Soil and Environmental Analysis: Modern Instrumental Techniques - Chapter 9 docx

Table 1 Tracer and Fractionation Approaches ComparedTracer approach Fractionation approach Isotope abundance investigation Usually pot or small plot, but for lightly enriched tracers, sm

N/ 14 N, 18 O/ 16 O, and 34 S/ 32 S Variations in isotope abundances can reveal and quantify processes in which these elements are involved Such processes include photosynthesis, respiration, evaporation, organic matter turnover, and C, N, and S metabolism Stable isotopes can also be used in activities

as diverse as monitoring pollution events, tracking animals’ food sources, reconstructing past climates, identifying plants’ water sources, and untangling biochemical pathways.

Valuable general references include Fritz and Fontes (1980), Vose (1980), O’Leary (1981, 1988, 1993), Hoefs (1987), Raven (1987), Rundel

et al (1989), Coleman and Fry (1991), Griffiths (1991, 1998), Krouse and Grinenko (1991), Robinson and Smith (1991), Handley and Raven (1992), O’Leary et al (1992), Ehleringer et al (1993), Engel and Macko (1993), Knowles and Blackburn (1993), Lajtha and Michener (1994), Boutton and Yamasaki (1996), Handley and Scrimgeour (1997), Kendall and McDonnell (1998), Bingham et al (2000), Mook (2001), Robinson (2001), and Dawson et al (2002) The Internet is being used increasingly as a source

of the latest stable isotope information The ISOGEOCHEM website at

Campbell (1998) list others.

Many of the approaches described in these references rely on isotopes being used as tracers An isotopically distinct, but chemically indistingui- shable, material, the tracer, is introduced into an experimental system, and isotope abundances are later measured in particular compartments of that system Sometimes the tracer is a naturally occurring substance that happens to be isotopically distinct from others in the system Increasingly, however, use is being made of isotope fractionations These can report on the operation of chemical and physical processes that change the natural isotopic abundances of substrates and products involved in those processes The tracer and fractionation approaches are conceptually distinct and capable of addressing different research questions (Table 1).

Each approach demands its own theory and protocols, but both require similar instrumentation Most of this chapter (Secs III and IV) describes current instrumentation and analytical techniques used for stable isotope analysis It relies heavily on our experience of automated, continuous-flow mass spectrometers to analyze the isotopic contents of soil, plant, and animal samples Examples of tracer and fractionation applications are discussed in Sec.V Section VI is, finally, a brief preview of future developments We begin, however, with an overview of terminology.

Table 1 Tracer and Fractionation Approaches Compared

Tracer approach

Fractionation approach Isotope abundance

investigation

Usually pot or small plot, but for lightly enriched tracers, small catchment studies are feasible (Nadelhoffer and Fry, 1994)

Conditions required Isotopic composition of tracer

greater than natural range.

Steady-state labeling achieved within sinks (Dele´ens et al., 1994)

Reliable and distinct ences in isotopic composition

differ-of all potential source pools

Reliable measurements

of isotopic tions of all potential source pools

Table 1 Continued

Tracer approach

Fractionation approach Information required Isotopic composition of tracer

before addition to system; system components before addition;

system components after tion Amount of tracer added

addi-Isotopic composition of all potential tracer sources;

of system containing no tracer; of components of system containing tracer.

Fluxes among pools if more than two sources are involved

Isotopic composition of all important pools Amounts

of element in each pool Fractionation factors for candidate processes

Information obtained Amounts and rates of mixing of

tracer in nontracer pools

Amounts and (possibly) rates of mixing of tracer

Aand Am are related by

X ¼XðAsample
AbackgroundÞ½mLð100
A tracerÞ þmHAtracer

ðAtracer
AbackgroundÞ½mLð100
A sampleÞ þmHAsample ð5Þ

where X is the total amount of the element in the sample, Asample is the sample’s atom % (Eq 2), Atracer is the atom % of the tracer originally added, and Abackground is the background atom % in the system before the tracer was added.

For a given isotope pair, Eq 5 is simplified considerably by substituting the appropriate values for mL and mH Let us suppose that our N sample for which Asample¼5 atom % is from an experiment to which

a tracer containing 7.5 atom % 15 N had been added (Atracer), and assume that the background 15 N abundance in the system (Abackground) is 0.3663 atom % (cf Table 2; see Sec V.A.1) If the sample contains a total of

100 g N (X), then the amount of N in the sample that is derived from the tracer (X*) is 65.1 mg.

The term atom percent enrichment or atom percent excess (APE) is used frequently This is simply the difference in atom % between a sample and background In Eq (5), the terms (Asample
Abackground) and (Atracer

Abackground) are APE values.

The natural abundances of D, 13 C, 15 N, 18 O, and 34 S range from only 0.01 to

4 atom % (Fig 1) Close to natural abundance, it is more convenient to express the isotope ratio of a sample as the relative difference from that of a standard; this is the notation values are expressed in parts per thousand

or ‘per mille’ ( ø) The value of a sample, sample, is given by

sample¼1000Rsample
Rstandard

where Rsample and Rstandard are the isotope ratios of sample and standard, respectively [Eq (1)] Values of Rstandard are listed in Table 2, with the corresponding atom % values In practice, working standards are calibrated against these primary standards using materials supplied by the interna- tional Atomic Energy Agency (Vienna), the Los Alamos National Laboratory (U.S.A.), and other agencies By definition [Eq (6)], each standard in Table 2 has a value of 0 ø.

Samples with negative values are ‘‘depleted’’ in the heavier isotope relative to the standard; those that are positive are ‘‘enriched’’ (see Kendall and Caldwell, 1998) For example, if a sample has a 13 C/ 12 C ratio of 0.0111372, this differs by only 0.0001 from the standard (Table 2) This is

Table 2 Heavy : Light Isotope Ratios (R standard ) and Atom % Values (A) in the International Standards Used for the Analysis of D/H, 13 C/ 12 C, 15 N/ 14 N, 18 O/ 16 O, and 34 S/ 32 S By Definition, the Value of Each is 0ø

D/H Vienna Standard mean Ocean Water

equivalent to 13 C ¼
8.9 ø in the sample, i.e., it is 13 C-depleted compared with the standard.

For a particular isotope pair, and A are related by

Rstandardð100
AÞ
1

where Rstandard is the value in Table 2 appropriate for the isotope pair.

D, 13 C, 15 N, 18 O, and 34 S occur naturally in varying amounts in different materials These variations reflect isotopic fractionations of the heavier and lighter isotopes in a pair Fractionations occur because more energy is needed to break or form chemical bonds involving the heavier isotope of a pair (Atkins, 1998, p 833).

For a reaction occurring over an infinitesimal time interval, a

substrate divided by that of the product for that time interval:

When a > 1, the heavier isotope accumulates in the substrate as a reaction proceeds; when a < 1, it accumulates in the product If a ¼ 1, Rsubstrate¼

Rproduct and there is no fractionation (Note that some authors define a as

Rproduct/Rsubstrate: e.g., Mariotti et al., 1981) An expression that translates a values onto a ø scale for direct comparison with a values is

where " is the instantaneous isotopic enrichment factor; see Mariotti et al (1981).

a and " values are not strictly constants, but depend on temperature,

the identities of the reactants (including any enzymes that mediate the reaction), and bond energies (Atkins, 1998, p 833) This is true whether the fractionation occurs during a unidirectional kinetic reaction (e.g., NHþ4þ

OH
!NH 3þH 2 O) or in a system at equilibrium (e.g., NHþ

4 þOH

$NH 3þH 2 O) In each of these examples, the NHþ4 becomes more 15 enriched than the NH3 For a given reaction under defined, closed conditions, however, a is effectively constant irrespective of substrate availability and may be characteristic of the reaction a values for some biologically important reactions are tabulated in Friedman and O’Neill (1977), Leary et al (1992), Handley and Raven (1992), O’Leary (1993), Nordt et al (1996), Wada and Ueda (1996), and Handley et al (1999) Most

N-of these indicate the magnitudes N-of fractionations when substrate availability is not limiting and other conditions are favorable They do not necessarily indicate the fractionations that occur in vivo and which are often smaller than those in vitro.

As a reaction proceeds, the values of substrates and products change

in a predictable way, as described by Rayleigh equations (Mariotti et al 1981; Hoefs, 1987) The value of the substrate (S) depends on its initial value (0

consumed in the reaction:

Figure 2 illustrates how S, Pi and  P

strictly to a unidirectional single-step reaction in a closed system In such a reaction, the final product has the same isotopic composition as the initial

P tends to 0 Equations 10–12 can, however, also be applied to natural systems comprising multiple open reactions, especially if one fractionating process dominates (O’Leary, 1988; Sec V.C.3) Open reactions involve the addition of new substrate and/or the removal of accumulated product, and are never ‘‘completed’’ Isotopic differences between substrates and products persist (although the differences are not necessarily constant) Such differences are termed isotope

Figure 2 Changes in values of substrate and product in a closed system as a substrate in this example is 0 ø The values of substrate, instantaneous product, and accumulated product are calculated using Eqs (10–12) " is the instantaneous isotope fractionation factior [Eq (9)], which is constant and, in this example, ¼ 10 ø Discrimination (
; Eq (14)] is not constant but approximates to the difference

¼ ".

discriminations (symbolized as
) If substrate availability is effectively

Fig 2), then

Combining Eqs 6, 8, 9, and 13 gives (O’Leary, 1981)

¼ substrate
product

1 þ ð product=1000Þ substrate
product ð14Þ

A positive discrimination indicates that the heavier isotope lates in the substrate (substrate> product); a negative discrimination indicates the opposite.

accumu-The most extensive biological use of
has been to compare criminations against 13 C during photosynthesis by C3 plants (Sec V.C.2) Unfortunately, other systems are not so amenable For example, it is not yet possible to calculate
for N assimilation by plants growing in soil This is because the availability and 15 N values of putative substrates [e.g., NO

dis-3 ,

NHþ

4 , dissolved organic-N (DON)] at the assimilatory site(s) or metabolic branch points (O’Leary, 1981) where 15 N/ 14 N fractionations may occur cannot be assumed or measured reliably by current methods (Sec V.C.3).

One of the most useful and frequently encountered isotope equations is the isotope mass balance The relates the value of a composite sample to those

of its components, each weighted by its mass If a sample has two components of mass X and Y with values Xand Y, respectively, then value of the composite sample (  ) is

an unknown value or mass.

For example, suppose one wished to enrich 1 kg of C3 plant material with 13 C so that its 13 C value was about 500 ø How much 13 C-enriched CO2 containing 5 atom % 13 C would be needed? 1 kg (fresh weight) of plant material would contain about 40 g C (X) with a 13 C value (Y) of about

By setting  to 500 ø, Eq (15) can be solved for Y, which, in this

example, is 6.6 g C, or about 0.5 mol The plants should, therefore, be exposed to about 1 mol of 13 C-enriched CO2 to allow for inefficient C assimilation, leakages from the chamber enclosing the plants, and other losses during exposure This is a rough calculation, but it is sufficient to estimate the likely amounts (and costs) of an isotope that will be required.

If absolute masses of each component are not known, but their fractional contributions are, Eq (15) becomes, for a two-component mixture,

Measuring natural abundance isotope ratios or low tracer enrichments of the biologically important light elements is a challenge The natural range in abundance of the heavier isotope in a pair varies from twofold for D to

<10% for 15 N, 13 C, and 18 O (Fig 1) Kinetic or equilibrium fractionations

or mixing of isotopically distinct sources (Sec II.D) may change net isotope abundance by only a small fraction of the natural range The analytical problem is to measure changes as small as one part per thousand or less in a ratio of 1/10000, as is the case for D/H At the other extreme, S, with  4%

as 34 S, is less of a challenge in this respect but is more difficult to handle chemically.

Certain spectroscopic techniques such as nuclear magnetic resonance and infrared spectroscopy can detect and measure the abundance of stable isotopes However, only mass spectrometry is capable of measuring natural isotopic variations in H, C, N, O, and S with a large sample throughput and high precision, and at a modest cost All of these are now essential requirements in most analytical laboratories that serve environmental research Even with a mass spectrometer, the problem is not easily solved, and specially designed isotope ratio mass spectrometers (IRMS) have been developed for this purpose over the past 50 years.

All mass spectrometers contain three essential components: an ion source, a mass analyzer, and an ion detector (Fig 3) These perform the following basic functions in any mass spectrometer The sample is first introduced to the ion source, where the substance is converted into positive

or negative ions These ions are focused into a beam that then enters the

either in time or in space before entering the ion detector This produces an output signal proportional to the abundance of each m/z species separated

by the mass analyzer The output is generally referred to as the mass spectrum The ion source, mass analyzer, and detector are contained in a high vacuum system to minimize dispersion of the ion beam by collisions with air molecules Here we are concerned with IRMS Before describing the two basic types (dual-inlet and continuous-flow), we consider some general principles that underlie the operation of each.

The measurement of ion beam intensities with sufficient precision to determine isotope natural abundances requires purpose-built IRMS The basic IRMS design has changed little since it was first developed Only

Figure 3 Schematic diagram of the essential components of any mass meter.

spectro-major developments in materials, electronics, and data handling distinguish modern automated IRMS from their predecessors.

An IRMS for low atomic weight elements can analyze only low molecular weight ‘‘fixed gases,’’ irrespective of the nature of the original sample H 2 is used for D/H measurement, N 2 for 15 N/ 14 N, CO 2 for both 13

C/ 12 C and 18 O/ 16 O, and SO2 or SF6 for 34 S/ 32 S The gas is admitted to the mass spectrometer from a reservoir through a fine capillary This gives a steady supply of gas to the ion source and avoids diffusive fractionation of the isotopes in the inlet In the source, the gas is ionized by an electron beam produced from a hot filament of rhenium or thoriated tungsten The ion stream is accelerated through 3–5 kV before entering a magnetic sector mass analyzer The ion beam passes through a magnetic field at 90 to its direction of travel This causes the beam to bend Ions of different m/z leave the source with equal velocity, but the heaviest have most momentum and are deflected less easily by the magnetic field Once separated in this way, ions of different m/z are focused into different ion beams at the end of the mass analyzer or ‘‘flight tube’’ (Fig 4).

Figure 4 Schematic diagram of a triple-collector IRMS designed for low molecular weight gases The parallel arrangement of collectors, gain resistors, and voltage-to-frequency converters (VFC) allows simultaneous measurement of the isotopomer ion currents.

The geometry of the source and flight tube gives low resolution of the ion beams, each beam having a constant intensity over a significant portion

of its peak width These ‘‘flat-topped’’ ion beams are each detected by separate Faraday cup collectors Separate collectors are required to cope with the large intensity range (up to 1 : 10,000) between the most and the least abundant ion beams, and allow the isotopomer ion currents to be measured simultaneously The ion beams are focused by adjusting the accelerating voltage and/or the magnetic field strength so that the middle of the flat top of each beam enters a Faraday cup In this way, small drifts in the focusing parameters do not alter the measured intensity ratio between the ion beams, as would be the case if the beams had sharp peaks.

The cups are connected to ground through a large resistance, completing the circuit from the source The ion current flowing through the resistor creates a voltage that is the output from the mass spectrometer The voltage is fed into a computer-based data system via an impedance- matching amplifier (Fig 4) The ion current through an IRMS is  10
8 A for the most intense beam and 10
11 A or less for the other beams To produce a useful output voltage for the data system (a range of 1–10 V), resistors of 10 8 to 10 12  are required for the most and least intense beams,

respectively.

By using a higher resistor for the less abundant ion beams, the output entering the data system can be brought into the same voltage range for each beam The respective ion beam intensities are then measured by integrating the output voltages over a time period using parallel voltage frequency converters (VFCs) and counter circuits An important design feature is that the gain resistors and amplifiers must be very stable and produce a minimum

of spontaneous noise, thereby minimizing drift during sequential measuring periods.

Although an IRMS measures isotope ratios for a particular fixed gas (H2, N2, CO2, or SO2: Sec II.A), the information usually required is the isotope ratio of a particular element Further processing of the IRMS output is required to derive this information The need to apply corrections to the measured isotope ratios is not a major drawback of the method compared with the significant advantages of analyzing stable and readily prepared gases Providing the corrections are fully understood and carefully used, precise and accurate results can be obtained by applying a standardized measurement method to a few gases derived from a wide variety of samples.

In modern IRMS, these ‘‘ion corrections’’ are normally carried out

automatically by the instrument software, and it should be remembered that assumptions may be involved in the calculations.

CO2 When analyzing CO2, we measure the isotope ratios for m/z ¼ 45/44 and 46/44 These correspond to the significant isotopomers of COþ

2

We wish to know either 13 C/ 12 C or 18 O/ 16 O but must allow for the presence

of 17 O More information is required to calculate the desired ratios than is available (only two measured isotope ratios for three independent isotope ratios) Only by assuming that 17 O abundance covaries with the 18 O abundance can 13 C/ 12 C and 18 O/ 16 O abundances be estimated from the experimental measurement (Mook and Grootes, 1973) This correction may

be invalid for certain samples, e.g., when even small amounts of enriched O isotopes are present.

H2 Different corrections are required for H2 analysis, where the measured isotope ratio at m/z ¼ 3/2 is a combination of the required D/H ratio and the H3þ/H2þratio H3þis unavoidably formed in the ion source and has the same mass as dihydrogen containing 2 H and 1 H Careful source design can minimize the amount of H 3þ

formed, but prior calibration of the IRMS is required to correct for this interfering signal.

N2 With N2, no ion correction is required at natural abundance, but correction for residual air in the IRMS may be required With 15 N-enriched samples, the possibility of 15 N2 (m/z ¼ 30) being formed must be allowed for Corrections may be applied above a threshold 15 N enrichment of

5 atom%.

SO2 Correction for the contribution of 18 O to the m/z ¼ 66/64 ratio

is required This is usually done by assuming a fixed value for 18 O/ 16 O (Eriksen, 1996) With SF6 no correction is required.

Despite all the above adaptations to cope with large differences in ion currents and to achieve stability, it is not possible to make absolute measurements of isotope ratios sufficiently accurate for natural abundance studies Differential measurements against a defined standard are used to achieve this and to minimize the effect of instability during measurement Differential measurement compares the isotope ratio of a reference gas with that of the sample, each measured under the same conditions and within a short time period of each other The conventional way of arranging this is to use a dual-inlet (DI) system.

Gas is held in separate reference and sample reservoirs From these, gas flows through matched capillaries to a system of crossover valves These

valves allow the gases to enter alternately the IRMS or a waste vacuum line (Fig 5) The crossover valves are designed to perturb the gas flow as little as possible during the switchover, and to avoid mixing of sample and reference gases The pressure in each reservoir can be adjusted and matched by altering reservoir volumes using bellows This ensures that the reference and sample gases are measured at the same ion current Such a degree of controlled matching is possible only with gaseous samples.

Using a DI, the reference and sample signals are each integrated for 10–20 s following a settling period of 5–15 s after each changeover This is repeated for several (3–10) cycles and the data averaged over each cycle and over the set In this way, drifts in the detector system can be compensated for as far as possible and aberrant measurements caused by transient noise

development of an alternative sample inlet system known as a

continuous-flow(CF) inlet Here, pulses of gas are introduced to the source in a steady flow of He carrier Up to ten samples can be analyzed between pulses of reference gas (Fig 6) The CF inlet is considerably simpler (and cheaper) than the DI and suited to more rapid analyses The precision of modern CF-IRMS can approach that of many DI-IRMS in routine use.

CF-IRMS systems are designed to be integrated with a sample preparation device to produce regular pulses of analyte gas The original and most common sample preparation device is a Dumas combustion ele- mental analyzer or ANCA (automated C and N analyzer: see also Chap 6),

the combination often being called an ANCA-MS (Sec IV.A) Other sample preparation systems for gas analysis and trace gas concentration are available for integrated CF-IRMS systems.

The ultimate exploitation of CF-IRMS is in systems that first rate individual compounds from a mixture by GC and then convert them to

sepa-an IRMS-compatible gas This technique has already acquired sepa-an tunate variety of names and acronyms: compound-specific isotope analysis, stable isotope ratio monitoring–GC/MS, GC-combustion IRMS; or just GC-IRMS This is still a specialized area, but it will undoubtedly lead to a much more detailed understanding of C and N metabolism in biological systems.

Sample preparation is a nontrivial part of isotope analysis and may require as much time and care as the final IRMS measurement All samples—animal,

Figure 6 Schematic diagram of a CF-IRMS, consisting of an elemental analyzer and gas IRMS After each solid sample is dropped into the elemental analyzer, pulses

of purified analyte gas (e.g., N 2 or CO 2 ) are carried by the continuous flow of He into the IRMS.

vegetable, or mineral—must be converted into a gas suitable for isotope analysis by IRMS Each gas must be pure to enable sample and reference matching and to avoid interfering reactions in the ion source For example,

a trace of CO2 in N2 will produce some COþ

in the ion source COþ

has

m/z ¼ 28, the same as for N2 It is equally important that the isotope ratio of the prepared gas truly reflects that of the original sample This means that sample conversion must be complete to avoid isotope fractionation or that

an equilibrium is set up under identical conditions for all samples.

It is often possible to integrate and automate sample preparation systems with an IRMS, and this has great practical advantages Automated systems can operate unattended overnight, making efficient use of instrument time, and can produce better sample-to-sample and batch-to-batch reproduci- bility than the most patient and careful operator An important example of such an integrated system is the ANCA-MS (Sec IV.A).

Even with automation, considerable labor may be needed to provide samples Approximately the same amount of material must be analyzed for each sample This requires careful dispensing of liquid samples or weighing of solids Solid samples must also be finely ground before analy- sis to ensure representative subsampling For example, the amount of plant material required for an elemental analyzer is  1 mg oven-dry weight These subsamples must be weighed accurately into a tinfoil cup The time-consuming steps of grinding and weighing have been a charac- teristic of all elemental analyzer use for many years and are largely unavoidable.

Most studies of natural abundance variations in C and N have used bulk samples, with little or no chemical separation of the components of the sample Detailed understanding of the mechanisms controlling the isotopic composition of the material will increasingly require such separation The methods used will vary with the compounds being studied, but the fundamental requirement is for those that are quick and efficient Complete separation of a compound from its matrix ensures that no isotope fractionation will occur (although the risks of fractionation decrease as the molecular weight increases).

We turn now to the CF-IRMS analysis of particular isotopes in different sample types The procedures that we describe have evolved from our experience of analyzing both solid and gas samples at the SCRI laboratory Slight modifications to accommodate different instruments and applications are to be expected.

A ANCA-MS for Carbon and Nitrogen

The CF inlet is particularly suited (and indeed was developed) for use with

an elemental analyzer Elemental analyzers oxidize samples of organic material to give a mixture of N2 and CO2 In an ANCA-MS, this mixture is carried by the He carrier into a gas chromatograph (GC) There the gases are separated and emerge as two peaks that can be fed sequentially into the IRMS (Fig 7).

1700C, ensuring complete combustion of the sample The He flow sweeps

Figure 7 Typical layout of an ANCA-MS system (1) Continuous flow of He into the elemental analyzer and autosampler (2) Autosampler holding solid samples in tinfoil cups (3) Combustion tube containing chromium trioxide at 1000C (4) Reduction tube containing copper at 600C (5) Water trap containing magnesium perchlorate (6) Optional CO 2 trap containing Carbosorb (7) Gas chromatograph to separate N 2 and CO 2 (8) Open split where a small portion of the He flow enters the IRMS through a crimped capillary (9) Open capillary vent for remainder of He (10) IRMS Helium flows continuously from (1) to (9).

the combustion products first through a copper reduction furnace at 600C where N oxides are reduced to N2 and then through magnesium perchlorate

at room temperature to remove water Optionally, the gases may be passed through a Carbosorb trap to remove CO2 The gases are then separated on a

GC column, to give fully resolved peaks of N2 and CO2 in the He carrier flow.

Only a fraction of the effluent enters the IRMS, to keep the analyzer pressure at  10
6 mbar This is achieved with a narrow crimped capillary and three-way valve or concentric capillaries—sometimes referred to as an open split The bulk of the effluent passes to atmosphere, through a long capillary to minimize back-diffusion of atmospheric gases.

After combustion, but before the sample gas reaches the mass spectrometer, the background signals are measured As the gas pulse enters the IRMS, the appropriate mass signals are integrated, m/z 28, 29, and 30 for N2 and 44, 45, and 46 for CO2 (Fig 8) Following the peak, the background is again measured, and the mean background subtracted from the integrated areas Blank values, obtained when no sample is introduced, are also subtracted from the peak areas; this is particularly important when traces of N in the O2 pulse interfere with the N produced by combustion.

Figure 8 ANCA MS trace showing the timing of a sample analysis (1) O 2 pulse (2) Sample drops (3) N 2 zero (4) Measure N 2 (5) Switch source to CO 2 (6) CO 2

zero (7) Measure CO 2

Calibration is made against reference material introduced before and after batches of (usually ten) samples.

A run of samples is set up after a daily check procedure This consists

of a background scan to check that there are no interfering signals or air leaks, and peak centering to ensure stable ratio measurement The water and CO2 traps are checked and replaced if necessary The ash collection tube in the combustion furnace is checked and replaced if a bright red glow is not visible After venting the O2 supply for 30 s to remove any air, three blanks are run with no samples in the autosampler This allows correction for the inevitable small N2 signal from the O2 pulse The C blank should be negligible.

The sample identifiers and weights are entered into the sample table of the data system Samples and standards are put in the autosampler wheel in the same order as in the sample table The analysis sequence starts with two

or three working standards used as dummies, followed by a working standard, and then ten samples, and then a pair of working standards The first working standard (sometimes referred to as a check standard) is used for quality control and the second as a standard The check standard can also be substituted for the standard if there is a problem such as an electrical spike while the standard is being measured The pattern of ten samples and pairs of working standards is continued until the set is complete A practical limit to the number of samples in a run is set by the analysis time and the capacity of the autosampler When the number of samples is more than the autosampler can hold, the run can be started some hours before the end of the working day The remaining samples are added

to the autosampler once sufficient spaces are free In addition to the check standards, further quality control standards may be included at the end or during the run.

Once the analysis is complete, the data can be replayed rapidly to check the traces for spikes or other anomalies Any suspect samples are noted, and if need be, changes to timing windows or selected standards are made and the data reprocessed The final report gives the signal size, elemental composition (based on that of the working standards), and isotopic composition (in or atom %) for each sample The data are available as hard copy or as data files These can be imported into a spreadsheet for more convenient data reduction.

CF-IRMS measurements of 13 C and 13 N using an ANCA-MS are now the method of choice for many applications requiring bulk isotopic data

on plant, animal, and other ecological samples ANCA-MS can also be used

for individual chemical species, providing they can be isolated in sufficient amount and purity (and preferably routinely) ANCA-MS offers high throughput and a precision that is adequate for most purposes Indeed, with newer instruments, the precision for 13 C, in particular, is almost as good as can be achieved by on- or off-line sample conversion and a DI system Unlike manual methods, the rapid analysis makes replication of samples practical, and realistic estimates of both analytical precision and biological variation can be made.

The SCRI laboratory processes up to 25,000 samples per year, using two ANCA-MS systems (Tracermass þ Roboprep and 20-20 þ ANCA-SL, both from PDZ Europa Ltd., Crewe, U.K.) The philosophy used to carry this out is discussed below, followed by some practical examples of analytical methods and supporting techniques This approach achieves satisfactory results for a range of plant, soil, and animal tissue samples, using only a few standard robust analytical methods Two guiding principles

in all these analyses are (1) the amount of analyte element is kept within

20% of that in the working standards, and (2) the standards reflect the chemical composition of the samples.

The precision that can be achieved depends on the kind of sample being analyzed and on how this analysis is done Some samples containing little of the analyte element will be unsuitable for ANCA-MS analysis All isotope ratio measurements are more or less subject to sample size effects These have many causes, of which ion-source behavior may be regarded as the most significant, but background signals, electronic offsets, and amplifier linearity may all contribute Further, IRMS operate successfully only over a small range of sample size Large ion currents saturate the detectors, while small ones result in excessive noise The design and operation of DI systems aims to minimize these problems by keeping sample and reference signals both equal and constant from one measure- ment to the next.

Sample conversion may also introduce variable background ination, which becomes more serious as samples get smaller Since ANCA-

contam-MS combines sample conversion and measurement, the causes of sample size effects are less easy to establish than with a DI system It is generally easier to maintain a constant amount of analyte in the samples than to eliminate or even minimize sample-size-dependent shifts in measured isotope ratio Where a range of sample sizes is unavoidable, a set of calibration standards can be run with the samples and a suitable correction made These standards consist of different amounts of a material of the same known isotope ratio The small increase in the number of analyses that this causes should not be a problem with CF systems Such additional calibration samples would be a considerable burden with manual measurements.

We use analytical methods designed for approximately equal amounts

of analyte (N, C), not of sample This is achieved from knowing the typical composition of the sample ( 40% C in plant dry matter,  10% N and 50% C in proteins and animal samples) Alternatively, a preliminary analysis of the sample is done using the ANCA-MS for elemental composition only Only samples of similar type are run together.

The working standards used are matched to the sample composition For plant samples either flour or, more conveniently, a synthetic mixture of

2% N and 40% C can be used For animal samples, an amino acid such as leucine is suitable The sample size is chosen to give a large enough signal for good precision, and for most purposes this is  100 mg of analyte element Samples too large for the autosampler or which cause unnecessary ash buildup in the combustion tube are avoided.

Most ANCA-MS can switch elements during a run and operate in a dual-isotope mode, and 15 N and 13 C can be measured from the same sample This can produce good results for both isotopes when there is a sufficiently low C/N ratio, as in protein or animal samples As the C/N ratio increases, it is increasingly difficult to get good 15 N data from the sample This is probably due to increased CO2 entering the MS and being incompletely pumped away before the next N2 peak is measured.

For most plant samples, we determine 13 C and 15 N as follows First,

in the dual-isotope mode and using 1 mg dry subsamples (Table 3), we determine % C, 13 C, and % N Then, in single-isotope mode, in which the CO2 is trapped before it enters the IRMS, a second subsample is analyzed for 15 N The subsample’s weight is determined by its % N such that we have a constant amount of N in each sample, usually 100 mg.

When measuring light tracer enrichments (i.e., above 100 ø), there are

less stringent requirements for precision, and the constraints on the amount

of analyte can be relaxed 13 C and 15 N can be measured together on 1 mg dry plant samples (containing 20 to 50 mg N) This reduces the amount of ash formed, as well as the potential for memory effects between samples Natural abundance and enriched samples should not be run together as there is the possibility of memory affecting the precision It is probably wise

to replace the combustion tube if natural abundance samples are to be run after many enriched samples.

In summary, it is desirable

To use a few standard methods

To use a constant amount of analyte element

To match standards to samples in both amount and composition

To only use the dual isotope mode when the C/N ratio is low ( < 5)

Table 3 Specific Methods for C and N Determination The Precisions are Realistic Estimates of What can be Achieved Routinely Over an Extended Period For Plant and Soil 15 N, the Precision Deteriorates as Sample % N Falls

Working standards

Precision (1s) ( ø)

13 C 15 N Dual isotope C and

trap not used

1 mg dry wt has sufficient N and

C for quantitation and isotope analysis in the dual isotope mode

1 mg dry wt plant and 10 mg dry wt soil has sufficient N and

C for quantitation and sufficient

C for isotope analysis in the dual isotope mode N isotope values should be ignored

1 mg 1 : 4 leucine : citric acid mix- ture (2% N)

<0.1 A 0.6 B

Calculated weight containing

100 mg N, obtained from % N from dual isotope analysis (above) if required

5 mg 1 : 4 leucine : citric acid mix- ture (2% N)

CO 2 trap used

Calculated weight containing

25 mg N, obtained from dual tope analysis (above) if required

iso-1 mg iso-1 : 4 leucine : citric acid mix- ture (2% N).

Other instruments will have different strengths and options, such as adjusting the proportion of sample entering the IRMS between elements However, to optimize any analysis, foreknowledge of the sample composi- tion and choosing a suitable sample size remain important.

It is also important to maintain a constant and comfortable laboratory temperature to achieve satisfactory and consistent performance The cost of air conditioning is modest compared with that of CF-IRMS instruments and is quickly repaid in reliability of both instruments and results.

The analytical methods that we use for particular types of samples, along with the appropriate standards and realistically achievable precision, are summarized in Table 3.

The check standards included at regular intervals throughout an analytical run indicate the precision of the data produced and how this compares from run to run Since these standards are of similar composition to the samples, matrix effects in the sample conversion are unlikely to produce great differences in precision between samples and check standards We calculate the mean and standard deviation of the check standards for each run and plot those on quality control charts for each analytical protocol These charts provide a check on the day-to-day performance of the whole ANCA-

MS system and a realistic estimate of the quality of the data being produced When the precision is significantly poorer than on previous runs, some remedial action (checking the water/CO2 traps, replacing the ash collection tube, etc.) is indicated The quality control charts also show the extent to which running enriched samples alters the precision of the results and any memory effects on subsequent runs.

Further quality control standards can be included in the run, and we routinely use a bulk supply of flour for this purpose, particularly for plant samples Two flour standards are included at the end of each run and the results again recorded on quality control charts Since these standards are weighed out (rather than freeze-dried like the working standards), they also provide a check on the accuracy of the elemental composition.

The accuracy of the isotopic results depends on the calibration of the working standards Herein lies a problem that is becoming increasingly common The reference materials for isotopic analysis come in a limited number of chemical forms, and some (e.g., metal carbonates) may not be suitable for ANCA-MS Others may have a chemical composition so different from the samples and working standards as to raise doubts about their direct comparability This is not really a new problem but was less obvious when sample conversion and DI-IRMS were two separate steps.