient per row foot used for agricultural purposes on approved crops. One box was planted without treatment as a control. The radishes were harvested 6 1/2 weeks later. (Slide 15) The mature radishes were then chopped and analyzed by the Luke Method to determine the amount of pesticide residues present. The Luke Method basically involved blending 50 grams of chopped sample with 100 ml of acetone in a blender, filtering, transferring 40% of the filtrate to a separatory funnel with the addition of 50 ml petroleum ether and 50 ml methylene chloride. The organic layer was removed and the remaining aqueous fraction was repartitioned 2 more times with 50 ml portions of methylene chloride. (Slide 16) The organic fractions were dried with sodium sulfate before reducing the volume of solvent and exchanging out the methylene chloride for hexane before cleanup and /or GC analysis. Overall we used over 300 ml to extract 50 grams of tissue. (Slide 17) Analysis of 7 samples provided data by which to evaluate the efficiency of the microwave extractions. We found PCNB at a level of 0.36PPM and HCB at 0.05PPM. No PCNB or HCB was detected when the control radishes were analyzed by the same procedure.

6 (Slide 18) For the microwave extraction method, we weighed grams of the chopped sample into the Teflon liners, added 25 ml of hexane, sealed the vessels and heated in the microwave unit to 110 C for 2, 5, and 10 minutes. (Slide 19) Since the water released from the sample settled to the bottom of the liner during cooling, it was possible to take an aliquot of the hexane directly from the vessel, dry it over sodium sulfate, then pass it through a syringe filter directly into a GC vial for analysis. As an alternative, the entire vessel contents could also be filtered and the water removed prior to analysis. Total solvent usage was ml. (Slide 20) Recoveries from the microwave method required 10 minutes to equal or exceed those by the standard method with the values for the aliquot analysis slightly exceeding those that involved more sample handling such as filtering. Recoveries of HCB greatly exceeded those by the Luke Method, perhaps due to significantly less sample handing involved with the microwave extraction. Since only a non-polar solvent was used the co-extraction of many polar interferences could be reduced. (Slide 21) The chromatograms of the GC analysis of these radish samples by the Luke method with no clean-up, and one from the microwave method clearly show the type of interferences that can be avoided. (Slide 22) In conclusion, the microwave solvent extraction of chlorinated pesticides from soil is accomplished in 20 minutes. For simultaneously run samples, moisture levels up to 37.5% will result in maximum temperature differentials of 12%. Microwave PCNB and HCB recovery from vegetable material is greater than by the Luke Method. Moisture present in a sample can be used to heat nonpolar solvents with microwave energy.

7 Microwave Solvent Extraction Techniques for Various Regulatory Compounds Presented by CEM Corporation 1

8 Development of Microwave Extraction Methods Thermal Stability of Compounds Polarity of Target Compounds Dielectric Constants of Solvents Water: Aid or Interfere Co-extractives with Certain Solvents 2

9 3

10 Soil With Matrix Spike: Effect of Extraction Temperature on Recovery Recovery (%) C 110 C 120 C 4 0 Lindane Heptachlor Aldrin Dieldrin Endrin 4,4' DDT Pesticides

11 5 Chlorinated Pesticide Residues in Certified Reference Material Aldrin α Chlordane β BHC DDE Heptachlor α BHC Lindane Dieldrin Endrin DDD DDT % Recovery Microwave Extraction b EPA Methods c Mean % RSD 10 Lab Means a. PriorityPollutnT/CLP-Soil (Environmental Resource Associates) b. 3:2 acetone/hexane, 50 ml; heated at 120 C for 20 minutes c. n = a

12 Temperature Variation Within a Set of Samples Containing Various Moisture Contents 6

13 Temperature Measurement Conditions Oven dried soil Add water to represent moisture levels of 0%, 7.5%, 15%, and 22.5% 35 ml 1:1 Acetone:Hexane Heat control vessel to 120 C and hold for 5 minutes Monitor temperature on 3 other vessels 7

14 Heating Curve for Soils with 1:1 Acetone:Hexane Temperature ( C) Vessel 1; 0.0% moisture (control) Vessel 2; 7.5% moisture Vessel 3; 15.0% moisture Vessel 4; 22.5% moisture Control temperature: 120 C Power: 920 watts, 5 g Sample Time (minutes)

15 Temperature Measurement Conditions Oven dried soil Add water to represent moisture levels of 0%, 12.5%, 25%, and 37.5% 35 ml 1:1 Acetone:Dichloromethane Heat control vessel to 120 C and hold for 5 minutes Monitor temperature on 3 other vessels 9

16 Heating Curve for Soils with 1:1 Acetone:Dichloromethane Temperature ( C) Vessel 1; 0.0% moisture (control) Vessel 2; 12.5% moisture Vessel 3; 25.0% moisture Vessel 4; 37.5% moisture Control temperature: 120 C Power: 920 watts, 5 g sample Time (minutes)

17 Procedure Soil + water to equal 5 grams + Matrix Spike * in microwave extraction vessel liner Stir with glass rod, cover & allow to equilibrate 1 hour Add 35 ml solvent mixture, seal vessel Microwave extraction 20 minutes at 120 C Cool and filter through Whatman #40 filter paper Process for GC analysis per EPA Method 8081 * Matrix Spike: lindane, heptachlor, aldrin, dieldrin, endrin, DDT ( ppm) 11

18 120 Spiked Soil Pesticide Recovery Versus Moisture Level Lindane Heptachlor Aldrin Dieldrin Endrin DDT Pesticides % Moisture 12.5% Moisture 25.0% Moisture 37.5% Moisture

19 High Moisture Vegetable Matrix Traditional methods Employ water-miscible solvents to aid in breaking down sample matrix May remove undesirable polar compounds Solvents may be incompatible with element specific GC detectors 13

20 Matrix Spiked Plant Tissue Recovery by Microwave Hexane Extraction % Recovery Potatoes Green Beans Lindane Heptachlor Aldrin Dieldrin Endrin DDT 14

21 Incurred Samples PCNB -pentachloronitrobenzene HCB -hexachlorobenzene Soil fungicides: good control of application Stable, nonpolar chlorinated compounds Easily resolved on ECD: short run times Taken up by radishes 15

22 Luke Method Blend chopped sample with acetone Filter Transfer 40% of filtrate to separatory funnel, adding petroleum ether and methylene chloride Remove organic layer Repartition aqueous layer 2 times with methylene chloride 16

23 Luke Method (Continued) Dry organic fractions with sodium sulfate Reduce solvent volume and exchange out methylene chloride for hexane Proceed with clean-up and/or GC analysis Total solvent usage: 300 ml for 50 g sample 17

24 Pesticide Recovery by Luke Method from Incurred Radish Tissue Pesticide PPB %RSD * PCNB HCB * n=7 18

25 Microwave Extraction Method Weigh 25 g chopped sample directly into Teflon liner Add 25 ml hexane and seal vessel Heat in microwave unit for 2, 5, and 10 minutes at 110 C Teflon is DuPont s registered trademark 19

26 After Microwave Extraction Option A: Take aliquot from hexane layer, dry over anhydrous sodium sulfate and syringe filter directly to GC vial Option B: Filter hexane layer through anhydrous sodium sulfate or vacuum filter for separatory funnel processing Reduce volume if needed and proceed with GC analysis. Total solvent usage: 25-30mL 20

27 Microwave Extraction PCNB and HCB recovery from incurred radishes as percent of Luke Method Extraction Time PESTICIDE 2 min 5 min 10 min PCNB -aliquot PCNB-whole HCB-aliquot HCB- whole

28 Chromatograms * for Incurred Radish Extractions HCB PCNB HCB PCNB 22 Luke Method *GC/ECD Microwave Extraction Method

29 Conclusions Microwave solvent extraction of pesticides from soil is accomplished in 20 minutes Moisture levels up to 37.5% will result in maximum temperature differentials of 12% Microwave pesticide recovery from vegetable material is greater than Luke Method Sample moisture allows for nonpolar solvent heating 23