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Sample Preparation by Immobilized Liquid Extraction
Robert Gullett
Immobilized liquid extraction (ILE) is a clean and simple technique for preparing aqueous samples (biological or environmental) for chromatographic analysis. ILE extractions are fundamentally similar to traditional liquid-liquid extractions (LLE). In traditional LLE, a compound partitions between two immiscible liquid phases, usually an aqueous sample and an organic solvent, based on its affinity for each of the liquids. ILE separations are very similar; however, the 'organic solvent' is substituted by a thin layer of polymer.
 Figure 1: ILE methodology. Click to enlarge. | A portion of each ILE device (i.e., cap septum, bottom of a 96-well plate) is coated with a sorptive elastomeric polymer, such as polydimethylsiloxane (PDMS), which acts as the extraction medium. A targeted compound in a sample is extracted into the device's coating, from which it is desorped into a small amount of solvent.
The extracting phase is a completely non-porous (no clogging) elastomeric polymer above its glass transition temperature (Tg) that exhibits the extractive characteristics of a liquid while maintaining complete matrix independence (no mixing or emulsions).
ILE methodologyWhen an aqueous sample is directly exposed to the immobilized phase, compounds partition between the two phases based on their relative affinity for each. Compounds that partition into the immobilized liquid are 'back-extracted' (eluted) into a small amount of GC or HPLC solvent to complete the extraction process. ILE extractions are taken to equilibrium, use small volumes of solvent (100 to 200 mL), and minimize requirements for continuous human support.
Generic ILE procedure (depicted in Figure 1):
1. Dispense sample into the ILE
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device (pH pre-adjusted).
2. Seal and agitate the ILE device, using a plate vortexer, orbital shaker, etc., until determined equilibrium time is reached.
3. Remove sample from the ILE device.
4. Dispense and remove rinse solution (optional interferant removal: i.e., water, buffer, etc.).
5. Dispense back-extraction (elution) solvent into the ILE device.
6. Seal and agitate to equilibrium.
7. Resulting solvent extract is ready for analysis.
Extraction time, efficiency, and reproducibilityExtraction time and efficiency were investigated by extracting a known quantity (2,500 ng/mL) of chlorpheniramine from 100-mL goat serum samples with a polyacrylate (Acrylate) ILE 96-well plate. The MS response given for each extracted sample was compared to the MS response of a non-extracted spike that correlates to 100% recovery.
Two series of 100-mL serum samples (n=7) were extracted for 10 and 20 minutes, respectively, to determine the required extraction time. At 10 minutes, an absolute extraction efficiency of 71.4% (3.7% RSD) was achieved, while a 20-minute extraction yielded an efficiency of 94.3% (3.3% RSD). Figure 2 displays the extraction efficiency of chlorpheniramine from a 100-mL serum sample as a function of time.
 Figure 2: Extraction efficiency of chlorpheniramine. Click to enlarge. | The time required for a targeted analyte to partition to equilibrium is specific to each analyte, matrix, and set of extraction conditions. If one was to plot extraction efficiency as a function of time, as in Figure 2, he would find that the quantity of extracted analyte approaches a maximum, and that as he gets closer to this maximum, the rate of extraction decreases (i.e., the targeted analyte has reached, or is near, equilibrium between the sample and extracting phase).
These maximum quantities depend on the targeted analytes' relative affinities for both the sample matrix and extracting phase, as well as the volumes of each phase. From this it may be deduced that, for a specific application, only a minimum extraction time must be determined (assuming that extractions are taken to, or near, equilibrium). As analyte partitioning nears equilibrium, the quantity of analyte in the extracting phase changes both negligibly and slowly. Hence, if a suitable extraction time has been determined, minor inconsistencies in the practice of this extraction time will result in unnoticeable data variability.
Standard curveA series of experiments was performed to show a linear correlation between sample concentration and MS response at a range of concentrations. 100-mL goat serum samples were diluted 1:1 with water and adjusted to pH 11 with 10M NaOH. Each sample was spiked with a known concentration of methadone. Five replicate samples were prepared at each of the following six concentrations: 10 ng/mL, 1,000 ng/mL, 2,500 ng/mL, 5,000 ng/mL, 10,000 ng/mL, and 20,000 ng/mL. Each sample was extracted using a trifluoropropylmethylsiloxane (Fluoro) phase ILE 96-well plate for 30 minutes, and then 'back-extracted' (eluted) into 150 ml acetonitrile. The results are depicted in Figure 3.
Direct serum extraction  Figure 3: Standard curve. Click to enlarge | Many extraction techniques require sample dilution to ensure rapid, efficient, and reproducible extractions. Consequently, samples with volumes that approach the capacity of an extraction device may not be diluted because the resultant diluted sample exceeds the device's capacity. ILE 96-well plates may extract directly from complex undiluted biological matrices like serum and plasma without negligible effects on extraction time, efficiency, and reproducibility.
A series of experiments was conducted to show this characteristic. This experiment compared two data sets of 100-mL samples (n=7). The first set consisted of 100-mL serum, the second of 50-mL serum, diluted 1:1 with water to 100 mL. Both sets of samples were spiked with 2,500 ng/mL chlorpheniramine and adjusted to pH 11 with 10.0N NaOH. All samples were extracted for 20 minutes using a trifluoropropylmethylsiloxane (Fluoro) phase, from which they were 'back-extracted' (eluted) into acetonitrile.
Extraction efficiency and reproducibility were measured under each set of conditions. The pure serum sample exhibited nearly the same extraction efficiency (94.3%) and reproducibility (3.3% RSD) as the diluted sample (95.4% and 2.6%).
EvaluationILE extractions do not require many of the common sample preparation steps that tend to introduce variability to results. Sorbent conditioning, protein precipitation, sample dilution, and solvent exchange/reconstitution are all avoided. The completely non-porous extraction media maintains matrix independence throughout the extraction, so neither clogged media nor emulsions are possible.
The ILE method has experimentally demonstrated precise (±2.6 to 3.7%) and efficient (94.3 to 95.4%) extractions. A linear correlation between sample concentration and MS response was defined across a wide range of analyte concentrations (10 to 20,000ng/mL). Extraction efficiency, reproducibility, and time are affected negligibly by complex sample matrices.
The simplified procedure requires little to no human attention or interaction, and is amenable to current trends toward fully automated, high-throughput applications. ILE offers an efficient approach to high-throughput extractions of small molecules directly from complex biological and environmental matrices.
Robert Gullett is the Operations Manager at ILE Incorporated. He may be contacted at ChromatographyTechniques@advantagemedia.com.
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