Hydrogen isotope ratios of leaf wax n-alkanes in grasses are insensitive to transpiration

We analyzed hydrogen isotope ratios of high-molecular weight n-alkanes (δD_l) and oxygen isotope ratios of α-cellulose (δ¹⁸O_C) for C₃ and C₄ grasses grown in the field and in controlled-environment growth chambers. The relatively firm understanding of ¹⁸O-enrichment in leaf water and α-cellulose was used to elucidate fractionation patterns of δD_l signatures. In the different relative humidity environments of the growth chambers, we observed clear and predictable effects of leaf-water enrichment on δ¹⁸O_C values. Using a Craig-Gordon model, we demonstrate that leaf water in the growth chamber grasses should have experienced significant D-enriched due to transpiration. Nonetheless, we found no effect of transpirational D-enrichment on the δD_l values. In field samples, we saw clear evidence of enrichment (correlating with relative humidity of the field sites) in both δ¹⁸O_C and δD_l. These seemingly contrasting results could be explained if leaf waxes are synthesized in an environment that is isotopically similar to water entering plant roots due to either temporal or spatial isolation from evaporatively enriched leaf waters. For grasses in the controlled environment, there was no enrichment of source water, whereas enrichment of grass source water via evaporation from soils and/or stems was likely for grass samples grown in the field.Based on these results, evaporation from soils and/or stems appears to affect δD_l, but transpiration from leaves does not. Further evidence for this conclusion is found in modeling expected net evapotranspirational enrichment. A Craig-Gordon model applied to each of the field sites yields leaf water oxygen isotope ratios that can be used to accurately predict the observed δ¹⁸O_C values. In contrast, the calculated leaf water hydrogen isotope ratios are more enriched than what is required to predict observed δD_l values. These calculations lend support to the conclusion that while δ¹⁸O_C reflects both soil evaporation and transpiration, δD_l appears to only record evaporation from soils and/or stems. Therefore, the δD of n-alkanes can likely be used to reconstruct the δD of water entering a leaf, supporting the soil-enrichment model of Smith and Freeman (2006). In both the field and controlled studies, we found significant photosynthetic pathway effects on n-alkane δD suggesting that biochemical pathways or plant phylogeny have a greater effect on leaf wax δD than leaf-water enrichment in grasses.     

Characterisation of n-alkanes and their hydrogen isotopic composition in sediments from Lake Qinghai, China

The distributions of n-alkanes and their hydrogen isotopic composition (δD) in surface and core sediments from the saline Qinghai Lake were measured to assess whether or not biological source information was recorded in the δD values of n-alkanes. The results indicate that the n-alkane distributions between shallow water surface and core sediments were similar, and closer to those of terrestrial herbaceous plants from the Qinghai Lake surrounding areas, rather than the aquatic plants living in the lake. The n-alkanes in the surface and core sediments had similar mean δD values, ranging from −185‰ to −133‰ and −163‰ to −142‰, respectively. The mean δD values of n-alkanes in the sediments showed that the even n-alkanes were heavier in D compared with the odd homologues.     

Fundamental studies on kinetic isotope effect (KIE) of hydrogen isotope fractionation in natural gas systems

Based on quantum chemistry calculations for normal octane homolytic cracking, a kinetic hydrogen isotope fractionation model for methane, ethane, and propane formation is proposed. The activation energy differences between D-substitute and non-substituted methane, ethane, and propane are 318.6, 281.7, and 280.2 cal/mol, respectively. In order to determine the effect of the entropy contribution for hydrogen isotopic substitution, a transition state for ethane bond rupture was determined based on density function theory (DFT) calculations. The kinetic isotope effect (KIE) associated with bond rupture in D and H substituted ethane results in a frequency factor ratio of 1.07. Based on the proposed mathematical model of hydrogen isotope fractionation, one can potentially quantify natural gas thermal maturity from measured hydrogen isotope values. Calculated gas maturity values determined by the proposed mathematical model using δD values in ethane from several basins in the world are in close agreement with similar predictions based on the δ¹³C composition of ethane. However, gas maturity values calculated from field data of methane and propane using both hydrogen and carbon kinetic isotopic models do not agree as closely. It is possible that δD values in methane may be affected by microbial mixing and that propane values might be more susceptible to hydrogen exchange with water or to analytical errors. Although the model used in this study is quite preliminary, the results demonstrate that kinetic isotope fractionation effects in hydrogen may be useful in quantitative models of natural gas generation, and that δD values in ethane might be more suitable for modeling than comparable values in methane and propane.     

Carbon isotope analyses of n-alkanes in dust from the lower atmosphere over the central eastern Atlantic

Atmospheric dust samples collected along a transect off the West African coast have been investigated for their lipid content and compound-specific stable carbon isotope compositions. The saturated hydrocarbon fractions of the organic solvent extracts consist mainly of long-chain n-alkanes derived from epicuticular wax coatings of terrestrial plants. Backward trajectories for each sampling day and location were calculated using a global atmospheric circulation model. The main atmospheric transport took place in the low-level trade-wind layer, except in the southern region, where long-range transport in the mid-troposphere occurred. Changes in the chain length distributions of the n-alkane homologous series are probably related to aridity, rather than temperature or vegetation type. The carbon preference of the leaf-wax n-alkanes shows significant variation, attributed to a variable contribution of fossil fuel- or marine-derived lipids. The effect of this nonwax contribution on the δ¹³C values of the two dominant n-alkanes in the aerosols, n-C₂₉ and n-C₃₁ alkane, is, however, insignificant. Their δ¹³C values were translated into a percentage of C₄ vs. C₃ plant type contribution, using a two-component mixing equation with isotopic end-member values from the literature. The data indicate that only regions with a predominant C₄ type vegetation, i.e. the Sahara, the Sahel, and Gabon, supply C₄ plant-derived lipids to dust organic matter. The stable carbon isotopic compositions of leaf-wax lipids in aerosols mainly reflect the modern vegetation type along their transport pathway. Wind abrasion of wax particles from leaf surfaces, enhanced by a sandblasting effect, is most probably the dominant process of terrigenous lipid contribution to aerosols.     

海相干酪根与原油裂解气甲烷生成及碳同位素分馏的差异研究

利用封闭金管高压釜体系对海相原油和成熟干酪根进行了热解生气实验,获取了两类裂解气的组分和甲烷碳同位素数据,对比研究了两类母质在生气机理上的差异,并借助碳同位素分馏动力学参数讨论了甲烷碳同位素分馏的异同点.结果表明,原油裂解气富含C2-5重烃,其后期裂解是甲烷的重要来源;而干酪根裂解气中C2-5的含量较低,其后期裂解对干酪根甲烷气的贡献较小.这是两类甲烷气体生成的最大差异之一.两类裂解气甲烷碳同位素都有随着热解温度增高,碳同位素值先变轻再变重的特点,但原油裂解气甲烷碳同位素的最小值对应的温度较高;在相同热解温度下,干酪根裂解气甲烷碳同位素值要重于原油裂解气甲烷碳同位素值,这与后者前系物经过多次碳同位素分馏有关.因此,生气机理的差异是造成同位素分馏差异的根本原因,两类甲烷气体碳同位素分馏动力学参数的差异也是有成因意义的.     

A steady-state model for sulphur isotope fractionation in bacterial reduction processes

A model is developed to explain the isotope fractionation effects produced in laboratory experiments involving the reduction of sulphate to hydrogen sulphide by the bacterium Desulfovibrio desulfuricans. The approach differs from previous ones in its use of zero-order kinetics to describe the uptake of sulphate by the bacterium. Expressions are developed which relate the overall isotope effects produced by the bacterium to the ratios of backward to forward flows between its internal sulphur reservoirs. Other applications of this type of model are discussed, including the differences of isotopic composition between sulphate and hydrogen sulphide in the Black Sea and an unusual isotope effect observed in the course of bacterial nitrate reduction.     

A note on hydrogen isotope fractionation involving acidic and basic solutions

The dissociation constant of D₂O is significantly lower than that of H₂O, hence hydroxyl and hydronium ions have very much lower deuterium contents than the water from which they form. If the concentration of either of these ions becomes significant, its presence will effect the hydrogen isotope fractionation between any hydrogen bearing phase and the total solution.     

Anomalous carbon isotope fractionation between atmospheric CO2 and dissolved inorganic carbon induced by intense photosynthesis

A stable isotope mass-balance of dissolved inorganic carbon during a blue-green algae bloom in a softwater lake demonstrates that at low partial pressure of carbon dioxide there must be a large net negative carbon isotope fractionation between atmospheric CO₂ and the CO₂ absorbed by lake water at pH = 9.5. The net fractionation of CO₂(g) with respect to HCO⁻₃ was about −13%. compared with about +8%. for water at equilibrium with atmospheric CO₂ at pH ≈ 7. Chemical enhancement of CO₂ invasion at high pH by the reaction CO₂ + OH⁻→ HCO⁻₃ at large apparent film thicknesses may result in carbon isotope fractionation approaching that for a hydroxide solution. This phenomenon, coupled with a decrease in the photosynthetic fractionation, forced the surface water of a softwater lake to achieve increasingly negative δ¹³C values during an algal bloom, which is in the opposite sense to the trend that results from photosynthesis under less extreme conditions. This and other similar systems must operate under non-equilibrium (kinetic) conditions, causing a large kinetic fractionation during CO₂ invasion at pH > 8 and relatively large film thicknesses (i.e., low wind stress).     

The variations of stable carbon isotope ratio of land plant-derived n-alkanes in deep-sea sediments from the Bering Sea and the North Pacific Ocean during the last 250,000 years

Two piston cores, one located far from the continents (The North Pacific Ocean: ES core), and another located comparatively closer to the continents (The Bering Sea: BOW-8a core) were investigated to reconstruct environmental changes on source land areas. The results show significant contribution of terrestrial organic matter to sediments in both cores. The δ¹³C values of n-C₂₇, n-C₂₉, and n-C₃₁ alkanes in sediments from the North Pacific ES core show significant glacial to interglacial variation whereas those from the Bering Sea core do not. Variations of δ¹³C values of land plant n-alkanes are related to the environmental or vegetational changes in the source land areas. Environmental changes, especially, aridity, rainfall, and pCO₂ during glacial/interglacial transitional periods can affect vegetation, and therefore C₃ / C₄ plant ratios, resulting in δ¹³C changes in the preserved land plant biomarkers. Maximum values of δ¹³C as well as maximum average chain length values of long chain n-alkanes in the ES core occur mostly at the interglacial to glacial transition zones reflecting a time lag related to incorporation of living organic matter into soil and transportation into ocean basins via wind and/or ability of C₄ plants to adapt for a longer period before being replaced by C₃ plants when subjected to gradual climatic changes. Irregular variations with no clear glacial to interglacial trends in the BOW-8a core may result from complex mixture of aerosols from westerly winds and riverine organic matter from the Bering Sea catchments. In addition, terrestrial organic matter entering the Bering Sea could originate from multiple pathways including eolian, riverine, and ice rafted debris, and possibly be disturbed by turbidity and other local currents which can induce re-suspension and re-sedimentation causing an obliterated time relation in the Bering Sea biomarker records.     

Distributions of n-alkanes and their hydrogen isotopic composition in plants from Lake Qinghai (China) and the surrounding area

Various aquatic plants from Lake Qinghai, the largest inland saline lake in China, and terrestrial plants from the surrounding area were investigated for the distribution of n-alkanes and their δD values. The n-alkanes in the samples range from C₁₅ to C₃₃ with C preference index (CPI) values of 4.0–29.7. The n-C₂₃ or n-C₂₅ alkane is the dominant compound in the aquatic submerged plants. The aquatic emergent and terrestrial plants have an abundance maximum at n-C₂₇, n-C₂₉ or n-C₃₁. The average chain length (ACL) values, ranging from 26.0 to 29.6, are closely related to the plant species. The n-alkanes from the aquatic plants have mean δD values of −169‰ to −121‰ and those from the terrestrial plants values of −173‰ to −109‰. The H isotopic composition (δD) and fractionation differ significantly among the plants studied. Comparison shows that additional evaporative enrichment of the lake water associated with saline lakes and humidity influence the δD values of the n-alkanes in aquatic and terrestrial plants, respectively. The mean δD values of n-alkanes in the plants decrease with increasing ACL value. The n-alkanes from the different types of plants are more depleted in D relative to environmental water and those from aquatic plants (with a mean value of −143‰) have a greater isotopic fractionation than terrestrial plants (mean value −113‰).     

Different types of methane monooxygenases produce similar carbon and hydrogen isotope fractionation patterns during methane oxidation

We determined the stable carbon and hydrogen isotope fractionation factors for methane oxidation under oxic conditions using strains with known degradation pathways. The aerobic oxidation of methane can be initiated by two different forms of enzymes known as methane monooxygenases (MMO). The expression of these enzymes is type-specific and dependent upon the adjusted copper concentration in the medium (or environment). In this study, the expression of either the soluble MMO or the particulate MMO was supported by adjusting the copper concentrations in the growth medium. Taxonomically different aerobic methanotrophic strains, mainly belonging to the alpha- and gamma- classes of Proteobacteria, produced methane isotope enrichment factors (ε_bulk) ranging from −14.8 to −27.9‰ for carbon, and from −110.0 to −231.5‰ for hydrogen. The ratio of hydrogen versus carbon discrimination (Λ = (α_H⁻¹ − 1)/(α_C⁻¹ − 1) ≈ Δ(δ²H)/Δ(δ¹³C)) were similar for all tested cultures, and are also identical to values calculated from previously published enrichment factors for aerobic and anaerobic methane degradation. In contrast, Λ-values for the abiotic oxidation of methane with OH radicals (this process is considered as the main removal process for methane from the atmosphere) were significantly higher than the values derived from biotic oxidation. Due to the low variability of microbial methane isotope fractionation patterns, we propose that combined carbon and hydrogen isotope fractionation analyses can be used to monitor and assess the occurrence of microbial methane oxidation in marine or terrestrial environments. However, it is not possible to distinguish distinct aerobic or anaerobic methane-oxidation pathways by this approach.     

A revised isotope fractionation model for dissimilatory sulfate reduction in sulfate reducing bacteria

Sulfur isotope fractionation during dissimilatory sulfate reduction has been conceptually described by the widely accepted Rees model as related to the stepwise reduction of sulfate to sulfide within the cells of bacteria. The magnitude of isotope fractionation is determined by the interplay between different reduction steps in a chain of reactions. Here we present a revision of Rees’ model for bacterial sulfate reduction that includes revised fractionation factors for the sulfite-sulfide step and incorporates new forward and reverse steps in the reduction of sulfite to sulfide, as well as exchange of sulfide between the cell and ambient water. With this model we show that in contrast to the Rees model, isotope fractionations well in excess of −46‰ are possible. Therefore, some of the large sulfur isotope fractionations observed in nature can be explained without the need of alternate pathways involving the oxidative sulfur cycle. We use this model to predict that large fractionations should occur under hypersulfidic conditions and where electron acceptor concentrations are limiting.     

A global model of mass independent mercury stable isotope fractionation

Mass independent fractionation (MIF) of stable isotopes associated with terrestrial geochemical processes was first observed in the 1980s for oxygen and in the 1990s for sulfur isotopes. Recently mercury (Hg) was added to this shortlist when positive odd Hg isotope anomalies were observed in biological tissues. Experimental work identified photoreduction of aquatic inorganic divalent Hg^II and photodegradation of monomethylmercury species as plausible MIF inducing reactions. Observations of continental receptors of atmospheric Hg deposition such as peat, lichens, soils and, indirectly, coal have shown predominantly negative MIF. This has led to the suggestion that atmospheric Hg has negative MIF signatures and that these are the compliment of positive Hg MIF in the aquatic environment. Recent observations on atmospheric vapor phase Hg⁰ and Hg^II in wet precipitation reveal zero and positive Hg MIF respectively and are in contradiction with a simple aquatic Hg^II photoreduction scenario as the origin for global Hg MIF observations.This study presents a synthesis of all terrestrial Hg MIF observations, and these are integrated in a one-dimensional coupled continent-ocean-atmosphere model of the global Hg cycle. The model illustrates how Hg MIF signatures propagate through the various Earth surface reservoirs. The scenario in which marine photoreduction is the main MIF inducing process results in negative atmospheric Δ¹⁹⁹Hg and positive ocean Δ¹⁹⁹Hg of −0.5‰ and +0.25‰, yet does not explain atmospheric Hg⁰ and Hg^II wet precipitation observations. Alternative model scenarios that presume in-cloud aerosol Hg^II photoreduction and continental Hg^II photoreduction at soil, snow and vegetation surfaces to display MIF are necessary to explain the ensemble of natural observations. The model based approach is a first step in understanding Hg MIF at a global scale and the eventual incorporation of Hg stable isotope information in detailed global mercury chemistry and transport models.     

地质样品正构烷烃组分分离纯化的部分问题探究

用实验室自配的标准正构烷烃样品分别经硅胶或氧化铝柱层析后发现,等量溶剂洗脱的情况下硅胶对长链正构烷烃无吸附而氧化铝具有一定吸附性。进一步通过用植物样品测试4种层析柱填充方法,发现不论是只用氧化铝填充还是上部硅胶下部氧化铝和上部氧化铝下部硅胶,都会对长链正构烷烃产生一定的吸附,且这种吸附效果随着碳链的增长而增强。在实验条件下,当碳链加长到C36时,用硅胶加氧化铝填充层析柱的吸附量已达到20%左右,而只用氧化铝填充层析柱的吸附量则高达50%。故建议对研究高碳数正构烷烃的地质样品组分提取时用单一的硅胶柱层析方法。同时,实验显示对于一些杂质多的正构烷烃样品经过尿素络合后比络合前"干净"得多。18个黄土-古土壤和植物样品平均回收率为50%左右,经过尿素络合后的样品正构烷烃各组分相对含量基本不会发生改变,也不会产生明显的同位素分馏效应。因此在进行非正构组分干扰较大的正构烷烃各组分相对含量或同位素分析时,可以选择尿素络合的方法来将其纯化。     

相控和气洗分馏作用对油气组分及碳同位素组成的影响

油藏条件下的相控和气洗分馏作用使得油藏中油气的物理性质和化学组成发生了明显变化。通过不同温压条件下的ＰＶＴ实验及其产物测量为该观点提供了十分有意义的科学证据,其主要结论结下：（１）总的来看,气洗分馏作用较由温度和压力引起的相控分馏作用明显,气洗作用是油藏中原油性质（如：含蜡量、密度和粘度等）发生重大变化的最主要因素;（２）ＰＶＴ分馏不仅导致凝析油中饱／芳比值的异常高值,而且饱／芳比值在凝析油和正常     

Selective enrichment of steroid and triterpenoid hydrocarbons from crude oil using gel permeation chromatography for stable carbon isotope analysis

Gel permeation chromatography (GPC) using a high performance liquid chromatography (HPLC) system was studied for the separation and enrichment of steroid and hopanoid hydrocarbons from crude oil for stable carbon isotope analysis. A crude oil sample was pretreated using silica gel chromatography and 5A molecular sieve to remove polycyclic aromatic hydrocarbons and n-alkanes. The GPC behavior of both the pretreated saturated hydrocarbon fraction of the oil and standard steroid [5α(H), 14α(H), 17α(H) C₂₇–C₂₉ steranes], hopanoid [17α(H) C₂₇ trisnorhopane, 17α(H), 21β(H) C₂₉–C₃₂ hopanes] and triterpenoid [18α(H)-oleanane, gammacerane] mixtures were examined. The results indicate that 17α(H), 21β(H) hopanes as well as steranes could be enriched efficiently using GPC and that they could be obtained without removing n-alkanes from the oil saturated hydrocarbon fraction. The GPC behavior of steroid and triterpenoid hydrocarbons was controlled by molecular size and shape.     

尿素络合法分离-气相色谱/同位素质谱法分析土壤和植物中低含量(ppm级)正构烷烃的碳同位素

尿素络合法和5A分子筛法是常用的分离富集环境样品中正构烷烃的方法,但由于复杂的处理流程对于低含量正构烷烃的回收率普遍较低。本文通过优化尿素络合法分离富集正构烷烃的实验条件,建立了尿素络合法分离-气相色谱/同位素质谱分析土壤和植物中低含量正构烷烃单体碳同位素的方法。即采用正己烷-丙酮溶解样品,在4℃冰箱中与2 m L尿素-甲醇饱和溶液反应48 h;用去离子水溶解尿素络合物,加入正己烷后分离出有机相和水相,分别用正己烷、丙酮-正己烷萃取有机相和水相中的正构烷烃。中长链正构烷烃的回收率达79%~104%,高于5A分子筛法和已有尿素络合法的富集效果;单体碳同位素的分析精度为0.09‰~0.63‰(1σ)。利用改进的方法分析实际样品,大幅降低了未分峰和共流出物的干扰,提高了实际样品中ppm级中长链正构烷烃的回收率。     

Isotopic fractionation: A kinetic model for crystals growing from magmatic melts

Recent experiments in the fields of crystal growth, crystal-melt element partitioning, and diffusion in magmatic melts make it possible to estimate disequilibrium partitioning for many species between liquid and crystals and to compute quantitative models which take into account partition coefficients, diffusivities and rates of crystal growth. A slight difference in the diffusivities of two isotopes should lead to selective depletion (or enrichment) in the crystals as they grow, resulting in varying isotopic ratios as crystallization proceeds. Reasonable hypotheses permit under general kinetic conditions an estimation of magnitude of possible effects. The resulting isotopic fractionation for major elements (like oxygen) may exceed the per ml level; for trace elements the effects may be more significant (5–10 per ml), especially in the case of low partition coefficients, but analytical difficulties are formidable at present.     

Timescales determining the degree of kinetic isotope fractionation by evaporation and condensation

A first order characteristic of the relative abundance of the elements in solar system materials ranging in size from inclusions in primitive meteorites to planetary sized objects such as the Earth and the Moon is that they are very much like that of the Sun for the more refractory elements but systematically depleted to varying degrees in the more volatile elements. This is taken as evidence that evaporation and and/or condensation were important processes in determining the distinctive chemical properties of solar system materials. In some instances there is also isotopic evidence suggesting evaporation in that certain materials are found enriched in the heavy isotopes of their more volatile elements. Here model calculations are used to explore how the relative rates of various key processes determine the relationship between elemental and isotopic fractionation during partial evaporation and partial condensation. The natural measure of time for the systems considered here is the evaporation or condensation timescale defined as the time it would take under the prevailing conditions for evaporation or condensation to completely transfer the element of interest between the two phases of the system. The other timescales considered involve the rate of change of temperature, the rate at which gas is removed from further interaction with the condensed phase, and the rates of diffusion in the condensed and gas phases. The results show that a key determinant of whether or not elemental fractionations have associated isotopic effects is the ratio of the partial pressure of a volatile element (P_i) to its saturation vapor pressure (P_i,sat) over the condensed phase. Systems in which the rate of temperature change or of gas removal are slow compared to the evaporation or condensation timescale will be in the limit P_i ∼ P_i,sat and thus will have little or no isotopic fractionation because at the high temperatures considered here there is negligible equilibrium fractionation of isotopes. If on the other hand the temperature changes are relatively fast, then P_i ≠ P_i,sat and there will be both elemental and isotopic fractionation during partial evaporation or partial condensation. Rapid removal of evolved gas results in P_i ? P_i,sat which will produce isotopically heavy evaporation residues. Diffusion-limited regimes, where transports within a phase are not sufficiently fast to maintain chemical and or isotopic homogeneity, will typically produce less isotopic fractionation than had the phases remained well mixed. The model results are used to suggest a likely explanation for the heavy silicon and magnesium isotopic composition of Type B CAIs (as due to rapid partial melting and subsequent cooling at rates of a few °C per hour), for the uniformity of the potassium isotopic composition of chondrules despite large differences in potassium depletions (as due to volatilization of potassium by reheating in regions of large but variable chondrules per unit volume), and that the remarkable uniformity of the potassium isotopic composition of solar system materials is not a measure of the relative importance of evaporation and condensation but rather due to the solar nebula having evolved sufficiently slowly that materials did not significantly depart from chemical equilibrium.     

Experimental determination of carbon isotope fractionation between CaCO3 and graphite

Carbon isotopic exchange between graphite and three polymorphs of CaCO₃ was investigated at temperatures of 600-1400 °C and at pressures from 1.4 to 2.3 GPa. Fractionation factors at all temperatures were determined by the partial exchange treatment of Northrop and Clayton (1966).Graphite starting material for the majority of the experiments was milled in water for 20-25 h, producing aggregates of nanosheets. The sheets range in width from 50 to 1000 nm and in thickness from 20 to 30 nm, and they retain hexagonal symmetry.Isotopic exchange appears to be the sum of surface exchange and interior exchange. At 1100-1400 °C, interior exchange exceeded surface exchange, probably by a combination of grain growth, as determined by increase in crystallite size, recrystallization, as observed in FESEM images, and diffusion. In some runs at 1200 and 1400 °C with an isotopic contrast between the initial graphite and calcite of close to 50‰, equilibrium fractionation was actually overstepped due to a kinetic effect. A weighted regression of fractionation factors from the high-temperature runs yields the line of equilibrium interior exchange: