Main gas chromatographic detectors used in brewing analytics

Gas chromatography is a very important analytical technique used for the determination of flavors which content is significant not only for monitoring of quality of final product but also in testing of new technological procedures. In brewing analytics the most frequently used gas chromatographic detectors are flame ionization detector (FID), electron capture detector (ECD), and flame photometric detector (FPD). This work is focused on principles of these detectors, their advantages and limitations during using in brewing analytics. Gas chromatography is a very important analytical technique used for the determination of flavors which content is significant not only for monitoring of quality of final product but also in testing of new technological procedures. In brewing analytics the most frequently used gas chromatographic detectors are flame ionization detector (FID), electron capture detector (ECD), and flame photometric detector (FPD). This work is focused on principles of these detectors, their advantages and limitations during using in brewing analytics.

1 INTRODUCTION

Official methods of brewery institutions as European Brewery Convention (EBC), Mitteleuropäische Brautechnische Analysenkommission (MEBAK), The Institute of Brewing (IOB) or The American Society of Brewing Chemists (ASBC) include gas chromatographic procedures used for the determination of beer flavors. Beer flavors or some contaminants can be determined by this separation procedure as in intermediate products as in final beer. The review of these compounds including used detector is shown in Tab. 1.

Tab. 1 List of compounds determined by gas chromatography in brewing analytics according to detector type

Flame ionization detector (FID) is the most useful gas chromatographic (GC) detector available and by far that most commonly used in GC analyses. In comparison with FID electron capture detector (ECD) and flame photometric detector (FPD) are characterized by higher sensitivity and higher selectivity for selected analytes and so these detectors are also often used.

A detector that can selectively respond to compounds of real inte - rest in a mixture and ignore those compounds of little interest is potentially able to improve efficiency by two ways. Firstly the amount of sample clean-up required to remove unwanted and interfering compounds from the sample, may be reduced thereby simplifying preanalysis sample handling. Secondly, analysis time on the chromatograph may be shortened since it may no longer be necessary to achieve complete separation of all components in the mixture.

It is for these two reasons that the selective detectors are the most frequently used ones. However, while selective detectors can provide undoubted benefits, their use can cause other problems, particularly when their mode of operation is not fully understood or their capabilities not fully utilized.

In this paper the principles of above mentioned detectors are described together with their utilization in brewing analytics.

2 THE FLAME IONIZATION DETECTOR (FID)

The flame ionization detector FID has a very wide dynamic range, a high sensitivity and (with the exception of a few low molecular weight compounds) will detect all substances that contain carbon. The first FID was described about the same time by Harley and Pretorious (25), and McWilliams and Dewer (26). Hydrogen is mixed with the column eluent and burned at a small jet. Surrounding the flame is a cylindrical electrode and a relatively high voltage is applied between the jet and the electrode to collect the ions that are formed in the flame. The resulting current is amplified by a high impedance amplifier and the output fed to a data acquisition system or a potentiometric recorder. The detector usually requires three separate gas supplies together with their precision flow regulators. The gases normally used are hydrogen for combustion, helium or nitrogen as the carrier gas and oxygen or air as the combustion agent. The detector is normally thermostatted in a separate oven; this is not because the response of the FID is particularly temperature sensitive but to ensure that no solutes condense in the connecting tubes.

2.1 The response mechanism of the FID

The ionizing mechanism of the FID was carefully examined by Ongkiehong (27) and Desty (28) in 1960 and it would appear no such detailed evaluation of the detector has been carried out since. The ionization mechanism in the FID flame was originally thought to be similar to the ionization process in a hydrocarbon flame which was studied intensively by Calcote and King (29) and Schuler and Weber (30) in the mid–1950s. The present generally accepted explanation is that ions are not formed by thermal ionization but by thermal emission from microscopic carbon particles that are formed during the combustion process. Consequently the dominating factor in the ionization of organic material is not their ionization potential but the work function of the carbon that is transiently formed during their combustion.

The flame plasma contains both positive ions and electrons which are collected on either the jet or the plate depending on the polarity of the applied voltage. Initially, the current increases with applied voltage, the magnitude of which depends on the electrode spacing. The current continues to increase with the applied voltage and eventually reaches a plateau at which the current remains sensibly constant. The voltage at which this plateau is reached also depends on the electrode distances. Once electron/ion pair production is initiated the recombination starts to take place. The longer the ions take to reach the electrode the more recombination takes place. Thus, the greater the distance between the electrodes and/or the lower the voltage, the greater the recombination. This is substantiated by the curves obtained by Ongkiehong: the plateau is reached at a lower voltage when the electrodes are closer together. It is seen that the plateau level is the same for both electrode conditions and it is assumed that on the plateau, all ions and electrons being produced in the flame are collected. In practice the applied voltage would be adjusted to suit the electrode distance to ensure that the detector operates under conditions where all electrons and ions are collected.

2.2 The operation of the FID

The FID is probably the simplest, easiest and most reliable detector to operate. Generally the appropriate flow rates for the different gases are given in the detector manual. Hydrogen flows usually range between 20 and 30 ml per min, air flows are about 6 times the hydrogen flow e.g. 120 to 200 ml per min. The capillary column flow rate may be less than 1 ml per min for very small diameter columns. The mobile phase can be any inert gas–helium, nitrogen, argon etc. To some extent the detector is self-cleaning and rarely becomes fouled. However, this depends a little on the substances being analyzed. In case of brewing and malting analyses the possibility of contamination of FID is minimized.

3 THE ELECTRON CAPTURE DETECTOR (ECD)

The electron capture detector ECD utilizes the fundamental observation that the conductivity of gases in an ionization chamber can be drastically altered by the presence or absence of contamination in the gas. Thus the ECD consists of an ionization chamber containing a radioactive source, usually nickel – 63 (characterized by long lifetime and temperature stability up to 450 °C), with a stream of inert gas usually nitrogen, flowing through it. The β-ray emanation from the source causes ionization of the inert gas with a consequent liberation of free electrons. This may be written simple as (1):

(1) N₂ + β-ray ↔ N₂⁺ + e⁻

The ionization chamber is connected to a low voltage potential and a current measuring device. The applied potential causes the liberated electrons to migrate to the anode where collection occurs and a current flow is established. By careful adjustment of the applied potential, an equilibrium can be established whereby the electrons produce in the ionizing process are all collected at the anode. This can be achieved because the mobility of the free electrons is greater than that of the positive ions and hence free electrons migrate to the anode before recombination can occur.

Therefore, in the steady state a constant current known as the standing current, can be measured flowing in the chamber. The area between the source and the anode where electron liberation is occurring is known as the plasma region. If an electrophilic compound is now introduced to the chamber, a reaction will occur between this compound and the free electrons as follows (2,3):

(2) e⁻ + AB ↔ AB⁻ non-dissociative
(3) e⁻ + AB ↔ A⁰ + B⁻ dissociative

As a result of these reactions, the fast moving electrons are replaced by slow moving negative ions. These slow negative ions take longer to migrate to anode and so have a higher probability of recombination with positive ions before reaching the anode than the electrons. Thus the nett results of introducing an electrophilic compound will be a decrease in the amount of current flow measured in the chamber.

The free electrons may in fact be moving so fast to the anode after their production that these necessary collisions and reactions do not have time to occur before the electron reaches the anode. So it is necessary to slow down the free electrons. The make-up gas, usually nitrogen, is used for this. The mobility of electrons is reduced by nonelastic collisions with the electrons (31).

3.1 Effect of temperature

The reaction between the sample molecule and free electros can be of two types – dissociative and non-dissociative (see equation 2,3). The dissociative reaction requires considerable energy for the dissociation of the sample molecule to occur during the reaction. An increase of detector temperature will effectively increase the energy of both the sample molecule and the electron and so will increase the ease of the dissociative reaction. The non-dissociative reaction, however, results in an increase of the energy of the sample molecule which has to be dispersed before a stable complex can be formed. An increase of detector temperature in this case would result in less chance of a non-dissociative reaction occurring.

Very simply then, the detector temperature can influence the sensitivity of the detector. For non-dissociative reactions, as low a detector temperature as possible, as is compatible with column temperature, should be used. For dissociative reactions, the higher the detector temperature the greater its sensitivity.

The type of reaction involved can be easily identified by injection of the sample at different detector temperatures. A decision can then be taken on the two responses as to whether the detector should be operated at a low or high temperature.

The determination of vicinal diketones (diacetyl and 2,3-pentanedione) and chlorinated aliphatic hydrocarbons in beer are examples of these two different principles. Fig. 1 shows the effect of increasing temperature on the response of those two groups of compounds. During vicinal diketones analyse non-dissociative reactions take part and the lowest ECD temperature is recommended. While in aliphatic chlorinated hydrocarbons determination dissociative reactions are of use. The results were obtained in our laboratory using Thermo Scientific Trace Ultra gas chromatograph.

3.2 Practical notes

For high degree of sensitivity and selectivity of electron capture detector the following points should be noted and adhered to:

Always keep the detector temperature well above that of the column to prevent condensation of sample and/or stationary phase in the detector.
Use small-sized samples of not more than 1 nanogram of strongly electron capturing material. The detector is so sensitive to these compounds that they will overload and saturate the detector even for several hours.

Always maintain a high total flow through the detector (usually not less than 40–50 ml/min) to ensure that all dead volumes are swept out.

Never use columns at or above the maximum temperature that is recommended for the stationary phase in the column. Because of the great sensitivity of the electron capture detector, small amounts of stationary phase bleed will cause a loss of standing current with a consequent loss of sensitivity. Moreover, any bleed from the column is not combusted, as on the flame ionization detector, and will tent to condense in the ECD.

A loss of standing current which is noticed after each injection indicates that contamination of the detector by compounds in the sample is occurring. If this is experienced, it is advisable to clean the detector regularly overnight by purging at an elevated temperature until the original standing currents are recovered.

Many types of solvent can be electron capturing (e.g. acetone, alcohol, ether and any chlorinated solvent). Even small injections of these solvents will eventually saturate the ECD.

Since carrier gas and make-up gas is continually passing through the detector, any contamination in these gases will quickly build-up in the detector thereby reducing the detector standing current. It is necessary, therefore, to ensure that gases used with the ECD are quite clean. This can be achieved by using gases in ECD quality or by cleaning gases through special filters.

4 THE FLAME PHOTOMETRIC DETECTOR (FPD)

The flame photometric detector (FPD) was described by Grant (32) in 1958. The fundamental principle of the flame photometric detector is based on specific emission of light during combustion processes. This detector is used for the detection of sulphur and phosphorus compounds.

The end of the capillary column is led into the flame jet where the column eluent mixes with the hydrogen flow and is burnt. The jet and the actual flame is shielded to prevent light from the flame itself falling directly on to the photo-multiplier. The base of the jet is heated to prevent vapor condensation. The light emitted above the flame, first passes through two heat filters and then through the wavelength selector filter and finally on to the photo-multiplier. Under the correct conditions, the combustion of phosphorus and sulphur compounds produces two species, HPO and S₂, which give off characteristic emissions at 526 and 394 nm wavelengths respectively. The response of the detector to sulfur is fairly insensitive to changes in hydrogen flow rate. However, the response to phosphorus compounds shows a maximum at a particular hydrogen flow rate, the magnitude of which varies with the air flow.

4.1 Non linear response

The largest number of user problems with the flame photometric detector is caused by the square law dependence of the detector when used in the sulphur mode. This effect shows itself during changes in retention time in the detection of very small amounts of analytes. In linear detector a change in retention time will cause a decrease in peak height and hence a decrease in the limit of detection. With a square law detector this effect is exaggerated and in trace determination the peak could disappear into the general noise level.

From this reason it is important to pay attention to factors influenced detector’s noise. Excessive flame noise can be usually caused by not running the flame at the recommended gas flow rates, by using contaminated gas or by column bleeding caused by operating the column at a too high temperature. Another source of excess noise could be the photomultiplier tube because it is susceptible to water condensation. This problem could appear when temperature varies over a large range or when the flame is not alight. It is to be expected, therefore, that on startup from cold, the noise may be rather more than normal. In a short time, however, the condensation would clear and the noise decrease (31).

5 CONCLUSIONS

Developments of gas chromatography are now relatively slow. In the detector sector different types of special mass detector are extended because the prices of these units depreciate and so these equipments are easily available. Nevertheless the detectors FID, ECD and FPD are always wide-spread and for routine brewing and malting analyses are fully sufficient.