S AMPLE I NTRODUCTION The design of injection ports for GC has been constantly improved to achieve precise

3.3.1 S AMPLE I NTRODUCTION The design of injection ports for GC has been constantly improved to achieve precise

and accurate retention times and analyte response and also to handle large volume injections (LVI), either to lower detection limits or to simplify sample workup, and

to allow direct coupling with sample preparation techniques. 20 –22 An important issue when selecting an injector is the properties of the analyte, such as potential for

chemical instability, thermal degradation, or discrimination of high-boiling-point compounds within the injector. A number of problematic pesticides are prone to degradation in the GC injector, including phthalimide fungicides (e.g., captan), organochlorines (e.g., DDT and chlorothalonil), organophosphorus pesticides (e.g., dimethoate), and pyrethroids.

The major source of inaccuracy in pesticide residue analysis by GC-MS, especially with food, is related to the injection of coextractives from the sample, the so-called ‘‘matrix effect.’’ A buildup of coextractives in a GC inlet may lead to successive adverse changes in the performance of the chromatographic system such as the loss of analytes and peak tailing due to undesired interactions with active sites

64 Analysis of Pesticides in Food and Environmental Samples in the inlet and column. Analytes that give poor peak shapes or degrade have higher

detection limits, are more difficult to identify and integrate, and are more prone to interferences than stable analytes that give narrow peaks. For susceptible analytes, significant improvements in peak quality are obtained when matrix components are present because they fill active sites, thus reducing analyte interactions. However, this can lead to problems with quantification. These matrix effects can produce an overestimation of the analyte concentration if calibration has been performed with standards in solvent. The presence of matrix effects should be evaluated for all tested analytes. There are a number of approaches for preventing, reducing, or compensat- ing for the occurrence of matrix effects 23,24 including the use of matrix-matched calibrants, 25 which is recommended for the monitoring of pesticide residues within the European Union. 19

3.3.1.1 Splitless Injection Cold on-column (COC) injection is rarely used for food analysis due to contamin-

ation of the column inlet with nonvolatile materials. 26 The hot split=splitless injection technique is the most probably used for pesticide residue analysis by GC-MS.

Split and splitless injection 27 are techniques that introduce the sample into a heated injection port as a liquid, and then rapidly and completely vaporize the sample solvent

as well as all of the analytes in the sample. For most pesticide residue applications, the target analyte concentrations are so low that splitting the sample in the injection port will not allow an adequate signal from the detector; so the injector should

be operated in the splitless injection mode. In splitless mode, the split outlet remains closed during the so-called splitless period so that sample vapors are transferred from the vaporizing chamber into the column. Flow through the split outlet is turned on again to purge the vaporizing chamber after most of the sample has been transferred. Transfer into the column is slow (e.g., 30 –90 s), resulting in broad initial bands that must be focused by cold trapping or solvent effects. Although splitless injection is >30 years old, the vaporization process in the injector continues to be investigated and debated. 28,29 Splitless injection, however, is frequently performed incorrectly for a large number of reasons; vaporizing chambers can be too small, syringe needles too short, carrier gas supply systems poorly suited, sample volumes too large, needle technique inappropriate (cool versus hot) by slow instead of rapid injection with too low carrier gas flow rates, incorrect column temperature during the sample transfer, splitless periods that are too short, and liner packings at the wrong site. Some of these problems relate to a lack of understanding of the mechanisms involved (e.g., evaporation by ‘‘thermospray’’ (TSP) and ‘‘band forma- 30 tion’’). Although compromises have to be made when dealing with multiresidue determinations, there is considerable benefit in evaluating each step of the injection process.

The limitations of splitless injections, small injection volumes (i.e., up to 2 mL), the potential to thermally degrade components, and incomplete transfer of compounds with high boiling points, can be overcome somewhat by using pres-

sure-pulsed splitless injection. 31 The pulsed splitless technique uses high pressure (high column flow rate) during injection to sweep the sample out of the inlet rapidly.

Analysis of Pesticides by Chromatographic Techniques 65 After injection, the column flow rate is automatically reduced to normal values for

chromatographic analysis. The pulsing effect maximizes sample introduction into the column while narrowing the sample bandwidth. Additionally, the sample has a very short residence time in the liner, thus minimizing the loss of active compounds. 32 Moreover, the pulsed splitless technique has been shown to enable an increase in the

volume that can be injected 33 but this approach does not permit LVI (>10 mL) and compounds may still thermally degrade in the injector even when the injector temperature is lowered.

3.3.1.2 Programmed Temperature Vaporizing Injection Temperature-programmed sample introduction was first described by Vogt 34 and based on this idea Poy 35 developed the programmed temperature vaporizing injector

(PTV). Although the PTV injector closely resembles the classical split=splitless injector, the primary difference is temperature control. In PTV injectors, the vapor- ization chamber can be heated or cooled rapidly. Combining a cool injection step with a controlled vaporization eliminates a number of important disadvantages

associated with the use of conventional hot sample inlets. 36 This type of injector is highly versatile and can be operated with a number of different configurations. PTV

splitless (PTV SL) introduces the sample into a cold liner (temperature set below or near the solvent boiling point), the split exit is closed, and the chamber is rapidly heated. This technique offers more accurate and repeatable injection volumes, protection of heat sensitive materials, and more homogeneous evaporation for better analyte focusing. Some optimization of parameters and choice of liners are required for good performance. 37

3.3.1.3 Large Volume Injection Time-consuming and labor-intensive evaporation steps during sample preparation

can be replaced by LVI in which the solvent is evaporated in the GC system, in a more rapid, automated, and controlled process. LVI can of course also be used to improve analyte detectability. If the sample extract is sufficiently clean and=or the detector selectivity is sufficiently high, the detection limits will improve proportion- ally with the volume injected. There are two main techniques by which injection

volumes for GC can be increased: COC 38 and PTV. 39 Although COC techniques are very accurate, especially when thermally labile or volatile analytes are concerned, contamination of the column inlet with nonvolatile material is frequent and thus the number of samples that can be analyzed before disruption is limited. LVI using a PTV injector is based on selective evaporation of the sample solvent from the liner of the PTV injector while simultaneously trapping the less volatile components in the cold liner. During this stage of the sampling process, solvent vapors are discharged via the opened split exit of the injector. During solvent elimination, the split exit is closed and the components are transferred to the column in the splitless mode by rapid temperature-programmed heating of the injector. An advantage of using the PTV in the solvent vent mode is that it can also be used for the introduction of polar

solvents, such as acetonitrile used for QuEChERS. 14 As the solvent is vented before introduction into the GC column, no band distortion occurs. The use of PTV

66 Analysis of Pesticides in Food and Environmental Samples injection also enables injection of large volumes of water to be directly injected into

GC without any sample preparation. 40 One more recent modification of the PTV inlet is the conversion of the inlet to allow for accommodation of a direct sample introduction device such as direct sample introduction (DSI) 41 or difficult matrix introduction (DMI). 42 In this approach, the standard PTV is converted into an intrainjector thermal desorption device where a microvial containing an extract volume up to 20 mL is inserted into a PTV injector liner using a holder or probe. For DMI an automated, robotic system is used to inject sample into a PTV liner holding the microvial, and then the PTV liner is robotically inserted into the injector. As in conventional PTV protocols, the start temperature is kept near the pressure-corrected boiling point of the solvent to allow evaporation and removal of solvent from the sample. The inlet is then heated rapidly to transfer volatile and semivolatile analytes to the column, leaving behind the nonvolatile components in the liner. After the separation the microvial or liner containing the microvial is removed, still containing nonvolatile matrix components, thus reducing build up of undesirable compounds in the PTV inlet or on the column.

Some modern microextraction techniques, such as solid-phase microextraction

43 (SPME) 44 and stir bar sorptive extraction (SBSE), can be directly coupled to GC-MS as sample introduction devices allowing the extraction and concentration

steps to be focused into a single, solvent-free, automated step. Both provide high sensitivity because the whole extract can be introduced into the GC by thermal desorption rather than an aliquot of a liquid extract.

For laboratories faced with the determination of pesticides at levels significantly above the detection limits and where those pesticides are not thermally labile, injections of 1 –2 mL via a splitless injector will probably suffice. For pesticides with high boiling points or that are thermally labile, a PTV inlet offers a robust solution for the injection of conventional volumes but with the additional capability of injecting large volumes to cope with the growing demand for lower detection limits in pesticide residue analysis and for coupling with microextraction devices. It will be interesting to see whether the degree of automation offered by devices such as DMI finds its way into routine use.