NICHOLAS H. SNOW and GREGORY C. SLACK
OVERVIEW
Even though gas chromatography is a very powerful separation method, some GC analyses require sample preparation prior to injection. While sample preparation may be as simple as diluting the analyte(s) in an appropriate solvent or loading into a vial, or as complex as multistep extractions, the even-tual quality of the method may be more dependent on the sample preparation than on the chromatography. Most sample preparation approaches for gas chromatography involve moving the analyte(s) into a solvent phase (usually organic) appropriate for liquid injection using a syringe or into the vapor phase for introduction as headspace, with a sample loop or a gas tight syringe.
In gas chromatographic method development, sample preparation should be considered in concert with the injection technique and the required detection limits of the method.
To traverse the instrument, analytes must be volatile enough under the conditions of the inlet and column; ideally, the matrix interferences must also be volatile, so as to not contaminate the inlet or column. In most cases, liquid samples are dissolved in a volatile organic solvent. The basic goal of sample preparation is to ensure that the above conditions are met, with additional goals that the preparation be reproducible to meet quantitative analysis requirements and straightforward to perform, if the analysis is to be performed routinely, as in quality assurance and in other routine testing laboratories.
Table 12.1 provides an overview of common sample preparation techniques, arranged by the phase of the bulk sample. It is readily seen that there are numerous possibilities for a given sample type. This presents the choice of sample preparation technique as one of the more diffi cult choices in develop-ing a method. Nearly all sample preparation methods involve the transition of analyte(s) between phases, commonly either solid or solution to gas, or solid, liquid, or gas to liquid. In any event, gases and liquids are by far the most
183 Basic Gas Chromatography, Second Edition, by Harold M. McNair and James M. Miller Copyright © 2009 John Wiley & Sons, Inc.
CHAPTER 12
commonly injected sample phases. Our ability to accomplish this phase trans-fer is driven fi rst by chemical equilibrium, which determines the amount of analyte that may be transferred from the original phase to the fi nal phase, determining recovery, or the amount that is extracted.
Second, the kinetics involved in reaching that equilibrium often determine the reproducibility of the method and may affect the recovery if equilibrium in the extraction process is not reached. There are few comprehensive treat-ments of sample preparation in the literature; however, there are many books and articles describing specifi c techniques, which are referenced throughout this chapter [1, 2] .
There are several implications for all sample preparation methods.
1. Quantitative extraction (100% transfer of the analyte to the extracted phase) cannot happen, although a high partition coeffi cient and/or mul-tiple extraction steps may nearly achieve it. Extraction phases should generally be chosen to maximize the partitioning into the extract phase.
2. Some amount of analyte (or interference) is always extracted, no matter how low the partition coeffi cient.
3. Multiple extraction steps will result in a more effi cient extraction and will magnify the positive effect of small differences between analyte and interference partition coeffi cients.
TABLE 12.1 Overview of Sample Preparation Techniques by Sample Type Sample Type: Solid Sample Type: Liquid Sample Type: Gas Chromatography , 4th ed., John Wiley & Sons, Hoboken, NJ, 2004, with permission.
LIQUID–LIQUID EXTRACTION (LLE) 185
4. Kinetics must be considered to ensure that the extraction reaches equilibrium. If equilibrium is not reached, reproducibility may suffer.
LIQUID – LIQUID EXTRACTION ( LLE )
LLE usually involves extraction of analytes from a dilute aqueous phase into an organic phase, often with a concentration step to improve sensitivity.
Liquid – liquid extractions are either macro - extractions or micro - extractions, depending on the volume of extraction solvent used, with the dividing line about 1 mL of extraction solvent. Macro - liquid – liquid extraction is performed using a separatory funnel, test tubes or a continuous extraction device.
Micro - liquid – liquid extraction is often performed using a volumetric fl ask, conical test tube or directly in a sample vial. The fundamentals of macro - LLE are described extensively in the laboratory textbooks for college organic chemistry, so only important factors affecting LLE recovery are discussed here, along with the special cases of micro - LLE and single - drop micro - extraction [3, 4] .
Factors Affecting LLE Recovery
There are a number of techniques and considerations that can affect recovery in LLE and other extractions. These include agitation, salting out, pH, tem-perature, washing or back extraction, and solvent choice.
Agitation. Extraction requires intimate contact between the two phases, most often with agitation by shaking, stirring, or vortex mixing. Generally, higher agitation speed results in more rapid equilibration, and longer agitation time ensures that equilibrium has been reached. Agitation devices (shaking speed, vortex mixer RPM, stirrer velocity, etc.) should be operated as reproducibly as possible. It is important to adjust extraction timing to reach a plateau. This ensures that small variations in mixing speed, solvent viscosity, or matrix effects should not adversely affect the extraction.
Salting Out. Adding high concentration of a salt such as sodium chloride often enhances extraction recovery of organic compounds extracted from water into organic phases. Increasing the ionic strength often reduces solubility of organic compounds in water, thus increasing the value of K c and therefore the amount extracted. However, it is diffi cult to make general statements about whether recovery will be improved for a specifi c extraction scheme and analytes without testing this experimentally.
p H Adjustment. Many common analytes and interferences are weak organic acids and bases. Since solution pH for these compounds can drastically affect
their solubility in an aqueous phase, knowledge of their p K a and control of the solution pH can be used to affect the extraction. The aqueous solubility of acidic compounds will be enhanced in basic solution, while the solubility of bases will be enhanced in acid. In both cases, K c is reduced, thereby reducing extraction recovery. To improve extraction recovery of acids, the aqueous phase can be adjusted to lower the pH, ideally to at least 2 pH units lower than the p K a of the desired analyte. Likewise, for bases, the pH can be raised.
If there are multiple ionizible analytes and/or interferences, it may be neces-sary to adjust the aqueous solution pH by buffering, to provide more repro-ducible control of the original solution pH.
Temperature Adjustment. The equilibrium position of all chemical pro-cesses is affected by the temperature. Generally, to ensure extraction repro-ducibility, temperature should be controlled as carefully as practical. This may be as simple as ensuring that all solutions and samples have equilibrated at the laboratory room temperature, or as complex as performing the extraction within an oven or heating block.
An increase in temperature will decrease the distribution constant, K c , thereby reducing the amount extracted. However, at elevated temperature, kinetics are often faster, so extraction speed may be increased. Often, adjusting temperature provides a trade - off between lowered recovery and faster kinet-ics. Careful temperature control may be required for reproducibility and is especially critical in liquid – vapor (headspace) extraction.
Choice of Extraction Solvent. The ideal extraction solvent would show very high solubility for analytes of interest and very low solubility for interferences, generating a large difference in the partition coeffi cients. If the solubilities of analytes and interferences in the original phase and in the extraction phase can be estimated or are known, K c can be estimated as a ratio of these solubili-ties. Furthermore, the extraction phase must not be miscible or signifi cantly soluble in the original phase.
Micro - Liquid – Liquid Extraction
Because of the high sensitivity of gas chromatography, liquid – liquid extraction can often be carried out directly in small auto - injector vials, thereby saving time - consuming and error - producing concentration and transfer steps and consuming considerable less solvent. To show the possibilities, Figure 12.1 shows a comparison of extraction effi ciencies for several LLE techniques:
MLLE carried out with equal volumes of solvent and sample in an auto injector vial, SPME (solid - phase micro - extraction), macro - LLE with a high degree of concentration (High), and macro - LLE low degree of concentration (Low). It is clearly possible for micro - liquid – liquid extraction to be competi-tive with larger volume extractions, especially if employed in combination with large volume gas chromatographic injection.
LIQUID–LIQUID EXTRACTION (LLE) 187
Single - Drop Micro - extraction ( SDME )
The concept of SDME, introduced in 1996, is simple: A single drop of organic solvent is suspended from a syringe needle into the aqueous phase, and the system is agitated to drive organic compounds into the drop. The organic drop can then be transferred to the gas chromatograph using the syringe [5, 6] . Figure 12.2 shows SDME in which the organic drop is suspended directly from a common gas chromatographic micro - syringe [7] . The equilibrium theory of SDME is similar to that seen in LLE, with the equilibrium concentration of analyte in the organic phase at equilibrium given by
A K A V
where the subscripts 1 and 2 refer to the aqueous and organic phases, respec-tively. If V 2 < < V 1 and K c is small, this reduces to
A[ ]2=K Ac[ ]1 (2) In other LLE methods, “ salting out ” increases the amount extracted; however, the opposite has been observed with SDME [8 – 10] , due to the higher ionic strength of the aqueous phase decreasing the analyte diffusion rate, thus requiring longer extraction time to reach equilibrium. Typical equilibration times range from 5 to 10 min. Psillakis and Kalogerakis have thoroughly reviewed SDME [11] .
Fig. 12.1. Comparison of fraction of analyte extracted for several extraction techniques versus partition coeffi cient. An n org / n aq value of 1 indicates exhaustive extraction.
MLLE: 1 - mL sample, 1 - mL solvent; SPME: solid - phase micro - extraction; High: 1 - L sample, 3 × 60 - mL solvent; Low: 5 - mL sample, 3 × 1 - mL solvent.