changes the flow of carrier gas during temperature programming unless adequately controlled. From the elution data for water, alcohols, glycols, and hydrocarbons, it appears that the solubility in the polymer is the most important factor in determining the order of elution, and boiling point or volatility is of less consequence. As seen by the order of elution for ethylene glycol before propylene glycol and n-butyl alcohol before t-amyl alcohol, the addition of a methyl group greatly increases the retention time, although the boiling points are actually lowered. The effect of the permeability (Table I, Column 3); as measured by GPC, on the separations and/or utility has not been thoroughly studied. It is apparent that if the permeability is too great or too small, the polymer is of little use.
Highly efficient columns can be packed using good porous polymers. The measured efficiency for n-butyl alcohol (Figure 4) was greater than 800 plates per foot. Column efficiency is dependent partly upon the absence of tailing and partly upon fast equilibration. This is due to the structure, fast access to a large surface area, and absence of thick sections. Porous beads which combine this structure with low and linear sorption properties can then be given greater capacity and specific selectivity by using them as supports for the liquid phases commonly used in gas-liquid chromatography.
J. C. Moore for consultations on polymer synthesis, to W. L. Howard for
ACKNOWLEDGMENT
RECEIVEDfor review August 23, 1965. Accepted December 20, 1956. Third International Symposium, Advances in Gas Chromatography, Houston, Texas, October 1965.
Contributions of many other persons to these studies are gratefully acknowledged. Thanks are particularly due to
assistance in preparation of the manuscript, and to W. V. Hayes and 11. C. Arrington for technical assistance. LITERATURE CITED
(1) Billmeyer, F. W., Jr., “Textbook of
Polymer Science,” pp. 341-2. Interscience. New York. 1962. (2) DeGrazio, R.’P., J . Gas Chromatog. 3, 204 (1965). (3) Lloyd, W. G., Alfrey, T., Division of Polymer Chemistry, 139th Meeting, ACS, St. Louis, March 1961. (4) Lloyd, W. G., Alfrey, T., J . Polymer Sci. 62, 301 (1962). (5) Moore, J. C., Ibid., Part A, 2, 835 (1964).
Separation and Identification of Derivatives of Biologic Amines by Gas-Liquid Chromatography POMPEO CAPELLA’ and E. C. HORNING Department o f Biochemistry, Baylor University College of Medicine, Houston, Texas The separation of biologic amines, including catecholamines, by gas chromatographic techniques requires the preparation of suitable derivatives. A procedure has been developed which involves the treatment of amine mixtures with hexamethyldisilazanefollowed (after complete reaction) by the addition of an aliphatic ketone or cyclobutanone (not cyclopentanone or cyclohexanone). Hydroxyl groups are converted into trimethylsilyl ether groups and primary amines are converted into eneamines or Schiff bases. Secondary amino groups are unchanged. The structures of the reaction products were determined by gas chromatography-mass spectrometry. Effects of changes in the reaction conditions were studied by gas chromatographic and gas chromatographic-mass spectrometric analysis of reaction mixtures. By-products, including oxazolidines and N-trimethylsilylamines, were formed under some conditions. The separations were carried out with a 10% F-60 column with temperature programmed operation. Amines expected as products of human metabolic pathways gave appropriate derivatives and were separated under these conditions.
T
was to develop a method for the gas chromatographic separation of the bioHE OBJECTIVE OF THIS STUDY
316
ANALYTICAL CHEMISTRY
logic amines found in human or other mammalian tissues. Many pharmacological and physiological investigations have been concerned with the occurrence and physiological or biochemical action of these amines, particularly the catecholamines, and the literature of these fields bontains numerous descriptions of isolation and determination procedures. With few exceptions, these methods involve a purification or fractionation procedure leading to a final fraction containing one or more of the amines under study, followed by a colorimetric, fluorometric or spectrophotofluorometric determination of a single amine or of several amines as a group. However, these methods have little in common with gas chromatographic procedures. One of the major problems in the development of a GLC procedure for use with polyfunctional compounds, such as the catecholamines, is that multiple products may be formed at the stage of conversion to derivatives. If nonclassical reagents are used, leading to derivatives which are stable in the gas phase but which can not be isolated for classical analytical study, it may be impossible to characterize the reaction pathways by well established methods. I n the present study no reaction products were isolated. All compounds of uncertain structure were examined in a gas chromatograph-mass spectrometer (combined instrument), and structural
conclusions were drawn on the basis of gas chromatographic and mass spectrometric data. These conclusions mere used as a basis for developing a procedure applicable to all of the amines listed in Table I. This work, however, was not intended to be a systematic or exhaustive study of mass spectrometric behavior for amine derivatives. It would be desirable to carry out a more detailed mass spectrometric investigation if it is proposed to identify amines of unknown structure by this method. The reaction conditions described in the experimental section are suitable for many biologic amines. Hydroxyl groups, when present, are converted to trimethylsilyl ether groups. Primary amines are converted to ketone condensation products through reaction with acetone or cyclobutanone. Secondary amine groups are unchanged, although condensation products may be observed with certain ketones. Tertiary amines were not studied, but from other work it is known that the amine group would remain unchanged under these conditions. If only a few amines are under study, isothermal separation conditions may be used. If a number of amines are to be studied, a temperature programmed separation procedure is recommended. Examples of separations 1 Present address, Institute of Pharmacology and Therapy, University of Milan, Milan, Italy.
are shown in the figures and these results are discussed in relation to earlier work. EXPERIMENTAL
Gas Chromatography. A BarberColman Model 5000 instrument equipped with a hydrogen flame ionization detector was used; a Keithley Model 417 picoammeter was used for current amplification. Column packings were prepared according to the usual procedure of this laboratory (8). The support was acidwashed and silanized (dichlorodimethylsilane) Gas Chrom P, 80-100 or 100120 mesh. The liquid phases were 10% F-60 (a methyl polysiloxane containing a low percentage of p-chlorophenyl groups; Dow Corning Corp.) and a mixture of 7% F-60 and 1% EGSP-Z (a copolymer of ethylene glycol, succinic acid, and a methylphenylsiloxane monomer; Applied Science Laboratories, Inc.). The coatings were applied by the filtration technique ( 7 ) . Isothermal studies of components of reaction mixtures were carried out at 170’ C. with an F-60-Z column (6 feet X 4-mm. glass C-tube) ; the carrier gas was nitrogen (17 p.s.i.). Temperature programmed separations were carried out from 100’ to 200’ C. with an F-60 column (6-foot X 4-mm. glass U-tube) ; temperature rise 1.5’ C. per minute; nitrogen, 12 p s i . The detector compartment was held a t 300’ C. “Methylene unit” values (14,15) were measured by interpolation between retention times for pairs of even-numbered straight chain saturated hydrocarbons ranging from C14to Cmin a temperature programmed separation. For a study of response factors, octadecane was used as an internal standard, and the nitrogen pressure (12 p.s.i.), air pressure (40 p s i . ) and hydrogen pressure (20 p.s.i.) were chosen so that quantitative results (*1.5%) were obtained for a reference standard mixture of straight chain hydrocarbons containing evennumbered components from tetradecane to eicosane. Mass Spectrometry. An Atlas CH4 mass spectrometer was modified according to Ryhage (11) to include a fast scan system and a “molecule separator.” A glass-tube gas chromatograph with a 10% F-60 column packing was used for structural studies of reaction products; the carrier gas was helium, and the gas stream was introduced into the mass spectrometer after concentration in the Rghage “molecule separator” inlet system. The ionizing potential was 20 e.v. and the ion source temperature was 250’ C. hlass spectra were recorded in 1.5 to 2 seconds. Compounds. The amines listed in Table I were obtained from commercial sources and were used without additional purification. They were obtained as hydrochloride salts with the following exceptions: free base N-methyl-p-phenylethylamine; bitartrate, epinephrine. General Procedure. The following
Figure 1. Gas chromatographic separation of the reaction products from the condensation of cyclopentanone with N-methyl-/3-phenylethylamine Peak 1, unreacted N-methyl-p-phenylethylamine; peak 2, dotted line, cyclopentylidene-p-phenylethylamine; peak 3, condensation product from cyclopentanone and N-methyl-Pphenylethylamine. Column; 10% 660,temperature, 135’
Solid potassium carbonate (or potassium bicarbonate) (2 mg.) was added, followed by 0.5 ml. of acetone or other ketones. The mixture was shaken at room temperature for 3 hours. Samples of the mixture were injected directly into the gas chromatograph in order to follow the course of the reaction. In order to compare rates of reaction and compositions under equilibrium conditions, this reaction was carried out for p-phenylethylamine hydrochloride and a mixture of ketones in equimolar proportions. The ketones were acetjone, cyclobutanone, cyclopentanone, cyclohexanone, and cycloheptanone.
Table I. “Methylene Unit” Values for Amine Derivatives Obtained by Application of the General Procedure with Acetone or Cyclobutanone”
Ketone Csclo-
C.
method was developed after an extensive study of reaction products obtained from the amines under different conditions. I n separate experiments it was tested and found suitable for all amines listed in Table I. To a solution of the free amine (0.5 to 1 mg.), or an equivalent amount of the salt, in 0.05 ml. of dimethylformamide, there was added 0.15 ml. of hexamethyldisilazane (HMDS). The mixture was allowed to stand for 30 minutes a t room temperature. (When amine hydrochlorides are used, a precipitate of ammonium chloride is formed. This does not interfere in subsequent operations.) A ketone-HMDS mixture was prepared by adding 1 ml. of HMDS to 10 ml. of acetone or cyclobutanone (or other ketones); the mixture was heated to boiling and allowed to cool. A 0.4ml. portion of the ketone-HMDS mixture was added to the dimethylformamide solution, and the reaction mixture was allowed to stand for 12 hours. Precipitates, if present, were separated by centrifugation. Samples of reaction mixtures (usually 1 to 2 pl.) were injected directly into the GLC or GLC-MS systems. The reaction products were found to be stable during several days’ storage a t -5’ C. A number of observations with respect to changes in amounts or types of reaction products were made by varying the experimental conditions; the pertinent observations are summarized in the next section. It was also observed that ordinary glass surfaces apparently had a catalytic effect leading to by-products from norepinephrine and octopamine when the usual experimental conditions were used; these effects were abolished by employing glass vessels with the surface silanized with 5% dichlorodimethylsilane in toluene. Formation of Eneamines. A solution of 0.5 to 1 mg. of a primary amine hydrochloride was dissolved in 0.05 ml. of dimethylformamide.
Amine p-Phenylethylamine Ephedrine Norephedrine p-H ydroxy-p-p henyl ethylamine p-( 4-Methoxypheny1)ethylamine Tyramine Phenylephrine p-( 3,4-Dimethoxyphenyl)-ethylamine p-( 3-Methoxy-4 hydroxypheny1)ethylamine Octopamine Metanephrine Dopamine Epinephrine Normetanephrine Norepinephrine 5-Hydroxytryptamine a
Ace-
bu-
toneb 13.2 13.9 14.5
tanone* 14.5 13.9 16.3
14.9
16.6
15.6 16.5 16.7
17.3 18.2 16.7
17.4
19.1
18.0
19.7 20.0 18.2 20.3
18.2 18.2 18.6 18.8 19.5 20.0
21.8
18.8 21.1
21.6
...
Primary amino groups are converted
to eneamines. All substituent hydroxyl
groups are converted to trimethylsilyl ether groups. Secondary amino groups are unchanged, and the same derivative is formed for a secondary amine under both reaction conditions. * The “methylene unit” values were obtained by linear interpolation in a temperature programmed separation with even-numbered hydrocarbon reference compounds from tetradecane (14.0) to eicosane (20.0). Column, 10% F-60; temperature rise, 1.5’ C. per minute.
The formation of enamines was followed by direct GLC analysis of small samples. An unchanging composition was reached in 20 hours. Condensation of N - methyl p-
-
phenylethylamine with Cyclopentanone and Cyclohexanone. The ene-
amine condensation conditions were used for N-methyl-p-phenylethylamine (free base) and cyclopentanone. The reaction products were defined by gas chromatography; the composition of the reaction mixture with respect to condensation products is shown in Figure 1. VOL. 38, NO. 2, FEBRUARY 1966
317
n
3
I
I
I
5
10
15
MINUTES
Figure 2. Reaction products obtained from epinephrine, diisopropylketone and hexamethyldisilazane Peak 1, tritrimethylsilyl ether of epinephrine; peak 2, unknown; peak 3, ditrimethylsilyl ether of oxarolidine from epinephrine and diirapropylketone. Column: F-60-Z; temperature, 170' c.
A similar result was obtained when cyclohexanone was used. However, only the cyclopentanone condensation product was subjected to structural study by mass spectrometry. Formation of N - trimethylsilylp-phenylethylamine. The eneamine condensation conditions were used, with the addition of hexamethyldisilazane (1.0 ml.) in place of the ketone. The reaction was studied for p-phenylethylamine and N-methylp-phenylethylamine. The course of the N-trimethylsilyl exchange reaction with p-phenylethylamine was followed by gas chromatography, and the structure of the product was determined by mass spectrometry. No reaction was observed for N-methyl-p-phenylethylamine. Acetone was added to the reaction mixture in order t o determine the stability of N-trimethylsilyl-p-phenylethylamine in the presence of both acetone and hexamethyldisilazane. The course of the reaction was followed by gas chromatography (the eneamine was the sole product). No further investigation was made of the properties of N-trimethylsilyl-p-phenylethylamine. Oxazolidine Formation. The eneamine condensation conditions were employed, except that a ketone (acetone, cyclobutanone or diisopropylketone) and hexamethyldisilazane were added a t the same time. When this condition was employed for primary amines, the product in each instance was the TMSi derivative (if the amine contained one or more hydroxyl groups) of the expected eneamine. When the reaction condition 31 8
ANALYTICAL CHEMISTRY
was used for epinephrine, two major products were observed. It was evident that oxazolidine formation might occur under these conditions, and the reaction sequence was modified. The eneamine condensation condition was employed (shaking at room temperature for three hours after addition of the ketone) ; hexamethyldisilazane (0.4 ml.) was then added and the mixture was allowed to stand for 12 hours. A typical formation of an oxazolidine is shown in Figure 2 for the reaction of epinephrine with diisopropylketone and hexamethyldisilazane. Solvent Interference. Condensation reactions carried out with norepinephrine and octopamine yielded, in addition to the expected products, variable amounts of a compound derived from the amine but whose retention behavior did not vary with the ketone employed in the condensation reaction. Highest yields were obtained under the following conditions. A mixture of 2 mg. of octopamine hydrochloride, 0.1 ml. of dimethylformamide, and 4 ml. of hexamethyldisilazane was heated a t 80' C. for 1 hour. The products were separated by gas chromatography and the structure of the by-product was inferred from its mass spectrum. In separate experiments it was found that this compound was not obtained when the reactions were carried out a t room temperature in silanized glass tubes. RESULTS AND DISCUSSION
Reports dealing with gas chromatographic methods for the separation, identification, and estimation of biologic amines are not numerous, and a satisfactory general procedure has not been available. The chief problem involves the preparation of suitable derivatives of epinephrine (I) and norepinephrine (11) and their metabolites metanephrine (111)and normetanephrine (IV).
p-,
HO
CH,NH R
B
Rl 0
1: Ri=HI, R,=CH,
IT: R,R2=H
III: RiR,=CH$ W: R,=CH,, R,,=H These polyfunctional amines are highly reactive substances and multiple products are often obtained under relatively mild conditions. The most obvious routes to volatile derivatives are acylation (of both hydroxyl and amino groups), reaction with hexamethyldisilazane t o form 0-trimethylsilyl ethers, and reaction with ketones, with or without additional reagents, to form enearnines or oxazolidines. Acyl derivatives of biologic amines may be separated by gas chromatography (S), but it is difficult to obtain reproducible and quantitative conversion of catecholamines to derivatives, probably because of the high reactivity associated with the benzyl hydroxyl group. This problem is not present for tryptamines or p-phenylethylamines lacking a benzyl hydroxyl group. For example, acyl derivatives of p-(3, 4dimethoxypheny1)-ethylaniine are stable under GLC conditions ( I S ) . (A separation of this amine, currently of considerable interest in connection with studies of central nervous system effects produced by biologic amines, is shown in Figures 3 and 4.) The eneamine is formed readily under the conditions described in the experimental section. The wide use of hexamethyldisilazane as a reagent for preparing derivatives of hydroxyl-substituted compounds led to several investigations of the use of this reagent in work with phenolic amines (2,6,10,12) [some phenolic amines may also be chromatographed directly ( 5 )1.
ACETONE SB
7 I
3.4-DMPE
Figure 3.
MN
DO
UUU
I/
Temperature programmed separation of derivatives of amines
Primary amines were converted to eneaminer b y reaction with acetone; secondary amino groups were unchanged. All hydroxyl groups were converted to trimethylsilyi ether groups. The amines were 8-phenylethylamine (PE), norephedrine (NEP), 0-hydroxy-pphenylethylamine @OH), tyramine (TYR), @-(3,4-dImethoxyphenyl) ethylamine (3,4-DMPE), metanephrine (MN), dopamine (DO), epinephrine (E), normetanephrine (NMN) and norepinephrine (NE). Column: 10% F-60, temperature programmed at 1 .5' C. per minute.
Sen and McGeer (12) obtained TMSi ether derivatives from epinephrine, norepinephrine, metanephrine, and normetanephrine but it was found that the primary and N-methyl pairs of derivatives were not separable with ordinary columns. Linstedt (10) also showed that TMSi ethers could be obtained from human biologic amines. The structures were not studied by other than GLC techniques. Sen and McGeer (12) found that a compound believed to be N-trimethylsilyl-p-phenylethylamine was formed from p-phenylethylamine, suggesting that HMDS and primary amines will form N-trimethylsilyl derivatives. Brochmann-Hanssen and Svendsen (1) studied the condensation products of ephedrine and related amines with acetone. The derivatives were assumed to be eneamines or oxazolidines. A reaction leading to eneamines is a general one for primary amines ( 1 4 , and it has been used for some primary amines of the biologic amine series (3). The formation of an oxazolidine may occur for epinephrine and other amines of related structure. The combined use of acetone and HMDS was employed earlier in a study of tryptamine bases (3, 6). The reagents may be used simultaneously unless oxazolidine formation is likely; in these instances the products will be mixed, as indicated in the present study. In order to arrive a t a procedure which would lead to a single product from each component of a mixture of amines containing catecholamines, it was necessary t o study a numbei of reaction conditions. The experimental methods employed were those of gas chromatography and gas chromatography-mass spectrometry. Reaction products were separated by gas chromatography and their structures were studied by mass spectrometry with the use of a combined gas chromatographmass spectrometer and the techniques described by Ryhage ( 1 2 ) . One of the problems studied was the possible formation of N-trimethylsilyl amines in reaction mixtures containing hexamethyldisilazane and a primary or secondary amine. This was investigated for p-phenylethylamine and W-methyl-p phenylethylamine, and it was found that an N-trimethylsilyl amine was formed readily from the primary amine but not from the secondary amine. This reaction was not complete (gas chromatography showed some unchanged amine) but the result confirmed the finding of Sen and McGeer (12) that a reaction product is formed from HMDS and p-phenylethylamine. N trimethylsilyl-p-phenylethylamine was not isolated, but a mass spectrometric study with a gas chromatographmass spectrometer provided data in accord with the expected structure.
n
Figure 4.
NE
CYCLOBUTANONE SB
Temperature programmed separation of derivatives of amines
Primary amines were converted to enearnines b y reaction with cyclobutanone; secondary amino groups were unchanged, Ail hydroxyl groups were converted to trimethylsilyl ether groups. The amines were @phenylethylamine (PE), norephedrine (NEP), P-hydroxy-P-phenylethylamine (POH), tyramine (TYR), metanephrine (MN), epinephrine (E), @(3,4-dimethoxyphenyl)-ethylamine (3,4-DMPE), dopamine (DO), normetanephrine (NMN), and norepinephrine (NE). Column: 10% F-60, temperature programmed a t 1.5' C. per minute.
Ions were observed a t m/e 193 (MW: 193) and a t m/e 102. The latter value corresponds to a product of cleavage between the carbon atoms of the side chain, with retention of charge on the nitrogen-containing moiety. When acetone (or cyclobutanone) was added to the reaction mixture, a gas chromatographic analysis showed only unchanged N-methyl-p-phenylethylamine and a compound corresponding to the condensation product of p-phenylethylamine and the ketone. N-trimethylsilyl-&phenylethylainine was no longer present. It was therefore concluded that an N-trimethylsilylation reaction should not interfere when a ketone condensation reaction is carried out -ubsequent to treatment of an amine mixture (containing primary and secondary amines) with hexamethyldisilazane. Primary aromatic amines react with ketones to form Schiff bases which usually may be isolated by conventional methods. Compounds obtained from the condensation of acetone with nonaromatic primary amines are excellent derivatives for gas chromatographic work (14), but it is usually undesirable to isolate the reaction products. The first observations relating to the formation of primary amine-acetone condensation products in quantitative yield for gas chromatography were by Fales (4,and independent observations of the usefulness of the reaction for distinguishing primary from secondary amines were made in other laboratories. It has been assumed without experimental verification that aliphatic and alicylic ketones will not react readily with secondary amines. This problem was examined through a comparision of the reactions of p-phenylethylamine and N-methyl-0-phenylethylamine with acetone, cyclobutanone, cyclopentanone, cyclohexanone, and cycloheptanone. When p-phenylethylamine was allowed to react with a mixture of equimolar amounts of acetone and the four alicyclic ketones, it was not possible to find (by gas chromatography) unchanged amine in the reaction mixture. However, the composition of the mixture did not remain constant; about 20 hours a t
room temperature was required before an unchanging composition was attained. The relative peak areas were as follows for condensation products formed from ketones and p-phenylethylamine a t equilibrium : Ketone Acetone Cy clobutanone Cyclo entanone Cyclogexanone Cycloheptanone
Peak ratio, yo 5.2 38.2 43.5 11.7 1.5
When the condensation reaction was studied for N-methyl-p-phenylethylamine, with the reaction conditions described in the experimental section, it was found that no reaction occurred for acetone or cyclobutanone, but that cyclopentanone and cyclohexanone gave condensation products. Figure 1 shows the GLC analysis of a reaction mixture containing cyclopentanone, N-methyl+ phenylethylamine, and a trace amount of p-phenylethylamine. The compound responsible for peak 3 was studied by mass spectrometry. Ions were observed a t m/e 219 (MW for the carbinolamine: 219) and a t 201 (M-18). A strong peak a t m/e 91 corresponded t o cleavage between the carbon atoms of the side chain (charge remaining on the aromatic portion). The structure indicated by these results is:
CH,
Because of the possible formation of carbinolamines from secondary amines and cyclopentanone or cyclohexanone, these ketones were eliminated from further consideration as useful reagents. The condensation of an aliphatic or alicylic ketone with a catecholamine may lead t o an oxazolidine. This reaction pathway was studied for epinephrine with acetone, cyclobutanone, and the sterically hindered ketone diisopropyl ketone. The conditions are described in the experimental section; HMDS was added a t the start or later to convert all free hydroxyl groups to TMSi ethers. A typical analytical reVOL. 38, NO. 2, FEBRUARY 1966
* 319
179
300
200
m/e
Figure 5.
Mass spectrum of the tritrimethylsilyl ether of epinephrine
sult is shown in Figure 2 for the reaction of epinephrine with diisopropyl ketone, followed by reaction with HMDS. The retention time observed for peak 1 did not vary when different ketones were used, but the retention time for peak 3 was different for each ketone. This evidence suggested that peak 3 resulted from oxazolidine formation. The mass spectrum for the product obtained from epinephrine and diisopropyl ketone (peak 3 in Figure 2) showed ions with m/e 309 (base peak), 379, 364, 179, and 84. All three oxazolidines derived from epinephrine and acetone, cyclobutanone and diisopropyl ketone showed a strong peak a t m/e 309; the structure of this ion is probably:
It was not possible to obtain a quantitative conversion to the oxazolidine with the experimental conditions used here. When epinephrine was allowed to react with HhIDS in the absence of a ketone, a volatile derivative (peak 1 in Figure 2) was obtained, in agreement with previous work, and this compound was not altered by the subsequent addition of a ketone. When the general procedure described in the experimental section was employed, all of the amines in Table I gave rise to single derivatives, and oxazolidine formation was not observed. Analytical separations of a mixture of amines are shown in Figure 3 (for acetone eneamines) and in Figure 4 (for cyclobutanone enamines). Since secondary amines do not react with these ketones, the retention times for secondary amine derivatives are the same for both circumstances. A minor difficulty encountered in this work was the appearance of amine-solvent condensation products; the structure of the substance obtained from octopamine and dimethylformamide was studied by mass spectrometry, and the appearance of an ion peak a t m/e 352 (MW: 352) confirmed the structure : TMSi TMSiOe!H 320
4w
CH,N=CHN(CH3)2
ANALYTICAL CHEMISTRY
Solvent condensation reactions did not occur when the glass surfaces of the reaction tubes were silanized. The structures of the derivatives shown in Figures 3 and 4 are apparent from their properties and mode of formation, but a study of the structure of the eneamines from several amines was carried out by mass spectrometry. For the general structure :
where RI, RP, and R3 are H or ThfSiO, and Rh is methyl or part of a cyclic system, the compounds show increasing retention times with increasing size of the Rd group, and a molecular ion is observed in low yield in the mass spectrum. The major peak in the mass spectrum for all compounds with a TMSiO substituent a t RI,R2,or R3 results from cleavage between the carbon atoms of the side chain. When Rs is TMSiO, this peak corresponds to a benzyl or tropylium ion including the substituent R3. If R3is H, the cleavage occurs a t the same place, but the base peak is due to the nitrogen-containing moiety of the molecule. This ion contributes relatively little structural information, since R4 is known, but its formation (or nonformation) as the base peak of the spectrum is indicative of the nature of R3. (In cases where R3 is H, peaks of low intensity corresponding to the aromatic portion of the derivative are also found in the spectrum.)
When one or more ThfSiO groups are present, peaks are always found a t M-15 and a t m/e 73. The M-15 peak is apparently due to loss of a Si-methyl group. An ion a t m/e 75 is observed as a strong peak whenever R3 is TlISiO. Cyclobutanone derivatives give spectra that show additional effects; an example is shown in Figure 5. A n ion peak a t ill-28 was observed for all cyclobutanone condensation products. The structure of a TMSi ether of a secondary amine was also studied by mass spectrometry. Figure 6 shows the mass spectrum for the TMSi ether of epinephrine. All three hydroxyl groups are converted to TMSi ether groups. Table I gives the "methylene unit" retention values for derivatives of a series of biologic amines. These values correspond to the Kovats (9) retention index values divided by 100, and they are superior t o relative retention time information for purposes of interlaboratory comparisons of data. Quantification. With the hydrogen flame ionization detection system used in this work, it was found that the precision of an analytical separation carried out with reference compounds was about 4=0.5%, and the accuracy was about i O . 5 to 1.5% for components eluted over a temperature range of about 100' to 200° C. The conditions described in the experimental section mere used for the analytical separation of a hydrocarbon reference mixture with an accuracy of &1.5% for all components before an analytical separation of the biologic amine derivatives was carried out. The reliability of the flame ionization detector is well established, but response factors should be determined locally if quantitative studies are to be carried out. The response of the detector is not uniform for all amine derivatives. ACKNOWLEDGMENT
We are deeply appreciative of the help given by J. A. XIcCloskey in the mass spectrometric work, and of the assistance provided by Ragnar Ryhage in adapting a mass spectrometer for use with a gas chromatography system.
I
Figure 6. Mass spectrum of the tritrimethylsilyl ether-eneamine derived from norepinephrine by reaction with hexamethyldisilazane and cyclobutanone A typical separation of this derivative is shown in Figure 4.
We are also indebted to Miss J. McCune for assistance in the laboratory work. LITERATURE CITED
(1) Brochmann-Hanssen, E., Svendsen, A. B., J . Phawn. Sci. 51, 393, 938
(1962). (2) Brochmann-Hanssen, E., Svendsen, A. B., Ibid., 51, 1095 (1962); 52, 1134 (1963). (3) Brooks, C. J. W., Homing, E. C., ANAL.CHEM.36, 1540 (1964). (4) Fales, H. M., National Institutes of Health, Washington, D. C., private communications, 1960-1961. ( 5 ) Fales, H. M., Pisano, J. J., Anal. Bzochem. 3, 337 (1962).
(6) Horning, E. C., Homing, M. G., VandenHeuvel, W. J. A., Knox, K. L., Holmstedt. B.. Brooks. C. J. W.. ANAL.CHEM.36, 1546 (1964). (7) Horning, E. C., Moscatelli, E., Sweeley, C. C., Chem. and Ind., (London), 1959, 751. (8) Homing, E. C., VandenHeuvel, W. J. A., Creech, B. G., in “Methods of Biochemical Analysis,” Vol. XI, D. Glick, Ed., Interscience, New York, 1963.
(9) Kovats, E., Helv. Chim. Acta 41, 1915 (1958). (10) Linstedt, S., Clin. Chem. Acta 9, 309 (1964). (11) Ryhage, R., ANAL. CHEM.36, 759 (1964). ~ - ,(12).Sen, N. P., McGeer, P. L., Biochem. Bzophys. Res. Commun. 13,390 (1963).
(13) Sen, N. P., McGeer, P. L., Ibid., 14, 227 (1964). (14) VandenHeuvel, W. J. A., Gardiner, W. L., Horning, E. c., ANAL. CHEW 36, 1550 (1964). (15) VandenHeuvel, W. J. A., Gardiner, W. L., Homing, E. C., J . Chromatog. 19, 263 (1965). RECEIVED for review August 11, 1965. Accepted December 13, 196Ci. Work supported in part by Grant HE05435 of the National Institutes of Health, Grant Q-125 of the Robert A. Welch Foundation, and by a grant from the Loula D. Lasker estate. Third International Symposium, Advances in Gas Chromatography, Houston, Texas, October 1965.
Electrical Discharge Pyrolyzer for Gas Chromatography JAMES C. STERNBERG and ROBERT L. LITLE Beckrnan Instruments, Inc., Fullerton, Calif.
b A new pyrolytic method employing a low current, high voltage discharge for the fragmentation of solid samples is described. The sample is placed on a porous graphite felt electrode which serves as the downstream electrode in a flow-through tubular discharge chamber. The sample tubes are readily sealed in place and removed from the system by means of a pneumatic actuator through use of a gasketed removable electrode assembly. Breakdown fragments are swept immediately out of the discharge and into a sample loop. A sampling valve permits introduction of the breakdown fragments onto the head of the chromatographic column, and permits the introduction and removal of sample tubes without interruption of the column carrier flow. The system has been shown to give reproducible and highly characteristic breakdown patterns for samples in various states of subdivision. Results obtained for a variety of samples and possible areas of application are discussed. HE technique of gas chromatogT r a p h y has made possible the analysis of complex mixtures of gases and volatilizable materials through separation followed by measurement. Many classes of sample, however, consist of or include components of insufficient volatility for gas chromatographic analysis. Many organic compounds, especially those of biological interest, decompose a t temperatures which are insufficient for their analysis by gas chromat,ography. It has long been recognized that the decomposition of nonvolatile compounds
under controlled conditions could lead to breakdown patterns characteristic of the starting material. Fragmentation of molecules through electron bombardment under vacuum conditions, with mass-based separation of the ionic fragments produced, is the basis of mass spectrometry. With the development of gas chromatography, a technique became available for the separation and measurement of the stable molecular fragments formed during the decomposition of larger molecules. Originally (2, 4 ) , the pyrolysis was carried out in a separate system, with subsequent syringe injection of the volatile products formed into the gas chromatograph. The more convenient technique of carrying out the pyrolysis in the carrier flow stream feeding the chromatographic column was soon introduced (7, 1 1 ) . Since gas chromatography normally operates a t atmospheric pressure or above, fragmentation methods applicable under these conditions have been used in place of high vacuum electron bombardment. For the fragmentation of solids and plastics, most workers have employed thermal pyrolysis, using a hot wire, a heated tube, or a chamber heated to a high temperature. A large number of publications cited in two recent reviews (9,10) describe various versions of this technique and present the results obtained. I n addition, several commercial devices are available for the thermal pyrolysis of solids. Keulemans and coworkers (6) have also employed thermal pyrolysis for characterization of gaseous species, much as we have used discharge pyrolysis. Simon (12) describes the use of high frequency
heating to pyrolyze samples deposited on small wires introduced into a region where the field can be applied. In a prior study (13) we have used a high voltage, low current, glow discharge in an inert gas carrier a t atmospheric pressure or above for the fragmentation and characterization in the gas phase. Because of the usefulness and reproducibility found attainable with the electrical discharge fragmentation technique when applied to vapor samples, it was decided to attempt to adapt this technique to the fragmentation of solids. I n working with vapor samples, the electrical discharge method affords exceptionally efficient coupling of the input power, since the current is carried by the sample and carrier gas itself and very low power levels (about 1 ma. a t 300 volts d.c.) are sufficient to fragment a significant fraction (greater than 10%) of the sample ( I S ) . Coupling of the power to a nonconducting solid material, however, cannot be as efficient as to vapors, since only a fraction of the input power will be transferred to the solid by collision with electrons and with atoms and molecules of gases which have been energized by the discharge. .4t the higher current levels required for volatilizing the solids, fragments passing int,o the vapor phase will tend to undergo appreciable further breakdown, since they find themselves in an environment much more energetic than that normally employed for vapor fragmentation, Thus, the problem of application of electrical discharge fragmentation to solids became much more than simply one of inserting a solid sample in ~i. VOL. 38, NO. 2, FEBRUARY 1966
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