Gas chromatographic isolation of individual compounds from complex matrices for radiocarbon dating

This paper describes the application of a novel, practical approach for isolation of individual compounds from complex organic matrices for natural abundance radiocarbon measurement. This is achieved through the use of automated preparative capillary gas chromatography (PCGC) to separate and recover sufficient quantities of individual target compounds for (14)C analysis by accelerator mass spectrometry (AMS). We developed and tested this approach using a suite of samples (plant lipids, petroleums) whose ages spanned the (14)C time scale and which contained a variety of compound types (fatty acids, sterols, hydrocarbons). Comparison of individual compound and bulk radiocarbon signatures for the isotopically homogeneous samples studied revealed that Δ(14)C values generally agreed well (±10%). Background contamination was assessed at each stage of the isolation procedure, and incomplete solvent removal prior to combustion was the only significant source of additional carbon. Isotope fractionation was addressed through compound-specific stable carbon isotopic analyses. Fractionation of isotopes during isolation of individual compounds was minimal (<5‰ for δ(13)C), provided the entire peak was collected during PCGC. Trapping of partially coeluting peaks did cause errors, and these results highlight the importance of conducting stable carbon isotopic measurements of each trapped compound in concert with AMS for reliable radiocarbon measurements. The addition of carbon accompanying derivatization of functionalized compounds (e.g., fatty acids and sterols) prior to chromatographic separation represents a further source of potential error. This contribution can be removed using a simple isotopic mass balance approach. Based on these preliminary results, the PCGC-based approach holds promise for accurately determining (14)C ages on compounds specific to a given source within complex, heterogeneous samples.     

Thermoluminescent dating of archaeological pottery

This paper describes a thermoluminescent technique for dating archaeological pottery without isolating quartz from objects of dating. The technique employs TLD-K soil-equivalent thermoluminescent SiO₂-based detectors for measuring the test dose of artificial irradiation and the background dose rate on the dig site.     

Equivalent dose (palaeodose) estimation in thermoluminescence dating using a single aliquot of polymineral fine grains

A single aliquot protocol for the estimation of the natural dose in thermoluminescence dating (SATL) is presented that makes use of polymineral fine grains extracted from ceramic materials. The protocol is demonstrated using aliquots made from two Neolithic sherds and is compared with results from an additive dose polymineral technique. The results of both techniques are in close agreement and highlight the potential advantages of the new procedure, especially when sample availability is restricted, e.g. dating of small pottery fragments, or in cases of authenticity testing.     

Evaluation of gas chromatographic isotope fractionation and process contamination by carbon in compound-specific radiocarbon analysis

The relevance of both modern and fossil carbon contamination as well as isotope fractionation during preparative gas chromatography for compound-specific radiocarbon analysis (CSRA) was evaluated. Two independent laboratories investigated the influence of modern carbon contamination in the sample cleanup procedure and preparative capillary gas chromatography (pcGC) of a radiocarbon-dead 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) reference. The isolated samples were analyzed for their 14C/12C ratio by accelerator mass spectrometry. Sample Delta14C values of -996 +/- 20 and -985 +/- 20 per thousand agreed with a Delta14C of -995 +/- 20 per thousand for the unprocessed PCB 169, suggesting that no significant contamination by nonfossil carbon was introduced during the sample preparation process at either laboratory. A reference compound containing a modern 14C/12C ratio (vanillin) was employed to evaluate process contamination from fossil C. No negative bias due to fossil C was observed (sample Delta14C value of 165 +/- 20 per thousand agreed with Delta14C of 155 +/- 12 per thousand for the unprocessed vanillin). The extent of isotopic fractionation that can be induced during pcGC was evaluated by partially collecting the vanillin model compound of modern 14C/12C abundance. A significant change in the delta13C and delta14C values was observed when only parts of the eluting peak were collected (delta13C values ranged from -15.75 to -49.91 per thousand and delta14C values from -82.4 to +4.71 per thousand). Delta14C values, which are normalized to a delta13C of -25 per thousand, did not deviate significantly (-58.9 to -5.8 per thousand, considering the uncertainty of approximately +/-20 per thousand). This means that normalization of radiocarbon results to a delta13C of -25 per thousand, normally performed to remove effects of environmental isotope fractionation on 14C-based age determinations, also cor-rects sufficiently for putative isotopic fractionation that may occur during pcGC isolation of individual compounds for CSRA.     

Identification of archaeological adhesives using direct inlet electron ionization mass spectrometry

Adhesives made from natural substances such as resins, tars, and waxes are found during excavations on archaeological sites dating back to prehistoric periods. Until now, their analysis was mainly performed by gas chromatography, possibly coupled to mass spectrometry, after extraction, purification, and derivatization of the samples. To minimize sampling and sample preparation of ancient organic remains, which are often preserved in tiny amounts, we have directly analyzed archaeological samples from Bronze and Iron Age periods by direct inlet electron ionization mass spectrometry. A series of contemporary natural and synthetic substances, including pine and pistacia resins, birch bark tar, beeswax, and plant oils, possibly used for adhesive fabrication during ancient times, was also investigated with the same technique as reference materials. Despite the complexity of their chemical composition, pine resin and birch bark tar were clearly identified in archaeological samples. Furthermore, mass spectrometry has been shown to be efficient for the identification of glues made of a mixture of beeswax, presenting a series of mass spectral peaks assigned to long-chain esters, and birch bark tar, whose mass spectrum presents characteristic peaks of lupane compounds. The intentional mixing of birch bark tar and beeswax during prehistory is reported here for the first time.     

Elemental analysis of organic species with electron ionization high-resolution mass spectrometry

We present a new elemental analysis (EA) technique for organic species (CHNO) that allows fast on-line analysis (10 s) and reduces the required sample size to approximately 1 ng, approximately 6 orders of magnitude less than standard techniques. The composition of the analyzed samples is approximated by the average elemental composition of the ions from high-resolution electron ionization (EI) mass spectra. EA of organic species can be performed on organic/inorganic mixtures. Elemental ratios for the total organic mass, such as oxygen/carbon (O/C), hydrogen/carbon (H/C), and nitrogen/carbon (N/C), in addition to the organic mass to organic carbon ratio (OM/OC), can be determined. As deviations between the molecular and the ionic composition can appear due to chemical influences on the ion fragmentation processes, the method was evaluated and calibrated using spectra from 20 compounds from the NIST database and from 35 laboratory standards sampled with the high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS). The analysis of AMS (NIST) spectra indicates that quantification of O/C is possible with an error (average absolute value of the relative error) of 30% (17%) for individual species. Precision is much better than accuracy at +/-5% in the absence of air for AMS data. AMS OM/OC has an average error of 5%. Additional calibration is recommended for types of species very different from those analyzed here. EA was applied to organic mixtures and ambient aerosols (sampled at 20 s from aircraft). The technique is also applicable to other EI-HRMS measurements such as direct injection MS.     

Compound-specific stable isotope analysis of soil mesofauna using thermally assisted hydrolysis and methylation for ecological investigations

Stable isotope mass spectrometric approaches are proving to be valuable tools in unravelling biotic interactions in complex ecosystems, yielding information on trophic preferences and functional roles of individual species. Gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) provides considerable opportunities to assist in studies concerned with ecosystem processes mediated by soil invertebrates and microorganisms by determination of delta(13)C values of individual compounds, for example, lipids, amino acids etc. However, techniques conventionally adopted for "wet" chemical extractions and derivatizations necessary for compound-specific stable isotope determinations restrict the size of soil organism that can be studied and can limit investigations of individuals or even parts of individuals. We demonstrate here that individual soil mesofauna can be probed directly for their fatty acid stable isotope signatures by pyrolysis-GC/C/IRMS. A thermally assisted hydrolysis and methylation (THM) reaction is described for the determination of delta(13)C fatty acid values using trimethylsulfonium hydroxide (TMSH). Authentic fatty acids, acyl lipids, and individual Collembola (Folsomia candida) raised on C(3) and C(4) isotopically labeled yeast were analyzed initially by py-GC/MS with TMSH and then by py-GC/C/IRMS. A kinetic isotope effect (KIE) observed with the THM reaction prevents direct calculation of the fatty acid delta(13)C values by simple mass balance equations. However, the KIE is shown to be both reproducible and robust and can therefore be accounted for by the use of correction factors. The fatty acid methyl ester compositions of individual F. candida and their respective delta(13)C values were determined and shown to agree with those obtained by conventional "wet" chemical procedures applied to much larger numbers of Collembola, thus enhancing the scope to which stable isotopes can be applied to the study of invertebrates in complex food webs in any environment.     

Thermoluminescence dating of archaeological artefacts from the Middle Neolithic,Bronze Age and the Roman Empire period

Thermoluminescence (TL) dating was applied for artefacts found near the small village of Michelstetten, Lower Austria. Settlements in this region can be traced hack a long time and, according to archaeologists, the discovered artefacts may be as old as 6000 years. A modified sample preparation technique based on the fine-grain method was developed. This technique results in a higher reproducibility and reduces the overall preparation time. For some artefacts the new information of the TL dating leads to an unforeseen re-interpretation of the archaeological age. Furthermore, an iron furnace from the period of the Roman Empire could be dated. For the first time, it was possible to estimate correctly the point of time of the burn-down of an ancient wooden house via an analysis of the house's clay plaster. The fire took place in the sixth century; this was confirmed by dating ceramic artefacts.     

Using an optimised OSL single-aliquot regenerative-dose protocol for low-dose retrospective dosimetry on household salt

The single-aliquot regenerative-dose (SAR) protocols used in retrospective dosimetry for optically stimulated luminescence measurements have been mainly developed for archaeological and geological dating of quartz and feldspar. The aim in this study is to find a read-out protocol that can generate the most reproducible signal for household salt (NaCl) at absorbed doses below 100 mGy. The relation between the reproducibility of the signal, in terms of the ratio between given absorbed dose and SAR-calculated dose, and parameters such as test-dose pre-heat temperature has been studied. It was found that a temperature of 200 °C yielded the best reproducibility in the SAR-calculated dose, which is a somewhat higher pre-heat temperature than what is used for dating of quartz.     

An interface for direct analysis of (14)c in nonvolatile samples by accelerator mass spectrometry

We describe here apparatus and methods for direct analysis of (14)C in biological specimens by accelerator mass spectrometry (AMS). Liquid samples, including plasma and urine, are deposited by pipet into a bed of CuO powder that fills a space within a rigid, refractory support. Volatile components are removed under reduced pressure prior to analysis. The CuO matrix is locally heated with an infrared laser while it is contained within a sealed chamber that is swept with He carrier gas. Heating induces combustion of the applied sample, and the carrier gas transports the CO(2) that is formed to the AMS instrument's ion source, which is appropriately modified for use with CO(2). A rodent study of drug clearance with [(14)C]-acetaminophen was performed to provide plasma and urine specimens, which were analyzed with this overall approach and by liquid scintillation counting for comparison. Results presented here confirm the potential utility of laser-induced sample combustion as an alternative to graphite production for AMS analysis of (14)C. Anticipated benefits of the present approach include reduced risk of sample cross-contamination, decreased analysis time, and greater compatibility with robotics.     

Freezing to preserve groundwater samples and improve headspace quantification limits of water-soluble organic contaminants for carbon isotope analysis

Few systematic investigations have addressed the use of freezing for applications in analytical chemistry. Here, we tested its potential to preserve groundwater samples and to improve headspace quantification limits for compound-specific isotope analysis. Analysis of compound concentrations, as well as stable carbon isotope ratios, confirmed that trichloroethene was preserved in frozen suspensions of nanoscale zerovalent iron. In contrast, storage at 7 degrees C was ineffective, and complete degradation of TCE occurred in 4 weeks. Hence, freezing may stop even abiotic chemical reactions that would not be prevented by cooling or traditional preservation agents. In the absence of iron, we found that headspace concentrations of 14 organic contaminants were considerably higher over frozen solutions than at 25 degrees C, likely reflecting a freezing-out effect governed by Raoult's law. The observed enhancement depended on the salinity of the samples and was strongest for water-soluble, volatile compounds (values in brackets indicate the minimum observed effect out of six replicates): tert-butyl alcohol (TBA, 35-fold), methyl tert-butyl ether (MTBE, 14-fold), 1,2-dichloroethane (10-fold), or benzene (7-fold). In contrast, little enhancement was observed for less water-soluble compounds, such as tetrachloroethene. Although standard deviations of the measurements were too high for the method to be used for quantitative analysis of total compound concentrations, since we found that freezing introduces no measurable carbon isotope effect for TBA, MTBE, 1,2-dichloroethane, and benzene, the method is an effective way of increasing the sensitivity of compound-specific isotope analysis, particularly of water-soluble organic contaminants.     

Atmospheric pressure photoionization. 1. General properties for LC/MS

In this work, we describe the performance of an atmospheric pressure photoionization (APPI) source for sampling liquid flows. The results presented here primarily focus on the mechanism of direct photoionization (PI), as compared to the dopant mechanism of PI. Measured detection limits for direct APPI were comparable to atmospheric pressure chemical ionization (APCI; e.g., 1 pg for reserpine). The ion signal is linear up to 10 ng injected quantity, with a useful dynamic range exceeding 100 ng. Evidence is presented indicating that APPI achieves significantly better sensitivity than APCI at flow rates below 200 microL/min, making it a useful source for capillary liquid chromatography and capillary electrophoresis. Results are presented indicating that APPI is less susceptible to ion suppression and salt buffer effects than APCI and electrospray ionization (ESI). The principal benefit of APPI, as compared to other ionization sources, is in efficiently ionizing broad classes of nonpolar compounds. Thus, APPI is an important complement to ESI and APCI by expanding the range and classes of compounds that can be analyzed. In this paper, we also discuss the role of direct APPI vs PI-induced APCI using dopants.     

Integration of continuous-flow accelerator mass spectrometry with chromatography and mass-selective detection

Physical combination of an accelerator mass spectrometry (AMS) instrument with a conventional gas chromatograph-mass spectrometer (GC/MS) is described. The resulting hybrid instrument (GC/MS/AMS) was used to monitor mass chromatograms and radiochromatograms simultaneously when (14)C-labeled compounds were injected into the gas chromatograph. Combination of the two instruments was achieved by splitting the column effluent and directing half to the mass spectrometer and half to a flow-through CuO reactor in line with the gas-accepting AMS ion source. The reactor converts compounds in the GC effluent to CO2 as required for function of the ion source. With cholesterol as test compound, the limits of quantitation were 175 pg and 0.00175 dpm injected. The accuracy achieved in analysis of five nonzero calibration standards and three quality control standards, using cholesterol-2,2,3,4,4,6-d6 as injection standard, was 100 +/- 11.8% with selected ion monitoring and 100 +/- 16% for radiochromatography. Respective values for interday precision were 1.0-3.2 and 22-32%. Application of GC/MS/AMS to a current topic of interest was demonstrated in a model metabolomic study in which cultured primary hepatocytes were given [(14)C]glucose and organic acids excreted into the culture medium were analyzed.     

Thermal pre-treatment in the OSL dating of quartz: is it necessary?

Thermal treatment before optically stimulated luminescence (OSL) measurement is an important step in all widely accepted OSL dating procedures used with quartz. This is a major constraint on the design of a portable luminescence instrument for estimating ages in the field. Preliminary experiments show that using a standard dating protocol without heating caused a 30-50% underestimation of equivalent dose. This underestimate arises mainly from OSL derived from the 110 degrees C thermoluminescence (TL) trap; because of the thermal instability of this trap, this OSL contribution is only present in unheated laboratory-regenerated signals, but not in the natural signal. An alternative to thermal pre-treatment is investigated, based on the mathematical separation of the stable dosimetry OSL signal from the total OSL; the latter is the sum of signals from several traps, including the 110 degrees C TL trap. Our results show consistency with dose estimates obtained using a standard measurement protocol including pre-heating.     

Ultrafast sodium dodecyl sulfate micellar electrokinetic chromatography with very acidic running buffers.

A new micellar electrokinetic chromatography (MEKC) procedure has been developed that allows the use of the anionic surfactant sodium dodecyl sulfate (SDS) together with separation buffers at pHs as low as 1. The technique is based on the employment of a high molecular weight polyethylenimine (PEI)-coated capillary that provides strong cathodal electroosmotic flows at acidic pHs when SDS is added to the running buffer. The procedure is easy to implement since the capillary coating is done by just flushing a PEI aqueous solution through the capillary and the subsequent steps are the same as those for any MEKC protocol. Moreover, the coating renewal provides reproducible separations between injections (migration time RSD values lower than 1.82 and 3.44% were obtained for the same day and three different days, respectively). The good possibilities of this system are demonstrated by showing the separation of a group of eight polyphenolic compounds within a separation time shorter than 2 min. This procedure allows one to extend the optimization of SDS-MEKC separations to the very acidic pH range too.     

Mortar radiocarbon dating: preliminary accuracy evaluation of a novel methodology

Mortars represent a class of building and art materials that are widespread at archeological sites from the Neolithic period on. After about 50 years of experimentation, the possibility to evaluate their absolute chronology by means of radiocarbon ((14)C) remains still uncertain. With the use of a simplified mortar production process in the laboratory environment, this study shows the overall feasibility of a novel physical pretreatment for the isolation of the atmospheric (14)CO(2) (i.e., binder) signal absorbed by the mortars during their setting. This methodology is based on the assumption that an ultrasonic attack in liquid phase isolates a suspension of binder carbonates from bulk mortars. Isotopic ((13)C and (14)C), % C, X-ray diffractometry (XRD), and scanning electron microscopy (SEM) analyses were performed to characterize the proposed methodology. The applied protocol allows suppression of the fossil carbon (C) contamination originating from the incomplete burning of the limestone during the quick lime production, providing unbiased dating for "laboratory" mortars produced operating at historically adopted burning temperatures.     

Luminescence quartz dating of lime mortars. A first research approach

Lime mortars mixed with sand are well suited for connecting structural materials, like stones and bricks, due to the mechanical properties this material exhibits. Their extensive use in architectural and decorative works during the last 4000 years motivated the introduction of the 'Luminescence clock' for age determination of mortars. The same principles as for quartz optically stimulated luminescence (OSL) dating of sediments were applied for age estimation of a mortar fragment removed from a Byzantine church monument dated by archaeological means to 1050-1100 years ago (the first half of the 10th century). The OSL from the quartz was monitored under blue light stimulation and UV detection, using a single-aliquot-regenerative-dose protocol. The quartz-OSL dating of the mortar resulted in 870 +/- 230 a. TL polymineral fine grain dating was also performed on a brick fragment which was connected to the mortar, resulting in a TL age of 1095 +/- 190 a.     

Kinetics of the degradation of 2-chlorophenol by ozonation at pH 3

Prediction of byproduct distribution during ozonation is of importance to the design of treatment process. In this study, degradation products in direct ozonation of 2-chlorophenol in aqueous solution were identified by employing the chemical derivatization technique, specifically, silylation. Transient distribution of degradation products, in a semi-batch reactor under three ozone dosages were identified and determined by HPLC analysis. An empirical degradation pathway was proposed to describe the ozonation reaction. A mathematical protocol consisting of 11 equations and 12 rate constants was developed to solve and optimize the kinetic parameters. Modeling results revealed that the empirical pathway was capable of predicting the ozonation reaction at the beginning phase under a higher ozone dosage (e.g., greater than 6mg/L(g)). The degree of agreement between predicted and experimental data decreased as the ozone dosage decreased to 1.2mg/L(g). Results suggested that there was a dosage-dependent pathway in the direct ozonation of 2-chlorophenol.     

Selective determination of the doxorubicin content of individual acidic organelles in impure subcellular fractions

Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell lines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V(-1) s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V(-1) s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.     

Dating silk by capillary electrophoresis mass spectrometry

A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 μg or less of silk fiber. Except for a 2 h acid hydrolysis at 110 °C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids.