GAS CHROMATOGRAPHY

Prof. ARULSELVAN

SYLLABUS  Introduction  Instrumentation - Carrier gas supply - Sample injection system including Head space analysis - Columns (Packed, Open tubular columns, Capillary columns) and column ovens (Self study-0.5 hr) - Detectors -Thermal conductivity, - Electron capture, - Flame ionization - Applications, - Advantages and - Limitations of GC (Self study-0.5hr)

IntroductIon • In gas chromatography (GC), the sample is vaporized and injected onto the head of a chromatographic column via a heated injection port, where it evaporates. • Then condenses at the head of the column, which is at a lower temperature. • Once on the column, separation of a mixture occurs according to the relative lengths of time spent by its components in the stationary phase. • Elution is brought about by the flow of an inert gaseous mobile phase (The mobile phase does not interact with molecule of the analyte; its only function is to transport the analyte through the column). • Gas-liquid chromatography is based upon the partition of the analyte between a gaseous mobile phase and a liquid phase immobilized on the surface of an inert solid. • Monitoring of the column effluent can be carried out with a variety of detectors.

INSTRUMENTATION Instrumentation:1.Carrier Gas (Mobile phase) - Chemically inert, Purified and Compatible with detectors - Nitrogen, Helium, Argon, Carbon dioxide 2.Sample Injector - Micro syringe - Two mode of injection- Split or Split less 3.Column - Packed column - Capillary column (open tubular column) - wall-coated open tubular (WCOT) or - support-coated open tubular (SCOT) 4.Detectors - Thermal conductivity - Electron capture - Flame ionization

INSTRUMENTATION

Carrier Gas-Supply • Carrier gases, which must be chemically inert, include helium, nitrogen, and hydrogen. • It should flow continuously throughout instrument with linear flow rate • Flow rate differs as per the column – Packed column:-25-100ml/min – Capillary column:-µL/min to 1mL/min

• Carries the sample vapor through the column to detector • Viscosity of carrier gas increases with column size • Associated with the gas supply are pressure regulators, gauges, and flow meters. • In addition, the carrier gas system often contains a molecular sieve to remove water or other impurities.

Sample InjectIon SyStem •

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The most common method of sample injection involves the use of microsyringe to inject a liquid or gaseous sample through a self-sealing, silicone-rubber diaphragm or septum into a flash vaporizer port located at the head of the column (the sample port is ordinarily about 50oC above the boiling point of the least volatile component of the sample). The injector port is held at 150-250°C depending on the volatility of the sample and direct injection of 0.1-10 µL of sample is made onto the head of the column. The sample is injected by a hypodermic syringe, through a silicon rubber septum into the column packing or into the flash heater For packed columns, sample size ranges from tenths of a µL up to 20 µL. The amount of sample injected onto a packed column is 1-2 µg per component.

Sample InjectIon SyStem • •

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Capillary columns (open tubular) need much less sample, typically around 10-3 mL. The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vaporizes to form a mixture of carrier gas, vaporized solvent and vaporized solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column. In split mode, the sample is split into two unequal portions, the smaller of which goes onto the column. split ratio range between 10:1 and 100:1,with the larger portion being vented in the higher flow out of the split vent. In splitless mode, all the sample is introduced onto the column and the injector purge valve remains closed for 0.5-1 min after injection. In splitless mode the peak shapes are poor due to overloading of the column with too much sample.

Sample InjectIon SyStem -Sample split into two unequal portion 2:46 mL/min - 1mL/min reach the column, in that some may vent out through split valve

-Split valve is closed, almost all sample (mL/min) reach the column only

GC COLUMN •

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Chromatographic columns vary in length from less than 2 m to 50 m or more. They are constructed of stainless steel, glass, fused silica, or Teflon. In order to fit into an oven for thermostating, they are usually formed as coils having diameters of 10 to 30 mm. Two general types of columns are encountered in gas chromatography, packed and capillary (open tubular). PACKED COLUMNS contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 4mm. CAPILLARY COLUMNS have an internal diameter of a few tenths of a mm. They major types are; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT) and Porous layer open tubular (PLOT) columns . In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column; These have much thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils. They have the advantages of physical strength, flexibility and low reactivity.

GC COLUMN -Wall-coated columns (WCOT) consist of a capillary tube whose walls are coated with liquid stationary phase. - In support-coated columns (SCOT), the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.

- The Porous layer open tubular (PLOT) columns in which the inner surface of the column has an embedded layer that contains the stationary phase

The STaTionary PhaSe  Desirable properties for the immobilized liquid phase in a gas-liquid chromatographic column include: (1) low volatility (ideally, the boiling point of the liquid should be at 100oC higher than the maximum operating temperature for the column); (2) thermal stability; (3) chemical inertness; (4) solvent characteristics such that k` and  values for the solutes to be resolved fall within a suitable range.  The retention time for a solute on a column depends upon its distribution constant which in turn is related to the chemical nature of the stationary phase.  Analyte must show some degree of compatibility (solubility) with the stationary phase for retention. Here, the principle of “like dissolves like” applies, where “like” refers to the polarities of the solute and the immobilized liquid.

The STaTionary PhaSe  Polar stationary phases contain functional groups such as -CN,-CO and -OH.  Hydrocarbon-type stationary phase and dialkyl siloxanes are nonpolar, whereas polyester phases are highly polar.  Polar solutes include alcohols, acids, and amines; solutes of medium polarity include ethers, ketones, and aldehydes.

The STaTionary PhaSe

Column ovens •

Column temperature is an important variable that must be controlled to a few tenths of a degree for precise work. Thus, the column is ordinarily housed in a thermostated oven incorporated with a fan (uniform heat distribution).

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They can be programmed to produce a constant temperature, isothermal conditions or a gradual increase in temperature.

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The optimum column temperature depends upon the boiling point of the sample and the degree of separation required. Roughly, a temperature equal to or slightly above the average boiling point of a sample results in a reasonable elution time (2 to 30 min).

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For samples with a broad boiling range, it is often desirable to employ temperature programming, whereby the column temperature is increased either continuously or in steps as the separation proceeds.

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The column oven has the function to adjust the column temperature to an accurate and reproducible value. The advantages are that materials of widely differing volatilities can be separated in a reasonable time and also injection of sample can be carried out at low temperature.

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Column ovens

Detection SyStemS Characteristics of the Ideal Detector: The ideal detector for gas chromatography has the following characteristics: 1. Adequate sensitivity 2. Good stability and reproducibility. 3. A linear response to solutes that extends over several orders of magnitude. 4. A temperature range from room temperature to at least 400oC. 5. A short response time that is independent of flow rate. 6. High reliability and ease of use. 7. Similarity in response toward all solutes or a highly selective response toward one or more classes of solutes. 8. Nondestructive of sample.

Flame IonIzatIon Detectors (FID) • The effluent from the column is mixed with hydrogen and air and then ignited electrically produce ions and thus increase in current between the jet and the collector. • A potential of a few hundred volts is applied and the resulting current (~10-12 A) is then measured. • The flame ionization detector exhibits a high sensitivity (~10-13 g/s), large linear response range (~107), and low noise. • Detects carbon and hydrogen containing compounds. • ADVANTAGE:- In combination with Capillary GC it may detect as low as 100pg-10ng. • DISADVANTAGE – Destruction of sample. – Insensitive to carbon atoms attached to oxygen, nitrogen or chlorine.

Collector/ output

Ni63 foil

Thermal Electrons

Argon/CO2 make-up gas

Column effluent Electron Capture Detector Flame ionization Detector

ElEctron-Capture DeteCtors(eCD) •

The electron-capture detector most widely used detectors for environmental samples because this detector selectivity detects halogen containing compounds, such as pesticides and polychlorinated biphenyls.

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The effluent from the column is passed over a  emitter, usually nickel-63. An electron from the emitter causes ionization of the carrier gas and the production of a burst of electrons. In the absence of organic species, a constant standing current between a pair of electrodes results from this ionization process. The current decreases markedly, however, in the presence of those organic molecules that tend to capture electrons. The electron-capture detector is selective in its response being highly sensitive to molecules containing electronegative functional groups such as halogens, peroxides, quinones, and nitro groups.

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An important application of the electron-capture detector has been for the detection and determination of chlorinated insecticides and analysis of drugs in body fluids.

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It is insensitive to functional groups such as amines, alcohols, and hydrocarbons.

Thermal ConduCTiviTy deTeCTors(TCd) • The thermal conductivity detector, TCD, can be based on one of two concepts: the hot wire or the thermistor. The hot wire based detector (or katharometer) is the most common. • The flow of pure carrier gas (reference stream) through the wires removes heat at a specific rate. When the gas mixture is added, there is a decrease in the rate of heat removal, which raises the temperature of the wire. • The data processing unit reads and records the resistance change resulting from the change in temperature. • The heated element may be a fine platinum, gold, or tungsten wire or a semiconducting thermistor. • The advantage of the thermal conductivity detector is its simplicity, its large linear dynamic range(~105), its general response to both organic and inorganic species, and its nondestructive character, which permits collection of solutes after detection.

Thermal ConduCTiviTy deTeCTors(TCd)

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Used to determine water in some BP assays, e.g., water in the peptides menotrophin, gonadorelin and salcatonin

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Universal detector used to determine water vapour.

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A limitation of the katharometer is its relatively low sensitivity (~10-8 g solute/mL carrier gas).

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Other detectors exceed this sensitivity by factors as large as 104 to 107.

DETECTORS & ITS APPLICATION Detector

Type

Support gases

Selectivity

Flame ionization Mass flow Hydrogen and air Most organic cpds. (FID) Thermal conductivity Concentration Reference Universal (TCD) Halides, nitrates, nitriles, Electron capture Concentration Make-up peroxides, anhydrides, (ECD) organometallics NitrogenMass flow Hydrogen and air Nitrogen, phosphorus phosphorus Sulphur, phosphorus, tin, Flame Hydrogen and air boron, arsenic, photometric Mass flow possibly oxygen germanium, selenium, (FPD) chromium Aliphatics, aromatics, ketones, esters, aldehydes, Photo-ionization Concentration Make-up amines, heterocyclics, (PID) organosulphurs, some organometallics Hall electrolytic Halide, nitrogen, Mass flow Hydrogen, oxygen conductivity nitrosamine, sulphur

Detectability

Dynamic range

100 pg

107

1 ng

107

50 fg

105

10 pg

106

100 pg

103

2 pg

107

Qualitative analysis Qualitative Analysis:• Gas chromatograms are widely used as criteria of purity for organic Compounds. • Contaminants, if present, are revealed by the appearance of additional peaks; the areas under these peaks provide rough estimates of the extent of contamination. • The technique is also useful for evaluating the effectiveness of purification procedures. • Retention times should be useful for the identification of components in mixtures. Gas chromatography provides an excellent means of confirming the presence or absence of a suspected compound in a mixture.

Quantitative Analysis:• An accuracy of 1% relative is attainable under carefully controlled conditions. • Reliability is directly related to the control of variables; the nature of the sample also plays a part in determining the potential accuracy.

InterfacIng gc wIth SpectroScopIc Methods Gas Chromatography/Mass Spectrometry (GC/MS) • The flow rate from capillary columns is generally low enough that the column output can be fed directly into the ionization chamber of the mass spectrometer. • For packed columns and megabore capillary columns however, a jet separator must be employed to remove most of the carrier gas from the analyte.

APPLICATIONS 1. The characterization of some unformulated drugs (detection of impurity, Process impurities) 2. Limit tests for solvent residues and other volatile impurities in drug substances 3. Quantification of drugs in formulations, particularly if the drug lacks a Chromophore. 4. Characterization of raw materials used in synthesis of drug molecules 5. Characterization of volatile oils, proprietary cough mixtures and tonics and Fatty acids in fixed oils 6. Measurement of drugs and their metabolites in biological fluids. 7. Quantitative analysis of - Methyl testosterone in tablets - Atropine in eye drops - Quantification of Ethanol in a formulation. - Determination of manufacturing and degrading residues by GC

STRENGTHS 1. Capillary column is capable of performing much more efficient Separation than HPLC. 2. Readily automated. 3. Used to determine compounds which lacks Chromophore. 4. Requires only very small samples with little preparation. 5. Good at separating complex mixtures into components. 6. Results are rapidly obtained (1 to 100 minutes). 7. Very high precision. 8. Only instrument with the sensitivity to detect volatile organic mixtures of low concentrations. 9. Equipment is not very complex (sophisticated oven). 10. Mobile phase does not vary and does not require disposal, and even if helium is used as a carrier gas. 11. Mobile phase is cheap compared to the organic solvents used in HPLC.

LIMITATIONS •

Only thermally stable and volatile compounds can be analyzed.

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The sample may require derivitization to convert it to a volatile form.

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Quantitative sample introduction is more difficult because of the Small volumes of sample injected.

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During injection of the gaseous sample proper attention is required.

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Aqueous solution and salts cannot be injected into the instrument

MODEL QP Q1. Define the following 1 Mark i) A gas chromatographic detector used for sample containing 1) halides 2) Carbon & Hydrogen 3) Halogens, peroxides, quinones & Nitro groups ii) Name the technique used in GC for detection of residual solvents Q2. Explain in detail 4 Marks i) Discuss the working of thermal conductivity detector ii) Discuss various types of GC column based on distribution of stationary phase iii) Block diagram of GC and explain in detail about principle and instrumentation of GC.