Soil and Environmental Analysis: Modern Instrumental Techniques - Chapter 12 (end) pps

This chapter describes the main steps required in analysis of key organic pollutants in environmental samples, concentrating on soil analysis to provide illustrative examples, as soil is

Analysis of Organic Pollutants

in Environmental Samples

Julian J C Dawson, Helena Maciel, and Graeme I Paton

The University of Aberdeen, Aberdeen, Scotland

of technique to suit the particular matrix and determinant(s) Significant advances in instrument automation and reliability, precision of flow control, detector development, and competitive instrument pricing have greatly increased the number and range of laboratories capable of fulfilling reliable and routine organic pollutant analysis.

This chapter describes the main steps required in analysis of key organic pollutants in environmental samples, concentrating on soil analysis

to provide illustrative examples, as soil is one of the more challenging matrices Citations are made to references that provide specific information about instrumentation and the underpinning principles and scientific rationale Several widely used methods are described and discussed in detail to exemplify the considerations needed for techniques.

A Why Quantify and Identify Organic Contaminants?

The presence of organic pollutants in the environment is ubiquitous From the high arctic to the tropics (Jones and de Voogt, 1999), recalcitrant and volatile pollutants are detectable in all environmental spheres Soils and sediments are major sinks for organic pollutants and can retain the highest concentrations of released pollutants (Northcott and Jones, 2000) Drinking water contaminated with biocides from runoff into surface waters or by the leaching of agrochemicals through soil to aquifers is widely acknowledged (Stackelberg et al., 2001) Researchers and regulators need sensitive and routine techniques to identify and quantify these contaminants Scientists also need to be able to study samples for signs of degradation and the occurrence of metabolites and cocontaminants that may indicate the relative damage or indeed remediation in soil or sediment systems.

Once a representative sample has been obtained, there are three further stages that underpin organic analysis: (1) the preparatory (drying) and extraction stage, (2) the cleanup stage(s) and (3) the determination stage Some determinations may only be performed after derivatization, when the determinant needs to be chemically altered to improve analytical resolution Organotin determination, for example, requires extensive derivatization because the determinants are not sufficiently volatile for direct gas chromatographic analysis (Abalos et al., 1997) Each of these stages will

be dealt with separately, and using illustrative examples, the selection criteria for certain approaches will be justified.

The type of drying technique carried out is determined by the nature of the determinant(s) and the matrices It is usually inappropriate to dry a soil or sediment in an oven as may be done for inorganic analysis, as this may cause

a substantial loss of the determinants Instead, a sulfate salt is often used to remove the water (Hess et al., 1995; Guerin, 1999) After drying, the organic determinant present must be brought into an appropriate organic solvent prior to quantification by gas chromatography (GC) or high-pressure liquid chromatography (HPLC) Determinants in water samples can be extracted using sequential volumes of organic solvent, which are then passed through the sulfate salt to remove residual water (Meharg et al., 1999) The extraction technique also enables the sensitivity of the analysis to be

manipulated through sample concentration Depending upon the nature of the sample and the target determinant, an appropriate technique can be selected.

When a solvent is immiscible with water and the target determinant is more soluble in the solvent than in water, then this is an ideal technique The partitioning coefficient of the determinant material is equal to the ratio of its concentration in the solvent divided by that in water The partitioning coefficient is independent of the volume ratio of solvent : water but constant

at any given temperature; thus increasing the amount of solvent increases the amount of determinant extracted Repeated extractions with the same solvent will also increase the efficiency of determinant extraction Extraction efficiency can be further improved by heating of the sample-extraction mixture (Dean and Xiong, 2000).

This is a commonly used technique for quantifying total concentrations of semivolatile and nonvolatile hydrophobic contaminants A diagram of the main components of the Soxhlet apparatus is shown in Fig 1 The soil or

Figure 1 Soxhlet apparatus for solvent extraction of organic pollutants from soils and sediments.

sediment sample is placed in a porous extraction thimble Below this thimble

is a cup containing the solvent, which is heated and passed through distillation and condensation stages, ensuring that there is a rigorous mixing

of the solvent with the sample Although the procedure is slow, it is a harsh but effective technique This method continuously re-extracts the sample with the same quantity of solvent: the solvent is refluxed in a Soxhlet apparatus, condenses, and trickles down through the sample back to the bottom of the apparatus, where the determinant collects This method is used for nonvolatile and semivolatile organics, and it will not collect any compounds with a boiling point lower than, or close to, that of the solvent used The solvent is typically a mixture of a nonpolar compound such as dichloromethane (DCM) with about 10% of a water-miscible polar solvent such as methanol or acetone added.

A supercritical fluid (SCF) is a substance held above its critical temperature and pressure SCFs have many advantages over liquid solvents for use in extraction of environmental samples (Camel, 2001) Their physical proper- ties include low viscosity, high diffusion coefficients, low toxicity, low flammability, and negligible surface tension These allow SCFs to penetrate

an environmental matrix very quickly, allowing rapid extractions compared

to those with conventional solvents A further advantage is that SFE systems can be interfaced directly with a chromatography column, thus minimizing sample preparation Supercritical carbon dioxide, possibly modified by the addition of methanol or acetone, is the most common solvent used in environmental analysis; however, a SCF with a dipole moment may be more effective (Alonso et al., 1998) Hawthorne et al (1992) found that supercritical CHClF 2 (Freon-22) was more effective than CO 2 for the extraction of PAHs and PCBs from soils, consistently extracting 2–10 times more determinant SFE with CHClF 2 was also fast: 30–45 minutes were required to extract comparable amounts to that obtained by 18 hours ultrasonication in DCM, or 32 hours Soxhlet extraction in hexane/methanol and hexane/acetone mixtures SCF techniques are not routinely used in soil analysis because they are quite expensive to set up and to run routinely However, they may be more widely used in future, particularly if they are shown to be applicable to a range of determinants that are not routinely tested for at present.

This method is used in conjunction with a gas chromatograph (GC) and is suitable for volatile and semivolatile hydrocarbons The solid sample is

warmed to approximately 85C in an enclosed system to desorb and tilize the hydrocarbons, which are then purged, trapped, and subsequently transferred onto the column Volatile organic compounds, such as benzene, toluene, ethylbenzene, xylene (BTEX), methyl tertiary butyl ether (MtBE), and naphthalene from liquid environmental samples, e.g., fresh and marine waters, soil extracts, and wastewater, can also be extracted by purging of the sample using an inert gas and trapping the extracted determinants The contents of the trap are then injected directly onto the column of the GC Although slow and costly to set up, the method is the most reliable one for quantifying relatively water-soluble determinants.

This technique is also known as solid phase extraction (SPE) The process is simple and fast and may prove cost-effective for some users A measured volume of the sample is passed through a cartridge tube with a suitable solid material, which sorbs the target determinant The determinant is then eluted from the cartridge using an appropriate solvent Semiautomated SPE systems use vacuum pumps to speed up the solvent flow, enabling elution to take place much more quickly SPE is also extensively used as a cleanup technique to remove material that may damage a chromatography column

or slow down the chromatographic procedure Most of the large chromatographic suppliers sell SPE systems The selection of the packing material is based upon the polarity of the contaminants to be determined Nonpolar hydrophobic adsorbents retain the nonpolar determinants and allow polar substances to pass through the column, whereas hydrophilic adsorbents adsorb the polar components, allowing the nonpolar materials

to pass through.

sediment samples in a solvent such as DCM It is used for non- and semivolatile organics at various concentrations (Guerin, 1999).

mixtures as for Soxhlet extraction, but the sample is held at increased temperature and pressure, thus reducing extraction time and solvent volume required (Fisher et al., 1997; Hubert et al., 2001).

similar in function to ASE in that it enables reduced extraction times and solvent volumes compared to traditional techniques The equipment is quite

costly to purchase, and the technique is not widely reviewed in publications; hence comparative evaluation may prove to be problematic.

Sample cleanup of organic extracts is used to prolong the life of the instrument column, injector, and detector A purified sample will also produce clearer peaks with improved resolution that will prove easier to quantify Sample purification tends to be based on one of the following principles: (1) partitioning between immiscible solvents; (2) adsorption chromatography; (3) gel permeation chromatography; (4) chemical destruc- tion of interfering substances; or (5) distillation.

The simplest of the above is the acid/base partitioning, in which a solvent extract is shaken with dilute alkali that enables acidic organics to partition into the aqueous layer while the basic and neutral fractions remain

in the organic solvent The aqueous layer can then be acidified and extracted using DCM so that the organic layer will now contain the acid fraction This technique is widely used in cleanup procedures for determining phenols and associated herbicides from soils and sediments (Patnaik, 1997).

Cleanup columns, either as premanufactured SPE systems or as laboratory-produced columns, are the most common routine technique for cleanup For example, highly porous and granular aluminum oxide (alumina) can be used and is readily available in acidic, neutral, or basic forms (Polese et al., 1996) Target determinants can be differentiated by chemical polarity After the column is packed with the granular material it is covered in anhydrous sodium sulfate and the sample is placed on the column By using the appropriate solvent, this enables the determinants to

be separated from impurities that are present Basic alumina is used in purification of steroids, alcohols, and pigments (Cho et al., 1997); the neutral form is used for esters and ketones (Polese and Ribeiro, 1998), while the acidic form separates strong acids and acidic pigments Alumina is also ideal for the cleanup of hydrocarbons (Cho et al., 1997; Shen and Jaffe, 2000).

Amorphous silica gel is suitable for the removal of interfering compounds of differing polarities (Shamsipur et al., 2000) Activated silica gel is heated for several hours at 150C prior to use and is also well suited for the cleanup of hydrocarbons (Miege et al., 1999) Deactivated silica gel has significantly more water present and is used to separate plasticizers, lipids, esters, and some organometallic compounds (Shamsipur et al., 2000).

If used appropriately, high specificity for target herbicides can be achieved.

In addition, the selection of different solvents (Supelco, 2001) can be used to manipulate adsorbent activity of the SPE system.

Florisil is a form of magnesium silicate with acidic properties A packed column of Florisil is used the same way as silica and alumina columns The material is ideal for the separation of aliphatic compounds from aromatics (Abdallah, 1994) and is used for a wide range of pesticides (Smeds and Saukko, 2001) and halogenated hydrocarbons (Schenck and Donoghue, 2000).

Gel permeation is able to differentiate material on the basis of pore size using hydrophobic gels (Knothe, 2001) As with SPE, this system is capable of performing to a high level of specificity, though equipment and consumable costs will reflect this.

In some solid environmental samples, the presence of specific materials may impose analytical problems For example, sulfur may reduce the resolution of chromatograms Sulfur has a solubility that is similar to a range of organochlorine and organophosphate pesticides and cannot be resolved using Florisil (Patnaik, 1997) Commonly, copper turnings are shaken with the sample to remove sulfur from the solvent extract (Schulz

et al., 1989) Mercury or tetrabutyl ammonium sulfite (Duinker et al., 1991) are also used Table 1 describes the materials typically chosen for cleanup procedures of selected contaminants extracted from soils and sediments.

Chromatography is a simple concept in that analyte components become separated as they either move in the mobile phase or become sorbed in another phase The characteristics of the sorption phases determine the extent to which analyte components become separated The resolution can

be manipulated by using appropriate columns in consideration of the determinants sought The major factors to ensure high quality chromatog- raphy are (1) purity of the mobile phase, (2) a reliable flow rate, (3) an

Table 1 Suggested Cleanup Techniques for a Number of

Common Contaminant Groups

Nitrosamines Gel permeation, alumina, Florisil

Organochlorines Gel permeation, Florisil

Organophosphates Gel permeation, Florisil

Phenols Gel permeation, acid–base, silica gel

PAHs Gel permeation, alumina, silica gel

Source:Patnaik, 1997.

appropriate column, and (4) a suitably sensitive detector Regardless of the type of chromatography, these rules must be adhered to The most commonly used chromatographic techniques in environmental analysis are

GC and HPLC, and these methods will be described briefly and then dered in more detail, using representative case studies, later in this chapter For routine analysis it is important to consider the value of an autosampler Current microrobotic technology provides high precision and reproducibility In many instruments, sample vials can undergo heating and mixing (with slight modifications to the sampler), thus enabling some automated derivatization Automated dilution systems where available, are also very useful, as the system is capable of operating with small volumes The automated injection system resolves problems associated with manual techniques, which may cause excessive and variable peak broadening on the column Most significantly, the autosampler allows hundreds of samples to

consi-be systematically analyzed This is ideal, consi-because of the long retention times associated with some determinations.

Traditionally this has been called gas–liquid chromatography because samples being carried through a column undergo partition between a gas phase (mobile) and a sorbed liquid phase (stationary) For the purpose of this chapter, only capillary GC will be considered, but further details on packed columns can be found in Bruno (2000), and in Chap 10.

The main components of a GC are

ready for injection Most GC analysis will be carried out using split or splitless injection This means that the sample is injected into a chamber where, under heating, it expands and then moves in the gas flow onto the column The selection of the solvent used for injection is therefore very important, as different solvents have different expansion characteristics In the case of split injection, a proportion of the sample is discarded, as it may overload the column and detector and cause a reduction in resolution Common split ratios are between 15 : 1 and 40 : 1, and thus a large proportion of the sample is discarded Splitless analysis, on the other hand, enables expansion of the solvent vapor within a glass liner, but the entire sample is presented to the column On-column injection is required for trace analysis and has no pre-expansion stage for the sample The injector systems are usually tailor-made to suit the style of analysis.

chroma-tography do not have access to high-purity gases and thus have to use

supplies containing small amounts of impurities, e.g., oxygen, moisture, and carbon compounds In such circumstances, filters should be used to remove these impurities, to avoid damaging the column and affecting the response

of the detector A carrier gas ensures steady flow of sample through the column while often an additional ‘‘make-up’’ gas is required for the detector Any new GC will have highly sensitive electronic or manual gas controls, which can be altered according to column-specific requirements.

success or failure of the separation Users should be aware of the range of columns on the market and the relative merits of inexpensive and expensive purchases The selection of a column is governed by what is referred to as the ‘‘theoretical plates per meter’’ concept This parameter describes the chromatographic performance of a column There is a wide range of texts that consider the principles that underpin this parameter, and for more information, Marr and Cresser (1983) is a good source All the major capillary column suppliers have catalogs either available in paper format or from the internet These should be consulted prior to purchase, as they will enable the most appropriate column to be selected The columns are composed of fused silica, and a narrow-bore inside diameter (i.d.) (usually 0.20, 0.25, or 0.32 mm) will provide the best separation for closely eluting components and isomers In general, the smaller the i.d., the greater is the level of resolution that can be achieved Conversely, to avoid sample overload for analytes in high concentrations, a larger i.d may be more appropriate The characteristics of some typical columns are shown in Table 2.

particular determinant(s) and analytes However, it is possible to alter the analysis most effectively by the manipulation of temperature For determinants to be separated, they are differentially partitioned between the mobile and stationary phases: the proportion in the gas phase depends

Table 2 Examples of Some Available Columns and Their Characteristics

Phenyl cyanopropyl

methyl silicone

on temperature When analyzing a broad suite of hydrocarbons, for example, it is not possible to select a column that is capable of discriminating between all determinants The most volatile fraction will move rapidly along the column, while the larger molecules will trail significantly behind To speed up this process, the oven can be adjusted to produce a temperature ‘‘ramp’’ during which the column temperature changes across a predetermined regime This simply means that as the analysis is progressing, the oven temperature is progressively raised, which means that the sample begins to reach ‘‘vapor pressure’’ and elutes more readily through the column Without the use of this ramp, retention time would rise significantly if the temperature were set too low, whereas if the temperature were initially set too high, all the determinants would elute together.

ionization detectors (FIDs) are the most commonly used types Because of its lack of specificity, the TCD is more appropriate for gas analysis (see

Chap 10), and it will not be considered in more detail here The FID, however, is an excellent detector for a wide range of determinants because it responds to the presence of organic carbon compounds (but not to CO,

CO 2 , or CS 2 ) In the FID, the passage of the organic compounds through a hydrogen-rich flame results in the creation of ions and a corresponding electrical response The FID is sensitive at the mg L
1 level to a plethora of compounds (Marr and Cresser, 1983) It is also a very forgiving detector, as

it has a linear response to concentration over seven orders of magnitude and

is resistant to overload and damage Flame photometric detectors (FPDs) can be used to measure determinants containing specific groups, including organic S, P, and Sn compounds (Singh et al., 1996) The FPD has a range

of filters to suit the optical emission characteristics of the target determinants The halogen-specific detector or the electron capture detector (ECD) is an essential detector for the measurement of trace levels of organochlorine compounds (Schulz et al., 1989).

The most significant detector used for routine analysis now is the mass spectrometer This is an excellent tool for identifying a range of unknown determinants in the target matrix Over the last decade the application of this detector in water, soil, and sediment analysis has grown enormously, and as a consequence the cost has dropped After separation of components

in an appropriate column, the eluted fractions are subjected to electron impact or chemical ionization The fragmented and molecular ions are resolved from characteristic mass spectra and determinants identified from their distinctive primary and secondary ions Quantification is achieved by peak height, representing the total ion count, at each specific mass : charge

ratio Although widely used, there is no specific example in this chapter, but examples can be found in the literature (De la Guardia and Garrigues, 1998; Eriksson et al., 1998; Ragunathan et al., 1999; Choudhary et al., 2000) The mass spectrometer detector requires a high vacuum, while the gas chromatograph requires a gas flow Hence GC-MS coupling is achieved

by combining a low flow rate with the use of a fast pumping low-density carrier gas, usually helium The capillary GC can produce sharp peaks, which enables a rapid scan with the mass spectrometer, and it is generally acknowledged that a mass spectrometer detector is as sensitive as a flame ionization detector With the application of a mass spectrometer detector, a library of stored spectra makes it possible to identify unknown determinants.

computer-controlled, and the resultant chromatograms are generally managed and analyzed by an appropriate software system An older GC

is usually managed manually and the results calculated from an integrator output As with all other methods, calibration curves for the target determinant(s) must be prepared from at least four standards Calibration can be performed with external or internal standards, though it is most common to use an internal standard method This involves the addition of equal volumes of an internal standard to each of the calibration standards and the sample extracts, to ensure reproducibility of detector response Further details about standardization and quantification can be found in Harvey (2000).

Routine GC analysis for soil and/or sediment samples involves carrying out confirmatory calibration checks prior to sample analysis to verify consistency of response Variations in the gas flow, in the presence of impurities, in the consistency of injection, and in oven temperatures may cause substantial variations in the response It is also worth noting that the length and ‘‘plumbing’’ of the column will have an impact on the retention characteristics, so analytical setup time can be substantial for complex determinants.

In this instrument, liquid/sorbed-phase chromatography is the principle of separation The analyte is carried in a liquid that is supported (adsorbed) on

an inert solid The separation efficiency of a column can be expressed in terms of the theoretical plates in the column, which are defined by the physical structure of the column and the type of packing (Harvey, 2000) A sample is placed at the start of the column, and sample constituents are

flushed through the column by the carrier solvent(s) The component parts

of the instrument are

pump, two-solvent mixtures can be regulated A real-time pressure feedback and control system automatically provides solvent compressibility compen- sation for accurate and precise flow, regardless of solvent composition Consistent gradients and precise retention times are provided by proven control algorithms and high-speed proportioning valves.

a modern instrument This component ensures optimal performance of the HPLC pump system The removal of the gases from the solvents allows more stable baselines, improved gradient shape, and high temporal reproducibility Dissolved gases account for most of the common problems encountered

in routine analyses, such as bubble formation, pump cavitation, detector noise, baseline drift, and loss of gradient precision This solvent degaser ensures the optimum performance of the HPLC system by thoroughly removing these dissolved gases from the mobile phase All wetted materials in the degaser are chemically and biologically inert This ensures maximum corrosion resistance and compatibility with sensitive biomolecules.

incubator The column is selected according to the specific application A useful ‘‘general column’’ is a C18 reverse phase column, which is composed

of bonded silica The applications for this include a wide range of nonionic polar compounds and aromatics.

visible type The detector has a flowcell into which column-partitioned fractions of the determinant are passed Time-programmable functions enable optimization of separations or exchange The detector must be able

to respond to particularly small volumes of determinants separated by the column Accordingly, rapid response is required Photometric detectors provide the necessary sensitivity, and often the limitation may prove to be the subsequent integrator The main photometric detector is usually composed of a dual-lamp design, ensuring sensitivity across the entire UV/visible spectrum A modern system will have high-speed scan mechanisms capable of achieving a slewing speed of 30,000 nm s
1

and positional precision accuracy of less than 0.01 nm (Agilent, 2001) This scanning ability means that detection can be achieved at the peak of the absorption spectrum, offering a combination of selectivity and sensitivity The cell volume should not be greater than one tenth of the volume containing the determinant yielding the smallest peak likely to be encountered, and it should be designed so that any bubbles can be rapidly cleared; this is often achieved by using a restriction block downstream

(Agilent, 2001) Refractive index detectors are also available but have fewer applications for environmental samples, as they have notoriously unstable baselines The most significant developments at the moment are taking place

in LC- mass spectrometric detectors This type of detector is commercially available from a range of manufacturers and has been widely used in the pharmaceutical and biochemical industries Over the next decade it is likely

to have a significant effect on soil and sediment analysis, as GC-MS has had over the past decade Applications to soil analysis that have been published include, for example, the determination of organometal speciation and recalcitrant compounds (Dass, 1999; Mondello et al., 1999).

comple-mented by automation of peak integration Originally, when chromatograms were recorded on chart paper, researchers would cut out the peaks and weigh them to quantify the determinants Now the PC achieves high- resolution determination of peak areas with user-friendly software It can calculate the retention times and recognise peaks as required It also enables computerization of all of the data collation, which can be extrensive Chromatographic analysis features include use of wavelength ratios, baseline subtractions, and mathematical manipulations, including first and second derivatives.

in Soil Using FID-GC

Typically, this technique is used for comparative evaluation either in a spatial or a temporal context There is a need therefore to be able to put through a large number of samples and to have a relatively rapid extraction technique that has been developed and optimized specifically for TPHs Approximately 10 g soil (wet weight) is weighed accurately (0.01 g) and ground over anhydrous Na 2 SO 4 using a mortar and pestle, until the soil/

Na 2 SO 4 mixture is fluid The sample is transferred to a 250 mL conical flask equipped with a PTFE-lined screw cap, and 1 mL of internal standard solution (see below) added This mixture is then extracted by sonication in

50 mL of dichloromethane (DCM) (glass-distilled grade) for 10 minutes and filtered through Whatman No 4 paper The extraction is repeated with

25 mL of DCM, filtered through the same paper, and the two extracts combined An aliquot of the extract (5 mL) can then be stored at
20C in a foil-capped vial for future use; the remainder, for analysis, can be

evaporated under nitrogen at 40C to a volume of 1 mL The extract is then cleaned by liquid chromatography using 2 g of octadecyl-bonded silica (60 A˚, < 200 mm) conditioned with 10 mL of DCM The sample is then loaded onto the column and eluted with 2  10 mL of DCM The eluate can then be concentrated further as necessary by evaporation under a stream of nitrogen at 40C.

TPHs are measured by FID-GC Extract (1 mL) is injected using an autosampler onto a GC equipped with a Phenomenex ZB-1 (100% polydimethylsiloxane) capillary column (30 m  0.32 mm i.d  0.5 mm), split injector, and flame ionization detector GC conditions are as follows: column gas flow (N 2 ): 1 mL min
1

; split flow: 20 mL min
1

Injector temperature is varied to suit the associated hydrocarbon composition:

200C (kerosene); 250C (diesel and motor oil) The detector temperature is held at 250C for kerosene, 320C for diesel and 350C for motor oil As previously discussed, to cope with analyzing this complex matrix, a temperature ramp is used: the temperature is held initially at 80C for

2 minutes, then increased at 10C min
1 to 250C (for kerosene), 320C (for diesel), and 350C (for motor oil), after which it is held for a further

10 minutes at the final temperature.

The internal standard (IS) is a chemically related compound of known concentration, but not present in the environmental samples, used to test extraction and chromatographic performance as well as the reproducibility

of the techniques Ideally, an IS should be added at both the extraction and analysis stages Squalene is used as the IS for diesel, while pristane is used as

an IS for kerosene and motor oil (pristane elutes after the kerosene envelope [the mixture of components constituting kerosene] and before the motor oil envelope) The IS can be dissolved in DCM and stored in a UV-proof bottle with a PTFE-lined screw cap Routinely R F (response factor) values are calculated over a range of 5 concentrations, and nonlinear regressions are fitted of R F against concentration Quantification includes some unresolved complex mixtures (UCMs) as well as resolved peaks A typical resulting chromatogram is shown in Fig 2.

Estimation of TPHs is the most commonly performed quantification for petroleum contamination and typically is used to set regulatory levels and cleanup targets There are two techniques for measuring TPHs First, infrared spectroscopy can be used to measure the absorption at 2930 cm
1 (corresponding to the methylene C-H stretching frequency (Lambert et al., 2001) This has the disadvantage of poor sensitivity to aromatic compounds.

It is also necessary to use a solvent with no C-H bonds, e.g., a