CHROMATOGRAPHIC METHODS OF ANALYSIS. USING OF THIN-LAYER CHROMATOGRAPHY IN THE PHARMACEUTICAL ANALYSIS.
Analytical chemists have designed methods for the analysis of analytes at increasingly lower concentrations and in increasingly more complex matrices. Despite the power of these techniques, they often suffer from a lack of selectivity. For this reason, many analytical procedures include a step to separate the analyte from potential interferents. Two additional separation methods that combine separation qwertand analysis: chromatography.
Chromatographic methods of the analysis (thin-layer, paper, gas and ion-exchange chromatography) were applied for a long time in the qualitative and quantitative analysis of medical products and to an estimation of quantities of some impurity. Recently in connection with increase of requirements to quality of medical products methods of a gas and high-performance liquid chromatography in the analysis of substances and ready medical products became obligatory. Thin-Layer Chromatography is the important method of identity of medicinal substances.
In gas chromatography (GC) the sample, which may be a gas or liquid, is injected into a stream of an inert gaseous mobile phase (often called the carrier gas). The sample is carried through a packed or capillary column where the sample’s components separate based on their ability to distribute themselves between the mobile and stationary phases.
THEORETICAL BASES OF CHROMATOGRAPHIC METHODS.
§ Chromatography is a process which is based on multiple repetition of sorption and desorption of substances. It involves passing a mixture dissolved in a “mobile phase” through a stationary phase.
§ Chromatography (from Greek χρώμα:chroma, color and γραφειν:graphein to write) is the collective term for a family of laboratory techniques for the separation of mixtures.
Chromatographic separations are accomplished by continuously passing one sample-free phase, called a mobile phase, over a second sample-free phase that remains fixed, or stationary. The sample is injected, or placed, into the mobile phase.
As it moves with the mobile phase, the sample’s components partition themselves between the mobile and stationary phases. Those components whose distribution ratio favors the stationary phase require a longer time to pass through the system.
Given sufficient time, and sufficient stationary and mobile phase, solutes with similar distribution ratios can be separated.
§ Sorbtion is absorption process of the gaseous or dissolved substance (sorbate) by firm substance or liquid (sorbent), reverce process is called desorbtion.
Sorbtion is divided on:
§ Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or a liquid (adsorbent), forming a film of molecules or atoms (the adsorbate).
§ Absorption is a physical or chemical phenomenon or a process in which atoms, molecules, or ions enter some bulk phase – gas, liquid or solid material.
§ Chemisorption is a classification of adsorption characterized by a strong interaction between an adsorbate and a substrate surface, as opposed to physisorption which is characterized by a weak Van der Waals force.
At constant temperature adsorption increases:
– with increase of solution concentration;
– with increase in pressure of gas.
Adsorption is usually described through isotherms, that is, the amount of adsorbate on the adsorbent as a function of its pressure (if gas) or concentration (if liquid) at constant temperature. The quantity adsorbed is nearly always normalized by the mass of the adsorbent to allow comparison of different materials.
Langmuir isotherm (red) and BET isotherm (green)
Often molecules do form multilayers, that is, some are adsorbed on already adsorbed molecules and the Langmuir isotherm is not valid. In 1938 Stephan Brunauer, Paul Emmett, and Edward Teller developed a model isotherm that takes that possibility into account. Their theory is called BET theory, after the initials in their last names.
The Langmuir equation or Langmuir isotherm or Langmuir adsorption equation relates the coverage or adsorption of molecules on a solid surface to gas pressure or concentration of a medium above the solid surface at a fixed temperature. The equation was developed by Irving Langmuir in 1916. The equation is stated as:
where: n – quantity (mol) of the adsorbed substance at equilibrium;
n∞ – a maximum quantity of substance which can be adsorbed on sorbent;
b – constant;
C – concentration.
§ In the range of small concentration an isotherm of adsorption is straight line. It is really if bc <<1 so (1 + bc) → 1, then
This equation of straight line adsorption (Henry’s equation).
The quantity of the adsorbed substance will be defined:
– Concentration of substance;
– Sorbent affinity.
CLASSIFICATION OF CHROMATOGRAPHIC METHODS ON MODULAR CONDITION OF PHASES, ON WAY OF MOVING OF PHASES, ON SORPTION MECHANISM, ON PERFORMANCE TECHNIQUES.
1. On physical nature of mobile and stationary phase:
|
Mobile phase |
The gaseous |
The liquid |
|
stationary phase |
||
|
The firm |
gas-solid chromatography |
Liquid adsorption chromatography, thin layer, ion-exchange, ionic, precipitation (sedimentation) chromatography |
|
The liquid |
Partition gas-liquid chromatography |
Partition liquid chromatography, HPLC, gel-chromatography |
2. On depending of sorbtion mechanism:
§ Molecular (interaction between a stationary phase (sorbent) and divided mix components at the expense of intermolecular interaction (Van der Waals forces);
§ chemisorption (ion-exchange, sedimentation, chelation chromatography, oxidation-reduction).
3. Type separation:
§ frontal chromatography;
§ elution – more often a used chromatography;
§ displacement chromatography.
Advantage – concentration of solution does not decrease.
Lack – often bridging of components zone because they are not divided by solvent.
4. Techniques by chromatographic bed shape
§ Column chromatography (it is a separation technique in which the stationary bed is within a tube)
§ Planar chromatography:
The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography)
The mechanism by which solutes separate provides a third means for characterizing a separation. In adsorption chromatography, solutes separate based on their ability to adsorb to a solid stationary phase. In partition chromatography, a thin liquid film coating a solid support serves as the stationary phase. Separation is based on a difference in the equilibrium partitioning of solutes between the liquid stationary phase and the mobile phase. Stationary phases consisting of a solid support with covalently attached anionic (e.g., –SO3–) or cationic (e.g., –N(CH3)3+) functional groups are used in ion-exchange chromatography.
Ionic solutes are attracted to the stationary phase by electrostatic forces. Porous gels are used as stationary phases in size-exclusion chromatography, in which separation is due to differences in the size of the solutes. Large solutes are unable to penetrate into the porous stationary phase and so quickly pass through the column. Smaller solutes enter into the porous stationary phase, increasing the time spent on the column. Not all separation methods require a stationary phase. In an electrophoretic separation, for example, charged solutes migrate under the influence of an applied potential field. Differences in the mobility of the ions account for their separation.
Schematics showing the basis of separation in (a) adsorption chromatography, (b) partition chromatography, (c) ion-exchange chromatography, (d) size exclusion chromatography, and (e) electrophoresis. For the separations in (a), (b), and (d) the solute represented by the solid circle (•) is the more strongly retained.
CHROMATOGRAM AND ITS CHARACTERISTICS: RETENTION TIME, ADJUSTED TIME, PEAK WIDTH ON HALF OF HEIGHT.
A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
!!! adjusted time is proportional to chromatographic resolution of the given component of investigated mix.
adjusted time depends from:
§ The nature chromatographic compounds;
§ The nature of a mobile phase;
§ The nature and weight of stationary phase;
§ Speeds of mobile phase movement;
§ Column temperatures (in a gas chromatography);
§ Lengths of a column;
§ Partition coefficient (than it is more for substance, the more its retention time).
§ retention volume – the volume of mobile phase needed to move a solute from its point of injection to the detector (Vr).
§ baseline width – the width of a solute’s chromatographic band measured at the baseline (w).
§ Peak height h or h’ (from a point of crossing of tangents with a zero line).
§ Peak width µ0,5 – distance between peak points on half of height (or on any other mark on height).
Relative retention time tr¢ and relative adjusted time tr is calculated on equation:
tr¢=t/ts tr= (t – t0) / (ts – t0)
!!! Relative retention times:
• Less depend on external conditions, than retention time;
• Resolve the serial analysis without standard samples of defined substances.
§ Often retention time isn’t measured, but measure retention distance l – distance on chromatogram from a point which corresponds of introduction sample moment to an absciss which correspond of peak maximum.
§ The retention volume it depends on speed u of mobile phase movements
V = t × u
§ The retention factor (delay) R is a relation of moving speed w of the given component and the speed u of mobile phase movements:
R = w /u or R = t0 /t
The capacity factor k – is equal to ratio of relative retention time ¢t= t – t0 of given components to t0:
k = (t – t0) / t0
§ The more the capacity factor k, than more time of investigated component in stationary phase.
Nonideal asymmetrical chromatographic bands showing (a) fronting and (b) tailing. Also depicted are the corresponding sorption isotherms showing the relationship between the concentration of solute in the stationary phase as a function of its concentration in the mobile phase.
CHROMATOGRAPHIC PROCESS: THE THEORY OF THEORETICAL PLATES AND THE KINETIC THEORY.
Martin and Synge theory (theoretical plate)
At the beginning of a chromatographic separation the solute occupies a narrow band of finite width. As the solute passes through the column, the width of its band continually increases in a process called band broadening. Column efficiency provides a quantitative measure of the extent of band broadening.
In their original theoretical model of chromatography, Martin and Synge treated the chromatographic column as though it consists of discrete sections at which partitioning of the solute between the stationary and mobile phases occurs. They called each section a theoretical plate and defined column efficiency in terms of the number of theoretical plates, N, or the height of a theoretical plate, H
!!! A column’s efficiency improves with an increase in the number of theoretical plates or a decrease in the height of a theoretical plate.
The number of theoretical plates in a chromatographic column is obtained by combining equations:
Alternatively, the number of theoretical plates can be approximated as
where w1/2 is the width of the chromatographic peak at half its height.
Rate theory (kinetic theory)
Van Deemter equation is an equation showing the effect of the mobile phase’s flow rate on the height of a theoretical plate.
where:
A = Eddy-diffusion
B = Longitudinal diffusion
C = mass transfer kinetics of the analyte between mobile and stationary phase
u = Linear Velocity.
The constant A depends on the size of particles, their density or density of column filling. The constant B is connected with diffusion factor of molecules in a mobile phase. The constant C characterises kinetics of sorption – desorption process, material transfers and other effects.
The A factor is determined by a phenomenon called Eddy Diffusion. This is also called the multi-path term, as molecular particles of a certain compound have a multitude of options when it comes to finding a pathway through a packed column. The following figure helps in visualizing Eddy diffusion:
Because there is an almost infinite number of different paths that a particle can travel by through a column, some paths will be longer than others. The particles that find the shortest path through the column will be eluted more quickly than those that travel a longer way. In the figure, particle B will be eluted before particle C, and both will be eluted before particle A. Since it is improbable for all particles of one compound to find the shortest path, there will be fractions of the component that will behave like particles A, B, and C. This leads to the broadening of the band. There is little a scientist can do to minimize the Eddy Diffusion factor, as it is influenced by the nature of column being used and by the particles’ movement through that column. The A term is loosely affected by the flow rate of the mobile phase, and sometimes the affect of the flow rate is negligible. It is for this reason that sometimes the Van Deemter equation is written as such:
H = A + B/u + Cu
B/u is called the longitudinal diffusion term, and is caused by the components’ natural migration from a place of high concentration (the center of the band) to a place of lower concentration (either side of the band) within the column. Diffusion6 occurs because molecules in a place of high concentration will tend to spread out to areas of lower concentration to achieve equilibrium. Given enough time, diffusion will result in equilibrium of the diffusing fluid via random molecular motion. The figure below helps to visualize this phenomenon:
At time zero in the figure above, the particles of a compound are generally localized in a narrow band within the separating column. If the mobile phase flow rate is too small or if the system is left at rest, the particles begin to separate from one another. This causes a spread in the concentration distribution of that compound within the column, thus bringing about band broadening for the band of that particular compound. As the time that the system is left still approaches infinity, the compound reaches complete concentration equilibrium throughout the entire column. At this point, there is no definitive band for that component, as a single concentration of that compound is present throughout the entire column. Longitudinal diffusion is a chief cause of band broadening in Gas Chromatography, as the diffusion rates of gaseous species are much higher than those of liquids. It is for this reason that longitudinal diffusion is less of an issue in liquid chromatography. The magnitude of the term B/u can be minimized by increasing the flow rate of the mobile phase. Increasing the velocity of the mobile phase does not allow the components in the column to reach equilibrium, and so will hamper longitudinal diffusion. The flow rate of the mobile phase should not be increased in excess, however, as the term Cu is maximized when u is increased.
Cu is referred to as the mass transfer term. Mass transfer refers to when particles are so strongly adhered to the stationary phase that the mobile phase passes over them without carrying them along. This results is particles of a component being left behind. Since it is likely that more than a single particle of any given compound will undergo this occurrence, band broadening results. This results in a phenomenon called tailing, in which a fraction a component lags behind a more concentrated frontal band. Non-equilibrium effects can be caused by two phenomena: laminar flow and turbulent flow. Laminar flow7 occurs in tubular capillaries, and so is most prominent in Capillary Electrophoresis. Turbulent flow occurs as a result of particles becoming overwhelmed by the stationary phase and is more common in column chromatography. This occurrence can be visualized by observing the figure below:
In the above figure, particles of the adsorbent solid become occupied by particles of the sample. If too many particles of the adsorbent are occupied, particle A will have nothing hindering it from flowing through the column. So, the particles of a single compound separate from one another. Also, as the mobile phase moves through the column, particles of the sample leave the stationary phase and migrate with the mobile phase. However, if the flow rate of the mobile phase is too high, many of the sample particles are unable to leave the stationary phase and so get left behind. These occurrences result in band broadening, as the individual particles of a single compound become less closely packed. The high flow rate of the mobile phase makes it more difficult for the components within the column to reach equilibrium between the stationary and mobile phase. It is for this reason that the Cu term is also called the non-equilibrium factor. Minimization of this factor can be achieved by decreasing the flow rate of the mobile phase. Decreasing the flow rate of the mobile phase gives sample components more time to leave the stationary phase and move with the mobile phase, thus reaching equilibrium.
By observing the Van Deemter equation, it can be deduced that an ideal mobile phase flow rate must be determined to yield the best (lowest) value of H. Decreasing the flow rate too much will result in an increase of the longitudinal diffusion factor B/u, while exceedingly increasing the flow rate will increase the significance of the mass transfer term Cu. So, H can be minimized to a finite limit depending on the various parameters involved in the chromatography being performed.
There is some disagreement on the correct equation for describing the relationship between plate height and mobile-phase velocity.
§ At small mobile-phase velocity the height equivalent to a theoretical plate (HETP) decreases, and then starts to increase.
§ The optimum mobile-phase velocity corresponds to a minimum in a plot of H as a function of u.
§ Optimum velocity of division which gives us a considerable quantity of theoretical plates, and accordingly small HETP, is calculate:
So, the kinetic theory gives a basis for optimisation of chromatographic process.
CHARACTERISTICS OF DIVISION EFFICIENCY: SEPARATION DEGREE, RESOLUTION, NUMBER OF THEORETICAL PLATES, THE HEIGHT EQUIVALENT TO A THEORETICAL PLATE.
The goal of chromatography is to separate a sample into a series of chromatographic peaks, each representing a single component of the sample. Resolution is a quantitative measure of the degree of separation between two chromatographic peaks, 1 and 2, and is defined as
For two peaks of equal size, a resolution of 1.5 corresponds to an overlap in area of only 0.13%. Because resolution is a quantitative measure of a separation’s success, it provides a useful way to determine if a change in experimental conditions leads to a better separation.
Three examples of chromatographic resolution.
If peaks are mutually bridging, definition of peak width of each substance is impossible on separation degree Y :
In chromatography: the qualitative analysis is based on definition of retention time and quantitative – on definition of peak height or area.
THE MAIN PARTS OF CHROMATOGRAPHIC EQUIPMENT.
Requirements to adsorbent (Al2O3, silicagels, the activated coal, porous capillaries on the basis of styrene, divinyl benzene, synthetic zeolites):
Ø Necessary selectivity;
Ø Chemical inertness to mix components;
Ø Availability.
Differential (concentration change
appear instant); are often applied
Detectors
Integrated (fix concentration
change for whole time interval), are applied not often
To group of differential detectors belong:
Thermal Conductivity Detector (TCD)
flame ionization detector
electron capture detector (ECD)
Others depending on properties of system, a modular condition of phases.
GAS CHROMATOGRAPHY (AT CONSTANT TEMPERATURE AND WITH TEMPERATURE PROGRAMMING); THE QUALITATIVE AND QUANTITATIVE ANALYSIS; APPLICATION.
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 (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
In gas chromatography, the mobile phase (or “moving phase”) is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column (a homage to the fractionating column used in distillation). The instrument used to perform gas chromatography is called a gas chromatograph (or “aerograph”, “gas separator”).
The gaseous compounds being analyzed interact with the walls of the column, which is coated with a stationary phase. 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.
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 mobile phase, whereas in column chromatography the stationary phase is a solid and the mobile phase is a liquid. (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.
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-liquid chromatography (GLC), or simply gas chromatography (GC), is a common type of chromatography used in organic 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 (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound.
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 used in scientific literature. Strictly speaking, GLPC is the most correct terminology, and is thus preferred by many authors.
A gas chromatograph with a headspace sampler
GC analysis
A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase. As the chemicals exit the end of the column, they are detected and identified electronically. The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, column length and the temperature.
n The moving phase (or “mobile phase”) is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen.
n The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column.
n 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.
In a GC analysis, a known volume of gaseous or liquid analyte is injected into the “entrance” (head) of the column, usually using a microsyringe (or, solid phase microextraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column. The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time). A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column.
Physical components
Diagram of a gas chromatograph.
Autosamplers
The autosampler provides the means to introduce a sample automatically into the inlets. Manual insertion of the sample is possible but is no longer common. Automatic insertion provides better reproducibility and time-optimization.
Different kinds of autosamplers exist. Autosamplers can be classified in relation to sample capacity (auto-injectors vs. autosamplers, where auto-injectors can work a small number of samples), to robotic technologies (XYZ robot vs. rotating robot – the most common), or to analysis:
Ø Liquid
Ø Static head-space by syringe technology
Ø Dynamic head-space by transfer-line technology
Ø Solid phase microextraction (SPME)
Traditionally autosampler manufacturers are different from GC manufacturers and currently no GC manufacturer offers a complete range of autosamplers. Historically, the countries most active in autosampler technology development are the
Inlets
The column inlet (or injector) provides the means to introduce a sample into a continuous flow of carrier gas. The inlet is a piece of hardware attached to the column head.
Common inlet types are:
Ø S/SL (split/splitless) injector; a sample is introduced into a heated small chamber via a syringe through a septum – the heat facilitates volatilization of the sample and sample matrix. The carrier gas then either sweeps the entirety (splitless mode) or a portion (split mode) of the sample into the column. In split mode, a part of the sample/carrier gas mixture in the injection chamber is exhausted through the split vent.
Ø On-column inlet; the sample is here introduced directly into the column in its entirety without heat.
Ø PTV injector; Temperature-programmed sample introduction was first described by Vogt in 1979. Originally Vogt developed the technique as a method for the introduction of large sample volumes (up to 250 µL) in capillary GC. Vogt introduced the sample into the liner at a controlled injection rate. The temperature of the liner was chosen slightly below the boiling point of the solvent. The low-boiling solvent was continuously evaporated and vented through the split line. Based on this technique, Poy developed the programmed temperature vaporising injector; PTV. By introducing the sample at a low initial liner temperature many of the disadvantages of the classic hot injection techniques could be circumvented.
Ø Gas source inlet or gas switching valve; gaseous samples in collection bottles are connected to what is most commonly a six-port switching valve. The carrier gas flow is not interrupted while a sample can be expanded into a previously evacuated sample loop. Upon switching, the contents of the sample loop are inserted into the carrier gas stream.
Ø P/T (Purge-and-Trap) system; An inert gas is bubbled through an aqueous sample causing insoluble volatile chemicals to be purged from the matrix. The volatiles are ‘trapped’ on an absorbent column (known as a trap or concentrator) at ambient temperature. The trap is then heated and the volatiles are directed into the carrier gas stream. Samples requiring preconcentration or purification can be introduced via such a system, usually hooked up to the S/SL port.
The choice of carrier gas (mobile phase) is important, hydrogen has a larger range of flowrates that are comparable to helium in efficiency . However, helium, may be more efficient and provide the best separation if flow rates are optimised. Helium is non-flammable, and works with a greater number of detectors and older instruments. Therefore, helium is the most common carrier gas used. However, the price of helium has gone up considerably over recent years, causing an increasing number of chromatographers to switch to hydrogen gas. Historical use rather than rational consideration may contribute to its continued preferential use of helium.
Detectors
The most commonly used detectors are the flame ionization detector (FID) and the thermal conductivity detector (TCD). Both are sensitive to a wide range of components, and both work over a wide range of concentrations. While TCDs are essentially universal and can be used to detect any component other than the carrier gas (as long as their thermal conductivities are different from that of the carrier gas, at detector temperature), FIDs are sensitive primarily to hydrocarbons, and are more sensitive to them than TCD. However, an FID cannot detect water. Both detectors are also quite robust. Since TCD is non-destructive, it can be operated in-series before an FID (destructive), thus providing complementary detection of the same analytes.
Other detectors are sensitive only to specific types of substances, or work well only iarrower ranges of concentrations. They include:
Ø Thermal Conductivity detector (TCD), this common detector relies on the thermal conductivity of matter passing around a tungsten -rhenium filament with a current traveling through it. In this set up helium or nitrogen serve as the carrier gas because of their relatively high thermal conductivity which keep the filament cool and maintain uniform resistivity and electrical efficiency of the filament. However, when analyte molecules elute from the column, mixed with carrier gas, the thermal conductivity decreases and this causes a detector response. The response is due to the decreased thermal conductivity causing an increase in filament temperature and resistivity resulting in flucuations in voltage. Detector sensitivity is proportional to filament current while it’s inversely proportional to the immediate environmental temperature of that detector as well as flow rate of the carrier gas.
Ø Flame Ionization detector (FID), in this common detector electrodes are placed adjacent to a flame fueled by hydrogen / air near the exit of the column, and when carbon containing compounds exit the column they are pyrolyzed by the flame. This detector works only for organic / hydrocarbon containing compounds due to the ability of the carbons to form cations and electrons upon pyrolysis which generates a current between the electrodes. The increase in current is translated and appears as a peak in a chromatogram. FIDs have low detection limits (a few picograms per second, but they are unable to generate ions from carbonyl containing carbons. FID compatible carrier gasses include nitrogen, helium, and argon.
Ø Catalytic combustion detector (CCD), which measures combustible hydrocarbons and hydrogen.
Ø Discharge ionization detector (DID), which uses a high-voltage electric discharge to produce ions.
Ø Dry electrolytic conductivity detector (DELCD), which uses an air phase and high temperature (v. Coulsen) to measure chlorinated compounds.
Ø Electron capture detector (ECD), which uses a radioactive beta particle (electron) source to measure the degree of electron capture. ECD are used for the detection of molecules containing electronegative / withdrawing elements and functional groups like halogens, carbonyl, nitriles, nitro groups, and organometalics.[5][4] In this type of detector either nitrogen or 5% methane in argon is used as the mobile phase carrier gas. The carrier gas passes between two electrodes placed at the end of the column, and adjacent to the anode (negative electrode) resides a radioactive foil such as 63Ni. The radioactive foil emits a beta particle (electrode) which collides with and ionizes the carrier gas to generate more ions resulting in a current. When analyte molecules with electronegative / withdrawing elements or functional groups electrons are captured which results in a decrease in current generating a detector response.
Ø Flame photometric detector (FPD), which uses a photomultiplier tube to detect spectral lines of the compounds as they are burned in a flame. Compounds eluting off the column are carried into a hydrogen fueled flame which excites specific elements in the molecules, and the excited elements (P,S, Halogens, Some Metals) emit light of specific characteristic wavelengths. The emitted light is filtered and detected by a photomultiplier tube. In particular, phosphorus emission is around 510-536nm and sulfur emission os at 394nm.
Ø Atomic Emission Detector (AED), a sample eluting from a column enters a chamber which is energized by microwaves that induce a plasma. The plasma causes the analyte sample to decompose and certain elements generate an atomic emission spectra. The atomic emission spectra is defracted by a difraction gradient and detected by a series of photomultiplier tubes.
Ø Hall electrolytic conductivity detector (ElCD)
Ø Helium ionization detector (HID)
Ø Nitrogen–phosphorus detector (NPD),a form a thermionic detector where nitrogen and phosphorus alter the work function on a specially coated bead and a resulting current is measured.
Ø Infrared detector (IRD)
Ø Mass spectrometer (MS) – also called (GC-MS) highly effective and sensitive, even in a small quantity of sample.
Ø Photo-ionization detector (PID)
Another different kind of detector for GC is the photoionization detector which utilizes the properties of chemiluminescence spectroscopy. Photoionization detector (PID) is a portable vapor and gas detector that has selective determination of aromatic hydrocarbons, organo-heteroatom, inorganice species and other organic compounds. PID comprise of an ultrviolet lamp to emit photons that are absorbed by the compounds in an ionization chamber exiting from a GC column. Small fraction of the analyte molecules are actually ionized, nondestructive, allowing confirmation analytical results through other detectors. In addition, PIDs are available in portable hand-held models and in a number of lamp configurations. Results are almost immediate. PID is used commonly to detect VOCs in soil, sediment, air and water, which is often used to detect contaminants in ambient air and soil. The disavantage of PID is unable to detect certain hydrocarbon that has low molecular weight, such as methane and ethane.
Ø Pulsed discharge ionization detector (PDD)
Ø Thermionic ionization detector (TID)
Ø GC Chemiluminescence Detectors
Chemiluminescence spectroscopy (CS) is a process in which both qualitative and quantitative properties can be be determined using the optical emission from excited chemical species. It is very similar to AES, but the difference is that it utilizes the light emitted from the energized molecules rather than just excited molecules. Moreover, chemiluminescence can occur in either the solution or gas phase whereas AES is designed for gaseous phases. The light source for chemiluminescence comes from the reactions of the chemicals such that it produces light energy as a product. This light band is used instead of a separate source of light such as a light beam.
Like other methods, CS also has its limitations and the major limitation to the detection limits of CS concerns with the use of a photomultiplier tube (PMT). A PMT requires a dark current in it to detect the light emitted from the analyte.
Some gas chromatographs are connected to a mass spectrometer which acts as the detector. The combination is known as GC-MS. Some GC-MS are connected to an NMR spectrometer which acts as a backup detector. This combination is known as GC-MS-NMR. Some GC-MS-NMR are connected to an infrared spectrophotometer which acts as a backup detector. This combination is known as GC-MS-NMR-IR. It must, however, be stressed this is very rare as most analyses needed can be concluded via purely GC-MS.
Methods
This image above shows the interior of a Geo Strata Technologies Eclipse Gas Chromatograph that runs continuously in three minute cycles. Two valves are used to switch the test gas into the sample loop. After filling the sample loop with test gas, the valves are switched again applying carrier gas pressure to the sample loop and forcing the sample through the Column for separation.
The method is the collection of conditions in which the GC operates for a given analysis. Method development is the process of determining what conditions are adequate and/or ideal for the analysis required.
Conditions which can be varied to accommodate a required analysis include inlet temperature, detector temperature, column temperature and temperature program, carrier gas and carrier gas flow rates, the column’s stationary phase, diameter and length, inlet type and flow rates, sample size and injection technique. Depending on the detector(s) (see below) installed on the GC, there may be a number of detector conditions that can also be varied. Some GCs also include valves which can change the route of sample and carrier flow. The timing of the opening and closing of these valves can be important to method development.
Carrier gas selection and flow rates
Typical carrier gases include helium, nitrogen, argon, hydrogen and air. Which gas to use is usually determined by the detector being used, for example, a DID requires helium as the carrier gas. When analyzing gas samples, however, the carrier is sometimes selected based on the sample’s matrix, for example, when analyzing a mixture in argon, an argon carrier is preferred, because the argon in the sample does not show up on the chromatogram. Safety and availability can also influence carrier selection, for example, hydrogen is flammable, and high-purity helium can be difficult to obtain in some areas of the world. (See: Helium—occurrence and production.) As a result of helium becoming more scarce, hydrogen is often being substituted for helium as a carrier gas in several applications.
The purity of the carrier gas is also frequently determined by the detector, though the level of sensitivity needed can also play a significant role. Typically, purities of 99.995% or higher are used. The most common purity grades required by modern instruments for the majority of sensitivities are 5.0 grades, or 99.999% pure meaning that there is a total of 10ppm of impurities in the carrier gas that could affect the results. The highest purity grades in common use are 6.0 grades, but the need for detection at very low levels in some forensic and environmental applications has driven the need for carrier gases at 7.0 grade purity and these are now commercially available. Trade names for typical purities include “Zero Grade,” “Ultra-High Purity (UHP) Grade,” “4.5 Grade” and “5.0 Grade.”
The carrier gas linear velocity affects the analysis in the same way that temperature does (see above). The higher the linear velocity the faster the analysis, but the lower the separation between analytes. Selecting the linear velocity is therefore the same compromise between the level of separation and length of analysis as selecting the column temperature. The linear velocity will be implemented by means of the carrier gas flow rate, with regards to the inner diameter of the column.
With GCs made before the 1990s, carrier flow rate was controlled indirectly by controlling the carrier inlet pressure, or “column head pressure.” The actual flow rate was measured at the outlet of the column or the detector with an electronic flow meter, or a bubble flow meter, and could be an involved, time consuming, and frustrating process. The pressure setting was not able to be varied during the run, and thus the flow was essentially constant during the analysis. The relation between flow rate and inlet pressure is calculated with Poiseuille’s equation for compressible fluids.
Many modern GCs, however, electronically measure the flow rate, and electronically control the carrier gas pressure to set the flow rate. Consequently, carrier pressures and flow rates can be adjusted during the run, creating pressure/flow programs similar to temperature programs.
Stationary compound selection
The polarity of the solute is crucial for the choice of stationary compound, which in an optimal case would have a similar polarity as the solute. Common stationary phases in open tubular columns are cyanopropylphenyl dimethyl polysiloxane, carbowax polyethyleneglycol, biscyanopropyl cyanopropylphenyl polysiloxane and diphenyl dimethyl polysiloxane. For packed columns more options are available.
Inlet types and flow rates
The choice of inlet type and injection technique depends on if the sample is in liquid, gas, adsorbed, or solid form, and on whether a solvent matrix is present that has to be vaporized. Dissolved samples can be introduced directly onto the column via a COC injector, if the conditions are well known; if a solvent matrix has to be vaporized and partially removed, a S/SL injector is used (most common injection technique); gaseous samples (e.g., air cylinders) are usually injected using a gas switching valve system; adsorbed samples (e.g., on adsorbent tubes) are introduced using either an external (on-line or off-line) desorption apparatus such as a purge-and-trap system, or are desorbed in the injector (SPME applications).
Sample size and injection technique
Sample injection
The rule of ten in gas chromatography
The real chromatographic analysis starts with the introduction of the sample onto the column. The development of capillary gas chromatography resulted in many practical problems with the injection technique. The technique of on-column injection, often used with packed columns, is usually not possible with capillary columns. The injection system in the capillary gas chromatograph should fulfil the following two requirements:
1. The amount injected should not overload the column.
2. The width of the injected plug should be small compared to the spreading due to the chromatographic process. Failure to comply with this requirement will reduce the separation capability of the column. As a general rule, the volume injected, Vinj, and the volume of the detector cell, Vdet, should be about 1/10 of the volume occupied by the portion of sample containing the molecules of interest (analytes) when they exit the column.
Some general requirements which a good injection technique should fulfill are:
1. It should be possible to obtain the column’s optimum separation efficiency.
2. It should allow accurate and reproducible injections of small amounts of representative samples.
3. It should induce no change in sample composition. It should not exhibit discrimination based on differences in boiling point, polarity, concentration or thermal/catalytic stability.
4. It should be applicable for trace analysis as well as for undiluted samples.
Column selection
The choice of column depends on the sample and the active measured. The main chemical attribute regarded when choosing a column is the polarity of the mixture, but functional groups can play a large part in column selection. The polarity of the sample must closely match the polarity of the column stationary phase to increase resolution and separation while reducing run time. The separation and run time also depends on the film thickness (of the stationary phase), the column diameter and the column length.
Column temperature and temperature program
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.)
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.
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.”
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.
Application
In general, substances that vaporize below ca.
Various temperature programs can be used to make the readings more meaningful; for example to differentiate between substances that behave similarly during the GC process.
Professionals working with GC analyze the content of a chemical product, for example in assuring the quality of products in the chemical industry; or measuring toxic substances in soil, air or water. GC is very accurate if used properly and can measure picomoles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.
In practical courses at colleges, students sometimes get acquainted to the GC by studying the contents of Lavender oil or measuring the ethylene that is secreted by Nicotiana benthamiana plants after artificially injuring their leaves. These GC analyses hydrocarbons (C2-C40+). In a typical experiment, a packed column is used to separate the light gases, which are then detected with a TCD. The hydrocarbons are separated using a capillary column and detected with an FID. A complication with light gas analyses that include H2 is that He, which is the most common and most sensitive inert carrier (sensitivity is proportional to molecular mass) has an almost identical thermal conductivity to hydrogen (it is the difference in thermal conductivity between two separate filaments in a Wheatstone Bridge type arrangement that shows when a component has been eluted). For this reason, dual TCD instruments are used with a separate channel for hydrogen that uses nitrogen as a carrier are common. Argon is often used when analysing gas phase chemistry reactions such as F-T synthesis so that a single carrier gas can be used rather than 2 separate ones. The sensitivity is less but this is a tradeoff for simplicity in the gas supply.
Qualitative analysis:
Generally chromatographic data is presented as a graph of detector response (y-axis) against retention time (x-axis), which is called a chromatogram. This provides a spectrum of peaks for a sample representing the analytes present in a sample eluting from the column at different times. Retention time can be used to identify analytes if the method conditions are constant. Also, the pattern of peaks will be constant for a sample under constant conditions and can identify complex mixtures of analytes. In most modern applications however the GC is connected to a mass spectrometer or similar detector that is capable of identifying the analytes represented by the peaks.
Pharmacopoeia offers three methods for identification:
1) comparison of retention times of analyzed substance in the investigated sample and in a comparison solution (a standard solution of investigated substance);
2) comparison of relative retention times of analyzed substance in the investigated sample and a comparison solution (if precision of condition chromotographic analyses isn’t possible);
3) comparison of chromatogram of investigated sample with chromatogram of comparison solution or with chromatogram, resulted in separate article (for preparations of herbal and animal origin).
n Separation process is based on differences in fugitiveness and solubilities separated components.
n The quantitative analysis can be made only in the event that the substance is heat-resistant, that it evaporates reproducibility and eluateed from column without decomposition. At substance decomposition on chromatogram are artificial peaks which belong to decomposition products.
Quantitive analysis:
n The area under a peak is proportional to the amount of analyte present in the chromatogram. By calculating the area of the peak using the mathematical function of integration, the concentration of an analyte in the original sample can be determined. Concentration can be calculated using a calibration curve created by finding the response for a series of concentrations of analyte, or by determining the relative response factor of an analyte. The relative response factor is the expected ratio of an analyte to an internal standard (or external standard) and is calculated by finding the response of a known amount of analyte and a constant amount of internal standard (a chemical added to the sample at a constant concentration, with a distinct retention time to the analyte).
n Pharmacopoeia demands to make definition of the quantitative contents through the areas of peaks, if symmetry factor of peak from 0.8 to 1.20 it is possible to apply in place of the areas by peak heights.
n In case of usage of temperature programming the quantitative analysis is made only through the peak areas.
Symmetry factor of peak: m0.05/2А,
where: m0.05 – width to peak on 1/20 of peak height;
A – distance between a perpendicular lowered from peak maximum and forward border of peak on 1/20 peak height.
Methods of quantitive analysis
1. Normalization method.
Accept the sum of all peak parametres (Sh, or SS, or S width of all peaks) for 100 %. Then the ration of height of individual peak to sum of heights or the ration of one peak area to sum of the areas and multiply on 100 will characterise W(component) (%) in a mix.
This method is used when it is identical dependence of value of the measured parametre from concentration of all components of a mix.
2. Normalization method with calibration factors
In this method accept the sum of all peak parametres for 100 % with due regard for sensitivity of the detector. Differences in sensitivity of the detector are considered with the aid of correction factors for each component. One of dominating components of a mix consider comparative and correction factor for it accept equal to one. Calibration factor Кі is calculated:
Сi – concentration of i-component in a modelling mix with standard substance;
Сstan – concentration of standard substance;
Пstan and Пi – parametres of substance peaks – standard and i-component.
For 100 % accept the sum of corrected parametres КіПі and result of the analysis is calculated such as in normalization method:
Advantage: 1) metered flow of sample isn’t necessary;
2) not absolute identity of analysis conditions at repeated definitions.
3. A method of absolute calibration (the most exact).
Experimentally define dependence of peak height or area from substance concentration and plot calibration chart. On calibration chart calculate concentration of analyzed substance.
It is the basic method of definition of impurity.
Advantage: does not demand separation of all components on sample, but only that substance are necessary for defining.
4. A method of the internal standard.
Is based on introduction in an analyzed mix of precisely known quantity of standard substance. As standard substance choose the substance similar on physical and chemical properties to components in mix, but not necessarily it is component of a mix.
To an investigated solution and a standard solution of investigated substance add strictly identical quantity of the internal standard.
After chromatography measure peak parametres of an analyzed component and the internal standard on chromatogram of investigated solution and the same parametres on chromatogram of standard solution of defined substance.
Advantage: 1) the account of chromatograph duty (t°, u mobile phase).
2) increase of analysis accuracy because of independence of reproducibility parallel chromatographic experiment series and chromatography of standard and investigated solutions.
Requirements to the internal standard:
ü Good solubility in sample and chemical inertness to components of an analyzed mix, stationary phase and the firm carrier.
ü The internal standard choose from compounds which similar to objects of the analysis on structure and fugitiveness.
ü Quantity of the internal standard in sample select so that the ration of peak areas of the standard and defined substance was close to 1.
ü The peak of the internal standard on chromatogram should take places affinity to peaks of compounds – objects of the analysis, not being imposed neither on them, nor on peaks of other substances.
ü The internal standard should not contain impurity which are imposed with peaks of defined substances-components of sample.
ü If define in sample two and more substances (which considerably differ retention times) so it is expedient to use 2 and more internal standards.
Application in the pharmaceutical analysis, technology and the toxicological analysis:
1. Quality assurance of substances and medicinal forms – identification and quantitative definition of the flying residual solvents, which may be in drugs after their reception.
2. Definition of ethanol content and other organic solvents in ready drugs.
3. Definition of preservatives (Nipagin, sorbic acid) in children’s syrups.
4. Definition of some preservatives in injection solutions
5. Quality assurance galena preparations.
