Gas Chromatography

 Two of the main decisions in setting up a chromatograph for an analysis are the stationary phase and the column temperature program. The selection of the stationary phase is less critical for open tubular columns than for packed columns, because of their much higher efficiency.

Rule respect length: Use the shortest useful column. This saves time, it is cheaper. If more resolution is required, consider to reduce the film thickness or the internal diameter. Rules respect internal diameter (i.d.): A)       Megabore (0.53 mm i.d.) are preferred for high carrier flow rates. It allows for simple and direct injection techniques. B)       Medium size columns (0.25-0.35 mm i.d.) are used for good compromise. C)       Narrow columns (0.10 mmd i.d.). Produced increased speed and separation efficiency. Shorter columns are possible, and hence, reduced times. Some limitations: high split ratios needed (500:1). Trace analysis is difficult. Higher carrier gas pressures required. Manipulation and equipment become critical. Rules respect to film thickness: Thick films produce higher retention and is frequently used for analysis of volatiles. Increased capacity; important for GC-MS or FTIR. Thin films produce maximum separation efficiencies, faster analysis and l

 Is very important to avoid void volumes in the inlet or the detector, because their existence can result in extra broad peaks, reducing efficiency. To minimize this problem, installation of the columns must be performed closely following the manufacturer’s instructions.

 Column bleed usually occurs when columns are temperature programmed near the maximum permitted column temperature. It is the process of evaporation or thermal decompose of the stationary phase. Column bleed can be seen as an increase in the baseline as the column is heated and it becomes constant when it is held constant. The effect can become more pronounced as the column ages. Column bleed can be diminished by using thinner films, or by selecting a stationary phase with greater temperature resistance or that whose interactions with the analytes is smaller.

 Even though fused silica columns were thought to be inert, it has active “hot spots” of silanol groups, and polar compounds, particularly basic amines, can be adsorbed strongly to its surface, producing peak tailing and poor quantitative results. The Grob test can be used to evaluate the effectiveness of the deactivation process. The test proposes a mixture of six classes of compounds, that would probe for any unwanted column adsorption: 1)       Hydrocarbons. They are neutral compounds and always should produce sharp and symmetrical peaks. If not, they indicate a poorly installed column. 2)       Fatty acid methyl esters. Used to determine separation efficiency of the column. Small peak heights indicate adsorption losses in the system. 3)       Alcohols. They interact if there is presence of silanol in the injection port liner or the column, producing reduced peak heights, due to hydrogen bonding. 4)       Aldehydes. Reduced heights or symmetrical peaks imply adsorption of al

With packed columns and thick film megabore columns, nitrogen is the chosen carrier gas, since the B term in van Deemter equation (which corresponds to longitudinal diffusion in the gas phase) dominates. Since nitrogen is heavier than helium, it minimizes this term and more efficiency is attained. In capillary columns, particularly those with thin films, hydrogen is the best carrier gas. Since the separations are usually good enough, the emphasis is placed on speed. Hydrogen can be used at faster-than-optimal flow rates, with a minimal loss in efficiency. High-speed analysis is not possible with packed columns or with thick capillary columns.

Currently, most commercial columns have been conditioned in the factory, thus requiring only minimal conditioning after that. A good conditioning practice is as follows: Eliminate any air in the column by making the gas to flow for several minutes before heating the column. Then program the temperature increase at a rate of 3-5ºC/min to slightly above your intended operation temperature. Never surpass the manufacturer’s recommended maximum column temperature. When a stable baseline is obtained, the column can be used.

  Stationary phases are usually liquids or very viscous polymers. The liquid phase must show high selectivity, α , for the compounds of interest. The capability of operating at high temperatures with minimal column bleed is also very important, particularly for sensitive detectors like FID, ECD and MS, used for trace analysis. The most important types of liquid phases as of 2018 are the siloxane polymers (mixtures of methyl, phenyl, and cyano) polysiloxanes like the following: ·          OV‐1, SE‐30 ·          DB‐1 (100% methyl polysiloxane) ·          OV‐17 ·          OV‐275 ·          DB‐1701 ·          DB‐710 Another popular type is the polyelthylene glycol (Carbowax 20M, Superox®, and DB‐WAX®) liquid phases. Contrary to packed columns, in capillary columns, the stationary phases are extensively cross-linked. Heating the freshly prepared capillary column at high temperatures and without gas flow in the column, the methyl groups form free radicals that readily cross-link, produ

  Thin films (less than 0.2 µm) provide high efficiencies and resolutions. This imply that shorter columns and reduced operating temperatures can be used, reducing column bleed. Bleeding is the process of losing stationary phase due to high temperatures and flow inside the column. A film thickness of 0.25 µm represents a compromise with high resolutions attainable with thin films and the high capacity of thick films. When capacity is higher, larger samples can be injected and the injection technique itself is simpler. Practical operating temperatures can be used and bleeding is not a big problem, because bleeding is proportional to the amount of the liquid phase in the column. With this film thickness and using fast flow rates, the column can be optimized for speed. Low flow rates allow to obtain high resolutions. Thick films (1.00 µm or greater) show increased retention of sample components. This feature is essential to separate volatile compounds. Their capacity is greater, and b

 The longer the column, the greater the plate number, N, and better separations are obtained. Resolution, Rs, however is proportional to the square root of column length. Resolution only increases by the square root of 2 (41%) if the length is doubled. Retention time, tR, is also proportional to column length. For fast analysis, reduced length columns are advised. Nevertheless, when resolution is critical, long columns are required. They are used for complex samples (50 components or more). The drawback of long columns are the long analysis times. Columns between 15 to 30 m are recommended for most applications, because they provide a good compromise between analysis speed and resolution.

For fused silica columns, internal column diameter (i.d.) ranges from 100 to 530 µ m (0.10-0.53 mm). Small i.d. columns (100 µ m) produce fast analysis and good efficiency, but they can only manage small sample sizes, thus they are not well-suited for trace analysis. Additionally, special sample techniques and high-speed data systems are required to take advantage of their full potential. Internal diameters between 250 to 320 µ m represent the best compromise between resolution, speed, sample capacity and ease of operation. They can be regarded as the reference columns. A column of an internal diameter of 250 µ m is a good starting point for general method development. On the other hand, 530 µ m or “wide-bore” columns have decreased resolution, but increased capacities and ease of operation. For example, direct on-column syringe injection is straightforward, and usually provides better quantitative results than packed columns.

  The five critical parameters for capillary columns are:   internal diameter column length film thickness stationary phase composition flow rate

 They are open tubes; hence the pressure drop through them is small. Long lengths, such as 60 m can be easily used. In classical packed columns, the material is tightly packed and, therefore, producing greater pressure drops. This fact also limits packed columns lengths, which is around 2 m. This difference makes capillary columns more efficient in terms of resolution. The capillary columns have typical efficiencies of 3000-5000 theoretical plates per meter. On the other hand, efficiencies in packed columns are around 2000 plates per meter, due to thicker and nonuniform films. Thus, total plates available in long capillary columns is huge compared with packed columns, resulting in greater resolutions.

F used silica has high tensile strength and typical wall thickness is around 25 µ m. This makes columns flexible and easy to handle. Nevertheless, this material is subject to rapid corrosion and breakage, even at normal laboratory conditions. Therefore, a thin polyimide protective coating is applied outside the tubing as it emerges from the drawing oven. This polyimide covering, protects the fused silica from moisture and corrosion. This material, in fact, determines the maximum operating temperatures for columns, which is around 360ºC, and around 380ºC for short term operations. This coating darkens with age. Higher operation temperatures require the use of stainless steel-clad fused silica.

Fused silica is flexible and easy to handle. Additionally, it is a very inert material for tubing and readily produces high-resolution columns. Due to surface energy considerations, silicone phases “wet” these tubing very well, producing uniform and thin films and very efficient columns. Fused silica is obtained by the reaction of SiCl 4 and water in a flame. The product of the reaction is (almost) pure SiO2, containing 0.1% superficial hydroxyl or silanol groups and less than 1 ppm of impurities such as Na, K, Ca, etc. The high purity obtained is responsible for its very inert chemical nature. Temperatures of about 1800ºC are required to soften the material and draw silica into capillary dimensions. This process is made using advanced fiber-optic technology and expensive machinery.

Stainless steel columns very active for highly reactive compounds and not very efficient. The more common are the fused silica types.

 The tubes can be made of fused silica, glass, or stainless steel, but the fused silica columns are, by far, the most common. One type of column and the more common, is the wall-coated open tubular column (WCOT). Capillary columns provide the highest resolutions. Internal diameters of 0.10, 0.20, 0.25, 0.32 and 0.53 mm are commercially available. Typical lengths are between 10 to 60 m, although lengths of up to 100 m are commercially available. Long columns result in longer analysis times. Film thickness is between 0.1 and 5.0 µ m. Thin films provide fast analysis and resolutions, but low sample capacities. The contrary is true for thicker films, and they are only used for very volatile compounds. There are also two other types of capillary columns. The support-coated open tubular (SCOT) column and the porous layer open tubular (PLOT) column. SCOT columns contain an adsorbed layer of very small solid support, coated with a liquid phase. Their capacity is increased, compared with WC

Almost all new GCs uses a personal computer or laboratory-wide data systems to collect and analyze data, because a good data system must measure signal with rapid sampling rates. Computers have greater flexibility in acquiring data, controlling the GC, data reduction, display and transfer the data to other devices. The detector output of most GCs is analog, and an analog to digital converter (A/D converter) is used to convert the data to a digital format for storage, analysis and display by the computer. All data systems can perform basic calculations for chromatography, including the start, apex, end and area of each peak, area percent, height percent, external standard and normalization calculation.

The detector senses the effluents from the column and provides a signal which is proportionate to the quantity of each solute or analyte, allowing to perform quantitative analysis. The record of this signals is used to generate the chromatogram. Flame ionization detector (FID) is the most common detector. Its most outstanding features are its high sensitivity, wide linear range and low detection limits. It is also relatively simple and inexpensive. Other popular detectors are the thermal conductivity detector (TCD) and electron capture detector (ECD).

The detector and its connections from the column exit must be hot enough to avoid sample condensation but cool enough to not degrade the sample or the stationary liquid phase of the column. Peak broadening and loss of peaks due to condensation are a possibility at low detector temperatures. Temperature control is dependent on the type of detector used. For instance, a thermal conductivity detector (TCD) needs to be controlled at ±0.1ºC or better to obtain good baseline stability and maximum detectivity. Flame ionization detectors (FID) do not need a strict temperature control. A temperature should just be high enough to avoid condensation of sample or water. A reasonable minimum temperature for an FID is 200ºC.

Isothermal analysis is performed at a constant column temperature. On the other hand, in temperature programmed, a linear increase of column temperature with time is used. This technique is very useful for wide boiling sample mixtures.

High enough temperatures for the sample to pass through the column at a reasonable speed should be used. Nevertheless, it is not necessary the temperature to be higher than the boiling point of the sample. It might seem illogical, but it must be remembered that the column operates at temperatures where the sample is in vapor state and not need to be in the gas state. Thus, in GC, the column temperature must be higher than the “dew point” of the sample, but not higher its boiling point. A good rule of thumb for temperature is that the initial temperature should be low enough that the first peak of interest elutes with a k value of at least 1.0. High temperatures provoke the retention times to decrease, reducing the analysis time. However, better resolutions are achieved at low temperatures.

The inlet must be hot enough to produce a fast vaporization of the sample without loss of efficiency from the injection technique. At the same time, very high inlet temperatures should be avoided, because thermal decomposition or chemical rearrangement can be produced. A general rule for flash vaporization is to have an inlet temperature 50ºC above the boiling point of the sample. Tests of the inlet temperature can be performed. If increasing the temperature produce better peak shapes or improves efficiency, the temperature was too low. If drastic changes are observed in retention times, peak areas or peak shapes, the temperature was too high, probably leading to thermal decomposition or rearrangements. On the other hand, in on-column injections, the inlet temperature can be lower.

The control of temperature is one of the easiest and most effective ways to influence separation. The control of temperature is necessary to achieve a good separation in a reasonable amount of time.

Capillary columns are columns that are not filled with packing material. Instead, they are internally covered by a thin film of a liquid phase. They are formally called “wall-coated open tubular” or open tubular (WCOT or OT columns). Their resistance to flow is very low, and long lengths are possible, up to 100m, which also permits efficient separation of very complex sample mixtures. Fused silica columns are the more inert.