3.1: Principles of Gas Chromatography (2024)

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    Archer J.P. Martin (Figure \(\PageIndex{1}\) ) and Anthony T. James (Figure \(\PageIndex{2}\) ) introduced liquid-gas partition chromatography in 1950 at the meeting of the Biochemical Society held in London, a few months before submitting three fundamental papers to the Biochemical Journal. It was this work that provided the foundation for the development of gas chromatography. In fact, Martin envisioned gas chromatography almost ten years before, while working with R. L. M. Synge (Figure \(\PageIndex{3}\) ) on partition chromatography. Martin and Synge, who were awarded the chemistry Nobel prize in 1941, suggested that separation of volatile compounds could be achieved by using a vapor as the mobile phase instead of a liquid.

    3.1: Principles of Gas Chromatography (2)
    3.1: Principles of Gas Chromatography (3)
    3.1: Principles of Gas Chromatography (4)

    Gas chromatography quickly gained general acceptance because it was introduced at the time when improved analytical controls were required in the petrochemical industries, and new techniques were needed in order to overcome the limitations of old laboratory methods. Nowadays, gas chromatography is a mature technique, widely used worldwide for the analysis of almost every type of organic compound, even those that are not volatile in their original state but can be converted to volatile derivatives.

    The Chromatographic Process

    Gas chromatography is a separation technique in which the components of a sample partition between two phases:

    1. The stationary phase.
    2. The mobile gas phase.

    According to the state of the stationary phase, gas chromatography can be classified in gas-solid chromatography (GSC), where the stationary phase is a solid, and gas-liquid chromatography (GLC) that uses a liquid as stationary phase. GLC is to a great extent more widely used than GSC.

    During a GC separation, the sample is vaporized and carried by the mobile gas phase (i.e., the carrier gas) through the column. Separation of the different components is achieved based on their relative vapor pressure and affinities for the stationary phase. The affinity of a substance towards the stationary phase can be described in chemical terms as an equilibrium constant called the distribution constant Kc, also known as the partition coefficient, \ref{1} , where [A]s is the concentration of compound A in the stationary phase and [A]m is the concentration of compound A in the mobile phase.

    \[ K_{c} = [A]_{s}/[A]_{m} \label{1} \]

    The distribution constant (Kc) controls the movement of the different compounds through the column, therefore differences in the distribution constant allow for the chromatographic separation. Figure \(\PageIndex{4}\) shows a schematic representation of the chromatographic process. Kc is temperature dependent, and also depends on the chemical nature of the stationary phase. Thus, temperature can be used as a way to improve the separation of different compounds through the column, or a different stationary phase.

    3.1: Principles of Gas Chromatography (5)

    A Typical Chromatogram

    Figure \(\PageIndex{5}\) shows a chromatogram of the analysis of residual methanol in biodiesel, which is one of the required properties that must be measured to ensure the quality of the product at the time and place of delivery.

    3.1: Principles of Gas Chromatography (6)

    Chromatogram (Figure \(\PageIndex{5}\) a) shows a standard solution of methanol with 2-propanol as the internal standard. From the figure it can be seen that methanol has a higher affinity for the mobile phase (lower Kc) than 2-propanol (iso-propanol), and therefore elutes first. Chromatograms (Figure \(\PageIndex{5}\) b and c) show two samples of biodiesel, one with methanol (Figure \(\PageIndex{5}\) b) and another with no methanol detection. The internal standard was added to both samples for quantitation purposes.

    Instrument Overview

    Components of a Gas Chromatograph System

    Figure \(\PageIndex{6}\) shows a schematic diagram of the components of a typical gas chromatograph, while Figure \(\PageIndex{7}\) shows a photograph of a typical gas chromatograph coupled to a mass spectrometer (GC/MS).

    3.1: Principles of Gas Chromatography (7)
    3.1: Principles of Gas Chromatography (8)

    Carrier Gas

    The role of the carrier gas -GC mobile phase- is to carry the sample molecules along the column while they are not dissolved in or adsorbed on the stationary phase. The carrier gas is inert and does not interact with the sample, and thus GC separation's selectivity can be attributed to the stationary phase alone. However, the choice of carrier gas is important to maintain high efficiency. The effect of different carrier gases on column efficiency is represented by the van Deemter (packed columns) and the Golay equation (capillary columns). The van Deemter equation, \ref{2} , describes the three main effects that contribute to band broadening in packed columns and, as a consequence, to a reduced efficiency in the separation process.

    \[ HEPT\ =\ A+\frac{B}{u} + Cu \label{2} \]

    These three factors are:

    1. the eddy diffusion (the A-term), which results from the fact that in packed columns spaces between particles along the column are not uniform. Therefore, some molecules take longer pathways than others, and there are also variations in the velocity of the mobile phase.
    2. the longitudinal molecular diffusion (the B-term) which is a consequence of having regions with different analyte concentrations.
    3. the mass transfer in the stationary liquid phase (the C-term)

    The broadening is described in terms of the height equivalent to a theoretical plate, HEPT, as a function of the average linear gas velocity, u. A small HEPT value indicates a narrow peak and a higher efficiency.

    Since capillary columns do not have any packing, the Golay equation, \ref{3} , does not have an A-term. The Golay equation has 2 C-terms, one for mass transfer in then stationary phase (Cs) and one for mass transfer in the mobile phase (CM).

    \[ HEPT\ =\ \frac{B}{u} \ +\ (C_{s}\ +\ C_{M})u \label{3} \]

    High purity hydrogen, helium and nitrogen are commonly used for gas chromatography. Also, depending on the type of detector used, different gases are preferred.

    Injector

    This is the place where the sample is volatilized and quantitatively introduced into the carrier gas stream. Usually a syringe is used for injecting the sample into the injection port. Samples can be injected manually or automatically with mechanical devices that are often placed on top of the gas chromatograph: the auto-samplers.

    Column

    The gas chromatographic column may be considered the heart of the GC system, where the separation of sample components takes place. Columns are classified as either packed or capillary columns. A general comparison of packed and capillary columns is shown in Table \(\PageIndex{1}\). Images of packed columns are shown in Figure \(\PageIndex{8}\) and Figure \(\PageIndex{9}\).

    Column Type Packed Column Capillary Column
    History First type of GC column used Modern technology. Today most GC applications are developed using capillary columns
    Composition Packed with silica particles onto which the stationary phase is coated. Not packed with particulate material. Made of chemically treated silica covered with thin, uniform liquid phase films.
    Efficiency Low High
    Outside diameter 2-4 mm 0.4 mm
    Column length 2-4 meters 15-60 meters
    Advantages Lower cost, larger samples Faster, better for complex mixtures
    Table \(\PageIndex{1}\) A summary of the differences between a packed and a capillary column.
    3.1: Principles of Gas Chromatography (9)
    3.1: Principles of Gas Chromatography (10)

    Since most common applications employed nowadays use capillary columns, we will focus on this type of columns. To define a capillary column, four parameters must be specified:

    1. The stationary phase is the parameter that will determine the final resolution obtained, and will influence other selection parameters. Changing the stationary phase is the most powerful way to alter selectivity in GC analysis.
    2. The length is related to the overall efficiency of the column and to overall analysis time. A longer column will increase the peak efficiency and the quality of the separation, but it will also increase analysis time. One of the classical trade-offs in gas chromatography (GC) separations lies between speed of analysis and peak resolution.
    3. The column internal diameter (ID) can influence column efficiency (and therefore resolution) and also column capacity. By decreasing the column internal diameter, better separations can be achieved, but column overload and peak broadening may become an issue.
    4. The sample capacity of the column will also depend on film thickness. Moreover, the retention of sample components will be affected by the thickness of the film, and therefore its retention time. A shorter run time and higher resolution can be achieved using thin films, however these films offer lower capacity.

    Detector

    The detector senses a physicochemical property of the analyte and provides a response which is amplified and converted into an electronic signal to produce a chromatogram. Most of the detectors used in GC were invented specifically for this technique, except for the thermal conductivity detector (TCD) and the mass spectrometer. In total, approximately 60 detectors have been used in GC. Detectors that exhibit an enhanced response to certain analyte types are known as "selective detectors".

    During the last 10 years there had been an increasing use of GC in combination with mass spectrometry (MS). The mass spectrometer has become a standard detector that allows for lower detection limits and does not require the separation of all components present in the sample. Mass spectroscopy is one of the types of detection that provides the most information with only micrograms of sample. Qualitative identification of unknown compounds as well as quantitative analysis of samples is possible using GC-MS. When GC is coupled to a mass spectrometer, the compounds that elute from the GC column are ionized by using electrons (EI, electron ionization) or a chemical reagent (CI, chemical ionization). Charged fragments are focused and accelerated into a mass analyzer: typically a quadrupole mass analyzer. Fragments with different mass to charge ratios will generate different signals, so any compound that produces ions within the mass range of the mass analyzer will be detected. Detection limits of 1-10 ng or even lower values (e.g., 10 pg) can be achieved selecting the appropriate scanning mode.

    Sample Preparation Techniques

    Derivatization

    Gas chromatography is primarily used for the analysis of thermally stable volatile compounds. However, when dealing with non-volatile samples, chemical reactions can be performed on the sample to increase the volatility of the compounds. Compounds that contain functional groups such as OH, NH, CO2H, and SH are difficult to analyze by GC because they are not sufficiently volatile, can be too strongly attracted to the stationary phase or are thermally unstable. Most common derivatization reactions used for GC can be divided into three types:

    1. Silylation.
    2. Acylation.
    3. Alkylation & Esterification.

    Samples are derivatized before being analyzed to:

    • Increase volatility and decrease polarity of the compound
    • Reduce thermal degradation
    • Increase sensitivity by incorporating functional groups that lead to higher detector signals
    • Improve separation and reduce tailing

    Advantages and Disadvantages

    GC is the premier analytical technique for the separation of volatile compounds. Several features such as speed of analysis, ease of operation, excellent quantitative results, and moderate costs had helped GC to become one of the most popular techniques worldwide.

    Advantages of GC

    • Due to its high efficiency, GC allows the separation of the components of complex mixtures in a reasonable time.
    • Accurate quantitation (usually sharp reproducible peaks are obtained)
    • Mature technique with many applications notes available for users.
    • Multiple detectors with high sensitivity (ppb) are available, which can also be used in series with a mass spectrometer since MS is a non-destructive technique.

    Disadvantages of GC

    • Limited to thermally stable and volatile compounds.
    • Most GC detectors are destructive, except for MS.

    Gas Chromatography Versus High Performance Liquid Chromatography (HPLC)

    Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins.

    GC HPLC
    Sample must be volatile or derivatized previous to GC analysis Volatility is not important, however solubility in the mobile phase becomes critical for the analysis.
    Most analytes have a molecular weight (MW) below 500 Da (due to volatility issues) There is no upper molecular weight limit as far as the sample can be dissolved in the appropriate mobile phase
    Can be coupled to MS. Several mass spectral libraries are available if using electron ionization (e.g., http://chemdata.nist.gov/) Methods must be adapted before using an MS detector (non-volatile buffers cannot be used)
    Can be coupled to several detectors depending on the application For some detectors the solvent must be an issue. When changing detectors some methods will require prior modification
    Table \(\PageIndex{2}\) Relative advantages and disadvantages of GC versus HPLC.
    3.1: Principles of Gas Chromatography (2024)

    FAQs

    3.1: Principles of Gas Chromatography? ›

    During a GC separation, the sample is vaporized and carried by the mobile gas phase (i.e., the carrier gas) through the column. Separation of the different components is achieved based on their relative vapor pressure and affinities for the stationary phase.

    What are the principles of gas chromatography? ›

    Principle of gas chromatography: The sample solution injected into the instrument enters a gas stream which transports the sample into a separation tube known as the "column." (Helium or nitrogen is used as the so-called carrier gas.) The various components are separated inside the column.

    What are the 4 principles of chromatography? ›

    Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography.

    What are the three important aspects of gas chromatography? ›

    There are three main GC system components: the sample injection unit, which heats the liquid sample and vaporizes it; the column, which is used to separate each compound; and the detector, which detects the compounds and outputs their concentrations as electrical signals.

    What are the fundamentals of gas chromatography? ›

    The basics of a gas chromatograph include an autosampler, an injector port, a column, and a detector. This analysis can be done for a number of applications including industrial, energy, food beverage analysis, environmental analysis, toxicology, and more.

    What is gas chromatography basic theory? ›

    The Working Principles of Gas Chromatography

    The sample is injected into the instrument where it is vaporized and mixes with the carrier gas to become a part of the mobile phase. This mobile phase is then carried through the chromatographic column where it interacts with the stationary phase of the column.

    What is the principle of separation in GC chromatography? ›

    During a GC separation, the sample is vaporized and carried by the mobile gas phase (i.e., the carrier gas) through the column. Separation of the different components is achieved based on their relative vapor pressure and affinities for the stationary phase.

    What is the principle of chromatography experiment? ›

    The principle involved can be partition chromatography or adsorption chromatography. Partition chromatography because the substances are partitioned or distributed between liquid phases. The two phases are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper.

    What is GC analysis? ›

    Gas chromatography (GC) is an analytical technique used to separate and detect the chemical components of a sample mixture to determine their presence or absence and/or quantities. These chemical components are usually organic molecules or gases.

    What are the three components involved in the principle of chromatography? ›

    Three components thus form the basis of the chromatography technique. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”

    What is the gas chromatography technique? ›

    Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture.

    What elutes first in gas chromatography? ›

    When predicting retention order in gas chromatography, the overriding factor is a comparison of the boiling points. The compound with the lowest boiling point elutes first.

    What are the gas chromatography standards? ›

    Gas Chromatography standards are reference substances used to calibrate and validate GC instruments. These compounds are injected into the GC system to establish precise reference points in the analysis. Standards facilitate the identification and quantification of unknown analytes in samples.

    What is gas chromatography and its principles? ›

    Gas Chromatography or Gas Liquid Chromatography is a technique applied for separation, identification and quantification of components of a mixture of organic compounds by selective partitioning between the stationary phase and mobile phase inside a column followed by sequential elution of separated components.

    What are the general principles of chromatography? ›

    What is the basic principle of chromatography? Chromatography is based on the concept of separating molecules in a mixture added to the ground or solid and liquid stationary state (stable phase) when travelling with the aid of a mobile phase.

    What is gas chromatography for dummies? ›

    Gas Chromatography – The Basics. Gas chromatography (GC) is a powerful analytical technique that can be used to separate, identify, and quantify individual chemical components in complex mixtures.

    What is the basic principle of GC MS? ›

    It works by heating a liquid sample until it converts into a vapor that can be carried by a gas like helium or hydrogen. The gas (called a carrier gas or mobile phase) transports the sample through a long, thin glass or metal tube (column) that is coated with a chemical (stationary phase).

    What are the main components of gas chromatography? ›

    The gas chromatography process has many applications, from food analysis to forensics and air quality measurements. However, no matter the application, gas chromatography has four components: injection port, carrier gas, column oven, and a detector.

    What is the working principle of gas liquid chromatography describe its application? ›

    The basic principle in Gas Liquid Chromatography (GLC) is that it exploits the differences in partition coefficients of volatilized sample between mobile gaseous phase and stationary liquid phase as the samples passes through the column.

    What is the principle of HPLC and GC? ›

    The HPLC detection method is non-destructive, typically using either an ultraviolet-visible (UV/Vis) spectroscopic detector or a refractive index detector (RID). While GC detection uses more destructive principles, like in a flame ionization detector (FID) that's effective when analysing hydrocarbons.

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