Trace Organic Analysis
Frequently Asked Questions
An organic compound (made up of one or more atoms of different types) always contains carbon and other elements such as hydrogen, oxygen, nitrogen, and sulfur. If the compound contains a metal or a nonmetal such as Se, then the compound is generally regarded as an organometallic compound. Methane (CH4) is considered an organic compound but carbon dioxide (CO2) is not. Thus, organic compounds usually contain hydrogen as well as carbon. Organic chemistry is the study of the chemistry of carbon (or organic compounds as we have defined them).
Generally, we will define trace organic analysis as analyses for organic compounds present at or below the part-per-thousand level in a sample. The analysis provides the identity (qualitative result) and the amount (quantitative result). The challenge is to perform the analysis under conditions where the relative amounts of other substances in the sample are vastly greater than that of the analyte. Often, we will find it necessary to separate the analyte from the bulk of the other substances (potential interferences) in the sample (matrix) before a final determination (amount) can be accomplished. Generally, the final determination involves a final separation/detection. For an operational definition of trace organic analysis (real application) see How do you do trace organic analysis?
Environmental measurements are often reported in dimensionless units such as part per million (ppm). If 1 liter (1.06 quarts) of water were involved, then 1 ppm would amount to 1 milligram (mg) per kilogram (kg) (1 L). This could also be expressed as 1 microgram per g. Other commonly used units include parts per billion (1 microgram per kg) and parts per trillion (1 nanogram per kg).
The gram is a basic unit of mass measurement that corresponds to the mass (weight) of one milliliter (mL) of water (a mL is about 1/1000th of a quart). The units of scale then go as powers of ten such that 1000 mL equals 1 liter (L), but 1/1000th of a mL is a microliter (1 µL). The same powers relation is used for the gram scale; thus, 1000 g is 1 kilogram (kg) and 1/1000th of a gram is a milligram (mg) and 1/1000th of a mg is a microgram (µg).
Concentration units (molar, M) may also be employed based on moles per liter (M) of a compound. The moles unit is dimensionless and is calculated by dividing the grams present by the gram molecular weight (the molecular weight expressed in grams) of the compound. The reason for the usefulness of this number is its ability to refer to a known number of molecules per unit volume rather than a weight per unit volume. Thus if a compound is present in 1 L of water at 1 M, then there are 6.023 X 1023 molecules of the compound present in the liter. In 100 mL, there would be 6.023 X 1022 molecules present, a still formidable number. (See: Environmental Chemistry: Measurement - Methods - Quality Assurance - Statistics.)
Separations are physical methods used to divide a sample into discrete components over a time or distance scale from an arbitrary starting time or distance. Thus, for example, in a horse race all horses start even and together. At the end of the race after circling the track they cross the finish line at different elapsed times and are also physically separated from one another. Many separation techniques work similarly by having the mixture start at the beginning of a column of separating material and components of the mixture exit the column one at a time as some flowing material (liquid or gas) helps to sweep them along. The ideal separation is fast, efficient (narrow peaks), and separates everything (selective). In complex real samples the ideal separation is rarely, if ever, achieved. Gas chromatography is a favorite separation technique in environmental analysis. Other techniques include high performance liquid chromatography, capillary electrophoresis, and thin-layer chromatography. A variety of animations are available on the web and which portray the principles of separation and detection.
Gas Chromatography (GC)
High Performance Liquid Chromatography
Capillary Electrophoresis (CE)
Mass Spectrometry/Mass Spectrometry
High Resolution Mass Spectrometry
Substances must somehow be recognized in the sample or in the separation technique applied to the sample. The process is called detection and can be based on a great variety of chemical and physical means. Light is often used to detect compounds because different compounds absorb light differently, and these differences can be measured. Thus, measurements are involved in this stage of the analysis where results are reported in numerical format with appropriate units.
Mass spectrometry is a favorite technique of detection because of its
specific, sensitive, and quantitative nature. Additional detector examples
include flame ionization, electron capture, infrared absorption, fluorescence
intensity, and electrical conductivity. Mass spectrometry itself depends
on making and separating ions from the sample (based on one of several
ion optics designs) and detecting them via an electron multiplier or photomultiplier
Diode Array Detector (PhotoDiodeArray)
Electron Capture Detector (ECD)
The basic information in a mass spectrum consists of the m/z ratios corresponding to the various fragments or whole molecular ions and their relative abundances. Data concerning these mass spectra form the backbone of mass spectral libraries where spectra obtained can be searched against what is in the database. Nowadays, spectral libraries containing more than 275,000 spectra are available.
The sequence of events follows this order. Samples are collected in the field based on a rational sampling plan (Figure 12) resulting from some concern about human health or the environment. The samples are analyzed directly or are extracted into a solvent, cleaned up (removal of nontarget substances or interferences to analytes), and subjected to the final separation/detection technique. For example, a sampling plan that follows a plume of a known contaminant could be designed (e.g., pyrene), soil samples taken, extraction (e.g., Soxhlet or rapid extraction) and cleanup (e.g., GPC and silica get cleanup) into a final solution of methylene chloride, and determination by GC/MS using an appropriate internal standard and calibration curve (based on appropriate ions from the target analyte and internal standard). The apprehension of untargeted analytes follows a similar course but is more tenuous due to the potential of failing to recover compounds from unexpected chemistry behavior.
|Name||Boiling Point||Density||Solubility (Water)||Si Polarity|
#chloroform/methanol/acetone in the w/w/w of 47/23/30 corresponding to
31.50/29.08/37.93 v/v/v form an azeotrope that extracts compounds with
a broad range of polarities.
Solvent Strengths on C18
Solvent Strengths on Carbon
First examine the molecular ion region (M+.) and the isotopes visible here (m/z 58). Usually, M+1 and M+2 are most important but in the case of halogenated compounds or organometallic compounds, for example, significant (in terms of relative abundance) isotope peaks occur at even higher m/z values. In the case of acetone the M+1 peak at m/z 59 is about 3-4 % relative abundance and the M+2 is 0.2-0.3% relative abundance to the M+. peak. The presence of Si or S is usually evident in the M+2 peak relative abundance. The presence of O or N is not obvious from relative abundances. The first major loss of 15 corresponds to loss of CH3. and production of (CH3CO)+ at m/z 43. There is also an ion at m/z 42 corresponding to (CH2=C=O)+. known as ketene. The ion at m/z 15 is CH3+. Ions such as m/z 43 and 42 are common in mass spectra and as neutral losses as well but the identity may be hydrocarbon (CH3CH2CH2+) instead of an oxygen-containing moiety. This is a general problem in fragmentation studies of identifying the elemental composition of ions. Additional collisional study of ions or linked scans allows mapping of fragments to their parent ions. One final note before leaving this example: the absence of ions at m/z values is also important with regard to specificity.
Consider next the mass spectrum of the methyl ester of stearic acid. The molecular ion region supports the presence of 18-20 carbon atoms in the compound. The fragmentation produces two homologous series of ions. One set contains the carbonyl function (m/z 74 and m/z 87, 101, 115, 129, 143, 157, 171, 185, 199, and 213) and while the other contains pure hydrocarbon series (43, 57, 71, 85, 99, 113 etc and the same ions associated also with loss of H2 such as m/z 55, 69, 83).
Interpretation of mass spectra then includes an examination of the molecular ion region, the consideration of rational cleavages and ion series, the recognition of particular m/z values that are correlated with commonly observed ions, and knowledge of the specific fragmentation pathways of compound classes and functional groups. This process is enormously aided by the existence of computer searchable libraries of spectra that now number more than 275,000 spectra. The hypothesis of a structure for a complete unknown is difficult and frequently benefits from the exact mass measurements of the molecular ion and key fragment ions. Even so, the identification may require purchase of standards for careful study, synthesis, or other spectroscopic data for a complete elucidation.Ref and links:
F. W. McLafferty and F. Turacek, Interpretation of Mass Spectra, 4th
ed., University Science Books, Sausalito, CA, 1993.
Look at the last step of the analysis. What determinative step is going to be used or is suggested by the properties of the analytes? Will derivatization be used?
2. Final separation/detection choice:
A. Target solubilities, volatility-stability. GC or GC/MS or derivatization and GC/MS. Ionization mode: EI, PICI, NICI (EC)B. HPLC (detector) reverse phase or normal phase; LC/MS.
C. CE (detector) free zone, MEKC, or other; CE/MS.
D. TLC (detector) and solvent system for separation.
3. Extraction and cleanup choices depend on matrix:
Water (liq/liq extraction, SPE, freeze dry)
Soil/sediment (Soxhlet, rapid extractions, sonication, shake outs)
Other (e.g., monomers in a polymer)Cleanup: normal phase (polar adsorbent) or reversed phase (hydrophobic adsorbent). Normal phase on Si uses a hexane, methylene chloride (or ether), acetone or methanol mixtures to elute. Reverse phase uses water and methanol or acetonitrile to elute. Adsorbents include silica, alumina, florisil, and carbon in normal phase and C18, C8, C2, and several others in reverse phase. GPC cleanup and HPLC cleanup may be used in multidimensional approaches.
The isotopic distribution is governed by the binomial distribution when calculating what the isotope pattern will be for a given set of elements. The general formula is:
For one chlorine the pattern is simple: 0.7577 / 0.2423 or 1.0 to 0.320 is the ratio of the relative abundances in the spectrum.
For two chlorines, we need to consider that we may have two 35Cl2,
35Cl37Cl, and 37Cl2 compositions
possible. In order to calculate the distribution, we use the coefficients
and powers dictated by the binomial theorem and the isotope abundances
in the calculation:
It is interesting to note that for organic compounds, the carbon isotope is always a major consideration. When the carbon numbers start to get very high, the isotope distribution becomes dominated by the carbon content. For example, a 100 carbon molecule already has as base peak the M+1 ion as illustrated for C100H100:
Data is acquired by digitizing the signal from the mass spectrometer at the rate of 40 kHz to 500 KHz, depending on sampling intervals dictated by mass resolution and scan speeds. Peak detection algorithms process the digital signal and result in centroided data that allow the assignment of a mass by means of a calibration that associates time with a given mass value (there could also be other mass assignment approaches such as field probe based assignments). Individual mass ions can be called out and displayed in a chromatographic format so that a particular ion current or m/z can be viewed as a function of time on the chromatographic time scale (i.e., retention time for ion chromatograms). These displays can be processed and areas of peaks obtained to use for quantitative determinations using peak detection algorithms. These algorithms usually take as parameters a peak detection sensitivity parameter for sensing peak start, a peak width parameter, and sometimes a peak end parameter for terminating the tail.
A calibration plot can be obtained in a number of ways. Illustrated below are some data for the calibration plot for a response of an ion from clofibric acid versus an ion of an internal standard. The data are handled by plotting the ratio of the response of the analyte to that of the internal standard versus the ratio of the amount of analyte to that of the internal standard. A linear regression of some order (usually linear, quadratic, or cubic) is performed on the data to obtain a calibration line.
The equation of this line can then be used to calculate the amount of the target analyte (s) in a sample in the form of an extract that has been added at a known level with the internal standard. For example, the internal standard is added at 10 ng for a 1 g sample the level is 10 ppb for the internal standard (is). From this value the amount of target can be calculated from the equation of the calibration curve. Usually, these calculations are transparent and handled by the data system based on the calibration curve. But for clarity let us assume the equation was:
y = 2 x + 0.001
y = (resp)s / (resp)is
(amt)s = (y * (amt)is - 0.001) / 2
In the case of the real sample we know the equation of the line, and all values except (amt)s. The value of (amt)is is 10 ppb for the example above and the resp values are the areas of the peaks from the ion chromatograms of target and is. Note that for quadratic and cubic fits the calculation is more complicated because a quadratic or cubic equation must be solved. A root finding numerical approach can be used rather than exact analytical expressions.In the case of an identification that is based on the correspondence of the mass spectrum and retention time of a standard and a response in a sample, the retention times should be identical within reproducibility and the spectrum should also be quite similar. The specificity of the particular mass spectrum under consideration is also a critical element in the confirmation of identity.
Gas ChromatographyThe phase ratio is useful in comparing retention between columns:
Kc = kb = k (r / 2df)
where r is column radius (micron), df film thickness (micron), k is partition ratio, and Kc is the distribution constant. The column with the smaller phase ratio will be the most retentive.The number of theoretical plates (N) will be inversely proportional to the radius of the column and will be given by
N = 5.545 (tr / wh)2
where tr is the retention time and wh is the peak width at half height.
Capacity Factor is given by the following equation and describes the retention of a component:
k = (tr -t0) / t0
where tr is the elution time of the component and t0
is the elution time of the unretained substance.
Separation Factor or Selectivity (a) is the ratio of the retention factors of two components:
a = k2 / k1
Resolution is defined by the separation of two adjacent peaks:
Rs = (1/4)(a - 1) (N)1/2 [k / (1 + k)]
where k is the average retention factor of the two components. This equation is interesting because it shows that resolution is a linear function of the relative retention but only improves by the square root of the number of theoretical plates.
Ref:C. F. Poole and S. K. Poole, Chromatography Today, Elsevier, Amsterdam, 1991.
ue = ueof - ua
where: ueof = the apparent mobility of the system peak which results from the displaced background ion moving with the velocity of the electroosmotic flow,
ua = lL/tV
where: V = applied voltage (V)
l = effective capillary length to the detector (cm)
L = total capillary length (cm)
t = migration time (seconds)
The velocity of an ion is given as:
v = ue E
where v is the velocity in cm/sec and E is the applied electric field.
Dispersion or the zone spreading results from differences in the velocity of the solutes within a zone and may be defined as the peak width at base (wb) which for a Gaussian peak is
wb = 4 o
where o is the standard deviation of the peak.
The number of theoretical plates, N, is given by
N = (l / o) 2
where l is the effective capillary length.
If the only source of peak broadening (longitudinal diffusion) is molecular diffusion, then
o2 = 2 Dt = (2 DlL) / (ueV)
where D is the diffusion coefficient of the solute.
Then the expression for N is
N = (ueVl) / (2DL) = (ueEl)
Experimentally, N is obtained from
N = 5.545 (tw1/2) 2where w1/2 is the peak width at half height.
Ref: D. N. Heiger, High Performance Capillary Electrophoresis, Hewlett-Packard, Walbronn, Germany, Publication No 12-5091-6199E.
Some compounds will not go through a gas chromatographic column without derivatization. The choice of making a derivative or using an alternative separation is dependent on the context of analysis and perhaps on available instrumentation as well as the detection limit needed. Derivatization may also be detector-oriented in that the derivative may enhance the ability to detect the analyte. The use of halogenated or fluorinated derivatizing reagents in GC/ECD is well known. The same approach has been used in GC/ECNIMS as well. In HPLC, the use of dansyl derivatives of amines is chosen to enable fluorescence detection of the derivatized analytes.
There are many analytes that are not easily derivatized that must be subjected to a separation that is consistent with their properties. High molecular weight proteins are obviously one such class but compounds as simple as sulfonyl urea herbicides and fluoroquinolone antibiotics are usually approached by liquid chromatography because in the former case they do not derivatize easily and in the latter case are amphoteric.
The most useful derivatizing reagents are silylating reagents and methylating reagents for facilitating gas chromatography. Acetylating reagents are also useful for amine-containing analytes. The derivatives that enhance electron capture processes are also well-developed. Approaches used in liquid chromatography usually are attempting to make use of fluorescent detection although some derivatives are used to enhance UV detection (e.g. ).
D. R. Knapp, Handbook of Derivatization Reactions, Wiley-Interscience, New York, 1979.
C. F. Poole and S. K. Poole, Chromatography Today, Elsevier, Amsterdam,
When using buffer solutions, the most effective buffering range of any weak acid or base is ± 1 pH unit. However, in practice, one may find them used at ± 2 pH units or more with little apparent buffering capacity left.
Compounds listed as bases are understood to be in their protonated form (conjugate acid) so that the pKa clearly indicates that they give up their proton at higher pH (> 7.0) and are therefore 50% protonated at their pKa, becoming more fully ionized at lower pH. Acids are listed in their unionized form such that the pKa represents the point at which they are 50% ionized and they become more fully ionized at higher pH.
|Bases||T(°C)||pKa of conjugate acid|
ACES 2-[(2-amino-2-oxoethyl)amino]ethanesulphonic acid
ADA N-(2-acetamido)-2-iminodiacetic acid
BES N,N-bis( 2-hydroxyethyl)-2-aminoethanesulphonic acid
BICINE n,n-bis(2-hydroxyethyl) glycine
BIS-TRIS-Propane 1 ,3-bis[tris(hydroxymethyl)methylamino] propane
CAPS 3-(cyclohexylamino) propanesulphonic acid
CHES 2- (cyclohexylamino) ethanesulphonic acid
HEPES N-2-hydroxyethylpiperazine-N'-2-ethane-sulphonic acid
HEPPS N-2-hydroxycthylpipcrazine-N'-3-propane-sulphonic acid
MES 2-(N-morpholino) ethanesulphonic acid
MOPS 3-(N-morpholino) propanesulphonic acid
PIPES piperazine-N-N-bis (2-ethanesulphonic acid)
TAPS 3-[tris (hydroxymethyl) methyl] amino propanesulphonic acid
TES 2-[tris (hydroxymothyl) methyl] amino ethanesulphonic acid
Tricine N-[tris (hydroxymethyl) methy] glycine
TRIS tris (hydroxylmethyl) aminomethane
Developments up to the present include accelerated solvent extraction, temperature-pressure-assisted solvent extraction, supercritical fluid extraction, SoxTec extraction, sonication extraction, and shakeout extraction. These techniques have intended to speed up the process while maintaining the excellent recovery figures of Soxhlet extraction and in some cases reduce solvent usage as well as reduce the amount of concentration that is necessary after extraction. Generally, levels as low as parts-per-billion are achievable. In some cases, levels as low as parts-per-trillion have been reached, usually with extensive sample cleanup (e.g., polychlorinated dibenzo-p-dioxins) and a small final volume.
In the case of aqueous matrix, the techniques of liquid/liquid extraction or a form of solid phase extraction are typically used. Because relatively large sample sizes can be used, typical detection limits in water can be 1000 times lower than those of a soil or sediment, for example. Thus, levels in the parts-per-quadrillion range are achievable with sensitive and selective detection and some sample cleanup.What are acceptable recoveries? This is an analyst's decision but should be known and reproducible. The accepted range for RCRA methods (SW-846) are often stated in the range from about 30-110% for multiresidue methods.
Recovery is not the precise issue in a technique such as solid-phase microextraction. Here a steady-state partition is established that is reproducible and is properly accounted for in producing a quantitative number. Exhaustive extraction is not the outcome or goal of this approach. Another example of using a non-exhaustive extraction approach is the vacuum distillation technique for volatiles.
C. F. Poole and S. K. Poole, Chromatography Today, Elsevier, Amsterdam,
Solid phase extraction is the presentation of an adsorbent in a convenient vehicle for the extraction of analytes from a matrix such as water. The adsorbent allows a fairly rapid flow through of the media. This is a solventless extraction and therefore reduces concentration requirements as well as solvent usage. Some solvent is required to flush the analytes from the adsorption bed unless SPME is used where the coated needle can be inserted directly into the injection port of the gas chromatograph and the analytes desorbed there.
N. Simpson and K. C. Van Horne, eds., Handbook of Sorbent Extraction Technology, Varian Sample Preparation Products, Harbor City, CA, 1993.
The following compounds are purported to be endocrine disrupting compounds: Benzenehexachloride (BHC); Chloroform; Dioxins and furans; Octachlorostyrene; PBBs; PCBs; PCB, hydroxylated; PBDEs; Pentachlorophenol; Butylated hydroxyanisole (BHA); Acetochlor; Alachlor; Aldrin; Allethrin, d-trans; Amitrol; Atrazine; Carbaryl; Chlofentezine; Chlordane; Cypermethrin; DDT; Dicofol (Kelthane); Dieldrin; Endosulfan; Ethylene thiourea; Fenarimol; Fenbuconazole; Fenitrothion; Fenvalerate; Fipronil; Heptachlor; Heptachlor-epoxide; Iprodione; Kepone (Chlordecone); Ketoconazole; Lindane (Hexachlorocyclohexane); Linuron; Malathion; Mancozeb; Maneb; Methomyl; Methoxychlor; Metribuzin; Mirex; Nitrofen; Nonachlor, trans-; Oxychlordane; Pendimethalin; Pentachloronitrobenzene; Permethrin; Procymidone; Prodiamine; Pyrimethanil; Sumithrin; Tarstar; Thiazopyr; Thiram; Toxaphene; Triadimefon; Triadimenol; Tributyltin; Trifluralin; Vinclozolin; Zineb; Ziram; Butyl benzyl phthalate (BBP); Di-n-butyl phthalate (DBP); Di-ethylhexyl phthalate (DEHP); Diethyl Phthalate (DEP); Benzophenone; Bisphenol A; Bisphenol F; Benzo(a)pyrene; Carbendazim; Ethane Dimethane Sulphonate; Perfluorooctane sulfonate; Nonylphenol, octylphenol; Resorcinol; Styrene dimers and trimers;