Quantitative Aspects of SPME

JANUSZ PAWLISZYN

1 Introduction

The objective of this chapter is to introduce the basic concepts facilitating accurate and precise quantitation using SPME technology. The information presented below is a summary of the comprehensive discussion of the topic covered in the recently published book.

Solid Phase Microextraction (SPME) was introduced as a solvent-free sample preparation technique. The basic principle of this approach is to use a small amount of the extracting phase, usually less than 1 µL. Sample volume can be very large, when the investigated system is sampled directly, for example air in a room or lake water. The extracting phase can be either high molecular weight polymeric liquid, similar in nature to stationary phases in chromatography, or it can be a solid sorbent, typically of a high porosity to increase the surface area available for adsorption.

To date, the most practical geometric configuration of SPME utilizes a small fused silica fibre, usually coated with a polymeric phase. The fibre is mounted for protection in a syringe-like device. The analytes are absorbed, or adsorbed, by the fibre phase (depending on the nature of the coating) until an equilibrium is reached in the system. The amount of an analyte extracted by the coating at equilibrium is determined by the magnitude of the partition coefficient (distribution ratio) of the analyte between the sample matrix and the coating material.

In SPME, analytes typically are not extracted quantitatively from the matrix. However, equilibrium methods are more selective because they take full advantage of the differences in extracting-phase/matrix distribution constants to separate target analytes from interferences. Exhaustive extraction can be achieved in SPME when the distribution constants are large enough. This can be accomplished for most compounds by the application of internally cooled fibre. In exhaustive extraction, selectivity is sacrificed to obtain a quantitative transfer of target analytes into the extracting phase. One advantage of this approach is that, in principle, it does not require calibration, since all the analytes of interest are transferred to the extracting phase. On the other hand, the equilibrium approach usually requires calibration for complex samples. This is usually accomplished by using surrogates, or standard addition technique, to quantify the analytes and to compensate for matrix-to-matrix variations and their effect on distribution constants.

Since equilibrium rather than exhaustive extraction occurs in the microextraction methods, SPME is ideal for field monitoring. It is unnecessary to measure the volume of the extracted sample, and therefore the SPME device can be exposed directly to the investigated system for quantitation of target analytes. In addition, extracted analytes are introduced to analytical instrument simply by placing the fibre in the desorbtion unit (Figures lb and lc). This convenient, solvent free process facilitate sharp injection bands and rapid separations. These features of SPME result in integration of the first steps in analytical process: sampling, sample preparation and introduction of extracted mixture to an analytical instrument.

The equilibrium nature of the technique also facilitates speciation in natural systems since the presence of a minute fibre, which removes small amounts of target analytes, is not likely to disturb the system. Because of the small size, coated fibres can be used to extract analytes from very small samples. For example, SPME has been used to probe for substances emitted by a single flower bulb during its lifespan.

Figure 1a illustrates the commercial SPME device, manufactured by Supelco, Inc. (Bellefonte, PA). The fibre, glued into a piece of stainless steel tubing, is mounted in a special holder. The holder is equipped with an adjustable depth gauge, which makes it possible to control repeatably how far the needle of the device is allowed to penetrate the sample container (if any) or the injector. This is important, as the fibre can be easily broken when it hits an obstacle. The movement of the plunger is limited by a small screw moving in the z-shaped slot of the device. For protection during storage or septum piercing, the fibre is withdrawn into the needle of the device, with the screw in the uppermost position. During extraction or desorption, the fibre is exposed by depressing the plunger, which can be locked in the lowered (middle) position by turning it clockwise (the position depicted in Figure la). The plunger is moved to its lowermost position only for replacement of the fibre assembly. Each type of fibre has a hub of a different colour. The hub-viewing window enables a quick check of the type of fibre mounted in the device.

If the sample is placed in a vial, the septum of the vial is first pierced with the needle (with the fibre in the retracted position), and the plunger is lowered, which exposes the fibre to the sample. The analytes are allowed to partition into the coating for a predetermined time, and the fibre is then retracted back into the needle. The device is next transferred to the analytical instrument of choice. When gas chromatography (GC) is used for analyte separation and quantitation, the fibre is inserted into a hot injector, where thermal desorption of the trapped analytes takes place (Figure 1c). The process can be automated by using an appropriately modified syringe autosampler. For HPLC applications, a simple interface mounted in a place of the injection loop can be used to re-extract analytes into the desorption solvent (Figure 1b).

The SPME device is capable for both spot and time-averaged sampling. As described above, for spot sampling, the fibre is exposed to a sample matrix until the partitioning equilibrium is reached between the sample matrix and the coating material. In a time-average approach, on the other hand, the fibre remains in the needle during the exposure of the SPME device to the sample. The coating works as a trap for analytes that diffuse into the needle, resulting in an accumulated mass of analyte proportional to an integral of concentration over time (see equation 10).

SPME sampling can be performed in three basic modes: direct extraction, headspace extraction, and extraction with membrane protection. Figure 2 illustrates the differences between these modes. In direct extraction mode (Figure 2a), the coated fibre is inserted into the sample and the analytes are transported directly from the sample matrix to the extracting phase. To facilitate rapid extraction, some level of agitation is required to transport the analytes from the bulk of the sample to the vicinity of the fibre. For gaseous samples, the natural flow of air (e.g. convection) is frequently sufficient to facilitate rapid equilibration, but for aqueous matrices, more efficient agitation techniques, such as fast sample flow, rapid fibre or vial movement, stirring or sonication are required to reduce the effect of a 'depletion zone' produced close to the fibre as a result of slow diffusional transport of analyte through the stationary layer of liquid matrix surrounding the fibre.

In the headspace mode (Figure 2b), the analytes are extracted from the gas phase equilibrated with the sample. The primary reason for this modification is to protect the fibre from adverse effects caused by non-volatile, high molecular weight substances present in the sample matrix (e.g. humic acids or proteins). The headspace mode also allows matrix modifications, including pH adjustment, without affecting the fibre. In a system consisting of a liquid sample and its headspace, the amount of an analyte extracted by the fibre coating does not depend on the location of the fibre in the liquid phase or in the gas phase, therefore the sensitivity of headspace sampling is the same as the sensitivity of direct sampling as long as the volumes of the two phases are the same in both sampling modes. Even when no headspace is used in direct extraction, a significant sensitivity difference between direct and headspace sampling can occur only for very volatile analytes. However, the choice of sampling mode has a very significant impact on the extraction kinetics. When the fibre is in the headspace, the analytes are removed from the headspace first, followed by indirect extraction from the matrix. Therefore, volatile analytes are extracted faster than semivolatiles. Temperature has a significant effect on the kinetics of the process, since it determines the vapour pressure of analytes. In general, the equilibration times for volatile compounds are shorter for headspace SPME extraction than for direct extraction under similar agitation conditions, because of the following three reasons: a substantial portion of the analytes is present in the headspace prior to the beginning of the extraction process, there is typically large interface between sample matrix and headspace, and the diffusion coefficients in the gas phase are typically higher by four orders of magnitude than in liquids. As the concentration of semivolatile compounds in the gaseous phase at room temperature is small, headspace extraction rates for those compounds are substantially lower. They can be improved by using very efficient agitation or by increasing the extraction temperature.

In the third mode (SPME extraction with membrane protection, Figure 2c), the fibre is separated from the sample with a selective membrane, which lets the analytes through while blocking the intereferences. The main purpose for the use of the membrane barrier is to protect the fibre against adverse effects caused by high molecular weight compounds when very dirty samples are analysed. While extraction from headspace serves the same purpose, membrane protection enables the analysis of less volatile compounds. The extraction process is substantially slower than direct extraction because the analytes need to diffuse through the membrane before they can reach the coating. The use of thin membranes and an increase in the extraction temperature result in shorter extraction times.

2 Theoretical Aspects of Solid Phase Microextraction Optimization and Calibration

Thermodynamics

Solid phase microextraction is a multiphase equilibration process. Frequently, the extraction system is complex, as in a sample consisting of an aqueous phase with suspended solid particles having various adsorption interactions with analytes, plus a gaseous headspace. In some cases specific factors have to be considered, such as analyte losses by biodegradation or adsorption on the walls of the sampling vessel. In the discussion below we will only consider three phases: the fibre coating, the gas phase or headspace, and a homogeneous matrix such as pure water or air. During extraction, analytes migrate between all three phases until equilibrium is reached.

The mass of an analyte extracted by the polymeric coating is related to the overall equilibrium of the analyte in the three-phase system. Since the total mass of an analyte should remain constant during the extraction, we have:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (1)

where C0 is the initial concentration of the analyte in the matrix; C∞f, C∞h, and C∞s are the equilibrium concentrations of the analyte in the coating, the headspace, and the matrix, respectively; Vf, Vh, and Vs are the volumes of the coating, the headspace, and the matrix, respectively. If we define the coating/gas distribution constant as Kfh = C∞ f/C∞h, and the gas/sample matrix distribution constant as Khs = C∞h/C∞s, the mass of the analyte absorbed by the coating, n = C∞fVf can be expressed as:

n = KfhKhsVfC0Vs/ KfhKhsVf + KhsVh + Vs (2)

Also

Kfs = KfhKhs = KfgKgs (3)

since the fibre/headspace distribution constant, Kfh can be approximated by the fibre/gas distribution constant Kfg, and the headspace/sample distribution constant, Khs, by the gas/sample distribution constant, Kgs, if the effect of moisture in the gaseous headspace can be neglected. Thus, equation 2 can be rewritten as:

n = KfsVfC0Vs/ KfsVf + KhsVh + Vs (4)