Gas Chromatography

The self-sealing septum provides a way to seal the injector and at the same time, allow the users to inject the samples into the GC. They are usually made of polymeric silicone. To select the correct septum, several properties should be considered: high-temperature stability, decomposition (also called “bleeding”), size, lifetime and cost. The self-seal is conserved during 50 injections or more for most septa. It should be replaced on a regular basis.

When using an autosampler, it should be programmed to inject the sample as fast as possible. For manual injections, some guidelines are useful: Exclude all air initially: can be achieved by repeatedly drawing and expelling out liquid sample into the syringe. Special care must be taken with very viscous liquid samples. A better alternative is to dilute them with an appropriate solvent. When handling the syringe manually, fill it with more liquid than the desired volume for the injection. After that, hold the syringe pointing up and the air in the syringe will go to the top of the barrel. Soft taps can be used to aid in this process. After that, press the plunger up to the desired injection volume. Wipe off the needle with a tissue and now add some air to the syringe. This serve two purposes: first it will often give a peak in chromatograms, which can be used to measure tM. Second, it prevents losing sample if the plunger is accidentally pushed. Injection technique require the us...

 Autosamplers are mechanical devices placed on top of the GC that automatize the process of injection. The main advantages of their use is that they provide rapid and highly reproducible injections, and of course, it does not need a person to be making every injection. The autosampler takes care of all the process, taking the sample from sealed vials, injection, flushing the syringe with a solvent after injection. Trays with large amounts of samples, standards and solvents are used and thus, they can run unattended overnight. The precision of an autosampler is greater than the manual injection with a typically 0.2% relative standard deviation (RSD).

 The barrel is usually made of glass, and the needle and plunger of stainless steel. The needle is epoxied into the barrel. Other models have removable needles that are screwed onto the end of the barrel. Some models also have a small wire inside the syringe needle, extending to its tip, so there is no dead volume after the injection. It is better to use a syringe whose total sample volume is at least two times the larger than the volume to be injected.

hey must be dissolved in an appropriate solvent, and the solution injected with a micro syringe.

The universal method for sampling liquids is the use of syringes. The most common syringes are of 1, 5 and 10 µ L. Small samples are desirable due to the great expansion of liquids after they are vaporized. Care must be taken to avoid overheating of the sample and thermal decomposition during injection.

These methods require the entire sample to be in the gas phase before the analysis. Mixtures of gases and liquids are problematic because in that case the analyst should convert all the sample to a gas, by heating the sample or to a liquid by increasing pressure. But this is not always possible. There are two main methods two introduce gas samples. The first one is the use of gas­-tight syringes. This method is cheaper, more flexible and the most frequent method used. The other method is the use of gas-sampling valves, which is used frequently with packed columns; give better repeatability, requires less skill from the analyst and can be more easily automated.

The most common way to introduce a sample is using a micro syringe and the sample is a liquid. The sample system should permit the sample to be introduced rapidly and quantitatively onto the column. Three types of inlets are the most common for capillary columns: split, split less and on-column. In practice is impossible to introduce the whole sample instantaneously, but it is desirable to introduce it as a sharp symmetrical band. The difficulty of achieving this can be seen by analyzing an example. A 1.0 µ L liquid sample of benzene vaporizes to 600 µ L after heating in the injector. If the flow rate is 1 mL/min, then 36 seconds would be needed to carry the whole sample onto the column. Hence, sampling and the size of the samples are critical aspects of chromatography. The smallest possible sample size should be used to obtain the maximum resolution and peak shape. On the other hand, the more components present in the sample, the larger the sample size may need to be. For trace work...

There are two ways of measuring the flows independently. The first one is an inexpensive soap-bubble flowmeter which consists of a calibrated tube (usually a modified pipet or buret) through which the carrier gas flows. With a rubber bulb we can create a bubble, which is raised into the path of the gas. After that, the ascension of a particular bubble to a defined volume is measured with a stopwatch. The carrier gas in mL/min is easily obtained from this measurement. There are also available electronic soap film flowmeters at a cost around $50. The second alternative is the use of a sophisticated device, composed of a solid-state sensor and a microprocessor to accurately flow measurements without using soap bubbles. Silicone-on-ceramic sensor can be used to measure flow rates of 0.1-500 mL/min for air, nitrogen, oxygen, helium, hydrogen and 5% argon in methane. The cost for this device is around $700. Very small flow rates, like the ones found in tubular columns, cannot be measured rel...

Two-stage regulator is connected to the cylinder as a first flow control system, which reduces the tank pressure of up to 2500 psig (psi gauge, or above the atmospheric pressure), down to a level useable level of 20-100 psig. The system should include a filter to prevent particulate matter from entering the regulator and a safety valve. The first valve indicates the pressure in the gas cylinder. With the second valve, one can increase the pressure delivered to the GC. The second stage regulator works better at pressures at least 20 psi higher than the maximum inlet pressure on the GC. Constant pressure is sufficient to provide a constant flow rate for isothermal operations if the pressure drop in the column is constant. In temperature-programmed operations, the flow rate decreases at constant temperature as a consequence of an increased viscosity of the gas at higher temperatures. Differential flow controllers are used to ensure a constant mass flow rate. Modern research-grad capil...

 The measurement and control of carrier gas flow is very important for GC. Column efficiency depends on the linear gas velocity. As a guideline, a typical column of 0.25-mm inner diameter (i.d.) open tubular (OT) column has an optimum of 0.75 mL/min. Nevertheless, the optimum for a given column should be determined experimentally. For qualitative analysis, it is crucial to have a constant and reproducible column flow rate. If this is accomplished, retention times can be reproduced, and the comparison of retention times is the quickest and easiest technique for compound identification. Two solutes can have the same retention time. However, a solute cannot have 2 different retention times in the same column. Thus, the retention times are characteristics of the solutes, but not unique.

The purity of the gas is very important in GC. Impurities such as oxygen and water can chemically attack the stationary phase of the columns and destroy it. Polyester, polyglycol and polyamide phases are particularly susceptible. Water impurities can lead to desorption of other column contaminants, producing high detector background or “ghost peaks”. Trace amounts of hydrocarbons can cause high background or noise signal in most ionization detectors and thus worsen their detection limits. Several ways to obtain the gas with the desired purities are possible. One of them is to purchase ultrahigh purity gas cylinders, which is very expensive. Another possibility is to use gas generators, especially for hydrogen and air. This possibility is economically feasible, but require and initial high investment and maintenance. The more common practice is to purchase high-purity gases and further purify them. Water and trace hydrocarbons can be removed by using a 5 Armstrong molecular sieve filt...

 Its main objective is to carry the sample through the column. It is mobile and it is inert, therefore it does not interact chemically with the sample or the stationary phase. It is also an appropriate matrix for the detectors to analyze the samples components.

 The main components of the chromatographic system are (1) carrier gas, (2) flow control, (3) sample inlet and sampling devices, (4) columns, (5) controlled temperature zones (ovens), (6) detectors, and (7) data systems. A gas chromatograph functions essentially as follows: An inert gas flows continuously from a gas cylinder through the inlet, the column and the detector. The flow rate of the carrier gas is controlled to ensure reproducible retention times and minimize detector drift and noise. The sample is injected into the inlet, where it is vaporized and carried into the column by the carrier gas. The capillary column has a typical length of 15-30 m and is coated inside with the stationary phase; a thin layer (0.2-1 µm) of a high boiling liquid. The sample interacts with the stationary phase, and partitions between the mobile and stationary phases. The sample is separated into individual components due to their differences in solubilities in the liquid and their vapor pres...

To analyze of the separation of two peaks is achieved, the following equation is used: Rs = d / Wb Where Rs is the resolution, d Is the distance between the maxima of two adjacent peaks and Wb is the peak width. Peak width, Wb, increases as the square root of the column length, L, and d increases directly with L. In other words Rs is proportional to L / L^(1/2) = L^(1/2) Resolution is proportional to the square root of the column length. If we plot d or Wb versus L, d will surpass Wb at a sufficient large value of L and the separation is achieved. The conclusion is that the chromatographic process in effective even if it produces peak broadening. Of course, in practice there are many methods to achieve a separation and the analyst rarely relies only in the column length.

A comparison of the effect of the carrier gas on the rate equation for a capillary column (H) is made. The following analysis holds for isothermal operation. The process can be optimized for column efficiency (plate number) or the analysis time. For a given column, the solute diffusivity is minimized (term B) using a higher-molecular-weight gas and more plates are generated. Nitrogen shows the minimum H, at the expense of slower analysis. To optimize for speed, however, it is better to choose a lighter carrier gas, like hydrogen or helium. The minima in the figure of H versus average linear velocity for nitrogen, helium and hydrogen are around 12 cm/s, 20 cm/s and 40 cm/s, respectively. On the other hand, hydrogen has the smallest slope beyond the minimum. Thus, an increase in hydrogen flow rate produces only a small loss in efficiency, while considerably speeding up the analysis. Regarding film thickness, high efficiencies are good to separate high-boiling compounds. However, the sa...

Plots depicting the Height plate (H, usually in mm) versus the linear velocity (u, typically in cm/s) are called Van Deemter plots. In such a picture, the three terms of the Golay equation are plotted. In the equation for H, one of the terms is multiplied by u and another is multiplied by it. This leads to the presence of a minimum in the curve for H, an optimal point of velocity which provides the highest efficiency and smallest plate height. Chromatographers manipulate the Van Deemter equation to get the best performance while minimizing the analysis time. The CM term is the most important contribution as the velocity increases. Plots are used to calculate H, and the van Deemter equation is rarely used to achieve this.

Mass transfer in the mobile phase can be related to the solute profile due to the nonturbulent flow through a tube. The molecules in the center of the tube move faster than those near the wall. Therefore, inadequate mixing (slow kinetics) result in band broadening. Columns with small diameter minimizes broadening because the mass transfer distances are reduced. The CM term for the Golay equation is: CM = (1+6k+11k^2)*rc^2 / (24*(1+k)^2*DG) Where rc is the radius of the column, k is the retention factor and DG is the diffusion coefficient. The relative importance of the two terms CM or Cs is dependent on the column radius and film thickness. In columns of small inner diameter, the term CM is less dominant than Cs. On the other hand, for thin films (<0.2 µm) the mass transfer in the mobile phase (CM) controls the C term; in thick films (2-5.0 µm), the C term is controlled by the stationary phase mass transfer (Cs); for intermediate films (0.2-2.0 µm) both factors are important. ...

A figure is used to describe the mass transfer in the stationary phase. We have a mid-horizontal line representing the interface. Above, we have the mobile phase and below, the stationary phase. At a given moment, an equilibrium is stablished, and two peaks with the base in the mid-horizontal line, but pointing to the top and to the bottom are depicted. An instant later the mobile gas moves the upper curve downstream, which gives rise to a situation of base size increase and the broadening of the overall zone of molecules. The solute molecules that moved ahead now participate in another partition between both phases, and vice versa for those that are in the stationary phase. The band broadening is inversely proportional to the speed of this process. The Cs term in the Golay equation is: Cs = (2*k*df) / (3*(1+k)^2*Ds) Where df is the average film thickness of the liquid stationary phase, Ds is the diffusion coefficient of the solute in the stationary phase and k is the retention f...

 Capillary columns are open tubes; hence their rate equation does not have the A term from Van Deemter equation. Golay introduced a new term to account for the diffusion process in the gas phase for the open tubular columns. His equation additionally has two C terms: one for mass transfer in stationary phase, Cs and another for mass transfer in the mobile phase, CM. The simple Golay equation is: H = B/ µ + (Cs + CM) µ The term B accounts for molecular diffusion. The equation for molecular diffusion is: B = 2DG Where DG is the diffusion coefficient for the solute in the carrier gas. A small value for the diffusion coefficient is desired to decrease the value of B and for H. The usage of gases of high molecular masses like nitrogen or argon as carrier gases aids in this sense. Additionally, a high linear velocity also decreases the time a solute spends in the column and the time available for molecular diffusion drops. On the other hand, the C terms are related to mass trans...

Van Deemter identified three effects that contribute to peak broadening in packed columns: Eddy diffusion (term A), longitudinal molecular diffusion (the term B) and mass transfer in the stationary phase (the term C). The Van deemter Equation is: H = A + B/ µ + C µ Where H is the plate height, and µ the linear gas velocity. A small value indicates a narrow peak (the desired condition). Minimize each term is the way to maximize column efficiency.

Column resolution Rs is another measure of efficiency and represents the degree to which two adjacent peaks are separated. Rs is defined by dividing the distance between the peak maxima for two solutes A and B by the sum of the baseline lengths of both peaks. This equation is strictly valid when both peaks have the same height. The larger the resolution, the better the separation. To produce a complete baseline separation, a resolution of at least 1.5 is required.

The plate height, H, is defined as: H = L / N Where L is the column length and N is the plate number. H has units of length and is better suited for comparing efficiencies of columns of different lengths. It is also called height equivalent to one theoretical plate (HETP). A good column will have a large N and a small H

A broadening of peaks with retention time is a natural phenomenon of the chromatographic process. Due to this fact, the most common measure of efficiency is the plate number, N, defined as: N = (tR/S)^2 = 16(tR/Wb)^2 = 5.54(tR/W_(1/2))^2 Where W_(1/2) is the width of the peak measured at half its height N is unitless. A high value of N indicates an efficient column, a very desirable quality. If the chromatogram has several peaks, the value of N for each compound will vary. However, it is common practice to assign a value to a column, based only on one measurement.

Theoretical discussion assumes an ideal Gaussian peak. Some of its characteristics are the following: The inflection points occur at 0.607 of the peak height. The tangents to those points produce a triangle with a base width Wb, equal to four standard deviations, 4σ, and a width at half height, Wh of 2.354σ. The width of the peak at the inflection point is 2σ.

The ideal peak shape, or the distribution of the molecules of a compound, can be approximated as being normal or Gaussian. The presence of asymmetry in peaks are an indication of the presence of undesirable interactions during the chromatographic process. In packed columns, broad peaks are common and usually indicate that mass transfer kinetics are slow. Asymmetric peaks can be classified as tailing or fronting depending on whether the peak seems to be compacted on the right side (fronting) or on the left side (tailing). The tailing factor (TF) is used to evaluate the extent of the asymmetry. It is defined as TF = b / a Where a and b are the peak width at the left and at the right of the maximum intensity of the peak. a and b are usually measured at 10% of the peak height. US Pharmacopeia recommends 5% of the peak height and the following formula: TF = (a+b) / 2a Care must be taken when using tailing factor for column comparisons, because there are several common definition...

The selectivity (α) of a separation is calculated as the ratio of the adjusted retention times of two adjacent peaks: Alpha = t’R(2) / t’R(1) The numbers 1 and 2 are for the earlier (1) and later (2) eluting peaks. Selectivity can also be written as the equality between the ratio of the retention factors (k1 and k2) and equal to the two partition coefficients (K1 and K2). The selectivity is a consequence of the differences on intermolecular interactions between the compounds and the stationary phases. Selectivity increases as the differences in those interactions increase. In GC capillary columns, the selectivity required to perform a separation can be as low as 1.02 or less.

  The retention factor, k, is the ratio of the mass of solute (not its concentration) in the stationary phase to the mass in the mobile phase: The retention factor is measured experimentally as the ratio of the adjusted retention time, t′R, to the gas hold‐up time tM The larger the value of k, the longer the solute will be retained in the column. The relevance of this factor is that can be obtained easily from the chromatogram. The retention factor and the distribution constant are related through the phase ratio (beta). Kc can be broken down into two terms: Kc equals the product of the retention factor and the phase ratio In formula: Kc = k x beta Beta is defined as the ratio between the volume of the mobile phase (VM) and the stationary phase (Vs). In formula Beta = Vm / Vs For capillary columns, with a known value of film thickness (df), beta is given by: Beta equals the square difference between the radius of the capillary column (rc) and the film thickness (d...

  The distribution constant, Kc, is a parameter that defines how fast a solute moves down the column. For a solute A that moves between the mobile phase and the stationary phase, in an equilibrium reaction, we can write the equilibrium constant Kc as the ratio between the concentration of A in the stationary phase and its concentration in the mobile phase. Larger values of this coefficient mean the solute is retained for longer periods because its sorbs more readily in the stationary phase. Even though an equilibrium constant is used, that does not imply the process is in equilibrium, because the mobile gas phase is continuously moving solute molecules down the column. However, the partition coefficient is adequate as a descriptor when the kinetics of mass transfer are fast, because the system will operate near to equilibrium conditions. Another assumption in the development of the theory is that the solutes do not interact with one another. Thus, the formation of azeotropes an...

  The main components of the gas chromatographers are: carrier gas supply, injection ports or inlets, column (inside an oven), flow controllers, detector and a data system. The column is the essential part of the technique because it is where the separation occurs. The first columns were the packed columns, metal tubes of 1-2 meters length and 0.2-0.4 cm of inner diameter, packed with inert supports which were coated with the stationary liquid phases. Today, the fused silica columns are of widespread use and are open tubes called capillary columns, due to their small inner diameter (0.1-0.53 mm). The stationary liquid phase is coated on the inner surface of the tube, forming a coating with a thickness of around 0.1-5 µ m.

  Only volatile samples can be analyzed Difficult to implement for thermally labile samples Not appropriate for preparative scale or large samples Usually requires additional techniques, like mass spectrometry, for confirmation of peak identities.

  Efficiency is a measure of how well the separations can be performed and is expressed in plate numbers. Capillary columns are known to possess several hundred thousand plate numbers. The efficiency of a column increases with column length. In theory, having very long columns can make analysis very efficient, however and unlimited increase in length is not practical and nowadays complex separations are performed by using comprehensive two-dimensional GC.

  GC provides fast separations, and current commercial instrumentation permit analysis to be performed in seconds. A technique called comprehensive two-dimensional GC (GCxGC) has been developed recently, which implies the injection of the eluent from a traditional separation into a very short column, a second column. The second analysis requires just a few seconds to be completed. The result obtained is a GCxGC contour plot where we have the second dimension, represented in a y-axis.

  Fast analysis, in the order of minutes High resolution and efficient High sensitivity, can detect ppm and ppb. Is a nondestructive technique, can be coupled to a mass spectrometer Quantitative analysis is very accurate with RSDs in the order of 1-5%. Only small samples are needed (microliters) Reliable and relatively simple technique Inexpensive

  In a typical chromatogram for a single solute A, we can see a small peak at the beginning, short time after the injection (which is performed at time zero). The solutes are characterized by their retention times or retention volumes (tr, or Vr). These quantities are depicted as the distance between the injection point and the peak maximum. The formula to calculate the retention volume Vr is: Retention volume equals (“retention time” multiplied by “gas flow rate”) Or we can express it in terms of the retention time as: retention time equals (“Retention volume” divided by “gas flow rate”) Or in formula Vr = tr x Fc   or   tr = Vr / Fc; where Fc is the gas flow rate The small initial peak corresponds to a component that does not is sorb into the stationary phase, thus it is an un-retained component. The IUPAC defines Vm, the holdup volume as: “the volume of the mobile phase (MP) required to elute the un‐retained compound from the chromatographic column and report...

  Distribution constant Kc: defined as the concentration of a solute in the stationary phase, divided by its concentration in the mobile phase. Is a thermodynamic value, dependent on temperature. Different migration rates through the column among compounds are a consequence of the differences in distribution constant values.

 Symbols recommended by the IUPAC are indicated with (IUPAC) after their definition. The terms without this clarification are other symbols and names in use. Kc: distribution constant (for GLC) (IUPAC) Kp: partition coefficient KD: distribution coefficient k: retention factor (IUPAC) k′: capacity factor; capacity ratio; partition ratio N: plate number (IUPAC) n: theoretical plate number; no. of theoretical plates H: plate height (IUPAC) HETP: height equivalent to one theoretical plate R: retardation factor (in columns) (IUPAC) RR: retention ratio Rs: peak resolution R (IUPAC) alpha: separation factor (IUPAC) - Selectivity; solvent efficiency tR: retention time (IUPAC) VR: retention volume (IUPAC) VM: holdup volume (IUPAC) Volume of the mobile phase; VG: volume of the gas phase VO void volume; dead volume

  The distribution constant or partition coefficient Kc, is a measure of the tendency of a component to be attracted to the stationary phase. In chromatography, the greater this value, the greater the attraction of that component to the stationary phase. The differences in distribution constants, which are controlled thermodynamically, are the responsible for chromatographic separations. There are two types of sorption processes: Absorption: sorption into the bulk of stationary phase Adsorption: sorption on the surface of the stationary phase One of these processes is usually dominant, but both can be present.

  The output signal of the detector produces what is called a chromatogram. Its representation is a series of peaks at different times (independent variable) and the relative sizes of the peaks are indicative of the relative masses of each component. The retention factor “k” is provided by the ratio of the mass in the stationary phase to the mass in the mobile phase and is a very important chromatographic variable.

  At the beginning, a sample containing two components A and B, is introduced to the column in a narrow zone and then, it is carried through the column by the mobile phase. The components partition between the liquid and gas phase. The component A has greater affinity to the mobile phase, and is carried down the column faster than the component B, who has more affinity for the liquid phase. As a consequence of this process, component A leaves the column first and is detected. After that, B leaves the column and is detected.

  The use of a gas phase requires contained and leak-free systems. Such systems are metal or glass tubes, called columns. The columns contain the stationary phase. The columns are named using the name of the stationary phase. For instance, if the stationary liquid phase is polydimethylsiloxane (PDMS), then one can refer to it as a PDMS column.

  All techniques within GC can be classified according to the state of the stationary phase as follows Gas-solid chromatography (GSC): the stationary phase is a solid. Gas-liquid chromatography (GLC): liquid stationary phase. OT or capillary: are Open tubular or capillary columns and can be of GSC or GLC type. GLC is, by far, the most used technique.

  Elution: The process in which a mobile phase is continuously passed through or along the chromatographic bed and the sample is fed to the system as a finite slug. Chromatographic processes names are linked to the state of the mobile phase. Hence, the mobile phase is a gas in GC and a liquid in LC. In GC, the sample is vaporized and carried through the column by the carrier gas. The components of the sample equilibrate into and out the stationary liquid phase in the column. These equilibriums are dependent on temperature and the components separate from one another based on their affinities for the stationary phase and relative vapor pressures.

 Gas chromatography (GC) is a technique used for separation and analysis of volatile compounds. It can be used to analyze gases, liquids or solids, which are usually dissolved in volatile solvents. The molecular weight of the compounds analyze can vary in the molecular weight range from 2 to over 1000 Da. For example, it can be used to separate and analyze more than 450 components in coffee aroma.

LC - Liquid chromatography   LSC - Liquid‐solid chromatography   MDQ - Minimum detectable quantity   MP - Mobile phase MS - Mass spectrometry MSD - Mass selective detector OT - Open tubular PDMS - Polydimethyl siloxane PLOT - Porous layer open tubular PTV - Programmed temperature vaporization RSD - Relative standard deviation SCOT - Support coated open tubular SD - Standard deviation SEC - Size exclusion chromatography SP - Stationary phase TCD - Thermal conductivity detector TF - Tailing factor TOF - Time of flight (MS) TPGC - Temperature programmed gas chromatography WCOT - Wall coated open tubular