GAS CHROMATOGRAPHY : GAS CHROMATOGRAPHY BY SHEKHAR BHOSALE CONTENTS : CONTENTS HISTORY
ADDITION METHOD HISTORY : HISTORY Chromatography dates to 1903 in the work of the Russian scientist, Mikhail Semenovich Tswett. German graduate student Fritz Prior developed solid state gas chromatography in 1947. Archer John Porter Martin, who was awarded the Nobel Prize for his work in developing liquid-liquid (1941) and paper (1944) chromatography, laid the foundation for the development of gas chromatography and later produced liquid-gas chromatography (1950). PRINCIPLE : PRINCIPLE Gas chromatography - specifically gas-liquid chromatography - involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. PRINCIPLE : PRINCIPLE The gaseous compounds being analyzed interact with the walls of the column, which is coated with different stationary phases. This causes each compound to elute at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness. PRINCIPLE : PRINCIPLE Gas chromatography is in principle similar to column chromatography (as well as other forms of chromatography, such as HPLC, TLC), but has several notable differences. Firstly, the process of separating the compounds in a mixture is carried out between a liquid stationary phase and a gas moving phase, whereas in column chromatography the stationary phase is a solid and the moving phase is a liquid. PRINCIPLE : PRINCIPLE (Hence the full name of the procedure is "Gas-liquid chromatography", referring to the mobile and stationary phases, respectively.) Secondly, the column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography (typically) has no such temperature control. Thirdly, the concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas. PRINCIPLE : PRINCIPLE Gas chromatography is also similar to fractional distillation, since both processes separate the components of a mixture primarily based on boiling point (or vapor pressure) differences. However, fractional distillation is typically used to separate components of a mixture on a large scale, whereas GC can be used on a much smaller scale (i.e. microscale).
Gas chromatography is also sometimes known as vapor-phase chromatography (VPC), or gas-liquid partition chromatography (GLPC). These alternative names, as well as their respective abbreviations, are frequently found in scientific literature. Strictly speaking, GLPC is the most correct terminology, and is thus preferred by many authors The chromatographic process : The chromatographic process Two different substances are partitioned between two phases. Depending on their affinity (toward the stationary phase) will spent different times adsorbed by the stationary phase. Schematic diagram of a gas chromatograph: : Schematic diagram of a gas chromatograph: GASES : GASES The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities. CARRIER GAS : CARRIER GAS Common gases--- Helium, Hydrogen, Nitrogen, argon
Requirements----- 1) Suitable for particular detector in use. 2) Inert 3) Dry and pure 4) Free from organic impurities 5) Regulated TYPES OF INJECTORS : TYPES OF INJECTORS 1) Packed column injectors
2) Split / splitless injectors
3) On column Capillary injectors
4) Programmable split/splitless injectors Split / splitless injectors : Split / splitless injectors For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10-3 mL. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector; Split / splitless injectors : Split / splitless injectors 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 vapourises to form a mixture of carrier gas, vapourised solvent and vapourised 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 Schematic diagram : Schematic diagram COLUMNS : COLUMNS There are two general types of column, packed and capillary (also known as 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 COLUMNS : COLUMNS Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, 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. COLUMNS : COLUMNS In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column; COLUMNS : COLUMNS 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. COLUMNS : COLUMNS COLUMN TEMREATURE(OVEN) : COLUMN TEMREATURE(OVEN) Slide 24: A gas chromatography oven, open to show a capillary column
The column(s) in a GC are contained in an oven, the temperature of which is precisely controlled electronically. (When discussing the "temperature of the column," an analyst is technically referring to the temperature of the column oven. The distinction, however, is not important and will not subsequently be made in this article.) OVEN : OVEN The rate at which a sample passes through the column is directly proportional to the temperature of the column. The higher the column temperature, the faster the sample moves through the column. However, the faster a sample moves through the column, the less it interacts with the stationary phase, and the less the analytes are separated. OVEN : OVEN In general, the column temperature is selected to compromise between the length of the analysis and the level of separation.
A method which holds the column at the same temperature for the entire analysis is called "isothermal." Most methods, however, increase the column temperature during the analysis, the initial temperature, rate of temperature increase (the temperature "ramp") and final temperature is called the "temperature program." OVEN : OVEN A temperature program allows analytes that elute early in the analysis to separate adequately, while shortening the time it takes for late-eluting analytes to pass through the column. Van Deemter : Van Deemter H = A + B/û + C*û
H = height of the theoretical plate
A = Eddy diffusion term (packed column only)
B = Longitudinal band broadening
C = Resistance to mass transfer
u = Average linear gas velocity EDDY DIFFUSION-(A term) : EDDY DIFFUSION-(A term) Analyte molecules follow different pathways around the particles of the stationary phase, some shorter and some longer.
These variations cause residence time of gas molecules to vary giving rise to broadening
Broadening depends on the particle size and geometrical packing factor EDDY DIFFUSION : EDDY DIFFUSION Smaller particles of uniform spherical shape result in low values of term A
For open tubular columns, this term is zero MOLECULAR DIFFUSIONTerm (B) : MOLECULAR DIFFUSIONTerm (B) Molecules of any analyte dissolved in a fluid will diffuse in all the directions with time
If along the axis of the column ---Axial spreading
Extent of spreading is dependent on :- a)- coefficient of diffusion of analyte in MP b)- the total time the sample is in MP. ( Term RESISTANCE TO MASS TRANSFER C) : ( Term RESISTANCE TO MASS TRANSFER C) Transfer of molecules of the analyte can occur only at interface between the two phases, to mantain distribution ratio
Both phases have a finite thickness
At the front edge of the peak the M.P. is rich in analyte and the stationary phase is deficient RESISTANCE TO MASS TRANSFER ( Term C) : RESISTANCE TO MASS TRANSFER ( Term C) The extent of broadening depends on diffusion rates of analyte in the two phases
Diffusion is time dependent and the broadening will be worsened if the flow rate of the M.P. increases
A compromise between the values for A,B and C is required to achieve optimum column efficiency. Van Deemter : Van Deemter H is minimum for a specific value of u
H vs. u is the “Van Deemter Plot” Van Deemter Plot : Van Deemter Plot The detectors : The detectors Types of the detectors:
TCD: Thermal Conductivity Detector
FID: Flame Ionization Detector
ECD: Electron Capture Detector
FPD: Flame Photometric GC Detector
NPD: Nitrogen Phosphorus Detector
Principles of the detectors DETECTORS : DETECTORS There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors. DETECTORS : DETECTORS The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas. Have a look at this tabular summary of common GC detectors: FLAME IONISATION DETECTORS : FLAME IONISATION DETECTORS WORKING OF FLAME IONISATION DETECTORS : WORKING OF FLAME IONISATION DETECTORS The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample OUTSTANDING FEATURES OF FID : OUTSTANDING FEATURES OF FID HIGHLY SENSITIVE TO ORGANIC COMPOUND
LITTILE OR NO RESPONSE TO WATER,CARBONDIOXIDE,CARRIER GAS IMPURITIES & HENCE ZERO SIGNAL WHEN NO SAMPLE IS PRESENT
STABLE BASELINE IS NOT SIGNIFICANTLY AFFECTED BY FLUCTUATION OF CARRIER GAS,FLOW RATE & PRESSURE Basic Principles of Headspace Analysis : Basic Principles of Headspace Analysis Simple Definition: The 'headspace' is the gas space in a chromatography vial above the sample. Headspace analysis is therefore the analysis of the components present in that gas.
Headspace GC is used for the analysis of volatiles and semi-volatile organics in solid, liquid and gas samples. The popularity of this technique has grown over recent years and has now gained worldwide acceptance for analyses of alcohols in blood and residual solvents in pharmaceutical products Basic Principles of Headspace Analysis : Basic Principles of Headspace Analysis A headspace sample is normally prepared in a vial containing the sample, the dilution solvent, a matrix modifier and the headspace. Volatile components from complex sample mixtures can be extracted from non-volatile sample components and isolated in the headspace or gas portion of a sample vial. A sample of the gas in the headspace is injected into a GC system for separation of all of the volatile components. Slide 45: Phases of the Headspace Vial
G = the gas phase (headspace)The gas phase is commonly referred to as the headspace and lies above the condensed sample phase.
S = the sample phaseThe sample phase contains the compound(s) of interest. It is usually in the form of a liquid or solid in combination with a dilution solvent or a matrix modifier.
Once the sample phase is introduced into the vial and the vial is sealed, volatile components diffuse into the gas phase until the headspace has reached a state of equilibrium as depicted by the arrows. The sample is then taken from the headspace Headspace Gas ChromatographyWhen to Use It? : When performing quantitative analysis of volatiles
When entire sample should not be injected into GC
When minimum sample handling is desirable
When high sample throughput is desirable
For trace levels & low to medium concentrations Headspace Gas ChromatographyWhen to Use It? Headspace Gas ChromatographyProcedure : 1) Place Sample in vial and seal it.
2) Place vial in Headspace Autosampler.
3) Collect Analysis results from data station
- Automatic thermostatting to equilibrium for volatiles between the phases.
- Automatic injection onto GC separation column of the gas-phase “extract”. Headspace Gas ChromatographyProcedure Slide 48: Standby Pressure-Balanced Time-Based Sampling Slide 49: Standby
Pressurization Pressure-Balanced Time-Based Sampling Slide 50: Standby
Sampling Pressure-Balanced Time-Based Sampling Pressure-Balanced Time-Based Sampling : Pressure-Balanced Time-Based Sampling Standby
Standby Calibration & standards : Calibration & standards The most straightforward method for quantitative chromatographic analysis involves the preparation of a semi rise of standard solutions that approximate the composition of the unknown. Chromatograms for the standards are then obtained and peak heights or areas are plotted as a function of concentration. A plot of the data should yield a straight line passing through the origin, analyses are based on this plot. INTERNAL STANDARD METHOD : INTERNAL STANDARD METHOD The highest precision for quantitative chromatography is obtained by use of internal standards because the uncertainties introduced by sample injection are avoided. In this procedure, a carefully measured quantity of an Internal standard substance is introduced into each standard and sample, and the ratio of analyte to internal standard peak areas( or heights) serve as the analytical parameter. for this method to be successful, it is necessary that the internal standard peak be well separated from the peaks of all the other components of the sample. AREA NORMILIZATION METHOD : AREA NORMILIZATION METHOD Another approach that avoids the uncertainties associated with sample injection is the area normalization method. Complete elution of all components of the sample is required. In the normalization method, the areas of all eluted peaks are computed, after correcting these areas for differences in the detector response to different compound types, the concentration of the analyte is found from the ration of its area to the total area of all peaks. Slide 55: Thank You