On-Line and Off-Line Application of Micro-SPE (MEPS)

Peter Dawes, Ern Dawes, and Paul Wynne, SGE Analytical Science, Ringwood, Victoria, Australia.

Solid-phase extraction (SPE) has revolutionized sample preparation. Variations on the technique offer enhanced recovery, greater speciation and reduced solvent and sample consumption over other techniques. Micro-extraction packed sorbent (MEPS) is the miniaturization of conventional SPE from millilitre to microlitre bed volumes that allows SPE to be used with very small samples. The manipulation of the small volumes is achieved with a precision gas tight syringe. With a typical void volume of 7 μL, the volume of solvent eluted from MEPS is compatible with GC and LC inlets making it ideal for integration into an automated sampling system for on-line SPE.

In most instances, MEPS allows the same level of sample concentration that is possible with off-line conventional SPE while providing opportunities for truly hybrid multidimensional methods. MEPS methods may be readily adapted from established SPE methods including those based on mixed mode or complex chemistries. Similar to SPE, MEPS is for use with liquid samples (either normal or reversed phase) and yields four fractions: the unretained, weakly bound, strongly bound and irreversibly bound fractions. However, because MEPS is a double pass system (sample and solvent enter and exit from the bottom of the bed, the weakly bound fraction (commonly the interferences eliminated by washing) is less strongly bound. The irreversibly bound fraction affects MEPS and conventional SPE and is usually associated with sorbent wetting rather than sample purification and so the irreversible binding of matrix material from one sample does not preclude reuse of the device for a sample of the same type. Similar to conventional SPE, the number of times the device can be reused is dependent on the sample matrix. For simple applications, MEPS devices have been used successfully for >50 cycles.

Benefits of MEPS

• MEPS allows SPE methodology to be applied to small sample volumes.

• MEPS can be integrated into autosampler robotics and allows on-line use of SPE.

• MEPS can reduce sample and reagent consumption and waste disposal.

• Double pass flows can reduce the weakly bound fraction.

• MEPS is field portable for remote sampling with or without the use of automated equipment.

• MEPS is adaptable for other analytical techniques including immunoassay and off-line analysis by NMR, IR and other methods.

Plant extracts

The plant Phytolacca octandra is one of the Inkweeds or Pokeweeds. There is some interest in the genus as a potential source of anticancer agents and for other therapeutic agents. In this application, we use the plant to illustrate the use of MEPS to speciate complex phytochemical mixtures. The aerial portions of the plant were homogenized in methanol/ammonia (98:2), allowed to percolate for 12 hours, filtered and then extracted using a C2 MEPS barrel insert and needle assembly (BIN). The MEPS was conditioned with methanol (30 μL), water (30 μL) and then 100 μL of the plant extract was passed through the sorbent at 5μL/sec. The exhausted fraction was ejected at the same rate and the sorbent washed with 100μL water. The sorbent was dried with air (3 × 80 μL at 50 μL/sec) and eluted sequentially with hexane (10 μL), dichloromethane (10 μL) and methanol (10 μL). The eluates were analysed directly by GC–MS on a BPX5 column (Figure 1).

Figure 1

MEPS allowed the rapid analysis of the plant hydrolysate in either off-line or on-line mode. The C2 phase facilitated the single step isolation of a FAME fraction (hexane) with a discrimination free recovery of 80% on the first elution. The hexane fraction was free of other interfering components and the remaining 20% of FAME could be eluted by sequential elution with either hexane or more polar solvents. The elution with dichloromethane yielded a further FAME fraction (16%), sterol esters, long chain alcohols (predominantly phytol), glycerol and some free fatty acids with C18 acids eluted in preference to C16. Subsequent elution of the MEPS with methanol gave complete elution of the free fatty acids, monoglycerides, bound sugars and flavanoid glycones.

Comparison of the crude extract and retained fractions show the effectiveness of washing at removing weakly bound compounds. The weakly bound fraction was comprised of sugars, highly polar compounds and glycones. These compounds were available for analysis by collection of the pre-hexane wash effluent as was the exhausted sample if the MEPS was to be used in a stripping or trapping mode. Speciation of polar and non-polar analytes from a single sample digest was readily achieved without the need for off-line sample preparation.

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