Gas chromatography

A gaseous chromatography team.

Gas chromatography is a chromatographic technique in which the sample is volatilized and injected into the top of a burner of a chromatographic column. Elution occurs by the flow of an inert gas mobile phase. Unlike the other types of chromatography, the mobile phase does not interact with the analyte molecules; its only function is to transport the analyte through the column.

There are two types of gas chromatography (GC): gas-solid chromatography (GSC) and gas-liquid chromatography (GLC), the latter being the most widely used, and can be simply called gas chromatography. gas (GC). In the GSC the stationary phase is solid and the retention of the analytes in it occurs through the adsorption process. It is precisely this adsorption process, which is not linear, that has caused this type of chromatography to have limited application, since the retention of the analyte on the surface is semi-permanent and elution peaks with tails are obtained. Its only application is the separation of gaseous species of low molecular weight. GLC uses as stationary phase liquid molecules immobilized on the surface of an inert solid.

GC is performed in a gas chromatograph. This consists of various components such as the carrier gas, the sample injection system, the column (usually inside an oven), and the detector.

History

Chromatography dates back to 1903 in the work of the Russian scientist, Mikhail Tsvet. German doctoral student Fritz Prior developed solid-state gas chromatography in 1947. Archer John Porter Martin, who was awarded the Nobel Prize for his work developing liquid-liquid (1941) and paper (1944) chromatography), laid the foundations for the development of gas chromatography and later of gas-liquid chromatography (1950). Erika Cremer laid the foundation and oversaw much of Prior's work.

Carrier Gas

Diagram of a gas chromatograph

The carrier gas basically serves two purposes: to transport the components of the sample, and to create a suitable matrix for the detector. A carrier gas must meet certain conditions:

• It should be inert to avoid interactions (both with the sample and with the stationary phase)
• Must be able to minimize gaseous diffusion
• Easily available and pure
• E Economic and Social
• Suitable to the detector to use...

The carrier gas must be an inert gas, to prevent its reaction with the analyte or the column. Gases such as helium, argon, nitrogen, hydrogen or carbon dioxide are generally used, and the choice of this gas sometimes depends on the type of detector used. The storage of the gas can be in normal bullets or using a generator, especially in the case of nitrogen and hydrogen. Then we have a system of manometers and flow regulators to guarantee a stable flow and a gas dehydration system, such as a molecular sieve.

Generally, pressure regulation is done at two levels: a first manometer is located at the exit of the bullet or gas generator and the other at the entrance of the chromatograph, where the flow is regulated. Inlet pressures vary between 10 and 25 psi, resulting in flow rates of 25 to 150 mL/min in packed columns and 1 to 25 mL/min in capillary columns. To check the flow rate, a rotameter or a simple soap bubble meter can be used, which gives a very accurate measure of the volumetric flow rate entering the column.

The purity of the gases is extremely important, levels 4.5 or higher are required, that is, 99.995% purity. However, due to the care that must be taken with the active phase of the column, it is completely necessary to install traps at the entrance of the carrier gas, these traps obviously have a limited capacity, but are very important when using the chromatograph.. These traps prevent the entry of hydrocarbons, water and CO, among others.

Sample injection system

Sample injection is critical, as an adequate amount must be injected, and it must be introduced in such a way (like a "vapor plug") that it is rapid to avoid widening of the exit bands; this effect occurs with high amounts of analyte. The most commonly used method employs a microsyringe (with capacities of several microliters) to introduce the analyte into a flash chamber. This chamber is at 50 °C above the boiling point of the less volatile component, and is sealed by a septa or septum silicone rubber gasket.

Sample injector for a GC

If a reproducibility of the injected sample size is necessary, a six-way valve or injection valve can be used, where the amount to be injected is constant and determined by the size of the loop of said valve.

An automatic sampler to use Head Space or Head Space for GC (accessorium).

If the column used is packed, the volume to be injected will be around 20 μL, and in the case of capillary columns this amount is less, 1 μL, and depending on the type of capillary column (since there are different internal diameter) is that if the entire injected sample volume is used. To obtain less volume, a flow divider is used (injection is known as 'split' mode) at the column inlet which discards part of the introduced analyte. If the entire sample volume is used, the injection is of the "splitless" type. The splitless mode was used more to determine small or trace quantities (environmental determinations).

If 1 microliter of solvent is injected -water for example-, when it goes into the vapor phase its volume will multiply by a thousand. That is, a microliter of water would become 1 mL of water in gas; Because the volume of the injection port is limited, pulsed split or other configurations are used to ensure adequate sample entry.

In the case of solid samples, they are simply introduced as a solution, since in the flash chamber the solvent is lost in the purge stream and does not interfere with the elution.

According to the Van Demter curves (HEPT vs. Linear Velocity), the best gas to use in the chromatographic column as a carrier of the analytes is hydrogen, however, given its danger, it is more used as ignition gas in the FID detector, along with air. Next come, respectively, helium and nitrogen.

Hydrogen gas is the best carrier and the flows handled by chromatographs are not dangerous, in addition to these there are generally flame restrictors that prevent the spread of a possible fire. The use of hydrogen can be recommended due to, firstly, its low price compared to other gases and the resolution of the peaks shown in the chromatograms.

The ratio for ignition between hydrogen and air is 4.1% for the lower limit and 74.8% for the upper limit at 101.3Kpa and 298K (Safety Standard for Hydrogen and Hydrogen Systems, NASA), and you have to be in the presence of a spark or high heating zone (from 520 °C).

Columns and temperature control systems

In GC two types of columns are used: packed or filling and open tubular or capillaries. The latter are more common today (2005) due to their greater speed and efficiency. The length of these columns is variable, from 2 to 60 meters, and they are made of stainless steel, glass, fused silica, or Teflon. Due to their length and the need to introduce them into a kiln, the columns are usually rolled into a helical shape with lengths of 10 to 30 cm, depending on the size of the kiln.

Temperature is an important variable, since the degree of separation of the different analytes will depend on it. To do this, it must be set to a precision of tenths of a degree. Said temperature depends on the boiling point of the analyte or analytes, as well as the maximum operating temperature of the column (stationary phase), and is generally set to a value equal to or slightly higher than it. For these values, the elution time will range between 2 and 30-40 minutes. If we have several components with different boiling points, the so-called temperature ramp is adjusted, with which it increases either continuously or in stages. On many occasions, adjusting the ramp correctly may mean separating the different analytes well or not. It is advisable to use low temperatures for elution, since -although the elution is faster at higher temperatures- there is a risk of decomposing the analyte. The ramp can be programmed to both increase and decrease the oven temperature so that there is no overlap of the peaks.

Detectors

The detector is the part of the chromatograph that is responsible for determining when the analyte has left the end of the column. The characteristics of an ideal detector are:

• Sensitivity: You need to be able to determine precisely when analytic comes out and when only the carrier gas comes out. They have sensitivity between 10^-8 and 10^-15 g/s of analyte.
• Linear answer to analyte with a range of several orders of magnitude.
• Short response timeindependent of the output flow.
• Wide Working Temperature Interval, for example from ambient temperature to about 350-400 °C, typical working temperatures.
• Stability and reproducibility, that is to say, equal amounts of analyte should give equal signal outputs.
• High reliability and easy handling, or tested by inexperienced operators.
• Similar answer for all analyticsor
• Selective and highly predictable response for a small number of analytics.

Some types of detectors:

• Flame Ionization Detector.
• Thermal conductivity detector (TCD, Thermical Conductivity Detector).
• Thermoionic detector (TID, ThermoIonic Detector).
• Electron capture detector (ECD, Electron-Capture Detector).
• Atomic emission detector (AED, Atomic Emission Detector).

View of a FID type GC detector (disassembled).

Other minority detectors are the photometric flame detector (PFD), used in compounds such as pesticides and hydrocarbons that contain phosphorus or sulfur. In this detector, the eluted gas is passed through a hydrogen/oxygen flame where part of the phosphorus is converted into an HPO species, which emits at λ = 510 and 526 nm, and simultaneously the sulfur is converted into S₂, with emission at λ = 394 nm. Said emitted radiation is detected with a suitable photometer. Other elements have been detected, such as some halogens, nitrogen, tin, germanium and others.

In the photoionization detector (PID), the eluted gas at the end of the column is subjected to ultraviolet radiation with energies between 8.3 and 11.7 eV, corresponding to a λ = 106-149nm. By applying a potential to the ionization cell, an ion current is generated, which is amplified and recorded.

Columns and types of stationary phases

• Filling columns

The packed or packed columns consist of tubes of glass, metal (ideally inert such as stainless steel, nickel, copper or aluminum) or Teflon, 2 to 3 meters long and with an internal diameter of a few meters. millimeters, typically from 2 to 4. The interior is filled with a solid material, finely divided to have a maximum interaction surface and covered with a layer of thicknesses between 50 nm and 1 μm. So that they can be introduced into the oven, they are conveniently rolled up.

The ideal packing material consists of small, spherical and uniform particles, with good mechanical resistance, to have a maximum surface area where the stationary phase and analyte can interact. The minimum specific surface area must be 1 m²/g. Like all GC column components, it must be inert at high temperatures (~400°C) and be uniformly wetted by the stationary liquid phase during the manufacturing process. The currently (2005) preferred material is natural diatomaceous earth, due to its natural pore size. These species, already extinct, used a molecular diffusion system to take nutrients from the environment and expel their waste. Therefore, they are especially useful materials, because the surface absorption system of the analyte and the stationary phase are similar.

The size is critical when it comes to the interaction process of the analyte, and at smaller sizes the efficiency of the column is better. But there is the problem of the pressure necessary to circulate a stable flow of carrier gas through the column, since said pressure is inversely proportional to the square of the diameter of said particles. Thus, the minimum size to use maximum pressures of 50 psi is 250 to 149 μm.

• capillary columns

Capillary columns are of two basic types: coated-wall (WCOT) and coated-support (SCOT). WCOTs are simply capillary tubes where the inner wall has been coated with a very thin layer of stationary phase. SCOT columns have a thin layer of absorbent material on the inside, such as that used in packed columns (diatomaceous earth), where the stationary phase has adhered. The advantages of the WCOT compared to the SCOT is the greater load capacity, since greater amounts of stationary phase are used in their manufacture, as the exchange surface is larger. In order of effectiveness, first are the WCOTs, then the SCOTs, and lastly the packed columns.

WCOT columns are manufactured from fused silica, known as Fused Silica Open Tubular Columns or FSOT. These columns are manufactured from especially pure silica, with hardly any metal oxide content. Due to the inherent fragility of this material, in the same process of obtaining the tube it is covered with a layer of polyimide, in this way the column can be rolled up with a diameter of a few centimeters. These columns, with properties such as low reactivity, physical resistance and flexibility, have replaced the classic WCOT.

FSOT columns have variable internal diameters, between 250 and 320 μm (for normal columns) and 150-200 μm for high resolution columns. The latter require less amount of analyte and a more sensitive detector, as less gas is eluted. There are also macrocapillary columns with diameters of up to 530 μm, which admit amounts of analyte comparable to those of packing but with better performance.

In these columns there is a problem due to the adsorption of the analyte on the surface of the fused silica, adsorption due to the presence of silanol groups (Si-OH), which strongly interact with polar organic molecules. This drawback is usually overcome by inactivating the surface by silylation with dimethylchlorosilane (DMCS). The adsorption due to metal oxides is largely mitigated by the high purity of the silica used.

• Stationary phase

The necessary properties for an immobilized liquid stationary phase are:

1. Distribution characteristics (capacity factor κ' and selectivity factor α) suitable for analyte.
2. Low volatility, the boiling point of the stationary phase should be at least 100 °C greater than the maximum temperature reached in the oven.
3. Low reactivity.
4. Thermal stability, to avoid decomposition during eluction.

There are at most a dozen solvents with these characteristics. To choose one, the polarity of the analyte must be taken into account, since the higher the polarity of the analyte, the higher the polarity of the stationary phase. Some stationary phases currently used (2005) are:

• Polydymethyloxane, non-polar phase of general use for hydrocarbons, aromatics, polynuclear, drugs, steroids and PCBs.
• Poly(phenylmethyldiphenyl)siloxane (10% fenyl), for methyl esters of fatty acids, alkaloids, drugs and halogenated compounds.
• Poly(phenylmethyl)siloxane (50% fennel), for drugs, steroids, pesticides and glycols.
• Poly(trifluoropropildimethyl)siloxanefor chlorinated aromatic, nitroaromatic, alkyl benzene replaced.
• Polietilenglicol,sirve for polar compounds, also for compounds such as glycols, alcohols, ethers, essential oils.
• Poly(Dicianoalyldimethyl)siloxane, for polyunsaturated fatty acids, free acids and alcohols.

Generally, in commercial columns, the stationary phase is bound and crosslinked to prevent its loss during elution or washing operations. In this way, a monolayer chemically adhered to the surface of the column is obtained. The reaction involved is usually the addition of a peroxide to the liquid to be fixed, initiating a reaction by free radicals that results in the formation of a carbon-carbon bond that also increases its thermal stability. Another way is gamma irradiation.

Another type of stationary phase is chiral, which allows resolving enantiomeric mixtures. These types of phases are usually chiral amino acids or some derivative adapted to column work.

The thickness of the film varies between 0.1 and 5 μm; the thickness depends on the volatility of the analyte. Thus, a very volatile analyte will require a thick layer to increase the interaction time and more effectively separate the different components of the mixture. For typical columns (internal diameters of 0.25 or 0.32 mm) thicknesses of 0.25 μm are used, and in macrocapillary columns the thickness goes up to 1 μm. The maximum thickness is usually 8 μm

Applications

GC has two important fields of application. On the one hand, its ability to separate complex organic mixtures, organometallic compounds and biochemical systems. Its other application is as a method to quantitatively and qualitatively determine the components of the sample. For qualitative analysis, the retention time is usually used, which is unique to each compound under certain conditions (same carrier gas, temperature ramp and flow), or the retention volume. In quantitative applications, integrating the areas of each compound or measuring its height, with the appropriate calibrations, the concentration or quantity of each analyte is obtained.

Assembly techniques

The assembly of a GC analytical technique is purely empirical, the profile of the analytes to be determined, the choice of the mobile phase, the retention times (elution) will be given exclusively by the particular conditions of the column (stationary phase) in front of the equipment. The temperature ramps to be selected can either be isothermal or stepped.

The choice of gas will depend on the type of detector, the choice of the column (stationary phase) will depend on the polarity of the compounds to be separated, the detector will depend on the type of compounds to be detected.

Usually an analytical GC technique will consume many chromatographer hours to be developed and installed by trial and error method before being validated as real.

The choice of standards is fundamental in the development of the technique. Baseline stabilization of the mobile phase in the stationary phase (after the solvent front) over time is critical to establishing a method. An unstable or irregular baseline (solvent) that changes in intensity in front of the detector as it elutes must be refined and stabilized before introducing analytes.

The layout of the oven temperature range parameters, the proper choice of the column and its stationary phase (includes type, length, and diameter), the proper choice of detector type, detector and injector temperatures, The volumes of analyte must be established in such a way that the greatest efficiency is obtained in separating the analytes, and with the best possible resolution. The purity of the sample will depend on its previous preparation.

GC is a highly effective methodology and its performance allows a wide range of possibilities for analytical chemistry in organic compounds. A derivation of this technique is HPLC Chromatography which works on the basis of the affinity of the analyte for the liquid mobile phase instead of the gaseous one.

The sensitivity of the GC technique can even detect micrograms of the analyte if it is well mounted. Quantification is based on calculations of the area under the curve which is proportional to the concentration of the analyte. Commonly used in internal standard work.