The headspace equilibration technique has traditionally been used to estimate different gas concentrations in liquid samples. The main principle of this technique involves a small headspace volume (gas phase) and a large liquid sample volume (water phase) reaching a state of equilibrium within a closed vessel. Hitherto, the definition of what is small and what is big has varied from study to study. Syringes or bottles are often used as vessels 1-5. The volatile components diffuse into the headspace until a state of equilibrium is reached. The sample for subsequent gas chromatography or infrared gas analysis is taken from the equilibrated gas phase and from the air that was originally injected as headspace 1,5. Recently developed instruments like CONTROS HydroCTM probes already combine the principle of diffusive equilibration with modern techniques like non-dispersive infrared spectrometry directly 6. However, it is often not possible to analyse the samples directly in the field. In this case they are stored until analysis, for example in closed evacuated serum vials 7,8. The protocol presented here describes the use of vials since they are gas-tight and easy to store (in contrast to syringes). But since it is not possible to evacuate these vials completely the protocol includes a background correction to the sampling procedure and a precision test is described to ensure that data are of sufficiently high quality 8. Alternative approaches have been described in Aberg and Wallin 5, Lambert and Fréchette 4, or Demarty, et al. 9, for instance.
Henry’s law and the water temperature at the time of sampling are used to calculate the Henry constant kh 10,11. Since there are many different definitions of the Henry constant, the units and also definitions of all other abbreviations used (including units) are provided in Table 1.
kh (here k0) is then often used to calculate the ambient partial pressure and concentration of CO2 based on the gas concentration of the equilibrated gas and the volumes of gas and water that were equilibrated, in accordance with Fick’s first law 1-3,12,13. Fick’s first law of diffusion states that a diffusive flux is proportional to the concentration (or partial pressure) gradient between different phases. This calculation ignores the effect of ambient pressure on the solubility of gas as described in the simple gas equation. Details are described in Dickson 13 and Aberg and Wallin 5, for example. The calculation presented here is based on the simple gas equation, also known as combined gas law, since it combines the laws of Boyle and Mariotte and Gay-Lussac’s law. It is based on the assumption of ideal conditions, with p*V/T=nR (p=pressure, V=volume, T=temperature, R=gas constant, n=quantity of gas)21. In contrast to other approaches it takes the effects of both temperature and pressure on the solubility of the gas into account. This is especially important if sampling takes place at different depths or altitudes or under varying weather conditions.
Application of the method. The presented application have benefits for different research fields, including sport medicine. The used calculation is for instant relevant for divers that often use the laws of Boyle and Mariotte as basis for the calculation of the gas volume needed for each dive. These laws ignore the effect of temperature on pressure and volume. A correction of this approach could help to reduce known diver accidents like the decompression sickness.
However, the original purpose of this article was to address the application of this technique for the aquatic research community. In recent decades the number of greenhouse gas studies - done in freshwaters and oceans - has increased rapidly. For researchers seeking to quantify the anthropogenic proportion of CO2 emissions and assess their contribution to ocean acidification, obtaining accurate measurements and robust analytical techniques for measuring dissolved CO2 has become a high priority13. CO2 measurements in particular are increasingly based on automatically collected time series coming from newly developed instruments 1,4,9,14,15. These can be placed directly in the water or next to it. Details concerning the potential and limitations of the instrumentation are described in UNESCO/IHA 12, Lambert and Fréchette 4 and Dickson 13, for example. Although some of the commonly used instruments are fast and precise, with high data acquisition rates and storage capacities, most of them are expensive and consume a large amount of power 12. Furthermore, calibration problems hamper the comparability between time series from different probes. For this reason there is a need for a standardized, easy to handle and cheap tool that can bridge the gaps between studies based on different instruments.
One alternative to automatically gathered CO2 data is based on measurements of pH, temperature and concentrations of total alkalinity or dissolved inorganic carbon 12,13,16-18. This approach has its limitations, especially if it is used in conditions of low carbonate alkalinity and high DOC concentrations, as well as in acid conditions if the pH value exceeds 8 and carbon precipitation takes place 1,17,19. Furthermore, precise calculation of CO2 values is highly dependent on accurate pH and temperature measurements, and these are subject to considerable degrees of uncertainty (up to 50%), especially outside the laboratory 1,19.
In view of this, the classical headspace equilibration technique would appear to be a reliable means of comparing different techniques 4,12,17. For instance, Lambert and Fréchette 4 demonstrated the stability of air samples (CO2 standard of 10000 ppmv) stored in syringes for at least 48 hours, with less than 5% loss. Moreover, samples near ambient air concentration (350-530 ppmv) remained stable for more than 21 days. In contrast, water samples returned inconclusive results. This was probably related to microbial activity or chemical reactions taking place in the different samples. Thus more robust data can be expected if gas samples rather than water samples are stored. If water samples have to be used it is worth exploring options for sample preservation (for instance using chloride). The choice of the means of transport (by air or by car) appears to have no significant effect on data quality 4, so samples can be transported over long distances.
In some cases the headspace equilibration technique has already been used to compare instruments or to test the accuracy of probes. For instance, Abril et al. 14 showed that CH4 and CO2 values obtained using the classical headspace equilibration technique with subsequent gas chromatographic analysis correspond with values from an infrared photo-acoustic gas analyser coupled with an in situ equilibrator to approximately 15%. Recently, Aberg and Wallin 5 compared the more frequently used direct headspace method with the acidified headspace method (in which dissolved inorganic carbon (DIC) is measured from an acidified sample and the partial pressure is calculated from DIC, pH and temperature). They found no significant differences.
This paper presents a detailed protocol of the direct headspace equilibration technique, including significant improvements such as background correction and the application of a statistical quality control routine. The quality control is especially recommended for CO2 because it is a non-ideal gas and also the CO2 concentration can vary greatly depending on experimental or environmental conditions. However, CO2 is only one of several gases that can be analysed following this protocol. Other candidates are CH4, and N2O, for instance. Especially where low concentrations of CH4 are to be analysed, the currently available probes are only of limited use. In this case, the headspace equilibration technique is clearly more advantageous on account of the availability of accurate analytical instruments 9,12. Moreover, the approach presented here can easily be adapted for research for which the diffusive exchange between different phases has to be calculated, for example in the investigation of rising gas bubbles. In this case the hydraulic pressures at different depths have to be measured instead of the ambient pressure as described here.
Limitations. The headspace equilibration technique is not suitable for cases in which large amounts of high-frequency data are to be gathered over a long period of time (for instance where long-term, cyclical daily measurements are required). In such situations automated systems are more effective. Furthermore, although this protocol simplifies the sampling procedure and the data processing, the use of analytical instruments demands expert knowledge, so technicians need to receive training in the use of specific instruments.