traction process; there is typically a large interface between sample matrix and headspace; and the diffusion coeffi cients in the gas phase are typi- cally higher by four orders of magnitude than in liquids. The concentration of semivolatile compounds in the gaseous phase at room temperature is small, and headspace extraction rates for these compounds are substan- tially lower. They can be improved by using effi cient agitation or by increasing the extraction temperature.
In the third mode (SPME extraction with membrane protection), the fi ber is separated from the sample with a selective membrane, which lets the analytes through while blocking the interferences. The main purpose for the use of the membrane barrier is to protect the fi ber against adverse effects caused by high molecular weight compounds when dirty samples are analyzed. While headspace trapping serves the same purpose, mem- brane protection enables the analysis of less volatile compounds. Use of thin membranes and an increase in extraction temperature result in shorter extraction times.
9.3 Calibration, Optimization, Precision and
tial for volatile analytes because the equilibration times are shorter in this mode than in direct extraction. Fiber protection should be used only for dirty samples in cases where neither of the fi rst two modes can be applied.
9.3.3 Selection of the Agitation Technique
Equilibration times in the gaseous samples are short and fre- quently limited only by the rate of diffusion of the analytes in the coating.
When the aqueous and gaseous phases are at equilibrium prior to the begin- ning of the sampling process, most of the analytes are in the headspace. As a result, the extraction times are short even when no agitation is used. How- ever, for aqueous samples, agitation is required in most cases to facilitate mass transport between the bulk of the aqueous sample and the fi ber.
Magnetic stirring is most commonly used in manual SPME ex- periments. Care must be taken when using this technique to ensure that the rotational speed of the stirring bar is constant and that the base plate does not change temperature during stirring. This usually implies the use of high quality digital stirrers. Alternatively, with cheaper stirrers, the base plate should be thermally insulated from the vial containing the sample to eliminate variations in sample temperature during extraction. Magnetic stir- ring is effi cient when fast rotational speeds are applied.
9.3.4 Selection of Separation or Detection Technique
Most SPME applications have been developed for gas chroma- tography (GC), but other separation techniques, including high performance liquid chromatography, capillary electrophoresis (CE) and supercritical fl uid chromatography, can be used in conjunction with this technique. The complex- ity of the extraction mixture determines the proper quantitative device. Regular chromatographic and CE detectors can normally be used for all but the most complex samples, for which mass spectrometry (MS) should be applied.
9.3.5 Optimization of Desorption Conditions
Standard gas chromatographic injectors, such as the popular split-splitless types, are equipped with large volume inserts to accommodate the vapors of the solvent introduced during liquid injections. As a result, the linear fl ow rates of the carrier gas in those injectors are very low in splitless mode, and the transfer of the volatilized analytes onto the front of the GC col- umn is also slow. No solvent is introduced during SPME injection; therefore, the large insert volume is unnecessary. Opening the split line during SPME in- jection is not practical, since it results in reduced sensitivity. Effi cient desorp- tion and rapid transfer of the analytes from the injector to the column require high linear fl ow rates of the carrier gas around the coating. This can be ac- complished by reducing the internal diameter of the injector insert, matching it as closely as possible to the outside diameter of the coated fi ber.
9.3.6 Optimization of Sample Volume
The volume of the sample should be selected based on the es- timated distribution constant. The distribution constant can be estimated by using published values for the analyte or a related compound, with the coat- ing selected. The distribution constant can also be calculated or determined experimentally by equilibrating the sample with the fi ber and measuring the amount of analyte extracted by the coating.
9.3.7 Determination of the Extraction Time
The equilibration time is defi ned as the time after which the amount of analyte extracted remains constant and corresponds within the limits of experimental error to the amount extracted after infi nite time. Care should be taken when determining the equilibration time, since in some cas- es a substantial reduction of the slope of the curve might be wrongly taken as the point at which equilibrium is reached. Determination of the amount extracted at equilibrium allows calculation of the distribution constants.
When equilibrium times are excessively long, shorter extraction times can be used. However, in such cases the extraction time and mass transfer conditions have to be strictly controlled to assure good precision. At equilibrium, small variations in the extraction time do not affect the amount of the analyte extracted by the fi ber.
On the other hand, at the steep part of the curve, even small variations in extraction time may result in signifi cant variations of the amount extracted. Shorter is the extraction time, larger is the relative error.
Autosamplers can measure the time precisely, and the precision of analyte determination can be good, even when equilibrium is not reached in the system. However, this requires that the mass transfer conditions and the temperature remain constant during all experiments.
9.3.8 Optimization of Extraction Conditions
An increase in extraction temperature increases the extraction rate but simultaneously decreases the distribution constants. In general, if the extraction rate is of major concern, the highest temperature that still provides satisfactory sensitivity should be used.
Adjustment of the pH of the sample can improve the sensitiv- ity of the method for basic and acidic analytes. This is related to the fact that unless ion exchange coatings are used, SPME can extract only neutral (non-ionic) species from water. By properly adjusting the pH, weak acids and bases can be converted to their neutral forms, so that they can be extracted by the SPME fi ber.
9.3.9 Determination of the Linear Dynamic Range of the Method
Modifi cation of the extraction conditions affects both the sen- sitivity and the equilibration time. It is advisable, therefore, to check the pre- viously determined extraction time before proceeding to the determination of the linear dynamic range. This step is required if substantial changes in sensitivity occur during the optimization process.
SPME coating includes polymeric liquids, such as PDMS, which by defi nition have a broad linear range. For solid sorbents, such as Carbow- ax/DVB or PDMS/DVB, the linear range is narrower because of the limited number of sorption sites on the surface, but it still can span over several or- ders of magnitude for typical analytes in pure matrices. In some rare cases when the analyte has extremely high affi nity to the surface, saturation can occur at low analyte concentrations. In such cases, the linear range can be expanded by shortening the extraction time.
9.3.10 Selection of the Calibration Method
Standard calibration procedures such as external calibration can be used with SPME. The fi ber blank should fi rst be checked to ensure that neither the fi ber nor the instrument causes interference with the deter- mination. The fi ber should be conditioned prior to the fi rst use by desorption in a GC injector or in a specially designed conditioning device. This process ensures that the fi ber coating itself does not introduce interference. Fiber conditioning may have to be repeated after analysis of samples containing large amounts of high molecular weight compounds, since such compounds may require longer desorption times than the analytes of interest.
A special calibration procedure, such as isotopic dilution or standard addition, should be used for more complex samples. In these methods, it is assumed that the target analytes behave similarly to spikes during the extraction. This is usually a valid assumption when analyzing homogeneous samples.
9.3.11 Precision of the Method
The most important factors affecting precision in SPME are:
Agitation conditions
•
Sampling time (if non-equilibrium conditions are used)
•
Temperature
•
Condition of the fi ber coating (cracks, adsorption of high mo-
•
lecular weight species)
Geometry of the fi ber (thickness and length of the coating)
•
Sample matrix components (salt, organic material, humidity,
• etc.)
Time between extraction and analysis
•
Analyte loss (adsorption on the walls, permeation of Tefl on,
•
absorption by septa)
9.3.12 Suitability
SPME is well suited to the analysis of fl avor and fragrance com- pounds. The typically small, volatile compounds are easily extracted by the fi bers, and the simplicity of the method allows easy coupling to analytical instruments. Headspace trapping can reduce the potential for interference peaks and prevent contamination of both the needle and the instrument.
Loss of these volatile compounds during sample preparation steps is mini- mized or eliminated compared to conventional methods, and the method is amenable to fi eld sampling and analysis.
SPME has been shown to be useful for semivolatile com- pounds, even though these appeared more challenging in the early years.
With appropriate matrix modifi cation, one can take advantage of headspace trapping for these as well. SPME provides signifi cant convenience for fi eld and air analysis. Quantifi cation is relatively straightforward, even in the pres- ence of varying air temperature. Finally, the use of SPME for time-weighted average sampling provides simplicity in monitoring fl avor and fragrance con- centrations over time.