Gas Chromatography CHEM 313-5 Dr. Reem M. Alghanmi 2017 1 st term
17.7 Gas Chromatography Introduction There are two types of gas chromatography: Gas-solid (adsorption) chromatography. Gas-liquid (partition) chromatography (GLC). The most important of the two is GLC. Gas chromatography is widely used, particularly by organic chemists, and it certainly rank as one of the most important new analytical techniques since it development in 1952. The separation of benzene and cyclohexane (b.p. 80.1 and 80.8ºC) is extremely simple by gas chromatography, but it is virtually impossible by conventional distillation. Very complex mixtures can be separated by this technique. 2
Principles 3 In gas chromatography the sample is converted to the vapor state (if it is not already a gas) and the eluent is a gas (the carrier gas). The stationary phase is generally a nonvolatile liquid supported on an inert solid such as firebrick. There are a large number of liquid phases available, and it is by changing this phase rather than the mobile gas phase that different separations are accomplished. The analyte in the vapor state distributes between the stationary phase and the carrier gas. Gas phase equilibria are rapid and so resolution and the number of theoretical plates can be high.
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Principles 5 A schematic diagram of a gas chromatography is given in Fig. 17.9. The sample is rapidly injected by means of a hypodermic syringe through a rubber septum into the column. The sample injection port, column, and detector are heated to temperatures at which the sample has a vapor pressure of at least 10 torr. The injection port and detector are usually kept somewhat warmer than the column to promote rapid vaporization of the injected sample and prevent sample condensation in the detector. Liquid samples of 0.1 to 10 µl are injected, while gas samples of 1 to 10 ml are injected.
Principles 6 Separation occurs as the vapor constituents partition between the gas and the liquid phases in the same manner as in other chromatographic processes. The carrier gas is a chemically inert gas available in pure form, such as argon, helium, nitrogen or carbon dioxide. A high-density gas gives best efficiency, but a low-density gas gives faster speed. The choice of gas is often dictated by the type of detector. The sample is automatically detected as it emerges from the column (at a constant flow rate), using a variety of detectors whose response is dependent upon the composition of the vapor.
Principles 7 Usually, the detector contains a reference side and a sampling side. The carrier gas is passed through the reference side before entering the column and emerges from the column through the sampling side. The response of sampling side relative to the reference side signal is fed to a recorder where the chromatographic peaks are recorded as a function of time. By measuring the retention time (the minutes between the time the sample is injected and the time the chromatographic peak is recorded) and comparing this time with that of a standard of the pure substance, it may be possible to identify the peak (agreement of retention times of two compounds dose not guarantee the compounds are identical).
Principles 8 The area under the peak is proportional to the concentration, and so the amount of substance can be quantitatively determined. The peaks are often very sharp and, if so, the peak height is taken and compared with a calibration curve prepared in the same manner. Automatic detection of the analyte as they emerge from the column make measurements rapid and convenient. Retention times are used for qualitative identification. Peaks areas are used for quantitative measurements.
Principle The separation ability of this
Gas Chromatography Columns Chromatographic columns can be in any shape that will fit the heating oven. Common forms are U-shaped tube, a W-shaped tube, and a coiled tube. Types of gas chromatography columns: 1- Packed column. 2- Capillary column 1- Typical packed column It is 1 to 2 m long and 0.3 to 0.6 cm in diameter. Short columns can be made of glass, but longer columns may be made of stainless steel so they can be straightened out for filling and packing. 10
2- Capillary, or Golay, columns. These are open tubular columns about 0.01 cm in diameter and 20 or more meters long. The liquid stationary phase is coated on and supported by the walls of the tube, where the original capillary columns had a very thin liquid film coated on a smooth metal or glass surface. The columns could be made very long (15 to 35 m) since there was a low pressure drop in the open tube, and they possessed very high separation efficiency (large number or theoretical plates) but they could handle only very small samples, about 10-2 or 10-3 µl of liquid. This necessitated the use of special gas-splitting devices so that only a fraction of the injected sample was introduced into the column. Only the most sensitive detectors could be use, and analysis of trade constituents was difficult. Support-coated open tubular (SCOT) overcome some of the restrictions of the earlier columns, where tubing diameter is 0.05 to 0.15 cm. Capillary columns can provide very high resolution, compared with packed columns. 11
Gas Chromatography Capillary, or Golay, columns Videos about Gas Chromatography and columns https://www.youtube.com/watch?v=08ywhltjlfo https://www.youtube.com/watch?v=pigsgkcwfaw https://www.youtube.com/watch?v=dffeilgekx8 https://www.youtube.com/watch?v=q0pm-k0svoq 12
Gas Chromatography Detectors 13 1- The Thermal Conductivity Detector 2- Flame Ionization Detector 3- The Flame Thermionic Detector 4- The β-ray (Argon Ionization)Detector 5- The Electron Capture (EC) Detector 6- The Coulometric Detector 13
Gas Chromatography Detectors 1- Thermal conductivity (hot wire) detector As a gas is passed over a heated filament wire, the temperature and thus the resistance of the wire will vary according to the thermal conductivity of the gas. The pure carrier is passed over one filament, and the effluent gas containing the sample constituents is passed over another. These filaments are in opposite arms of a Wheatstone bridge circuit that measure the difference in their resistance. So long as there is no sample gas in the effluent, the resistance of wires will be the same, but whenever a sample component is eluted with the carrier gas, a small resistance change will occur in the effluent arm. The change in the resistance, which is proportional to the concentration of the sample component in the carrier gas, is registered on the recorder. Hydrogen and helium carrier gases are preferred with thermal conductivity detectors because they have a very high thermal conductivity compared with most other gases, and so the largest change in the resistance occurs in the presence of sample component gases (helium is preferred for safety reasons). 14
Thermal conductivity (hot wire) detector Wheatstone bridge 15
2- Flame ionization detector, FID The basis of an extremely sensitive detector is that most organic compounds form ions in a flame. The ions are measured (collected) by a pair of oppositely charged electrodes. The response (number of ions collected) depends on the number of carbon atoms in the sample and on the oxidation state of the carbon. This detector gives excellent sensitivity, permitting measurement of components in the ppb concentration range. The carrier gas is relatively unimportant. He, N 2 and Ar are most frequently employed. The flame ionization detector is insensitive to most inorganic compounds, including water and so aqueous solutions can be injected. 16
3- The Flame thermionic detector Is essentially a two stage flame ionization detector, which is designed to give an increased specific response for N and P containing substances. A second flame ionization detector is mounted above the first, with the flame gases from the first passing into the second flame. The column effluent enters the lower flame, which acts as a conventional FID whose response may be recorded. However, if a substance containing N or P is burned in the lower flame, the ions resulting from these greatly increase the volatilization of the alkali metal from the screen. This results in a response that is much greater (at least 100 times) than the response of the lower flame to the N or P. By recording the signals from both flames, one can obtain the usual chromatogram of a FID: a second chromatogram is obtained where the peaks corresponding to the N and P containing compounds are amplified over the others, which will be practically missing. 17
4- β-ray or argon ionization detector, AID The sample is ionized by bombardment with β rays from a radioactive source (Ex. Sr-90). The carrier gas is argon and it is excited to a metastable state by the β particles. Argon has an excitation energy of 11.5 ev, which is greater than the ionization potential of most organic compounds, and the sample molecules are ionized when they collide with the excited argon atoms. The ions are detected in the same manner as in the flame ionization detector. This detector is very sensitive but less accurate than others, and the β-ray source is a potential hazard, although with proper shielding, no danger exists. The sensitivity is about 300 times greater than that of the conventional thermal conductivity cell. 18
5- The Electron capture (EC) detector Is extremely sensitive for compounds that contain electronegative atoms and is selective for these. It is similar in design to the β-ray detector, except that N 2 or methane doped with Ar is used as the carrier gas. These gases have low excitation energies compared to argon and only compounds that have high electron affinity are ionized, by capturing electrons. The detector cathode consists of a metal foil impregnated with a β- emitting element, usually tritium or Ni-63. A 30% KOH solution is usually used to clean the sources. 19
6- The Coulometric detector It was developed in 1960 by Coulson and co-workers. It was originally designed for the selective detection of chloridecontaining substances and is widely used for pesticide analysis. The column effluent is automatically combusted by passing through an oven at 800ºC to give HCl, which is absorbed in a cell containing a solution of silver nitrate. The cell contains a potentiometric silver-indicating electrode that detects changes in the silver concentration as AgCl is precipitated. The variation in potential relative to a reference electrode is amplified and triggers a generating current circuit; the circuit contains a pair of generating electrodes, the anode being a silver wire. 20
Detector Application Sensitivity Linearity Remarks 1- Thermal conductivity General, responds to all substances Fair Good, except thermistors at higher temp. Sensitive to temp. and flow changes; concentration sensitive 2- Flame ionization All organic substances; some oxygenated products respond poorly Very good 10-11 g/ml in carrier gas Excellent, up to 10 6 Requires very stable gas flow; response for water is 104-106 times weaker than for hydrocarbons; concentration sensitive (organics) 3- Flame thermionic All N and P containing substances Excellent Need recoating of sodium salts on screen; masssensitive 21
Detector Application Sensitivity Linearity Remarks 3-Rubidium silicate bead Specific for N and P containing substances. Excellent Mass sensitive 4-Argon ionization (β-ray) All organic substances; with ultraure He carrier gas, also for inorganic and permanent gases. Very good 10-10 -10-13 g/ml in carrier gas Good Very sensitive to impurities and water; needs very pure carrier gas; concentration sensitive 5-Electron capture All substances that have affinity to capture electrons; no response for aliphatic and naphthenic hydrocarbons. Excellent for halogen containing substances Poor Very sensitive to impurities and temp. changes; quantitative analysis complicated; concentration sensitive. 22
Some Practical Aspects of Gas Chromatography 1- Solid Supports The solid support for a liquid phase should have a high specific surface area that is chemically inert but wettable by the liquid phase. It must be thermally stable and available in uniform sizes. The most commonly used supports are prepared from diatomaceous earth, a spongy siliceous material. They are sold under many different trade names. 2- Coating the Solid Support Column packing support material is coated by mixing with the correct amount of liquid phase dissolved in a low-boiling solvent such as acetone or pentane. After coating, the solvent is evaporated by heating and stirring; last traces may be removed in vacuum and by passing carrier gas through the column. Open tubular columns are coated by slowly passing a dilute solution of the liquid phase through the columns. 23
3- Selecting The Liquid Phase There are thousands of commercially available column materials, and several attempts have been made to predict the proper selection of liquid immobile phase without resorting exclusively to trial and error techniques. These methods attempt to group phases according to their retention properties, Ex. According to polarity. Liquid stationary phases are selected based on polarity, determined by the relative polarities of the solutes. The Kovats retention index is useful for identifying a compound from its retention time relative to those of similar compounds in a homologous series (those that differ in the number of carbon atoms in a similar structure, as in alkaline chains). The index I is defined as I, log tr( unk) log t 100 ns,, log tr( n ) log t l r where n s is the number of carbon atoms in the smaller alkane, and n l refers to the larger alkane. The Kovats index for an unknown compound can be compared with catalogued indices on various columns to aid in its identification., r( n ( n s s ) ) 24
4- Polar Versus Non-polar Liquids Non-ploar liquid phases are generally nonselective. The reason for this is that there are few forces between the solute and solvent, and so the volatility of the solute is determined primarily by its vapor pressure. Hence, separations will tend to follow the order of the boiling points of the solutes, the lower boiling being eluted first. With polar liquid phases, however, volatility is not so simply determined because of interactions between the solute and the solvent, such as dipole-dipole interactions, hydrogen bonds, and induction forces. 5- Column Length Column lengths of 2 to 3 m are commonly used with packed columns. The resolution generally increases only with the square root of the length of the column. Long columns require high pressure and longer analysis times and are used only when necessary. Separations are generally attempted by selecting columns in lengths of multiples of three, such as 1 m, 3 m and so on. If a separation isnʼt complete in the shorter column, the next longer one is tried. 25
5- Temperature Selection The proper temp. selection in gas chromatography is a compromise between several factors. The injection temp. should be relatively high, consistent with thermal stability of the sample, to give the fastest rate of vaporization to get the sample into the column in a small volume; decreased spreading and increased resolution result. Too high an injection temp., though, will tend to degrade the rubber septum and cause dirtying of the injection port. The column temp. is a compromise between speed, sensitivity, and resolution. At high column temp., the sample components spend most of their time in the gas phase and so they are eluted quickly, but resolution is poor. At low temp., they spend more time in the the stationary phase and elute slowly; resolution is increased but sensitivity is decreased due to increased spreading of peaks. The detector temp. must be high enough to prevent condensation of the sample components. The sensitivity of the thermal conductivity detector decreases as the temp. is increased and so its temp. is kept at the minimum required. Some separations can be facilitated by temperature programming. 26
Quantitative Measurement of Chromatographic Peaks We mentioned that the concentrations of eluted solutes are proportional to the areas under the recorded peaks. A variety of methods can be used to estimate the areas, including mechanical or electronic integrators (as a part of the recorder). We have mentioned the possibility of measuring the peaks height to construct a calibration curve. The linearity of a calibration curve always be established. The method of Standard Additions is a useful technique for calibrating, especially for occasional samples. One or more aliquots of the sample are spiked with a known concentration of standard, and the increase in peak area is proportional to the added standard. This method has the advantage of verifying that the retention time of the unknown analyte is the same as that of the standard. A more important method of quantitative analysis is the use of Internal Standards. Here, the sample and standard are spiked with an equal amount of a solute whose retention time is near that of the analyte. The ratio of the area of the standard or analyte to that of the internal standard is used to prepare the calibration curve and determine the unknown concentration. 27
17.9 GAS CHROMATOGRAPHY MASS SPECTROMETRY The appearance of chromatographic peak at a particular retention time suggests but dose not guarantee the presence of a particular compound. Spectral information, such as infrared or ultraviolet spectrometry, may be sought. A very powerful tool is the combination of gas chromatography with mass spectrometry, a technique known as gas chromatography mass spectrometry (GC-MS). Mass spectrometry is a sophisticated instrumental technique that produces, separate and detectors ions in gas phase. The basic components of a mass spectrometer are shown in Fig. 17.12. A sample with a moderately high vapor pressure is introduced in an inlet system, operated under vacuum (10-4 to 10-7 torr) and at high temp. (up to 300ºC). It vaporizes and is carried to the ionization source.. Analyte molecules are typically neutral and must be ionized. This is accomplished by various means but typically is done by bombarding the sample with high-energy electrons in an electron-impact source. 28
The ions are separated in the spectrometer by being accelerated through a mass separator. Separation is actually accomplished based on the mass to charge (m/e) ratio of the ions. The resolution is more limited than with other mass analyzers, but this is not usually a problem when combined with the gas chromatography information. The separated ions are detected by means of an electron multiplier, which is similar in design to photomultiplier tubes. Detection sensitivities at the nanogram level are common. The effluent from a gas chromatography may be connected to the sample inlet system of a mass spectrometer, forming a GC-MS system. The mass spectrometer then serves as the GC detector with high sensitivity and selectivity. 29
The mass spectrometer may be operated in various modes: 1- In the total ion current (TIC) monitoring mode, it sums the currents from all fragment ions as a molecule (or molecules) in a GC peak passes through the detector, to provide a conventional looking gas chromatogram of several GC peaks. 2- In the selective ion mode, a specific (m/e) ratio is monitored, and so only molecules that give a molecular or fragment ion at that ratio will be sensed. The mass spectrum of each molecule detected is stored in the systemʼs computer, and so the mass spectrum corresponding to a given GC peak can be read out. The mass spectrum is generally characteristic for a given compound (if only one compound is present under the GC peak), giving a certain fingerprint of peaks at various m/e ratios. 30