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An automatic device for the application of evaporated samples to the gas chromatogram
JOURNAL
OF CHROMATOGRAPHY
An automatic
131
device for the application
.of evaporated
samples to the gas
chromatogram
The periodic application by hand of samples to the gas chromatogram renders it unsuitable for routine batch analysis and inefficient in that only a limited number of samples may be applied in a working day. The apparatus described was devised so that a batch of samples may be loaded and without attention will be fed singly to the column at set intervals and the peaks assessed at the completion of the run. An overnight run is particularly useful. Essentially small ferrous metal cylmders containing the evaporated,samples are fed to and removed from the flash heater by application of appropriate electromagnetic fields, the current supplying the solenoids being controlled by a simple timing device.
.
Practical details The metal cylinders measure 16 mm by 4 mm diameter and are made from tinplate (thickness 0.03 mm, as used for canning), They are fashioned around a glass rod of appropriate diameter so that a I-mm slit occurs longitudinally along the cylinder. Heavier gauge metal tubing proved unsuccessful because of its excessive weight. Cylinders of the above dimensions can be drilled and turned, from iron rod if machining is done very meticulously. The cylinders are siliconised and conditioned at 300’ before use, samples of up to IOO ,ul solution are applied to the inside of the cylinder and the solvent evaporated. The glass section of the apparatus is made from 7-8 mm diameter glass (Fig. I),
i Solenoid
c
c Argon
I
-‘out’Solenoid
rgon
Fig, X. Apparatus for’the automatic addition and removal of sample containers. J. Ckromntog,, 20 (1963) 131-1.34
=32
NOTES
with a I37 joint leading to the top of the chromatogram tube. There is a constriction in the tube just above the I37 joint. The in and ozctportions of the apparatus are joined by pressure tubing, the upper arm being supported by a metal rod extension from the chromatograph body. The iqz portion of the apparatus is essentially a T piece on its side. It is glass blown with a joint so that when a series of metal cylinders are drawn to the glass stop, on releasing the magnetic field, only the first cylinder falls. The side arm is horizontal for 4 cm to I3 (Fig. I) and then inclines upwards at some ~---Io~to the horizontal. This facilitates satisfactory movement of the’ cylinders which should be tested using a horseshoe magnet before winding the solenoid. Using a straightforward T piece, a sample cylinder occasionally stuck at the glass stop, held by the inertia of the cylinders behind it so that it did not fall by gravity. This was overcome by placing a small glass covered ferrous core in the upper arm of the T piece actuated by a solenoid around this arm. It is wound in series with that around the side arm so that when current is passed, the core is raised and the sample cylinders are drawn to the glass stop. When the circuit is broken the core falls and knocks the leading cylinder down the entry tube. The core falls about a cm on to a constriction in the glass tubing just above the side arm. The solenoid is wound in series around the glass side arm and upper arm using about 25 metres 26 gauge (0.457 mm diameter) shellac covered copper wire. A single turn every 2 to 3 mm is made from C to I3 (Fig. I), the remaining wire is wound from A to 13 and at D. The glass tube at the top of the apparatus is sealed with a small bung, where if the core at D is removed, sample cylinders may be introduced by hand.
Fig. 2. Timing device for actuating I.
Ch’OWtUtOg.,
20
(1965)
131-134
the solenoids. This motor makes one I*evolution every 30 min.
133
NOTES
The number of sample containers the apparatus will take depends on the length of the side arms, the apparatus in use holds 25 with a side arm of 43 cm. The otit solenoid is wound using about 30 m of 24 gauge (0.559 mm diam.) shellac covered copper wire. Two turns are closely wound directly onto the glass from X to Y. It should be wound so that when a cylinder is in position in the flash heater only half the cylinder is within the magnetic field. This ensures that it is drawn into the magnetic field with added momentum. The remainder of the wire is wound so that the centre and strongest magnetic field occurs at the glass seal 2 (Fig. I). The electromagnets in and out, are connected to a 12 V 40 A/h battery through 2 make and break mechanisms actuated by a motor* which in this instance makes one revolution in 30 min (Fig. 2). Two Tufnol discs are mounted on the motor shaft and keep the contacts open. A small saw cut in each disc allows contact to be made once every revolution; if this cut is made with a fine saw, the current is only passed for 6-10 se& Each solenoid consumes up to IO A and because of heat generated the time the current passes should be kept as short as possible. A hand-operated switch is also incorporated in the circuit so that sample cylinders may be added or removed at will. The metal cylinders containing samples are loaded at C with the longitudinal slits uppermost. It is important that they are pushed along the side arm so that the leading cylinder enters the stronger electro-magnetic field at B to A and that there are no spaces between cylinders; otherwise the whole series will not move along the side arm. Carrier gas enters both side arms and after the; few min required to flush the apparatus, the bung is inserted in the uppermost arm. Disczcssion
The apparatus was devised so that routine steroid determinations may be adapted to the gas chromatograph. The use of liquid injection for quantitative work has many drawbacks, only a few ,~l of solvent may be injected so that relatively concentrated solutions of steroids must be prepared, this is difficult to do accurately when only a few ,ug of steroid may be available. Also the addition of solvent causes a large peak which may obscure or invalidate peaks due to fast moving materials. Using the present method up to IOO ~1 of dissolved steroid extract may be added to a cylinder and the solvent evaporated before application to the chromstograph. If necessary several additions may be made to the sample capsule, evaporating the solvent after each. addition. This means that steroidal extracts at concentrations usually met in usual methods are more easily dealt with. Another ad.vantage of the method is that the top of the column is not opened during addition and removal of samples. The present apparatus is designed for a 3o-min cycle of adding samples. The type of motor used is available at a variety of speeds so that any particular time’cycle may be chosen if the appropriate motor is fitted. A slower moving motor may be used instead and actuating discs designed with contact grooves cut at intervals around the discs. A given time cycle is then chosen by fitting the appropriate discs to the motor. The apparatus is easily removable so that columns may be changed. The ground glass joint occurring within the flash heater may be criticised; it has caused no trouble. A piece of wire around the ear of the column end extends up through the flash heater where it joins an extension spring anchored to the upper part of the apparatus; this keeps the joint in compression, * Obtainable from Venner hlotors Ltd. J.
ChYOWWtO~.,
20
(1965)
ISI--134
134
NOTES
Using a Pye Argon Chromatograph, a 4 ft. I % SE30 column at ZZ~“, flash heater 270°, pressure 15 psi. the reproducibility of the method was tested. Twelve samples of a pregnanediol diacetate solution, each 4 ,ug in 40 ,ul, were pipetted on to cylinders, evaporated, and the batch assayed using the automatic device described. Peak height and area by triangulation were determined and gave: height, mean 71 & s.d. 3.0; area, 1330 -& s.d. 46 mm2. The experiment was repeated the following day using the same column conditions but the evaporated samples in open glass cylinders were added and removed from the flash heater manually. This gave precision : height, mean 65 & s.d. 3.9; area, mean 1250 5. s.d. 70 ‘mm2. The apparatus has been used chiefly.for overnight runs.’ Samples”‘and standards are loaded in the evening and a separate time switch is set to stop the chart drive at the end of the run. The peaks are quantitated the following morning. Acknowledgement My thanks are due to Mr. H. ICESTEVENand Mr. J. BAMFORD making the timing’ device. De$artnzent of Chemical Pathology, The rioyal Infiwnar_y, She field (Great Britain) Received
March zgrd,
J. ChrOVnatOg.,
20 (1965)
Gas chromatography I. Separation
for their help in
D.A.
PODMORE
1965 131-134
of fluorinated
and identification
fatty acids
of lower 24uorofatty
acids
There has been considerable interest, in recent years, in the biological activity of fluoro-organic compounds related to aliphatic fatty acidsl. Fluoroacetic acid was found by MARAIS~ to be the toxic principle of Gifblaar. Several w-fluorofatty acids were identified in the seeds of Dichafietalum toxicariwn by PETERS and coworlcerssv”. In a study of the fate of Auoroacetic acid in nondividing yeast cells, ALDOUS~ tentatively identified a fluorobutyric acid and a fluorohexanoic acid as metabolic products, Positive identification could not be made for lack of authentic samples. As the result of our program on the synthesis of fluorinated molecules, a. series ’ of z-fluorofatty acids became available. The separation and identification of this series of lower fiuoroacids alone and in combination with a similar series of unfluori-, nated saturated aliphatic fatty acids’by gas chromatography are the subject of this report.
Apfiaratzcs. For the separation and identification of the z-fluorofatty acids alone, a Perkin-Elmer Model x54D Vapor Practometer fitted with a’thermal conductivity .. Ji
Ckt'OlVZatOg., 20 (1965)
134-137
OF CHROMATOGRAPHY
An automatic
131
device for the application
.of evaporated
samples to the gas
chromatogram
The periodic application by hand of samples to the gas chromatogram renders it unsuitable for routine batch analysis and inefficient in that only a limited number of samples may be applied in a working day. The apparatus described was devised so that a batch of samples may be loaded and without attention will be fed singly to the column at set intervals and the peaks assessed at the completion of the run. An overnight run is particularly useful. Essentially small ferrous metal cylmders containing the evaporated,samples are fed to and removed from the flash heater by application of appropriate electromagnetic fields, the current supplying the solenoids being controlled by a simple timing device.
.
Practical details The metal cylinders measure 16 mm by 4 mm diameter and are made from tinplate (thickness 0.03 mm, as used for canning), They are fashioned around a glass rod of appropriate diameter so that a I-mm slit occurs longitudinally along the cylinder. Heavier gauge metal tubing proved unsuccessful because of its excessive weight. Cylinders of the above dimensions can be drilled and turned, from iron rod if machining is done very meticulously. The cylinders are siliconised and conditioned at 300’ before use, samples of up to IOO ,ul solution are applied to the inside of the cylinder and the solvent evaporated. The glass section of the apparatus is made from 7-8 mm diameter glass (Fig. I),
i Solenoid
c
c Argon
I
-‘out’Solenoid
rgon
Fig, X. Apparatus for’the automatic addition and removal of sample containers. J. Ckromntog,, 20 (1963) 131-1.34
=32
NOTES
with a I37 joint leading to the top of the chromatogram tube. There is a constriction in the tube just above the I37 joint. The in and ozctportions of the apparatus are joined by pressure tubing, the upper arm being supported by a metal rod extension from the chromatograph body. The iqz portion of the apparatus is essentially a T piece on its side. It is glass blown with a joint so that when a series of metal cylinders are drawn to the glass stop, on releasing the magnetic field, only the first cylinder falls. The side arm is horizontal for 4 cm to I3 (Fig. I) and then inclines upwards at some ~---Io~to the horizontal. This facilitates satisfactory movement of the’ cylinders which should be tested using a horseshoe magnet before winding the solenoid. Using a straightforward T piece, a sample cylinder occasionally stuck at the glass stop, held by the inertia of the cylinders behind it so that it did not fall by gravity. This was overcome by placing a small glass covered ferrous core in the upper arm of the T piece actuated by a solenoid around this arm. It is wound in series with that around the side arm so that when current is passed, the core is raised and the sample cylinders are drawn to the glass stop. When the circuit is broken the core falls and knocks the leading cylinder down the entry tube. The core falls about a cm on to a constriction in the glass tubing just above the side arm. The solenoid is wound in series around the glass side arm and upper arm using about 25 metres 26 gauge (0.457 mm diameter) shellac covered copper wire. A single turn every 2 to 3 mm is made from C to I3 (Fig. I), the remaining wire is wound from A to 13 and at D. The glass tube at the top of the apparatus is sealed with a small bung, where if the core at D is removed, sample cylinders may be introduced by hand.
Fig. 2. Timing device for actuating I.
Ch’OWtUtOg.,
20
(1965)
131-134
the solenoids. This motor makes one I*evolution every 30 min.
133
NOTES
The number of sample containers the apparatus will take depends on the length of the side arms, the apparatus in use holds 25 with a side arm of 43 cm. The otit solenoid is wound using about 30 m of 24 gauge (0.559 mm diam.) shellac covered copper wire. Two turns are closely wound directly onto the glass from X to Y. It should be wound so that when a cylinder is in position in the flash heater only half the cylinder is within the magnetic field. This ensures that it is drawn into the magnetic field with added momentum. The remainder of the wire is wound so that the centre and strongest magnetic field occurs at the glass seal 2 (Fig. I). The electromagnets in and out, are connected to a 12 V 40 A/h battery through 2 make and break mechanisms actuated by a motor* which in this instance makes one revolution in 30 min (Fig. 2). Two Tufnol discs are mounted on the motor shaft and keep the contacts open. A small saw cut in each disc allows contact to be made once every revolution; if this cut is made with a fine saw, the current is only passed for 6-10 se& Each solenoid consumes up to IO A and because of heat generated the time the current passes should be kept as short as possible. A hand-operated switch is also incorporated in the circuit so that sample cylinders may be added or removed at will. The metal cylinders containing samples are loaded at C with the longitudinal slits uppermost. It is important that they are pushed along the side arm so that the leading cylinder enters the stronger electro-magnetic field at B to A and that there are no spaces between cylinders; otherwise the whole series will not move along the side arm. Carrier gas enters both side arms and after the; few min required to flush the apparatus, the bung is inserted in the uppermost arm. Disczcssion
The apparatus was devised so that routine steroid determinations may be adapted to the gas chromatograph. The use of liquid injection for quantitative work has many drawbacks, only a few ,~l of solvent may be injected so that relatively concentrated solutions of steroids must be prepared, this is difficult to do accurately when only a few ,ug of steroid may be available. Also the addition of solvent causes a large peak which may obscure or invalidate peaks due to fast moving materials. Using the present method up to IOO ~1 of dissolved steroid extract may be added to a cylinder and the solvent evaporated before application to the chromstograph. If necessary several additions may be made to the sample capsule, evaporating the solvent after each. addition. This means that steroidal extracts at concentrations usually met in usual methods are more easily dealt with. Another ad.vantage of the method is that the top of the column is not opened during addition and removal of samples. The present apparatus is designed for a 3o-min cycle of adding samples. The type of motor used is available at a variety of speeds so that any particular time’cycle may be chosen if the appropriate motor is fitted. A slower moving motor may be used instead and actuating discs designed with contact grooves cut at intervals around the discs. A given time cycle is then chosen by fitting the appropriate discs to the motor. The apparatus is easily removable so that columns may be changed. The ground glass joint occurring within the flash heater may be criticised; it has caused no trouble. A piece of wire around the ear of the column end extends up through the flash heater where it joins an extension spring anchored to the upper part of the apparatus; this keeps the joint in compression, * Obtainable from Venner hlotors Ltd. J.
ChYOWWtO~.,
20
(1965)
ISI--134
134
NOTES
Using a Pye Argon Chromatograph, a 4 ft. I % SE30 column at ZZ~“, flash heater 270°, pressure 15 psi. the reproducibility of the method was tested. Twelve samples of a pregnanediol diacetate solution, each 4 ,ug in 40 ,ul, were pipetted on to cylinders, evaporated, and the batch assayed using the automatic device described. Peak height and area by triangulation were determined and gave: height, mean 71 & s.d. 3.0; area, 1330 -& s.d. 46 mm2. The experiment was repeated the following day using the same column conditions but the evaporated samples in open glass cylinders were added and removed from the flash heater manually. This gave precision : height, mean 65 & s.d. 3.9; area, mean 1250 5. s.d. 70 ‘mm2. The apparatus has been used chiefly.for overnight runs.’ Samples”‘and standards are loaded in the evening and a separate time switch is set to stop the chart drive at the end of the run. The peaks are quantitated the following morning. Acknowledgement My thanks are due to Mr. H. ICESTEVENand Mr. J. BAMFORD making the timing’ device. De$artnzent of Chemical Pathology, The rioyal Infiwnar_y, She field (Great Britain) Received
March zgrd,
J. ChrOVnatOg.,
20 (1965)
Gas chromatography I. Separation
for their help in
D.A.
PODMORE
1965 131-134
of fluorinated
and identification
fatty acids
of lower 24uorofatty
acids
There has been considerable interest, in recent years, in the biological activity of fluoro-organic compounds related to aliphatic fatty acidsl. Fluoroacetic acid was found by MARAIS~ to be the toxic principle of Gifblaar. Several w-fluorofatty acids were identified in the seeds of Dichafietalum toxicariwn by PETERS and coworlcerssv”. In a study of the fate of Auoroacetic acid in nondividing yeast cells, ALDOUS~ tentatively identified a fluorobutyric acid and a fluorohexanoic acid as metabolic products, Positive identification could not be made for lack of authentic samples. As the result of our program on the synthesis of fluorinated molecules, a. series ’ of z-fluorofatty acids became available. The separation and identification of this series of lower fiuoroacids alone and in combination with a similar series of unfluori-, nated saturated aliphatic fatty acids’by gas chromatography are the subject of this report.
Apfiaratzcs. For the separation and identification of the z-fluorofatty acids alone, a Perkin-Elmer Model x54D Vapor Practometer fitted with a’thermal conductivity .. Ji
Ckt'OlVZatOg., 20 (1965)
134-137