The previous instalment of this column on headspace sampling for gas chromatography (GC) (1) discussed the basic physicochemical
processes that determine the contents of the gaseous headspace above a sample in a sealed vial. Although it is possible —
and sometimes even desirable — to sample and inject headspace directly into a GC system with a hand-held syringe, most of
the time analysts will call upon an automated headspace sampler to acquire and transfer appropriately sized fractions of the
headspace into a GC system's inlet. Aside from providing the benefits of better accuracy and repeatability similar to what's
gained with a liquid autosampler compared to manual injections, automated headspace samplers control other crucial variables
beyond timing. Temperature, pressure and flow rates in addition to the timing parameters, strongly influence the composition
of the headspace gas as it appears at the GC column or inlet. Headspace autosamplers and the associated sampling methods are
more complex than their liquid autosampler counterparts; they comprise a richer set of choices that chromatographers should
understand and manage to obtain the best possible results.
The Inside Story
Most analysts are familiar with the inner workings of a liquid autosampler. Basically, the device is a robotic arm that actuates
a microsyringe with better precision and accuracy than a mere human operator could achieve. Peering underneath the covers,
it's not difficult to see and understand the motions and gyrations that a liquid autosampler undergoes as it processes sample
A headspace autosampler is different. The mechanical tasks of vial positioning and needle movement in and out of the vial
septum are familiar, but there is much more going on than meets the eye. The inner workings of a headspace autosampler are
hidden underneath insulation and internal housings. It's not easy to observe the device's pressures and temperatures without
attaching thermocouples and gauges at inconvenient locations. Of course, the designers and engineers who created the autosampler
would have done so as part of the development process. For analysts, these physical properties are abstracted to the list
of settings and timings that make up the sampling method. What, then, are the relationships between the sampler settings and
the physical processes that result in sample injection?
Modern autosamplers that perform equilibrium headspace sampling enact three fundamental steps for sample injection: equilibration,
pressurization and sample transfer. Each sampler model supplements these three with additional nuances and enhancements. Certainly,
there is more to discuss about these details, but that extends beyond the scope of this article. The three principal steps
can be delineated as follows.
Equilibration is the most important headspace sampling step. Careful attention toward choosing appropriate equilibration temperatures
and times during method development helps ensure a robust and longlived procedure. Equilibration is the period between when a vial is filled with sample and sealed and when the sampler needle enters the vial to commence
sample acquisition. During equilibration, sample components partition between sample and headspace, and equilibrium is reached
after their concentrations attain constant values. Each component migrates at its own temperaturedependent rate, so the slowest-moving
component of interest determines the minimum equilibration time period.
Very volatile components start to migrate out of the sample phase immediately during sample preparation, and significant amounts
can be lost to the atmosphere with concomitant reduction in sample recovery along with poor repeatability. Sample handling
and preparation at reduced temperatures can help reduce such pre-vial losses. Careful attention and consistent sample handling
procedures aid in improving repeatability.
In addition to different migration rates across the sample-headspace interface, each component has its own temperature-dependent
solubility coefficient, K. For most commonly encountered solute-solvent systems the solubility coefficient decreases as the temperature increases,
thereby raising analyte concentrations in the headspace at higher temperatures. Thus, careful control of vial temperatures
during equilibration is important for obtaining both consistent quantitative results and equilibration times.
The contents of headspace vials may attain an initial equilibrium state at room temperature in an external sample tray while
waiting for the actual headspace process to begin, but they will cease to be in equilibrium once inserted into a heated carousel
before sampling. As soon as the temperature changes, the contents of the headspace will shift in response to the changes in
solubilities. In certain cases, the prethermostating wait time may influence the overall equilibration time; method developers
may need to take this into account and allow sufficient equilibration time for the shortest anticipated wait time between
vial preparation and the start of equilibration.
Vial heating is one of the most important aspects of a headspace sampler. Early samplers used water or even oil baths for
consistent results, but modern headspace devices employ either a solid metal heating carousel or, in some cases, a carefully
controlled air bath oven arrangement. A vialtovial temperature control precision of about ±1–2 °C should yield acceptable
results consistently. Absolute temperature accuracy influences inter and intralaboratory reproducibility of results. Headspace
samplers should be calibrated consistently to a standard reference thermometer about every six months if sample calibration
procedures cannot establish sufficient results comparability.
Headspace sampling temperature dependencies lead to the general use of elevated equilibration temperatures. Higher equilibration
temperatures are desirable for both greater headspace sensitivity and shorter equilibration times. Choosing an equilibration
temperature of at least 15 °C above room temperature will ensure good thermal control, but there are limits to how high equilibration
temperatures can be pushed.
Elevated temperatures can accentuate the breakdown of thermolabile materials, and a careful study of such effects should be
performed when there is any question of analyte stability. In addition, high temperatures may degrade the performance of headspace
vial septa that are not designed for hightemperature applications and cause both leaks and sample contamination with septum
Many headspace samplers stir or agitate the vial to speed up equilibration. As components leave the liquid phase at the gas–liquid
interface, the reduced solute concentrations in the interfacial liquid layer are replenished relatively slowly by molecular
diffusion from the bulk of the liquid. Convective stirring occurs naturally to some extent, but mechanical mixing is more
effective toward replenishing solute that has entered the gas headspace phase.
Another side effect of elevated equilibration temperatures is an increase in the internal or "natural" vial pressure before
the actual sampling step. Most of this pressure is created by the vapour pressure of the solvent. Water as a headspace solvent
has a vapour pressure of close to 1 atm (101 kPa) at 100 °C, and other more volatile solvents can create similar or higher
pressures if present in significant quantities. Internal vial pressures increase exponentially as temperatures increase, and
it is possible to generate pressures high enough to breach the vial septum seal or even burst the vial itself.
The natural vial pressure can be measured with a needle-mounted pressure gauge while a test vial with sample is heated in
a small external chamber. Most chromatography suppliers and manufacturers can provide such equipment. When high pressures
are anticipated, use a selfventing septum safety cap to prevent equipment damage. It is better to have to repeat analyses
than try and remove broken glass from a delicate piece of equipment.
Besides the danger of broken glass or lost sample, natural vial pressures that are too high can affect the pressurization
stage of headspace sampling by causing sample vapours to depart from the vial too early in the sampling process. Such an occurrence
causes doubled peaks, poor repeatability or both.