Gas Chromatography Lecture Notes PDF

# Gas Chromatography ## Chem 312, 316 ## Dr. Yasmeen Gaber Abou El-Reash # Gas Chromatography (GC) - Uses: A separation technique that can be used to separate either naturally volatile or can be converted to a volatile form substance in a mixture at high temperatures. - The gaseous mobile...

# Gas Chromatography ## Chem 312, 316 ## Dr. Yasmeen Gaber Abou El-Reash # Gas Chromatography (GC) - Uses: A separation technique that can be used to separate either naturally volatile or can be converted to a volatile form substance in a mixture at high temperatures. - The gaseous mobile phase is called a carrier gas. - The carrier gas carries the sample through packed or capillary columns where all solutes get separated. - There are two types of GC: - Gas-solid chromatography (GSC); and - Gas-liquid Chromatography (GLC). - Gas-solid chromatography: separation depends on the affinity of solutes to adsorb on solid stationary phase. - Gas-liquid chromatography: separation depends on partition of solutes between gaseous mobile phase and liquid stationary phase. # Instrumentation * It consists of - Injection Port or Sample introduction system (syringe / septum) - Carrier gas - Flow controller - A separation column (the stationary phase) - An oven - a detector - Recorder ## 1- Mobile Phase or Carrier Gas - It passes the solutes to and along the column under pressure. - He, Ar, and N₂ are widely used. N₂ is cheap comparing to He and Ar. - Hydrogen could be used but less preferred because it is explosive. - A carrier gas should have the following properties: - A higher density gas is preferred. - low molecular weight gases (He, H₂) → larger diffusion coefficients - low molecular weight gases → faster, more efficient separations - Inert so that no reaction with stationary phase or instrumental components can take place, especially at high temperatures. - O₂ can react solutes and stationary phase or with surfaces of the injector, connections and detector - The choice of carrier gas is often determined by the instrument's detector.(thermal conductivity detector requires H₂ or He) - Highly pure (> 99.9%), cheap and available carrier gas. ## 2- Flow controller - Controls the flow rate of the carrier gas. - The flow rate for packed columns is 25 - 150 mL/min. - The flow rate for capillary columns is 1 - 25 mL/min. ## 3- Injectors - Microsyringe: is used for manual injection of the sample into the column inlet - Auto-sampler: is used to automatically inject the sample into the column inlet. ## Principle - The injection port consists of a rubber septum through which a syringe needle is inserted to inject the sample. - These are composed of a glass tube where vaporization of the sample takes place because the injection port is maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture. - If the temperature of the injector is not high enough (at least 50 degrees above highest boiling component), band broadening will take place. ## 4- The sample - All constituents injected into GC must be volatile to move through the column. - Solutes of low volatility might be retained by the column and continue to elute during analysis of subsequent samples. - Non-volatile solutes condense in the column and destroys the column's performance. - Volatile analytes can be separated from non-volatile matrix by solid-phase extraction or liquid-liquid extraction. - Temperature is used to vaporize the liquid samples. - The sample should not decompose at the vaporization temperature. - Compounds that cannot be analyzed by GC - Compounds that do not vaporize (inorganic metals, ions, and salts). - Highly reactive compounds and chemically unstable compounds. ## 5- Chromatographic Columns - A column can either be a packed or open tubular. - **A. Packed column**: Columns containing a particulate packing material. - It is constructed from glass, stainless steel, copper or aluminum. - It is typically 2-6 m in length with an internal diameter of 2-4 mm. - The solid support is usually diatomaceous earth, which consists of silica skeleton. - Hydrolysis of diatomaceous earth gives silanol groups (-Si-OH) on the surface, which act as active sites to adsorb solute molecules in gas-solid chromatography. - In gas-liquid chromatography, the liquid stationary phase is coated on solid support (diatomaceous earth), and the silanol groups (-Si-OH) are deactivated by silanization to avoid unwanted adsorption of solutes on the support material. - **Silanization** is the covering of a surface with organofunctional alkoxysilane molecules. - More recently, solid supports made from glass beads or fluorocarbon polymers have been introduced. These supports have the advantage of being more inert than diatomaceous earth. - **Advantages** - higher capacity (higher conc). - **Disadvantages**: low resolution. - **B. Capillary or open tubular columns**: It is also called open tubular columns. - It does not contain a particulate packing material. - Column length might reach 100 m, and the internal dimeter is approximately 150 – 300 µm. - There are two types of capillary columns: 1- Wall-coated; and 2- Support-coated open tubular columns. - There are 2 types of Capillary column: - **A. wall-coated open tubular column** - An open tubular column in which the stationary phase is coated on the inner wall of the capillary column. - **B. support-coated open tubular column** - An open tubular column in which the stationary phase is coated on a solid support that is attached to the inner wall of the capillary column. - **Advantages of capillary columns over packed columns:** - They are longer than packed columns. - Have better separation efficiency. - Have large numbers of theoretical plates. ## Stationary Phases - There are two types of stationary phases used in GC: - Solid adsorbents (GSC) - Liquids coated on solid supports (GLC) - Selectivity in gas chromatography is influenced by the choice of stationary phase. - There are several important criteria for choosing a stationary phase: - It should be chemically inert. - Thermally stable (Has low volatility). - It must have a polarity that is appropriate for the sample's components. - Common adsorbents used as solid stationary phases include: - Alumina, silica, active carbon, molecular sieves (crystalline aluminosilicates [zeolites] and clay) ## 6- Temperature Control - Since the separation is dependent on temperature, the separation column is usually contained in a thermostat-controlled oven. - There are two approaches for separation (elution): 1- Isothermal separation; and 2- Temperature programming. - **A. Isothermal separation** - The column is maintained at constant temperature. - The temperature is set slightly below that for the lowest boiling point solute to increase solute's interaction with stationary phase. - **Drawback**: the temperature favoring separation of low boiling point solutes might lead to unacceptable long retention time for high boiling point solutes. - **B. Temperature programming** - There is a continuous or step change in column temperature. - The initial temperature is set below that for the lowest boiling solute. - During separation, the temperature is slowly increased in a uniform rate or in a series of steps. ## Practical steps - A sample containing the materials to be separated (which may be a gas or a liquid) is injected into the inlet part of the gas chromatograph. - The mobile phase (inert carrier gas) moves through a packed or capillary column that contains a wall coated or granular solid coated stationary phase. - As the carrier gas flows through the column, the components of the sample come in contact with the stationary phase the separates the sample components based on their ability to partition between the mobile phase and the stationary phase. - The different components of the sample have different boiling points and affinities for the stationary phase, which results in differential migration of solutes, thus leading to separation. - Gas chromatography can be used for both qualitative and quantitative analysis. - Identification of materials present in the tested sample can be done by comparing the peaks appeared at definite retention times in the chromatogram with other corresponding to retention times for standards. - Quantitative analysis is accomplished by measurement of area under peak as the concentration of samples directly proportional with area under peak. ## Mechanism of separation Process - The organic compounds are separated in the column due to differences in: - **Boiling point:** The substance that has lower boiling point (easily converted to gas) have greater affinity to remain with the carrier gas so can separated first. While substances have higher boiling point have higher affinity to remain in the liquid stationary phase, so it leaves the column later. - **Polarity:** according to the difference in polarity of samples and stationary phase (i.e. for polar stationary phase, the less polar substance will be separated first). In general, nonpolar solutes are more easily separated with a nonpolar stationary phase, and polar solutes are easier to separate using a polar stationary phase. - **Size:** If the substances to be separated have the same or nearly close boiling points, the substance that has smaller size or smaller molecular weight will be separated fist because the carrier gas can carry it easier and faster through the separation column to the detector. ## What factors affect retention time in GC? - **Volatility of the component:** Volatile components travel through a column faster than non-volatile components. The volatility is related to the boiling point and to the size of the molecules. This means that the smaller molecules within a homologous series have shorter retention times than the larger molecules - **Type of Stationary phase:** Not the stationary phase itself, but the affinity of the sample component for the stationary phase is important. The stronger this affinity, the stronger is the interaction, and therefore the longer the retention time. - **Amount of stationary phase:** The k-values of sample components are related to the amount of stationary phase. The more stationary phase is present, the larger the k-values and thus the longer the retention time. ## GC Detectors - After the components of a mixture are separated using gas chromatography, they must be detected as they exit the GC column. - The choice of detector will depend on the type of analyte. - There are several types of detectors such as: - Thermal-conduc (TCD). - Flame ionization (FID) detectors. - Electron Capture Detector (ECD). ## According to selectivity, detectors can be grouped into: 1. **Non-selective detector:** responds to all compounds except the carrier gas. 2. **Selective detector:** responds to a range of compounds with a common physical or chemical property. 3. **Specific detector:** responds to a single chemical compound. ## a. TCD Detector - **Function:** - Detect various organic and inorganic compounds (H2, N2, CO2 or CO). - He is the most commonly used carrier gas because it has a thermal conductivity 6-10 times higher than other organic vapors. - **Working principle:** - This detector depends on the reduction in the thermal conductivity of the carrier gas in the presence of analyte is detected. - The detector consists of two pairs of thermistors forming a Wheatstone bridge. - When pure carrier gas passes over both pairs of thermistors the bridge is balanced. - When pure carrier gas passes over one pair of thermistors and a mixture of carrier gas + eluted solutes passes over the other pair, the bridge is imbalanced. This because of unequal cooling of the two pairs of thermistors. - **Working mechanism** - The heart of the TCD detector is an electrically-heated tungsten-rhenium filaments or wires which is cooled by He carrier gas. - The filament's electrical resistance depends on its temperature, which, in turn, depends on the thermal conductivity of the carrier gas. - Under normal conditions there is a stable heat flow from the filament to the detector body. - When a solute elutes from the column, the thermal conductivity of the He gas decreases and the temperature of the wire filament rises, and thus its resistance, increases. - Changes in thermal conductivity when organic molecules displace some of the carrier gas (He), causes a temperature rise in the filament which is sensed as a change in resistance. - The thermal conductivity is measured using a themistor. - The measurement current and the reference current are compared with one another in a bridge circuit. - The resistance change is often sensed by a Wheatstone bridge circuit which produces a measurable voltage change. - The column effluent flows over one of the resistors while the reference flow is over a second resistor in the four-resistor circuit. The read out is proportional to concentration. - **Advantages:** - Universal detector and can be used for all kind of solutes. - It gives signals for any solutes whose thermal conductivity is different from helium. - It gives a linear response for solute concentrations over a range of 104 - 105 order. - Non-destructive detector (solute does not get destroyed). - **Disadvantage:** It has a poor detection limit in comparison with other detectors. - **Important Notes:** - Variations in flow rate, pressure, or electrical power, all of which may lead to a change in the filament's resistance. - Other carrier gases cant be used, since the drop in thermal conductivity in the presence of sample is too slight to be detected. - Since all compounds, organic and inorganic, have a thermal conductivity different from helium, all compounds can be detected by this detector. ## Electron Capture Detector (ECD) - A detector for GC that provides selectivity for solutes containing electronegative functional groups such as halogen and nitro functional groups and thus has found wide applications in the detection of pesticides and polychlorinated biphenyls. - The electron capture detector is used for detecting electron-absorbing components (high electronegativity) such as halogenated compounds. - The mechanism of sensing relies on the fact that electronegative atoms, like halogens, will capture electrons coming from a radioactive beta particle (electron) emitter consists of a metal foil holding 63Ni or Tritium (T or 3 H). - **Working mechanism** - The radioactive material emit electrons that collide with the N₂molecules, resulting in ionization of the carrier gas, producing ions and other electrons. - Then, electrons are accelerated towards a positively charged anode, generating a current. - When a solute containing electron withdrawing groups (Cl, NO₂) is eluted from the column, it captures the electrons, therefore, the electric current decrease. The decrease in electric current serves as the signal. - **Note:** - ECD is insensitive to amine, alcohols, and hydrocarbons. - Usually, N₂ or Ar are used as carrier gases, because it exhibits a low excitation energy, so it is easy to remove an electron from a nitrogen molecule. - **Advantage:** it has excellent detection limit. ## Flame Ionization Detector (FID) - This is the most widely used detector. - It used to detect the ions produced by the flame. - In order to be able to take these ions we need 2 electrodes and potential difference should be applied. - The detection principle is based on the change in the electric conductivity of H₂ flame in an electric field when feeding organic compounds (hydrocarbons). - **Advantages:** - Good detection limit. - Useful for analysis of atmospheric and aqueous environment samples. - **Note:** the sample is destroyed in FID. - **Mechanism:** - H₂ carrier gas get mixed with organic compounds escaping out of separating column (efluent) and get into the flame where it get burned. - During this process, organic matters get decomposed via heating into radicals. - These radicals get oxidized into ions within the flame by the O₂ stream which is fed into the stream from outside according to the following equation: - CH + O → CHO+ + e - At the bottom of the flame, we have the -ve electrode, and in the area where the collection occur we have the +ve electrode. - High voltage is applied between the 2 electrodes so the ions produced in the flame can be collected. - The produced ions are then collected by collector plate which is connected to a very sensitive ammeter. - Electrons emitted are attracted by a positive electrode, where an electric current is obtained. - The increase in current between the 2 electrodes when ions are formed in the flame is amplified and recorded. - The detected signal is converted to a peak. - The current across this collector is thus proportional to the rate of ionisation which in turn depends upon the concentration of HC in the sample gas. - The FID is sensitive to all compounds that contain C-C or C-H bonds. - Insensitive for carbonyl C=O, alchols OH, and halogens or amino groups. - Most inorganic compounds and many gases like H₂O and CO2 cannot be detected. ## Gas Chromatography Advantages - long column lifetimes - ability to separate some compounds not easily resolved by other Chromatographic methods. - Fast. - Sensitive. ## Applications 1) Separation of volatile compounds. 2) Quantitative analysis. ### External standard method - Measure the peak height or peak area for a series of standard solutions. - Plot a calibration curve between the concentration and peak height or peak area. - Measure the peak height or peak area for the unknown sample. - Determine the concentration of the unknown sample from the curve. | Concentration | Peak height or peak area | | :-------------------- | :----------------------- | | Solution 1 | C1 | | Solution 2 | C2 | | Solution 3 | C3 | | Solution 4 | C4 | | Solution 5 | C5 | ## 2) Identification of unknown compounds. - Comparing the retention time of unknown sample with that of reference sample. A graphic depicting the process is shown.