Use of Accelerated Solvent Extraction (ASE) with Centrifugal Evaporation to Automate Fat Determination in Chocolate Aaron Kettle Thermo Scientific, Sunnyvale, CA
Overview Purpose: To demonstrate automated fat determination in food matrices such as chocolate. Methods: Accelerated solvent extraction (ASE) technique with centrifugal evaporation (Rocket Evaporation) to determine fat content gravimetrically. Results: Accelerated solvent extraction technique greatly reduces the extraction time and amount of solvent require to remove fat from chocolate. The sample preparation workflow can be automated with the ASE and Rocket Evaporator to produce highly accurate and reproducible fat extraction from food matrices. Introduction Accelerated solvent extraction is a proven technique that speeds up the gravimetric fat determination in food products such as chocolate and greatly reduces the amount of solvent used. The Thermo Scientific Dionex ASE 350 Accelerated Solvent Extraction system uses a combination of elevated temperature and pressure to increase the extraction kinetics of lipids out of the matrix, thus decreasing the extraction time and solvent consumption. Current methods for determining the fat content of chocolate are labor-intensive and require large amounts of solvent and time (Table 1). For example, the Mojonnier ether extraction method takes 2.5 3.5 hours and requires 140 ml of solvent per samples. This method also requires an analyst to be present for the extraction and does not enable walk-away capability. By using the Dionex ASE System, extraction time is reduced to 18 minutes and solvent use is reduced to 20 ml per sample, demonstrating superior performance when compared to traditional extraction methods used for food matrices. The accelerated solvent extraction technique is fully automated, making it possible to extract up to 24 samples unattended. Furthermore, extracts can be transferred directly from the Dionex ASE system to the Thermo Scientific Rocket Evaporator system, eliminating manual sample transfer and cumbersome nitrogen evaporation techniques. In this application, fat is extracted from both milk and baking chocolate. The time required for extraction and amount of solvent used are compared to the Mojonnier Method, Soxhlet Extraction, and the Twisselmann Extraction. Table 1: Comparison of sample preparation techniques for fat extraction in chocolate Dionex ASE 350 System Mojonnier Method Soxhlet Extraction Twisselmann Extraction Solvent per sample Time per sample 20 ml 140 ml >100 ml >100 ml 18 min 2.5-3.5 hours 4 hours 2 hours 2 Use of Accelerated Solvent Extraction (ASE) with Centrifugal Evaporation to Automate Fat Determination in Chocolate
Equipment Dionex ASE 350 Accelerated Solvent Extractor System Stainless Steel Extraction Cells, 10 ml Collection Vials, 60 ml Cellulose Filters Rocket Evaporator Julabo Recirculating Chiller Thermo Scientific Dionex ASE Pucks Analytical Balance (read to the nearest 0.0001 g or better) Mortar and Pestle (Fisher Scientific) Drying Agents and Dispersants Thermo Scientific Dionex ASE Prep DE (diatomaceous earth) Ottawa Sand (Fisher Scientific) Samples Baking chocolate and milk chocolate bars purchased from a local grocery store Methods Extraction Conditions* Solvent Petroleum Ether (100%) Oven Temperature 125 C Static Time 3 min Static Cycles 3 Flush Volume 60% Purge Time 60 sec *Pressure is fixed at 1500 psi and does not need to be programmed in the method. Sample Preparation Chocolate bars were finely grated and mixed in a 1:2 ratio with diatomaceous earth (1g:2g) using a mortar and pestle. A cellulose filter was placed at the bottom of a 10 ml extraction cell and the sample was transferred directly into the cell. Void volume was topped off with Ottawa Sand. Extraction Procedure The 60 ml collection vials were weighted and labeled prior to being placed into the Dionex ASE 350 system. Filled extraction cells were loaded on the carousel and the extraction method was run using the present conditions. Once the extraction was complete, the resulting extracts were removed from the ASE 350 and placed into Dionex ASE Pucks (Figures 1 and 2). The Pucks were loaded into the Rocket Evaporator (Figures 3 and 4) and the preprogrammed evaporation method was run (Method 1 for Low Boiling Point Solvents). Note that samples can be directly transferred from the Dionex ASE 350 to the Rocket and the need for manual pipetting is eliminated. The Rocket Evaporator will evaporate the solvent to dryness Thermo Scientific Poster Note PN70996_PITTCON_2014_E_03/14S 3
FIGURE 1: 60 ml vial with extract FIGURE 2: ASE Puck holds 60 ml vials 4 Use of Accelerated Solvent Extraction (ASE) with Centrifugal Evaporation to Automate Fat Determination in Chocolate
FIGURE 3: Load Extracts into Puck FIGURE 4: Puck in Rocket Evaporator Thermo Scientific Poster Note PN70996_PITTCON_2014_E_03/14S 5
Results Table 2 displays the results following the extraction and evaporation. The fat content from three samples of each chocolate type were used to evaluate the effectiveness of the Dionex ASE system and the Rocket Evaporator to determine the content against the label claims. The extraction solvent was evaporated in the Rocket and the 60 ml collection vials containing a layer of fat were weighed. The percent fat of each sample was determined by dividing the weight of the fat residue by the sample weight. The use of the Dionex ASE system and the Rocket Evaporator demonstrates that automating fat extraction in these matrices can accurately extract fat content within 0.73% of label claim. Table 2: % Fat* Recovery from Chocolate Baking Chocolate Milk Chocolate Average (n=3) 52.27% 25.27% Standard Deviation 0.03 0.01 %RSD 0.13 0.07 Label Claim 53% 26% *%fat = (residue/sample weight) x 100% Conclusion The use of the Dionex ASE 350 system with the Rocket Evaporator automates the extraction of fat from chocolate. Accelerated solvent extraction technique eliminates much of the time and solvent required by conventional extraction techniques, using 20 ml of solvent and requiring only 18 minutes per sample. The use of Dionex ASE Pucks and the Rocket Evaporator eliminate the need for manual sample transfer and nitrogen stream evaporation. By using the Dionex ASE 350 system and Rocket Evaporator together, the sample preparation workflow for fat extraction can be automated for increased productivity and throughput. References 1. Klöck, G., Noke, A., and Christiansen, T. (2013). Soxhlet or Twisselmann, an Evidential Report. Separation, 1/2013., pp. 32-33 2. Thermo Scientific Application Note 344. Extraction of Fat From Chocolate Using Accelerated Solvent Extraction. 2004. Sunnyvale, CA. [Online] http://www.dionex.com/en-us/webdocs/114809-au344-extraction- Chocolate-ASE-AN70798_E.pdf (accessed Feb 11, 2014). 6 Use of Accelerated Solvent Extraction (ASE) with Centrifugal Evaporation to Automate Fat Determination in Chocolate
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