Sample introduction

A critical point in gas chromatography is sample inlet. It is important to introduce the sample onto the column within the shortest possible time and at the same time to have it in the gas phase. The so called fast evaporation technique is applied for this by heating the injector to high temperature. In theory, gas, liquid or even solid samples can be studied by GC but because of the slow evaporation of solids the samples injected are liquids or gases almost exclusively. Gas samples are injected by means of a six-port switching valve system (Figure 39.). These injectors contain a sample loop of calibrated volume. First the loop is filled with the sample gas by washing it with the sample gas of 5-10 times volume This is necessary to be sure that the loop contains only the components of the sample and no air or remaining eluent gas. Upon switching, the content of the sample loop is washed into the carrier gas stream and injected to the column (Figure 39.).

Figure 39.: six port gas sampling valve

Liquid samples can be injected by means of micro-syringes. The volume of the sample depends on the column parameters. The fast vaporizing inlet is the most often applied one but so called cold techniques such as cool-on-column, large volume injection (LVI) or programmed temperature vaporizer (PTV) are available, too. In case of the fast evaporation method the temperature of the injector should be 50-70 ^oC higher than the boiling point of the highest boiling point sample component. The volume of the injector is a few cm³ and sample vapor should fill this volume. Split/splitless injectors are applied in case of small diameter liquid film columns. The capacity of such columns is small and it is possible to over saturate the stationary phase if ‘high’ amount of sample is injected. To prevent this sample vapor is splitted and only a portion of it (1/10; 1/100) is passed onto the column and the rest of it exits through a split vent. The main disadvantage of the split technique is discrimination among sample components according to their boiling point and high split ratio causes smaller peak

column

area. To prevent discrimination or when the solute concentration is very small the so called splitless method is used. In a splitless inlet there is no split till the complete evaporation of the sample so there is no discrimination and then the excess volume of the sample is vented to prevent the overloading the column. Modern split/splitless injectors can work in direct mode, too, when the whole injected sample is washed onto the column. Of course, in such a case an appropriate column should be installed which is not overloading and the so called liner in the injector should be changed to an appropriate one. This liner (also called inlet or insert) (Figure 40.) is located in the injector where the evaporation occurs so it should be made of deactivated glass or quartz of suitable shape. As sample components can decompose on high temperature metal surfaces a liner is necessary to prevent the contact between sample vapors and high temperature metal parts inside the injector.

Figure 40.: liners

The next problem of fast evaporation is that the applied temperature is significantly higher than the boiling point of the sample components and lots of compound suffers thermal decomposition. If it is a problem then “cool-on-column” injection techniques can be applied.

Here the sample is injected into a column part of wider in diameter and length of a few cm.

This column portion is cooled and then the eluent flow brings the sample into the heated

column oven where the column is heated to the required temperature. Here the sample is vaporized and chromatographic separation starts.

The above description clearly shows that all techniques has advantage and computer controlled heating can rise its temperature up to 400-500 ^oC but can be cooled to the initiate temperature also quickly. Columns can be divided into two groups (Figure 41.). The so called packed columns are 1-5 m length and 2-6 mm internal diameter tubes filled with the appropriate stationary phase. As it has been mentioned already, in case of a separation based on adsorption the stationary phase could be a high surface area solid such as activated charcoal, Al2O3, silica, molecular sieve or organic polymer. In case of absorption based packed column the solid material is impregnated with the stationary liquid phase and then filled into the column.

Figure 41.: classification of GC columns GC columns

Capillary or open tube columns (Figure 42.) are capillaries with length of 10-100 m and internal diameter of 0.2-0.5 mm and their inner surface is covered with the phase. The separation can also be based on adsorption such as with Porous Layer Open Tube (PLOT) columns or partition such as Sorbent Coated Open Tube (SCOT) or Wall Coated Open Tube (WCOT) columns (Figure 41.). PLOT columns contain a porous adsorption layer on their internal walls. The walls are covered with the stationary liquid phase in case of WCOT columns while the wall of SCOT columns has been covered with a carrier layer and the stationary liquid phase is bonded to that coating

Figure 42.: capillary column

There are significant differences among the different column types in terms of capacity and applicability. Packed and the so called wide bore columns have the highest capacity and narrow bore columns have the smallest capacity. However, this order can be modified somewhat with the thickness of the layer of the stationary phase. Today one can choose among a wide variety of columns of different physico-chemical characteristics and the choice is driven by the analytical problem to be solved. Nevertheless we should keep in mind that choosing the best column is crucial in GC techniques as the set of eluents is small compared with HPLC. Main factors affecting GC separation are the followings:

1. length of the column

2. internal diameter of the column 3. type of stationary phase

4. film thickness of the stationary phase.

5. type of eluent gas 6. flow rate of the eluent 7. temperature.

From the point of view of oven temperature there are isotherm and temperature programmed separation methods. In the first case the column temperature is constant while in the second case the temperature is programmed by inserting several ramps during the measurement.

Isotherm analysis is applied usually in case of one component analysis or when the components have similar characteristics while temperature programmed analysis could be suggested in case of diverse multi component samples.

IV.1.4. Detectors

As it has been already discussed the components of the sample are separated on the column and get into the detector one after the other where a signal proportional with their concentration is generated by means of a physical method. The most often applied detectors in GC include Thermal Conductivity Detector (TCD), Flame Ionization Detector (FID), Electron Capture Detector (ECD), Mass Spectrometry (MS) detector and Infrared Spectrophotometric (IR) detectors. Other special detectors are also available such as Photoionization Detector (PID), Flame Photometric Detector (FPD), Pulsed Flame Photometric Detector (PFPD) or Atomic Emission Detector (AED).

The working principle of TCD is that a tungsten or rhodium wire in a small chamber is heated electronically to high temperature. The even eluent flow causes a constant heat transfer i.e.

cooling of the wire. However, when a sample component in the eluent reaches the wire this heat transfer is disturbed resulting stronger or less cooling of the wire so its electric resistance is changing. This resistance change is transformed into a current signal by means of a suitable electric circuit. The bigger the difference in heat conductivity of the eluent and the solute the higher the signal. As H2 and He have the highest heat conductivity these can be the usual choice of eluent for TCD. However, we can use N2 for example if the solute is H2. The TCD is a universal detector but its main disadvantage is its relatively small sensitivity (10^-6 g)

compared with other type of detectors. When hydrogen is used as eluent all safety precautions should be observed to prevent formation of explosive gas mixture with air.

In gas chromatography the most often applied high sensitivity (10^-11 g/s) detector is FID. In this detector a small hydrogen flame can be found with electrodes in it. This means that source of pressurized air is also necessary for a GC instrument equipped with FID detector.

Ions are formed in the flame which results a constant ion flow so electric current. However, when organic chemicals get into the flame large amounts of ions are formed and the ion current is significantly increases which can be detected after amplification. However, this type of detector is not applicable to detect inorganic or not flammable components.

IV.1.5. Application of GC

Gas chromatography techniques can be used in wide field of analytical applications.

They are equally useful in environmental research, food industry, laboratory diagnostics, toxicology and in different phases of drug manufacturing. GC can be applied to analytical investigations of drugs and metabolites extracted from biological samples (blood, plasma, serum, urine, tissues etc.), too. Different Pharmacopeias (Ph.Eur., Ph.Hg.) recommend GC analysis of solvents which include quality control of solvents as well as determination of their contaminants or solvent residues in active pharmaceutical ingredients, excipients and pharmacy products. Gas chromatography is the suggested method for the analysis of oils and fatty acids as fatty acid methyl ester (FAME). Although special columns are already available for analyzing fatty acids they can be determined more conveniently and accurately as their methyl ester. Fatty acid methyl esters are more volatile than the free acid and can be prepared by derivatization. The volatile derivatives can be easily injected and analyzed by gas chromatography. Other derivatization reactions include acylation of carboxylic acids and silylation of sugars, steroids, alcohols and amides.

This chapter has clearly demonstrated that gas chromatography is a high performance analytical technique with diverse applications. More detailed information on gas chromatography is available in the literature. As in the case of other analytical techniques very useful information can be gathered from the catalogs of instrument or column manufacturer companies. For further readings I suggest the following books: Robert L. Grob, Eugene F. Barry (eds.): MODERN PRACTICE OF GAS CHROMATOGRAPHY and Harold M. McNair, James M. Miller: BASIC GAS CHROMATOGRAPHY.