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The Application of Gas Chromatography to Forensic Science
T h e Application of Gas Chromatography to Forensic Science D. W. HILL Research Department of Anaesthetics, Royal College of Surgeo?zs of England
A brief descrifition i s given of the principles of gas chromatogra$hy, together with some design details of gas chromatographs. Mention i s made of possible applications of gas chromatografihy to forensic science. The great attraction of gas chromatography as an analytical technique lies in its versatility. Because of its wide range of application, it is only possible to give a gcncral idea of circumstances in which gas chromatography may prove to be of use. In chromatographic methods the sample to be analysed is carried past a stationary phase material by means of a moving phase. The components of the sample become distributed between the two phases. The principles of gas chromatography were first described by Martin and Synge in 1941. The idea of using a gaseous moving phase was not exploited until 1952 when James and Rlartin published tlieir paper on the separation of volatile fatty acids. The stationary phase in the form of a liquid is usually carried on an inert supporting material such as firebrick. This is contained in a tube, known as a column, through which flows the gas phase. As the sample is swept through the column, those components with tlie least affinity for the stationary phase emerge from the column first, and the coln~nnthus separates the components with respect to time. After the sample has traversed a certain length of column it is found that the concentration of the samplc in the gas phase is in equilibrium with the concentration in the stationary phase. The length of column involved in the equilibration is known as the "Height equivalent to one theoretical plate " or HETP. In the process of passing through one theoretical plate the equivalent of one equilibration between the gas and liquid phase occurs. The separation of sample components achieved with a column depends on two factors, the separation per plate and the number of plates in the column. The separation per plate is dependent on the nature of the components, the nature of the stationary pllase and tlie temperature. The number of plates depends on many factors, including the thickness of the liquid layer, uniformity of column packing, nature of carrier gas, flow rate of carrier gas and rates of diffusion in the two phases. The term "Chromatography" is due t o Tswett (1906). Using liquid-liquid chromatography lie separated the extract of green leaves into several components having colours of various shades of green and yellow. Because the separations involved coloured materials, Tswett called tlie process chromatography. Altllougll gas chromatography seldom involves colour, the term is well established. A good introduction to the principles of gas chromatography is given in the book "Principles and Practice of Gas Chromatography" edited by Pecsok (1959). A gas chromatograph comprises the following main parts : carrier gas stream, sample introduction device, cl~rornatographiccolurnn detector and recorder. These will now be described in detail. The Chromatograph Column A conventional column for a gas chromatograph consists of a tube having an internal diameter of about one quarter of an inch (6 mm) and a length usually in the range one t o thirty feet (0.3 m to 10 m). Glass or metal are the usual materials en~ployed. Copper refrigeration tubing which is supplied in sealed 32
clean lengths may be conveniently used. Partition columns which consists of such a tube packed with a supporting substance impregnated with a suitable liquid of low volatility, are the type which are in general use. For the separation of gases such as oxygen, nitrogen and carbon monoxide the column is packed with a solid adsorbent. This consists of a 'molecular sieve' material which is a form of artificial zeolite. For the separation of oxygen, carbon dioxide and cyclopropane the column can be filled with Davison Type 70 silica gel. Tlie usual stationary phase supporting materials for partition columns are Celite 545 and crushed firebrick Type C22, both by Johns-Manville Ltd. These materials are both basically diatomaceous earth. The material is sieved to a narrow mesh range, say 52-60 B.S.S. mesh for firebrick and 100-120 B.S.S. mesh for Celite. I t is then carefully washed to remove fines, this process is very important. A commonly encountered partition column is dinonyl pthalate on firebrick. The dry size-graded firebrick is covered in a dish with a 10% by volume solution of dinonyl ~ t h a l a t ein ether. After stirring, the solvent is evaporated leaving behind the impregnated brick. Tliis is packed into the column by shaking in small amounts and tapping the colun~nuntil no more packing will go in. The ends of the column are filled with a small plug of glass wool.
Operation of the column Through the column there flows a steady stream of some 30ml per minute of carrier gas. Into the gas stream a t the entrance to the column is injected a discrete amount or "slug" of the sample to be analysed. For gases the sample volume would be in the range 0.1 to 3 ml a t KTP. For liquids in the range 0.1 to 2 microlitres. As the sample is swept through the column the various sample components are removed from the gas stream a t rates dependent on their various affinities for the stationary phase. A band of the stationary phase a t the entry to the column is thus saturated with the components of the sample, and as carrier gas passes the rear of this band it tends to remove the components from the packing and to redeposit them further down the column. The result of the continual operation of this process is that the components of the sample move down the column a t characteristic rates, those with the greatest affinity for the stationary phase moving slowest. Tlie action of the column is thus to resolve the components of the sample with respect to time. Tlie emergence of the individual components irom the far end of the column is recorded by means of a suitable sensing device-the detector. The transfer processes in the column result in each of the separated components emerging with an approximately gaussian distribution of concentration in the carrier gas. Thus as they pass through the detector, a recorder driven from it is caused to trace out a peak on the chart for each component. The order of elution of the components can be influenced by the choice of stationary phase material, but is generally in the order of the boiling points, the lower boilers emerging first and the higher boilers last. As a general rule substances can be separated by gas chromatography provided that their boiling points are not more than 50 to 100°C higher than the temperature of the column. An auxiliary pyrolysis unit can be used to decompose thermally samples of substances such as alkaloids and barbiturates (Janak, 1960). The primary products of the pyrolysis are then swept on to the chromatographic column for analysis. The later peaks of a chromatogram tend to be more broad and squat than the earlier peaks. When a sample to be analysed consists of a wide boiling range of components, the later peaks can bc sharpened by steadily raising the column temparature throughout the run, Harrison et al. (1958). Sample introduction systems The main requirement of sample introduction systems is that the sample is introduced in the form of a discrete plug of material. In addition, the volume of tubing between the point where the sample is introduced and the start of the 33
column, and between the end of the column and the detector should be a minimum. Gaseous or vapour samples are convenientljr handled by means of a sampling valve. An electrically operated version which can be set to operate in steps of one minute from one to twenty minutes has been described by I-Ii11 and Hook (1960). Liquid samples can be handled with a micro-syringe either of the micrometer type or else fitted with a Chaney adaptor for reproducibility (Chaney, 1938). Another well tried system uses a micro-dipper (Tenney and Harris, 1957). A flash evaporator is often used before the column to evaporate liquid samples. Detectors The "katharometer" or thermal conductivity cell has been widely used as a detector in chromatographs where modest sensitivity requirements have to be met. Tlle cell consists of two separate platinum wires having a diameter of one tliousandth of an inch (25 microns) each suspended in its own gas channel. A stream of pure carrier gas flows through one channel and the other carries the output gas stream from the column. The two wires form arms of a Wheatstone bridge circuit and this is arranged to be balanced when the column is eluting pure carrier gas only. When a sample peak emerges from the column the thermal conductivity of the carrier gas plus the sample component is different from that of the carrier gas alone. The effect of this is to cause the bridge to unbalance and the recorder to trace out a peak. The sample concentration is proportional to the peak area, but for narrow peaks a calibration curve may be constructed of peak height against concentration. A simple device based on an integrating motor is often used for the measurement of peak area (Dal Nogare et al. 1958). A suitable bridge circuit is that of Harvey & Morgan (1956). The PARALLEL COLUMN GAS CHROMATOGRAPH PRESSURE CONTROL VALVES
HELIUM
VALVE PRFSSURE GAUGE SAMPLE 'A'+ INJECTION
SAMPLE 'El' INJECTION
COLUMN
COLUMN 'A) Dl-METHYL SULPHOXIDE
COLUMN '8' Dl -NONYL PHTHALATE
r~ ~,"r~s~
1-1
RECORDER
IONISATION CROSS-SECTION DETECTOR
Fig. 1. Schematic diagram of a parallel column gas chromatograph. 34
bridge may be powered from an accumulator or from a transistorised power unit capable of supplying an ampere at up to fourteen volts. As an alternative to a hot wire katharometer, thermistors may be used (Hill 1960). The bridge can then be powered from dry batteries. Thermistors have been found to work well in a chromatograph designed for the routine monitoring of anaesthetic atmospheres. Thermal conductivity detectors are best operated with hydrogen or helium as the carrier gas. The thermal conductivity of these gases is considerably higher than that of most sample components, and so the sensitivity of the device does not differ markedly for different components. When dealing with the analysis of a mixture of gases and vapours, it is convenient to separate the gases on one column and the vapours on another column in order to achieve minimum analysis times. (Hill, 1960). A schematic diagram of such a parallel column gas chromatograph is shown in Fig. 1. A simple ionisation chamber with a tritiated zirconium foil radioactive source can be used as an ionisation cross-section detector for gases and vapours in concentrations up to one hundred per cent (Boer, 1956). The analysis of a mixture of gases and vapours is shown in Fig. 2. OUTPUT
FROM BOYLE ANAESTHETIC
I , SAMPLE
I
I
MACHINE
INJECT ION l
l
I2
l
l
1
9
FLUOTHANt
J04
E CARBON
Fig. 2.
1
6
A . ETHER 12% B
1
1
3
1
1
0
TIME
- MlNS
C OXYGEN 2 0 % D NITROUS OXIDE b@% DIOXDE
Analysis of a mixture of anaesthetic gases and vapours.
The chromatograph was run off a t one gain setting. Normally the minor components would be enlarged on the recorder chart by increasing the setting of a bridge output voltage control. 35
High Sensitivity Detectors The application of gas chromatography to trace analysis has been greatly extended by the advent of the argon detector (Lovelock, 1958) and tlie flame ionisation detector (McWilliam & Dewar, 1958). With an argon flow rate of 20 ml. per minute through the detector Lovelock (1960) quotes a minimum detectable component concentration of 4 x 10-" gms per ml. of carrier gas. The argon detector consists of a small ionisation chamber containing a tritiated foil source. A voltage of some 1500 volts D.C. is placed across the chamber. The electric field thus produced accelerates electrons produced by ionisation from tlie source acting on the argon, and accelerates them sufficiently to produce excited metastable argon atoms. When a component peak is eluted from the column and enters the chamber its molecules are ionised by the argon metastable atoms to give a substantial increase in chamber current. In the flame ionisation detector the output from the column is passed into a small flame which is fed fro~rlan auxiliary mixture of hydrogen and air. Two small electrodes placed one on each side of the flame and polarised with about 120 volts D.C. collect the current arising from thermal ionisation processes in the flame. When a component is eluted from the column it is ionised in the flame to give a substantial increase in current. A flame ionisation detector chron~atographis shown in Fig. 3.
GAS CHROMATOGRAPHIC APPARATUS
MANOMETER
120
-2 4 0 V
- ...-. -
NEGRETTI
PRECISION
I
ELECTROMETER
REGULATOR
L VALVE
f5
TWO STAGE REGULATOR 0-30 PSI
CARRIER GAS
Fig. 3.
REGULATOR
IAv/ THERMOSTAT lC OVEN
Flamc ionisation dctcctor gas chromatogralrh
The advantage offered by these ionisation detectors is that in the absence of a component there is only a small signal from the detector. This contrasts with thermal conductivity detectors where the bridge must first be balanced with purc carrier gas alone flowing. At high sensitivity any drift occuring in 36
the bridge balance shows up a marked baseline wander. Ionisation detectors require an electrometer amplifier to measure the small chamber currents. Thompson (1959) gives details of a suitable balanced D.C. electrometer which will feed into a standard 1 mV potentiometric recorder. The recorder should be capable of responding to a signal equivalent to full scale deflection in one second. For most purposes chart speeds of one inch in three or five minutes are adequate. Faster speeds are required for the accurate determination of the number of theoretical plates in a column. For trace analysis work a vibrating reed type of amplifier is generally used. I t may not, however, be generally known that potentiometric recorders by George Kent 1,td. are available with an input resistance of 75 meg-ohms. These can be arranged to work directly with an ionisation detector thus eliminating the need for a separate electrometer. These high sensitivity detectors have greatly helped in the development of capillary columns (Golay 1958). These consist of narrow tubes of metal, glass or nylon of some ten or twenty thousandths of an inch (250 microns to 500 microns) internal diameter and often several hundred feet long. They have a very Iiigh efficiency, and this can often be traded for time to give very quick analyses, the chromatogram being displayed on a cathode ray tube. S o m e applications of g a s c h r o m a t o g r a p h y The flame ionisation detector is insensitive to water vapour and this fact makes it suitable for use with samples obtained from biological fluids. Butler and Hill (1961) have used it to determine trace amounts of volatile anaesthetic agents in blood or other tissues. The anaesthetic was extracted with n-heptane and a halothane concentration of ten parts per million by weight in n-heptane could be measured. Gloesener et al. (1959) have also analysed an anaesthetic mixture using a gas chromatograph. Other papers of interest in forensic science are "Gas Chromatographic Analysis of Alcohol and certain other volatiles in biological material for forensic purposes" Fox (1958) ; "A sensitive procedure for determining carbon monoxide in blood and tissue using gas-solid chromatography" Dominquex et al. (1959) ; "Gas chromatography as a new technique in Forensic Toxicology and Criminalistics" Weinig and Lautenbach (1958) ; "Gas chromatography, General characteristics and use in the detection of adulteration" Chovin (1959) ; " Identification of organic substances by the gas chromatographic analysis of their pyrolsis products" Janak (1960). Amongst other examples this paper discusses the estimation of veronal in urine. "Determination of nail lacquer sol\~entsby gas-liquid chromatography" Jones e t al. (1958). The analysis of crop extracts for traces of chlorinated pesticides by gas-liquid partition chromatography" Goodwin et al. (1960). This paper describes the use of a very sensitive electron capture method for detecting concentrations of well below one part per million of chlorinated compounds. Kade and Abernethy (1961) have used gas chromatography to identify cyclopropane, natural gas and argon in post mortem pulmonary air. Curry (1955) mentions the use of gas chromatography to idcntify lighter fuel added to a medicine containing chloroform and oil of anise. Cadman and Jones (1960)identified by gas chromatography arson materials, paint solvents and stolen petrol. Lucas (1960) decribes the identification of petroleum products in cases of suspected arson. I t is hoped that this short list will serve to illustrate the possibilities of gas chromatography in the forensic field. I t should be remembered that much can be done with apparatus of relative simplicity. "
Reference BOER,H., 1956, "A comparison of detection methods for gas chromatography including detection by beta ray ionisation." Vapour Phase Chromatography (ed. D. H . Desty) 9. 169, Butterworth, London. BUTLER,R. A. & HILL D. W., 1961, " Estimation of volatile anaesthetics in tissues by gas chromatography," Nature, 189, 488. 37
CADMAN, \V. J. & JONES, T., 1960, "Application of the gas chromatograph in the laboratory of criminalistics." J. Forensic sciences, 5, 369. CHANEY, A. L., 1938, "A syringe attachment for accurate volumetric work." Anal. E d . I n d . & Engr. Chem., 10, 326. CHOVIN, P., 1958, "Gas chromatography. General characteristics and use in the detection of adulteration." A n n . fals. et fraudes, 51, 253. CURRY, A. S., 1955, "Toxicological analyis." J. Pharm. & Pharmacol, 7, 969. DALWOGARE, S., BENNETT, C. E. and HARDEN, J. C., 1958, "A simple electromechanical integrator" in Gas chromatogruphy." ( E d . V . J . Coates) Academic Press, N e w Y o r k , 117. DOMINGUEZ, A. M., CHRISTENSEN, H. E., GOLDBAUM, L. R. & STEMBRIDGE, V. A. "A sensitive procedure for determining carbon monoxide in blood and tissues using gas-solid chromatography." Toxicol. A$@. Pharmacol, 1, 135. FOX,J. C., 1958, "Gas chromatographic analysis of alcohol and certain other volatiles in biological material for forensic purposes." Proc. Soc. Exptl. Biol. Med., 97, 236. GLOESENER, E., ~ ~ A P I E R EC., I,., VERSIE,J., 1959, " Toxicological Study of the Schleich anaesthetic mixture." J. Pharm. Belg., 13,365. GOLAY,M. J. E., 1958, " Theory and practice of gas-liquid partition chromatography with coated capillaries" in Gas chromatography. ( E d . V . J . Coates), Academic Press, N e w Y o r k . GOODWIN, E. S., GOULDEN, R., RICHARDSON, A., REYNOT.I)S, J. G., 1960, " The analysis of crop extracts for traces of chlorinated pesticides by gas-liquid partition chromatography." Chem. and I n d . , 1220. HARRISON, G. F., KNIGHT,P., KELLEY,R. P. & HEATH,M. T., 1958, " The use of multiple columns and programmtd column heating in the analysis of wide-boiling range halogenated hydrocarbon sample " in Gas chromatography 1958. ( E d . D. H . Destry) 216, Butterworths, London. HARVEY,D., MORGAN, G. 0. (1956), "Factors affecting thermal conductivity detectors in vapour phase partition chromatography in V a p o u r phase chromatogra$hy." ( E d . D. H . Desty) 74, Butterworths, London. HILL, D. W., HOOK,J. R., 1960, "Automatic gas-sampling device for Gas chromatography." J. Sci. Instrum., 37, 253. HILL, D. W., 1960, " The application of Gas chromatography to anaesthetic research" in Gus chromatography 1960. ( E d . R. P . W . Scott). 344, Butterworths, London. JAMES, A. T., MARTIN, A. J. P., 1952, "Gas-liquid partition chromatographv : The separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid." J. Biol. Chem., 50, 679. JANAK,J. 1960, " Identification of organic substances by the gas chromatographic analysis of their pyrolysis products " in Gas chromatography 1960. 387 ( E d . R. P. W . Scott) Butterworths, London. JONES,W. L. & KIESELBACH, R., 1958, " The determination of nail lacquer solvents by gas-liquid chromatography." J. Assoc. Ojfic. Agr. Chemists, 41. 673. KADE,'H., ABERNETHY, R. J., 1961, " Identification of noxious gases in post mortem pulmonary air." J. Forensic Sciences, 6 , 125. LOVELOCK, J. E., 1958, "A sensitive detector for Gas chromatography." J . Chromatog., 1, 35. LOVELOCK, J. E., 1960, "Argon detectors" in Gas chromatography 1960. ( E d . R. P . W . Scott), 16, Butterworths, London. LUCAS,D. M., 1960, " The identification of petroleum products in forensic science by Gas chromatography." J. Forensic Sciences, 5, 236. MARTIN,A. J. P., SYNGE,R.L.M., 1941, "A new form of chromatogram employing two liquid phases. 1-A theory of chromatography. 2-Application of the microdetermination of the higher monoaminoacids in proteins. J. Bio. Chem., 35, 1358. "
38
MCWILLIAM, I. G., DEWAR,R. A., 1958, " Flame ionisation detector for Gas chromatograpfiy" in Gas chromatography 1958. (Ed. D . H . Desty), 142, Butterworths, London. PECSOK, R. L., 1959, " Principles and practice of Gas chromatography." (Ed. R. L. Pecsok), Chapman & Hall, London. SUNNER, S., KARRMAN, K. J., SUNDEN, V., 1956, " Separation of mercaptans by gas-liquid partition chromatography." Mikrochim. Acta., 1144. TENNEY,H. M., HARRIS,R. J., 1957, " Sample introduction system for Gas chromatography." Anal. Chem., 29, 317. THOMPSON, A. E., 1959, "A flame ionisation detector for Gas chromatography." J . Chromatog., 2, 149. WEINIG,E., LAUTENRACH, I,., 1958, "Gas chromatography as a new technique in forensic toxicology and criminalistics.'; Arch. Kriminol, 122, 11.
A brief descrifition i s given of the principles of gas chromatogra$hy, together with some design details of gas chromatographs. Mention i s made of possible applications of gas chromatografihy to forensic science. The great attraction of gas chromatography as an analytical technique lies in its versatility. Because of its wide range of application, it is only possible to give a gcncral idea of circumstances in which gas chromatography may prove to be of use. In chromatographic methods the sample to be analysed is carried past a stationary phase material by means of a moving phase. The components of the sample become distributed between the two phases. The principles of gas chromatography were first described by Martin and Synge in 1941. The idea of using a gaseous moving phase was not exploited until 1952 when James and Rlartin published tlieir paper on the separation of volatile fatty acids. The stationary phase in the form of a liquid is usually carried on an inert supporting material such as firebrick. This is contained in a tube, known as a column, through which flows the gas phase. As the sample is swept through the column, those components with tlie least affinity for the stationary phase emerge from the column first, and the coln~nnthus separates the components with respect to time. After the sample has traversed a certain length of column it is found that the concentration of the samplc in the gas phase is in equilibrium with the concentration in the stationary phase. The length of column involved in the equilibration is known as the "Height equivalent to one theoretical plate " or HETP. In the process of passing through one theoretical plate the equivalent of one equilibration between the gas and liquid phase occurs. The separation of sample components achieved with a column depends on two factors, the separation per plate and the number of plates in the column. The separation per plate is dependent on the nature of the components, the nature of the stationary pllase and tlie temperature. The number of plates depends on many factors, including the thickness of the liquid layer, uniformity of column packing, nature of carrier gas, flow rate of carrier gas and rates of diffusion in the two phases. The term "Chromatography" is due t o Tswett (1906). Using liquid-liquid chromatography lie separated the extract of green leaves into several components having colours of various shades of green and yellow. Because the separations involved coloured materials, Tswett called tlie process chromatography. Altllougll gas chromatography seldom involves colour, the term is well established. A good introduction to the principles of gas chromatography is given in the book "Principles and Practice of Gas Chromatography" edited by Pecsok (1959). A gas chromatograph comprises the following main parts : carrier gas stream, sample introduction device, cl~rornatographiccolurnn detector and recorder. These will now be described in detail. The Chromatograph Column A conventional column for a gas chromatograph consists of a tube having an internal diameter of about one quarter of an inch (6 mm) and a length usually in the range one t o thirty feet (0.3 m to 10 m). Glass or metal are the usual materials en~ployed. Copper refrigeration tubing which is supplied in sealed 32
clean lengths may be conveniently used. Partition columns which consists of such a tube packed with a supporting substance impregnated with a suitable liquid of low volatility, are the type which are in general use. For the separation of gases such as oxygen, nitrogen and carbon monoxide the column is packed with a solid adsorbent. This consists of a 'molecular sieve' material which is a form of artificial zeolite. For the separation of oxygen, carbon dioxide and cyclopropane the column can be filled with Davison Type 70 silica gel. Tlie usual stationary phase supporting materials for partition columns are Celite 545 and crushed firebrick Type C22, both by Johns-Manville Ltd. These materials are both basically diatomaceous earth. The material is sieved to a narrow mesh range, say 52-60 B.S.S. mesh for firebrick and 100-120 B.S.S. mesh for Celite. I t is then carefully washed to remove fines, this process is very important. A commonly encountered partition column is dinonyl pthalate on firebrick. The dry size-graded firebrick is covered in a dish with a 10% by volume solution of dinonyl ~ t h a l a t ein ether. After stirring, the solvent is evaporated leaving behind the impregnated brick. Tliis is packed into the column by shaking in small amounts and tapping the colun~nuntil no more packing will go in. The ends of the column are filled with a small plug of glass wool.
Operation of the column Through the column there flows a steady stream of some 30ml per minute of carrier gas. Into the gas stream a t the entrance to the column is injected a discrete amount or "slug" of the sample to be analysed. For gases the sample volume would be in the range 0.1 to 3 ml a t KTP. For liquids in the range 0.1 to 2 microlitres. As the sample is swept through the column the various sample components are removed from the gas stream a t rates dependent on their various affinities for the stationary phase. A band of the stationary phase a t the entry to the column is thus saturated with the components of the sample, and as carrier gas passes the rear of this band it tends to remove the components from the packing and to redeposit them further down the column. The result of the continual operation of this process is that the components of the sample move down the column a t characteristic rates, those with the greatest affinity for the stationary phase moving slowest. Tlie action of the column is thus to resolve the components of the sample with respect to time. Tlie emergence of the individual components irom the far end of the column is recorded by means of a suitable sensing device-the detector. The transfer processes in the column result in each of the separated components emerging with an approximately gaussian distribution of concentration in the carrier gas. Thus as they pass through the detector, a recorder driven from it is caused to trace out a peak on the chart for each component. The order of elution of the components can be influenced by the choice of stationary phase material, but is generally in the order of the boiling points, the lower boilers emerging first and the higher boilers last. As a general rule substances can be separated by gas chromatography provided that their boiling points are not more than 50 to 100°C higher than the temperature of the column. An auxiliary pyrolysis unit can be used to decompose thermally samples of substances such as alkaloids and barbiturates (Janak, 1960). The primary products of the pyrolysis are then swept on to the chromatographic column for analysis. The later peaks of a chromatogram tend to be more broad and squat than the earlier peaks. When a sample to be analysed consists of a wide boiling range of components, the later peaks can bc sharpened by steadily raising the column temparature throughout the run, Harrison et al. (1958). Sample introduction systems The main requirement of sample introduction systems is that the sample is introduced in the form of a discrete plug of material. In addition, the volume of tubing between the point where the sample is introduced and the start of the 33
column, and between the end of the column and the detector should be a minimum. Gaseous or vapour samples are convenientljr handled by means of a sampling valve. An electrically operated version which can be set to operate in steps of one minute from one to twenty minutes has been described by I-Ii11 and Hook (1960). Liquid samples can be handled with a micro-syringe either of the micrometer type or else fitted with a Chaney adaptor for reproducibility (Chaney, 1938). Another well tried system uses a micro-dipper (Tenney and Harris, 1957). A flash evaporator is often used before the column to evaporate liquid samples. Detectors The "katharometer" or thermal conductivity cell has been widely used as a detector in chromatographs where modest sensitivity requirements have to be met. Tlle cell consists of two separate platinum wires having a diameter of one tliousandth of an inch (25 microns) each suspended in its own gas channel. A stream of pure carrier gas flows through one channel and the other carries the output gas stream from the column. The two wires form arms of a Wheatstone bridge circuit and this is arranged to be balanced when the column is eluting pure carrier gas only. When a sample peak emerges from the column the thermal conductivity of the carrier gas plus the sample component is different from that of the carrier gas alone. The effect of this is to cause the bridge to unbalance and the recorder to trace out a peak. The sample concentration is proportional to the peak area, but for narrow peaks a calibration curve may be constructed of peak height against concentration. A simple device based on an integrating motor is often used for the measurement of peak area (Dal Nogare et al. 1958). A suitable bridge circuit is that of Harvey & Morgan (1956). The PARALLEL COLUMN GAS CHROMATOGRAPH PRESSURE CONTROL VALVES
HELIUM
VALVE PRFSSURE GAUGE SAMPLE 'A'+ INJECTION
SAMPLE 'El' INJECTION
COLUMN
COLUMN 'A) Dl-METHYL SULPHOXIDE
COLUMN '8' Dl -NONYL PHTHALATE
r~ ~,"r~s~
1-1
RECORDER
IONISATION CROSS-SECTION DETECTOR
Fig. 1. Schematic diagram of a parallel column gas chromatograph. 34
bridge may be powered from an accumulator or from a transistorised power unit capable of supplying an ampere at up to fourteen volts. As an alternative to a hot wire katharometer, thermistors may be used (Hill 1960). The bridge can then be powered from dry batteries. Thermistors have been found to work well in a chromatograph designed for the routine monitoring of anaesthetic atmospheres. Thermal conductivity detectors are best operated with hydrogen or helium as the carrier gas. The thermal conductivity of these gases is considerably higher than that of most sample components, and so the sensitivity of the device does not differ markedly for different components. When dealing with the analysis of a mixture of gases and vapours, it is convenient to separate the gases on one column and the vapours on another column in order to achieve minimum analysis times. (Hill, 1960). A schematic diagram of such a parallel column gas chromatograph is shown in Fig. 1. A simple ionisation chamber with a tritiated zirconium foil radioactive source can be used as an ionisation cross-section detector for gases and vapours in concentrations up to one hundred per cent (Boer, 1956). The analysis of a mixture of gases and vapours is shown in Fig. 2. OUTPUT
FROM BOYLE ANAESTHETIC
I , SAMPLE
I
I
MACHINE
INJECT ION l
l
I2
l
l
1
9
FLUOTHANt
J04
E CARBON
Fig. 2.
1
6
A . ETHER 12% B
1
1
3
1
1
0
TIME
- MlNS
C OXYGEN 2 0 % D NITROUS OXIDE b@% DIOXDE
Analysis of a mixture of anaesthetic gases and vapours.
The chromatograph was run off a t one gain setting. Normally the minor components would be enlarged on the recorder chart by increasing the setting of a bridge output voltage control. 35
High Sensitivity Detectors The application of gas chromatography to trace analysis has been greatly extended by the advent of the argon detector (Lovelock, 1958) and tlie flame ionisation detector (McWilliam & Dewar, 1958). With an argon flow rate of 20 ml. per minute through the detector Lovelock (1960) quotes a minimum detectable component concentration of 4 x 10-" gms per ml. of carrier gas. The argon detector consists of a small ionisation chamber containing a tritiated foil source. A voltage of some 1500 volts D.C. is placed across the chamber. The electric field thus produced accelerates electrons produced by ionisation from tlie source acting on the argon, and accelerates them sufficiently to produce excited metastable argon atoms. When a component peak is eluted from the column and enters the chamber its molecules are ionised by the argon metastable atoms to give a substantial increase in chamber current. In the flame ionisation detector the output from the column is passed into a small flame which is fed fro~rlan auxiliary mixture of hydrogen and air. Two small electrodes placed one on each side of the flame and polarised with about 120 volts D.C. collect the current arising from thermal ionisation processes in the flame. When a component is eluted from the column it is ionised in the flame to give a substantial increase in current. A flame ionisation detector chron~atographis shown in Fig. 3.
GAS CHROMATOGRAPHIC APPARATUS
MANOMETER
120
-2 4 0 V
- ...-. -
NEGRETTI
PRECISION
I
ELECTROMETER
REGULATOR
L VALVE
f5
TWO STAGE REGULATOR 0-30 PSI
CARRIER GAS
Fig. 3.
REGULATOR
IAv/ THERMOSTAT lC OVEN
Flamc ionisation dctcctor gas chromatogralrh
The advantage offered by these ionisation detectors is that in the absence of a component there is only a small signal from the detector. This contrasts with thermal conductivity detectors where the bridge must first be balanced with purc carrier gas alone flowing. At high sensitivity any drift occuring in 36
the bridge balance shows up a marked baseline wander. Ionisation detectors require an electrometer amplifier to measure the small chamber currents. Thompson (1959) gives details of a suitable balanced D.C. electrometer which will feed into a standard 1 mV potentiometric recorder. The recorder should be capable of responding to a signal equivalent to full scale deflection in one second. For most purposes chart speeds of one inch in three or five minutes are adequate. Faster speeds are required for the accurate determination of the number of theoretical plates in a column. For trace analysis work a vibrating reed type of amplifier is generally used. I t may not, however, be generally known that potentiometric recorders by George Kent 1,td. are available with an input resistance of 75 meg-ohms. These can be arranged to work directly with an ionisation detector thus eliminating the need for a separate electrometer. These high sensitivity detectors have greatly helped in the development of capillary columns (Golay 1958). These consist of narrow tubes of metal, glass or nylon of some ten or twenty thousandths of an inch (250 microns to 500 microns) internal diameter and often several hundred feet long. They have a very Iiigh efficiency, and this can often be traded for time to give very quick analyses, the chromatogram being displayed on a cathode ray tube. S o m e applications of g a s c h r o m a t o g r a p h y The flame ionisation detector is insensitive to water vapour and this fact makes it suitable for use with samples obtained from biological fluids. Butler and Hill (1961) have used it to determine trace amounts of volatile anaesthetic agents in blood or other tissues. The anaesthetic was extracted with n-heptane and a halothane concentration of ten parts per million by weight in n-heptane could be measured. Gloesener et al. (1959) have also analysed an anaesthetic mixture using a gas chromatograph. Other papers of interest in forensic science are "Gas Chromatographic Analysis of Alcohol and certain other volatiles in biological material for forensic purposes" Fox (1958) ; "A sensitive procedure for determining carbon monoxide in blood and tissue using gas-solid chromatography" Dominquex et al. (1959) ; "Gas chromatography as a new technique in Forensic Toxicology and Criminalistics" Weinig and Lautenbach (1958) ; "Gas chromatography, General characteristics and use in the detection of adulteration" Chovin (1959) ; " Identification of organic substances by the gas chromatographic analysis of their pyrolsis products" Janak (1960). Amongst other examples this paper discusses the estimation of veronal in urine. "Determination of nail lacquer sol\~entsby gas-liquid chromatography" Jones e t al. (1958). The analysis of crop extracts for traces of chlorinated pesticides by gas-liquid partition chromatography" Goodwin et al. (1960). This paper describes the use of a very sensitive electron capture method for detecting concentrations of well below one part per million of chlorinated compounds. Kade and Abernethy (1961) have used gas chromatography to identify cyclopropane, natural gas and argon in post mortem pulmonary air. Curry (1955) mentions the use of gas chromatography to idcntify lighter fuel added to a medicine containing chloroform and oil of anise. Cadman and Jones (1960)identified by gas chromatography arson materials, paint solvents and stolen petrol. Lucas (1960) decribes the identification of petroleum products in cases of suspected arson. I t is hoped that this short list will serve to illustrate the possibilities of gas chromatography in the forensic field. I t should be remembered that much can be done with apparatus of relative simplicity. "
Reference BOER,H., 1956, "A comparison of detection methods for gas chromatography including detection by beta ray ionisation." Vapour Phase Chromatography (ed. D. H . Desty) 9. 169, Butterworth, London. BUTLER,R. A. & HILL D. W., 1961, " Estimation of volatile anaesthetics in tissues by gas chromatography," Nature, 189, 488. 37
CADMAN, \V. J. & JONES, T., 1960, "Application of the gas chromatograph in the laboratory of criminalistics." J. Forensic sciences, 5, 369. CHANEY, A. L., 1938, "A syringe attachment for accurate volumetric work." Anal. E d . I n d . & Engr. Chem., 10, 326. CHOVIN, P., 1958, "Gas chromatography. General characteristics and use in the detection of adulteration." A n n . fals. et fraudes, 51, 253. CURRY, A. S., 1955, "Toxicological analyis." J. Pharm. & Pharmacol, 7, 969. DALWOGARE, S., BENNETT, C. E. and HARDEN, J. C., 1958, "A simple electromechanical integrator" in Gas chromatogruphy." ( E d . V . J . Coates) Academic Press, N e w Y o r k , 117. DOMINGUEZ, A. M., CHRISTENSEN, H. E., GOLDBAUM, L. R. & STEMBRIDGE, V. A. "A sensitive procedure for determining carbon monoxide in blood and tissues using gas-solid chromatography." Toxicol. A$@. Pharmacol, 1, 135. FOX,J. C., 1958, "Gas chromatographic analysis of alcohol and certain other volatiles in biological material for forensic purposes." Proc. Soc. Exptl. Biol. Med., 97, 236. GLOESENER, E., ~ ~ A P I E R EC., I,., VERSIE,J., 1959, " Toxicological Study of the Schleich anaesthetic mixture." J. Pharm. Belg., 13,365. GOLAY,M. J. E., 1958, " Theory and practice of gas-liquid partition chromatography with coated capillaries" in Gas chromatography. ( E d . V . J . Coates), Academic Press, N e w Y o r k . GOODWIN, E. S., GOULDEN, R., RICHARDSON, A., REYNOT.I)S, J. G., 1960, " The analysis of crop extracts for traces of chlorinated pesticides by gas-liquid partition chromatography." Chem. and I n d . , 1220. HARRISON, G. F., KNIGHT,P., KELLEY,R. P. & HEATH,M. T., 1958, " The use of multiple columns and programmtd column heating in the analysis of wide-boiling range halogenated hydrocarbon sample " in Gas chromatography 1958. ( E d . D. H . Destry) 216, Butterworths, London. HARVEY,D., MORGAN, G. 0. (1956), "Factors affecting thermal conductivity detectors in vapour phase partition chromatography in V a p o u r phase chromatogra$hy." ( E d . D. H . Desty) 74, Butterworths, London. HILL, D. W., HOOK,J. R., 1960, "Automatic gas-sampling device for Gas chromatography." J. Sci. Instrum., 37, 253. HILL, D. W., 1960, " The application of Gas chromatography to anaesthetic research" in Gus chromatography 1960. ( E d . R. P . W . Scott). 344, Butterworths, London. JAMES, A. T., MARTIN, A. J. P., 1952, "Gas-liquid partition chromatographv : The separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid." J. Biol. Chem., 50, 679. JANAK,J. 1960, " Identification of organic substances by the gas chromatographic analysis of their pyrolysis products " in Gas chromatography 1960. 387 ( E d . R. P. W . Scott) Butterworths, London. JONES,W. L. & KIESELBACH, R., 1958, " The determination of nail lacquer solvents by gas-liquid chromatography." J. Assoc. Ojfic. Agr. Chemists, 41. 673. KADE,'H., ABERNETHY, R. J., 1961, " Identification of noxious gases in post mortem pulmonary air." J. Forensic Sciences, 6 , 125. LOVELOCK, J. E., 1958, "A sensitive detector for Gas chromatography." J . Chromatog., 1, 35. LOVELOCK, J. E., 1960, "Argon detectors" in Gas chromatography 1960. ( E d . R. P . W . Scott), 16, Butterworths, London. LUCAS,D. M., 1960, " The identification of petroleum products in forensic science by Gas chromatography." J. Forensic Sciences, 5, 236. MARTIN,A. J. P., SYNGE,R.L.M., 1941, "A new form of chromatogram employing two liquid phases. 1-A theory of chromatography. 2-Application of the microdetermination of the higher monoaminoacids in proteins. J. Bio. Chem., 35, 1358. "
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MCWILLIAM, I. G., DEWAR,R. A., 1958, " Flame ionisation detector for Gas chromatograpfiy" in Gas chromatography 1958. (Ed. D . H . Desty), 142, Butterworths, London. PECSOK, R. L., 1959, " Principles and practice of Gas chromatography." (Ed. R. L. Pecsok), Chapman & Hall, London. SUNNER, S., KARRMAN, K. J., SUNDEN, V., 1956, " Separation of mercaptans by gas-liquid partition chromatography." Mikrochim. Acta., 1144. TENNEY,H. M., HARRIS,R. J., 1957, " Sample introduction system for Gas chromatography." Anal. Chem., 29, 317. THOMPSON, A. E., 1959, "A flame ionisation detector for Gas chromatography." J . Chromatog., 2, 149. WEINIG,E., LAUTENRACH, I,., 1958, "Gas chromatography as a new technique in forensic toxicology and criminalistics.'; Arch. Kriminol, 122, 11.