In the Laboratory edited by
Topics in Chemical Instrumentation
David Treichel Nebraska Wesleyan University Lincoln, NE 68504
Using Single Drop Microextraction for Headspace Analysis with Gas Chromatography Daniel Riccio, Derrick C. Wood, and James M. Miller* Department of Chemistry, Drew University, Madison, NJ 07940; *
[email protected] Gas chromatographic (GC) analysis of complex samples containing both non-volatile and volatile components can often be simplified by headspace (HS) sampling if the analytes of interest are volatile. HS sampling prevents the non-volatiles from contaminating the GC column and produces a simplified chromatogram of only the volatiles. Methods used to obtain the HS sample are often classified as static, or dynamic (purgeand-trap) (1). A newer, static method makes use of a coated silica fiber that can be exposed to a HS sample (or be immersed in a sample solution). Known as solid-phase microextraction, SPME, it was first reported in 1989 (2, 3) and has since become popular. Different fiber coatings can be chosen to sorb desired sample components, thus providing selectivity for this sampling method. After exposure to the sample HS and selectively sorbing the desired components, the fiber is inserted in the heated inlet of a GC, the analytes are desorbed, and a conventional GC analysis is executed. Even more recent is a liquid extraction method that uses a microdrop of liquid, either in the HS or in a liquid sample. The procedure is very similar to SPME, except that a microdrop of liquid is used instead of a coated fiber. Many have called it
single-drop microextraction, SDME, to indicate its similarities to SPME. Other names are solvent microextraction (SME) or hanging drop microextraction. When used for HS sampling, which was first reported in 2001 (4, 5), the names headspace solvent microextraction (HS-SME or HSME) or headspace liquidphase microextraction (HS-LPME) are commonly used. Similar to SPME, SDME is simple, fast, and easy and can be automated. But, in addition it is less expensive because it does not require specialized equipment. High selectivity is possible by choosing an appropriate solvent extractant, often resulting in low detection limits. Unlike conventional liquid–liquid extraction (LLE), minimal quantities of solvent are required. There is no sample carryover since fresh solvent used each time (renewable drop) unlike the practice in SPME where the fiber is reused and needs to be desorbed after each use. In SPME, the fiber has an active coating, usually bonded to its surface. Consequently, the mechanism of sorption is mainly adsorption and, in some applications, it has been noted that the total quantity of analyte that can be sorbed is thereby limited. In SDME, the liquid drop is capable of not only adsorption, but also absorption, often giving it the capability to extract larger quantities of analyte than SPME. Principles and Practice of HS-SDME
Figure 1. Photograph of a 1 µL drop on a microsyringe in the headspace of a 20 mL vial.
In the simplest, manual method of using HS-SDME, a conventional microsyringe with a beveled needle is used to draw up some extracting liquid. Usually 1 to 3 µL are used; larger sizes improve detectivity but are more prone to fall off the needle. Some experimentalists recommend that the needle be roughened to improve retention of the drop. The sample is usually contained in a conventional headspace vial with a septum closure; the 20 mL size is convenient for manual use. The vial is heated in a water bath for 20 to 30 minutes and should be stirred to aid in the attainment of equilibrium between the phases—liquid and vapor. The loaded syringe is inserted through the septum into the HS and a microliter or two is expelled onto the tip of the needle (Figure 1). Extraction is allowed to proceed for a few minutes and then the drop is retracted into the syringe and taken to a GC and injected as is conventional with liquid samples, using split injection for capillary columns. The requirements for the extracting liquid are fairly obvious: high enough boiling point so that the solvent elutes after the volatile sample components; sufficient viscosity to prevent loss of the drop; high purity; and low toxicity. If reasonable care is exercised, drops up to 3 µL usually do not fall off the needle for the solvents we have tested. The main limitation has been to find a solvent of sufficient purity so that there are few, if any, GC impurity peaks from the solvent to interfere with the sample
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peaks. Some of the solvents that have been reported in the literature are shown in List 1. The most popular solvent, 1-octanol, is a polar compound with a fairly high boiling point. The use of polar SDME extractants has been popular, probably because SPME has not been too satisfactory with polar fibers. Gaining in popularity are the recently rediscovered room temperature ionic liquids (RTILs). While most of the articles cited in this publication use GC for the analysis step (4–38), RTILs are often analyzed by HPLC (39–43) owing to their high boiling points. Capillary electrophoresis has also been used (44). Full discussion of the many variables that need to be evaluated in setting up a quantitative method is not possible in this article, but they include the following:
• Selection of extractant including consideration of its polarity as well as those characteristics listed earlier
• Selection of internal standard
• Size of extracting drop
• Ratio of sample volume to HS volume
• Temperature
• Stirring speed
• Equilibration time (liquid–vapor)
• Ionic strength (desalting)
• Extraction time
• Chromatographic conditions
Most of the articles cited can provide details involved in the evaluation of these variables.
List 1. Solvents Used as Extractants for HS-SDME Benzyl alcohola Butyl acetate Cyclohexane n-Decanea Diethylphthalate Dimethylformamide (DMF) Dodecane Ethylene glycol n-Hexadecane 1-Hexanol N-Methylpyrrolidone (NMP) n-Octane 1-Octanolb Room temperature ionic liquids (RTILsa such as 1-butyl-3-methylimidazolium hexafluorophosphate) n-Tetradecane Toluenea Water o-Xylene a
Solvent often used. bMost popular solvent.
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HS-SDME is normally run as a static method, but it has been shown that liquid–vapor equilibrium need not be attained for a satisfactory analysis (7, 28). Sufficient equilibration time is necessary to get a significant quantity of analyte in the vapor state, so samples are usually allowed to equilibrate for a length of time, equivalent to the run time for a sample, so as not to delay the subsequent run. Most of the published articles contain experimental data evaluating the variables listed above. Several contain more complete discussions of the theories behind the present practices (6, 8, 28, 30). The expectation that a longer extraction time would produce lower detection limits is not always observed (40). The large number of variables to be evaluated could lead to the use of a statistical method of evaluation such as the Plackett– Burman design, which is reported in ref 43. The use of statistics in instructional analytical analysis is highly desirable and HS-SDME is an ideal class project to illustrate it; each student could contribute data for the statistical analysis performed by the whole class. The use of an internal standard (IS) is highly recommended for manual work, but may not be needed for automated analysis (17). Many experimenters have put the IS in the extracting drop, rather than in the sample. While they report success with this practice, we do not favor it for pedagogic reasons and for another reason discussed below. Typically the extractant is chosen following the principles used in LLE or in GC (in the choice of the stationary phase). For example, a polar solvent is often used for polar volatiles. SDME produces a large solvent peak in the chromatogram (not the case with SPME), which can be a disadvantage. However, the most commonly used solvents have rather high boiling points to prevent them from evaporating during the extraction and consequently they usually elute after the analytes of interest. If the solvent does evaporate, poor precision and sensitivity can result (32, 43). One way to avoid this problem would be to dissolve the sample in the same solvent as the one used as the extractant. It has been shown that this practice works well (17), but, of course, it does not result in a selective enhancement of the volatile extraction. At best, the concentration of an analyte in the extractant would be the same (at equilibrium) as its concentration in the sample. Conversely, the size of the drop can increase during extraction (25). We too have noted that some extractants, such as N-methylpyrrolidone (NMP), which is totally miscible with water, will take up water during the extraction. For example, an extraction with NMP over a water sample will result in an extractant drop that is composed of a mixture of NMP and water, growing substantially in size. Obviously the composition of the drop changes during the extraction, resulting in a change in solvent properties. This is another reason why we do not favor the practice of putting the IS in the extractant. In general, insufficient attention has often been paid to changes in drop size during extraction; the drop can get smaller or larger. These changes may explain some of the unexpected results reported in the literature (40). The extractant need not be a pure solvent, but can be a mixture, thus providing a specific selectivity. However, this practice could result in a change in drop composition during extraction owing to selective evaporation (due to boiling point differences as noted before). Another practice that can be used with SDME is similar to that performed in SPME, namely the use of a derivatizing agent in the extracting solvent (26, 27, 33, 36).
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The Literature of HS-SDME At least 44 articles have been published on HS-SDME, 35 of which used GC as the analysis step (4–39). References are presented in chronological order. In this article, we are concerned primarily with the GC articles. In addition, some related articles have also been included (40–51) since they contain useful information about the technique. Possible Student Experiments
Detector Response
It does not appear that any articles have been published reporting the use of HS-SDME in the academic instructional laboratory. Many possibilities exist as can be seen from the extensive list of publications, including the determination of chlorobenzenes, halomethanes, hydrocarbons, amines, and BTEX (benzene, toluene, ethylbenzene, and xylene). Wood, Miller, and Christ (17) have reported the use of HS-SDME with NMP for the qualitative and quantitative analysis of residual solvents in pharmaceutical products, but there is no report of its instructional use. One possibility is the analysis of the active (volatile) ingredients in a mouth wash such as Listerine. A preliminary trial of such an experiment produced the chromatogram shown in Figure 2. The experiment used GC with FID detection, and the six largest peaks (Table 1) were identified using GC–MS. The scaling used in Figure 2 also shows the favorable signal-tonoise ratio and the presence of at least five impurities.
1
3
4
2
5 6
A summary of the results is given in Table 1; the largest peak is ethanol. The extractant was water (not detected by the FID), 1 µL of which was exposed to a 10 mL sample at 45 °C for 5 min. Trial runs using NMP or octanol as the extractant were unsuccessful because both of them eluted chromatographically at the same time as several of the volatile analytes. Although not tested in an instructional laboratory, the experiment has been shown to be easily adapted for that purpose. Most laboratories would have all of the needed equipment and instrumentation. While this is an example of a qualitative analysis, the system could be studied further as has been described to illustrate: (a) a quantitative analysis using an IS, (b) a HS analysis, (c) the use of statistical methods in an analysis, or (d) heterogeneous equilibria. Another experiment we have tested was based on a published SPME analysis, the quantitative analysis of coffee vapor (52, 53). We chose to determine pyridine, one of the major volatile components, using NMP as the extractant and the solvent for the sample, ground coffee (not brewed). The aroma of pyridine is somewhat offensive, and the NIOSH guideline on levels in air that are immediately dangerous to life or health (IDLF) is 3,600 ppm (54). It is well recognized that virtually everyone is exposed to low levels in air, water, and food. Determining the level in coffee vapor, which is a function of the coffee type and the roast conditions, provides an interesting experiment for the student laboratory. In our experiment, NMP was used as the sample solvent and the extractant drop. A 0.5 g sample of ground espresso coffee beans was added to 10 mL of NMP with 3-picoline as an internal standard and sealed in a 20 mL headspace vial. After heating at 60 °C for 30 minutes, the HS was sampled with a 1 µL drop of NMP for 5 minutes and was then analyzed by GC. Using conventional calibration methods, a typical concentration of pyridine was found to be about 8 ppm (55). Virtually any SPME analysis method should be adaptable to SDME. In fact, HS-SDME could be used to illustrate the technique of HS-SPME (without requiring the use of the specialized equipment of SPME). Hazards
0
2
4
6
8
10
12
14
Time / min Figure 2. Chromatogram of Listerine analytes sampled with a water microdrop. Instrument: Hewlett-Packard (Agilent) 5890 Series II with FID. Conditions: 15 m × 0.53 mm RTX-5 (Restek Corp.) column. Split injection (4:1). Temp. Program: 35° for 2 min; programmed at 30 deg/min to 95°; then at 4 deg/min to 115°; hold for 5 min.
Table 1. Identities of Peaks in Figure 2 Peak Number
Retention/min
Compound
1
0.65
Ethanol
2
0.88
1-Propanol
3
5.68
Eucalyptol
4
7.99
Menthol
5
8.47
Methyl Salicylate
6
10.80
Thymol
The hazards are minimal with this experiment as the quantities of the organic solvents are small. N-Methylpyrrolidone is a skin, eye, and respiratory irritant. Conclusions We believe that HS-SDME has sufficient advantages to make it a desirable technique for the instructional laboratory. It can be used to illustrate both qualitative and quantitative analysis, the use of an IS in analysis, the use of HS in analysis, or as an example of a technique that resembles the popular SPME. It is affordable and unlike SPME, which requires the purchase of some specialized equipment, it only requires equipment available in a conventional GC laboratory. Anyone who can run GC can also run HS-SDME-GC. Acknowledgments The authors wish to thank the undergraduate students and chemistry faculty who aided in this work and our alumni colleagues Thomas Brettell and Jonathan Crowther.
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