A gas chromatograph essentially comprises of six vital components, namely :
(a) Carrier Gas Regulator and Flow Meter,
(b) Sample Injection System,
(c) Separation Column,
(d) Thermal Compartment,
(f) Recording of Signal Current, and
These components shall be discussed briefly in the sections that follow : Figure 29.2, gives the sche-matic diagram of a standard GLC equipment showing the various parts :
The sample is introduced into the vaporizer and enters the column along with the carrier gas at a constant flow through the detector oven. The reference sample also passes through the detector oven into the column which is maintained by column-oven heat control device. The detector picks up the signals of the sample as well as the reference substance one after the other which is duly amplified and the signal current recorded on a strip-chart recording device or other suitable means. After passing through the detector oven the vapours of the sample plus the carrier gas leaves the equipment through an exhaust pipe.
Note : Ultrapure N2 for use in flame-ionization devices may be generated by the Serfass Apparatus available commercially.
The various carrier gas used in GC along with their characteristic features are stated below :
H2 : It has a distinctly better thermal conductivity and lower density. Demerits are its reactivity with unsaturated compounds and hazardous explosive nature,
He : It has an excellent thermal conductivity, low density, inertness and it permits greater flow rates. It is highly expensive,
N2 : It offers reduced sensitivity and is inexpensive, and
Air : It is employed only when the atmospheric O2 is beneficial to the detector separation.
Importantly, the operating efficiency of a chromatograph is directly dependent on the maintenance of a highly constant carrier gas-flow-rate. Carrier gas passes from the tank through a toggle value, a flow meter, a few feet of metal capillary restrictors, and a 0-4 m pressure gauze. The flow rate could be adjusted by means of a needle value mounted on the base of the flow meter and is controlled by the capillary restrictors. On the downstream side of the pressure regulator, a tee (T) may split the flow and direct it to the sample and the reference side of the detector.
The sample injection system is very important and critical because GC makes use of very small amounts of the samples. A good and ideal sample injection system should be the one where the sample must not—
(i) be decomposed at the point of injection,
(ii) create pressure surges, and
(iii) undergo fractionation, condensation or adsorption of components during the course of transfer to the column.
There are different modes of handling liquid, solid and gaseous samples in a GC which will be discussed briefly here :
(a) Liquid Samples : They are usually injected by hypdermic syringes through a self-sealing silicon-rubber septum into a preheated-metal-block flash evaporator. The sample is vapourized as a ‘plug’ and carried right into the column by the respective carrier gas. Sample size ranges between 1–10 μ l.
(b) Solid Samples : These are either dissolved in volatile liquids (solvents) or temporarily liquefied by exposure to infra-red heat.
(c) Gas Samples : They are best handled and injected by gas-light syrings or a gas-sampling valve, usually termed as a stream-splitter. In the simplest form this is merely a glass-system (Figure 29.2A) made up of three stop-cocks, between two of which there is a standard volume wherein the ‘gas’ is trapped. Gas from this bypass-capillary-loop is introduced right into the column by sliding or rotating a valve to connect the loop with the stream of carrier gas.
It is also known as the ‘chromatographic column’. In reality the heart of a GC is the column duly packed or capillary in which the separation of constituents is materialized. The packed-column is usually a tubing having an internal diameter of 4.0 mm and made up of stainless-steel, copper, cupronickel or glass either bent in U-shape or coiled. Its length varies from 120 cm to 150 M.
The general requirements of a liquid phas are :
· Differential partitioning of sample components,
· Reasonably good solvent properties for components,
· High thermal stability, and
· A lower vapour pressure at the column temperature.
Table 29.1, illustrates the characteristic features of some typical liquid-phases used in GC :
A precise control of the column temperature is not only a must but also a requisite, whether it is intended to maintain an invariant-temperature or to provide a programmed-temperature. Importantly, the temperature of the column oven must be controlled by a system that is sensitive enough to changes of 0.01°C and that main-tains an accurate control to 0.1°C. In normal practice, an air-bath chamber surrounds the column and air is circulated by a blower through the thermal compartment. However, separate temperature controls are very much desirable for the vaporizer block as well as the detector-oven.
More recently, programmes are also available that features both in linear and non-linear temperature programming as sample and reference columns. The compartment temperature can also be raised at various rates upto a maximum of 60 °C min–1 in the lower-temperature ranges and about 35 °C min–1 at higher temperatures.
There are in all six different kinds of detectors used in ‘Gas Chromatography’, namely :
(i) Thermal conductivity detector (TCD),
(ii) Flame ionization detector (FID),
(iii) Electron capture detector (ECD),
(iv) Thermionic detector (NP–FID)
(v) Flame photometric detector (FPD), and
(vi) Photoionization detector (PID).
The first three detectors are invariably used in GLC and shall be discussed in details below ; whereas a passing reference shall be made with respect to the second three detectors.
The thermal conductivity detector, or katharometer, was the first ever detector employed for GLC; and is still being used today be virtue of its versatility, stability, simplicity and above all the low-cost.
Principle : The underlying principle of TCD is that the ability of a gas to dissipate heat, i.e., its thermal conductivity, from a heated body shall change with the composition of the gas. It may be further explained by the fact that each specific carrier gas shall have a characteristic thermal conductivity that is picked up first-and-foremost by the equilibrium temperature of the detecting element to afford a baseline signal. Evidently, the thermal conductivity of the mixture of carrier gas plus sample must be altogether different from that of pure carrier gas ; and while the mixture takes its course through the detector, an obvious change in the temperature of the detecting element is duly recorded as a signal.
Figure 29.3 shows a simple diagram of a thermal conductivity detector. It essentially consists of two cells of small volumes, made within a metal block, termed as reference cell and sample cell. Each cell has a resistance wire or thermister or filament that possesses a high temperature coefficient or resistance i.e., the resistance varies appreciably with slight variation in temperature. These two resistances, namely : reference cell (R) and sample cell (S) are included in two arms of a Wheatstone Bridge. Now, the carrier gas is passed into both the cells, but the column-effluents are allowed to enter only the sample cell. Thus, the temperature of the sample cell changes due to widely different thermal conductivity of the sample component than that of the carrier gas, thereby causing a change in resistance of (S) and the Wheatstone Bridge gets unbalanced. The off-balance current is transmitted to the recorder that finally draws the elution-curve for the sample(s) undergoing chromatographic separations.
(i) First turn the carrier gas on and then switch on filament-current/detector block heater, and
(ii) Do not off the carrier gas before switching off the detector current or before the detector block has attained ambient temperature. This saves the filament from being damaged and enhances its life-span considerably.
The general class of ‘ionization detectors’ comprise of the following important detectors, namely :
· Flame ionization detector,
· Electron capture detector,
· Thermionic detector, and
· Photoionization detector.
No other detector till date has surpassed the flame ionization detector (FID) as a universal gas chroma-tographic detector. It hardly meets, all the characteristic features of TCD in terms of simplicity, stability, and versatility besides having two distinctly positive plus points :
(i) Its linearity over a wider concentration range, and
(ii) It being more sensitive with less flow and temperature dependency.
Principle : First, the principles of operation for all ionization detectors shall be discussed briefly and then the actual principles with specific details would be described under each particular detector.
Generally, the fundamental physical process underlying the operation of an ionization detector is the conduction of electricity by gases. At normal temperatures and pressures a gas essentially behaves as a perfect electrical insulator. However, if electrically charged particles (ions and electrons) are produced in a gas, it becomes a conductor. In other words, their free motion in the direction of the electrical field renders the gas conducting. Assuming a situation, when a vapour is held between two electrodes to which a voltage is applied, practically and absolutely no current shall flow at all in the electrical circuit until and unless charged particles are introduced. The quantum of electric current thus generated would become the signal of the ionization detector. On applying adequate voltage to the electrodes, all of the ions would be collected, and hence the ion-current shall be directly proportional to the number of ions between the electrodes. As the presence of only a few ions are capable of exhibiting the conductivity of the gas; therefore, ionization detectors are usually very sensitive.
Principle of FID : The underlying principle of FID is that invariably a mixture of hydrogen-oxygen or hydrogen-air flame burns with the generation of comparatively fewer ions, but when an organic compound viz., most pharmaceutical substances is ignited in such a flame, ion production gets enhanced dramatically. There-fore, when such a flame is held between two electrodes to which a voltage ranging between 100-300 V is applied, it would instantly give rise to an ion current on burning an organic compound in the flame.
Figure 29.4, illustrates a schematic diagram of a flame-ionization detector. It comprises of a posi-tively charged ring (also referred to as cylindrical collector electrode), whereas the flame jet serves as the negative electrode. The flame jet has two inlets ; from the bottom of the column effluent is introduced and from the side H2 to form the fuel, whereas air is let in uniformly around the base of the jet.
In the domain of gas chromatography the electron capture detector (ECD) enjoys the reputation of being one of the most sensitive as well as selective detectors. However, this valuable detector needs to be handled with a lot of skill and expertise so as to achieve wonderful and dependable results.
Principle : ECD belongs to the general class of ionization detectors, the underlying principles of which have already been discussed. In ECD specifically a β-emitter serves as a source of radiation to generate the ions that helps in ionizing the carrier gas molecules to form positive ions and free electrons as expressed in the following Equation (e) :
C + radiation → C+ + e– ...(e)
In a situation when the said phenomenon is conducted between a pair of charged electrodes, the mobility of the lighter negative ions i.e., the electrons, would be much higher in comparison to the heavier positive ions
i.e., the charged carrier-gas-molecules, thereby ruling out the possibility of their ‘recombination’. Thus mostly the cations and electrons will be collected, while generating a standing current that forms the baseline-signal of the ECD detector. At this stage, if an organic molecule, (i.e., a pharmaceutical substance) possessing a com-paratively high electron affinity is introduced, a portion of the electrons shall be captured to produce negatively charged ions. These heavy-negative-ions will have less mobility as compared to the electrons ; therefore, they will have no other coice than to unite with positive ions. Thus, the net result would be fewer ions and electrons available to migrate to the electrodes, thereby causing a marked and pronounced reduction in the standing current of the detector. Ultimately, this observed current decrease represent as the ‘signal’ of the electron capture detector.
Figure 29.5, depicts the diagram of an electron capture detector. The metal block of the detector housing itself serves as a cathode, whereas an electrode polarizing lead suitably positioned in the centre of the detector housing caters for a collector electrode (anode). The radioactive source from a beta-emitter is introduced from either sides of the detector housing below the electrode polarizing lead.
The column-effluent is passed into the detector from the bottom whereas its exhaust goes out from the top.
The very name suggests, the thermionic detector functions on the principle of ion-current generated by the thermal production of ions. It may also be invariably termed as a nitrogen detector, a sulphur detector, a phophorus detector, and a halogen detector by virtue of the fact that its specificity in detecting organic compounds essentially containing these elements. Furthermore, it is also widely known as NP-FID because it is invariably employed for carrying out the analysis of N- or P-containing organic compounds.
Brody and Chaney* in 1966, were the first and foremost to describe the flame photometric detector (FPD) which unfortunately could not get enough recognition in the field of gas chromatographic analysis due to the following reasons, namely :
(i) Its selectivity, and
(ii) Its poor commercial availability.
It solely operates on the principle of photon emission. If P- or S-containing hydrocarbons are ignited in a hydrogen-rich flame, it gives rise to chemiluminescent species spontaneously which may subsequently be detected by a suitably photomultiplier device. Hence, FPD is regarded as a specific detector for P- or S-containing compounds.
Lovelock** in 1960, first introduced the photoionization detector but unfortunately its reported usages have been more or less scarce.
PID belongs to the generic class of ionization detectors whose principles have already been discussed earlier. As the very name signifies the PID induces ionization via photons emitted by an UV-lamp. A PID detector makes use of a photon energy of 10.2 electron volts (eV) emitted as a Lyman alpha line***. Only such compounds having ionization potentials less than 10.2 eV shall absorb the UV-radiation and be subse-quently converted to positive ions. Two-charged electrodes serve as an electric field in the detector, the cathode becoming the collector electrode for the ions. The ion-current thus generated, that will be directly proportional to the ion concentration, then becomes the signal of the detector.
In general, the signal from a gas chromatograph is recorded continuously as a function of time by means of a potentiometric device. Most frequently, a recorder of 1-10 mV full-scale deflection (~ 10 inches) and having a response time 1 second or less is quite adequate.
Variable chart speeds between the range of 5-50 mm. min–1 are most preferable in GC.
Essentially in a potentiometric recorder, the input signal is balanced continuously by a feedback signal making use of a servomechanism ; whereby a pen strategically connected to this system moves proportionally along the width of the chart paper, thus recording the signal, whereas simultaneously the chart paper keeps moving at a constant speed along its length.
The following important points should be noted before operating a recorder, namely :
(i) Its ‘zero’ must be adjusted (or synchronized) with the ‘input zero’ otherwise the baseline might shift with alterations in attenuation of the signal,
(ii) The amplifier gain must also be adjusted duly so as to avoid completely the dead-base and oscilla-tion,
(iii) A recorder with inadequate shielding from AC circuits would display shifting of its zero point, and
(iv) A reasonably good recorder having quality performance must be employed so as to achieve correct recording of analog-signal, a topmost priority towards quantitative accuracy and precision.
An ‘intergrator’ may be regarded as a device that essentially facilitates simultaneous measurement of areas under the chromatographic peaks in the chromatogram either by mechanical or electronic means. It is, however, pertinent to mention here that ‘manual techniques’ for determining peak areas are known, such as :
‘triangulation’, cutting and weighting of peaks, planimetry, but all these methods are quite time consuming, tedious and not accurate. Hence, based on the actual need, incorporation of an appropriate integrator in a reasonably good GC-set up is an absolute necessity.
There are two types of integrators generally employed in GC, namely :
(a) Ball and Disk Integrator : This is nothing but a purely mechanical device and installed at one end of the very strip-chart recorder itself. It carries a pen that writes along a span of about one inch, reserved for integrator on the recorder chart paper at the end. The zero line of the integrator moves almost parallel to the base line of the chromatogram and as soon as a peak appears on the recorder, the integrator-pen starts moving from right to left the vice-versa within its one-inch strip. Each one-inch traverse (counted along projection parallel to signal axis) is usually assigned a value of 100 counts ; the total number of counts corresponding to a peak are directly proportional to the area of the peak.
The type of mechanical integrator* affords fairly good accuracy and precision ; and above all it is quite cheap.
(b) Electronic Integrator : An ‘electronic integrator’ is definitely a much superior, accurate and dependable device wherein the GC-signal is converted to a frequency pulse that are accumulated corresponding to a peak and later on digitally printed out as a measure of the peak area. The main advantages of an electronic integrator are, namely :
(i) Provides a much wider linear range,
(ii) Changing the ‘attenuation’ is not required, and
(iii) Offers highest precision in peak-area measurement.
Of course, the electronic integrators are quite expensive
Precision of the TWO methods : The ‘electronic integrator’ is almost 3 times** more accurate and precise than the ‘ball and disc integrator’ :
GC-Computer System : Nowadays, a large number of data-processing-computer-aided instruments for the automatic calculation of various peak parameters, for instance : relative retention, composition, peak areas etc., can be conveniently coupled with GC-systems. A commercially available*** fairly sophisticated computer system of such type are available abundantly that may be capable of undertaking load upto 100 gas-chromatographs with ample data-storage facilities. In fact, the installation such as ‘multi GC-systems’ in the routine analysis in oil-refineries and bulk pharmaceutical industries, and chemical based industries have tre-mendously cut-down their operating cost of analysis to a bare minimum.