Characterization and Separation of Amines by Gas Chromatography

Characterization and Separation of Amines by Gas Chromatography W. J. A. VANDEN HEUVEL, W. L. GARDINER, and E. C. HORNING Lipid Research Center, Departmenf o f Biochemistry, Baylor University College of Medicine, Houston, Texas

b The separation, identification, and estimation of biologically important amines by gas chromatographic methods presents a number of unresolved problems. The use of appropriate derivatives and selective stationary phases permits a wide choice of conditions which may b e used to increase or decrease volatility of the compounds under study, and to improve both separation patterns and the quantitative aspects of analytical separations. These experimental variables have been less thoroughly investigated for amines than for many other substances. Accordingly, a study was carried out of several groups of amines as model substances with different kinds of structure (long chain and alicyclic monoamines, aliphatic diamines, and aromatic amines) with the aim of obtaining basic informotion which might b e used in biochemical separation problems. At the same time, observations were made with respect to relationships between gas chromatographic behavior and the structure of amines and their derivatives.

T

of gas chromatographic methods in the analysis of biological amines has not proceeded at as rapid a rate as has their application to problems in steroid and fatty acid analysis. Several publications have described analytical methods for aliphatic amines (8,10,I S ) , and the separation of long chain diamines and triamines has recently been reported (9). A variety of alkaloids and nitrogencontaining drugs have been chromatographed successfully (1, 6 ) , as have certain phenolic and aromatic amines (I, 2, 4,6,If). Free amines may be chromatographed directly in some cases, but in many instances it has proved advantageous to prepare derivatives ( I , 4,6,11). No general study of the preparation and gas chromatographic properties of amine derivatives has been reported, although similar studies in the steroid and in other fields have proved fruitful. The use of derivatives often improves gas chromatographic properties in general, and frequently heparation factors and quantitative aspects of separations are also improved.

I n addition, much structural information may often be obtained by observing retention behavior after treatment with functional group reagents (3, 14). Information concerning structure may also be obtained through the use of a variety of stationary phases, both selective and nonselective, with derivatives. The present investigation was carried out to obtain basic information relating to the gas chromatographic properties of amines, and to develop methods useful in separation, identification, and estimation problems involving biological amines. EXPERIMENTAL

Gas chromatographic retention data were obtained with the following instruments: Barber-Colman Models 10 and 5000, and EIR Model AI;-8 (all were equipped with argon ionization detection systems). Quantitative data were obtained with a Barber-Colman Model 5000 equipped with a hydrogen flame ionization detection system.

HE INTRODUCTION

1550 *

ANALYTICAL CHEMISTRY

The column support, 100 to 120 mesh Gas Chrom S, was acid-washed and silanized according to procedures developed in this laboratory ( 7 ) . The stationary phases were 5.5% (w./w.) of 20 mole per cent p-cyanoethyl n-ethyl polysiloxane (CKSi; General Electric Co.) ; ,5.% SE-52 (a methyl polysiloxane containing a low percentage of phenyl groups; General Electric Co.); a misture of 7 7 , F-60 (a methyl polysiloxane containing a low percentage of p-chlorophenyl groups; Dow Corning Corp.) and 1% polymer EGSS-Z (a copolymer of ethylene glycol, succinic acid, and a methyl phenyl siloxane monomer; Applied Science Laboratories, Inc.). The coatings were applied by the filtration technique (5, 7'). The column conditions (6-foot X 4-mm. glass [--tubes) for work with long chain amines and diamines were as follows: CIuSi, 195", 18 p.s.i.; SE-52, 1 8 9 O , 13.5 p.s.i.; F-GO-Z, 190", 13.5 p.s.i. For work with aromatic amines, the conditions were CSSi, 165", 11 p.s.i.; SE-52, 130°, 13.5 p.s.i.; F-60-Z, 140", 10.5 psi. Amines and reagents were obtained from commercial sources. Derivatives, except for the Schiff bases, were prepared on a milligram scale by a short procedure involving reaction with the appropriate reagent in ethyl acetate (pyridine catalyst) ; the reaction misture was chromatographed directly. Schiff bases were prepared by dissolving amines in an appropriate ketone which also served as a solvent. In a number of cases larger samples were prepared and isolated; the following representative compounds had satisfactory elemental analyses : S-n-hesadecylacetamide, A'-n-hexadecyltrifluoroacetamide, methyl iY-n-hexadecylcarbamate, A'-n-dodecylmethanesulfonamide, the eneamine condensation product of n-hexadecylamine and acetone, and the ditrifluoroacetyl and di-propionyl derivatives of 1,5-diaminopentane (cadaverine). RESULTS AND DISCUSSION

0

IS

MINUTES Figure 1 . Gas chromatographic separation of a mixture of four amines with F-60-Z a t 180" C. The compounds are n-dodecylamine (n-C12NH2), cyclododecylornine (cyclo-C~zNHz), n-tetrodecylamine (n-ClsNHz), and n-hexadecylamine (n-ClaNHJ.

Properties of Derivatives. The separation, on a molecular weight basis, of successive members of a homologous series of compounds by gas chromatography has been observed many times, and long chain primary amines show the same effect. This is illustrated in Figure 1. The separation of n-dodecylamine and the cor-

F-60-Z A,

/)CYCW-C12NHPr CYCW-CI~NHP~

B.

b

Figure 2.

Gas chromatographic separation

The pentafluoropropionamider (NHPFPr) (panel A ) and propionamides (NHPr) (panel 6) of n-dodecylamine (n-Cln), cyclododecylamine (cyclo-C~z), n-tetradecylamine (n-Cla), and n-hexadecylamine (n-Cl8) with F-60-Z

responding alicyclic primary amine is also shown. Free amines may show peak tailing and partial irreversible adsorption, even with relatively inert column packings, and for this reason it is oft'en desirable to work with derivatives. Among suitable derivatives of primary and secondary amines are amides of various kinds, carbamates, and sulfonamides, and the gas chromatographic propert'ies of a number of these compounds were investigat'ed with t'hree st'ationary phases differing in selectivity. Phase SE-52 may be considered to possess low selectivity toward most funct'ional groups, F-60-Z is slightly more selective, and the selectivity of CXSi for functional group retention is significantly enhanced over t,hat of F-60-2. Figure 2 shows the separation of the pentafluoropropionarnides (Panel A) and the propionamides (Panel 13) of n-dodecyl, cyclododecyl, n-tetradecyl and n-hexadecylamine with F-60-2. The cyclododecyljn-dodecyl separation is poorer for t'he fluoro derivatives, and the separation for both types of deriva-

tives is less than that observed for the free amines. The fluoropropionamides are strikingly more volatile, however, than the ordinary propionamides, as evidenced by the fact that N-n-hexadecylpentafluoropropionamide is eluted only slightly more slowly than S-ndodecylpropionamide, even though the former is higher in molecular weight by approximately 150 mass units. With CNSi the separation factor for the n-dodecyl and cyclododecylpropionamides is significantly reduced, and the elution order for the two corresponding pentafluoropropionamides is reversed. The relative retention times, with respect to n-docosane, for both sets of derivatives are greater with CNSi than with F-60-Z, indicating a greater selective retention effect of the CNSi phase for amide groups. The gas chromatographic behavior of methyl carbamates and methyl thiolcarbamates is given in Tables 1-111. Satisfactory properties were observed for these derivatives; the introduction of a sulfur atom results in an increase in retention time approxi-

mately equal to that found for the introduction of two methylene groups into the chain. The formation of a methanesulfonamide results in a large increase in retention time. For example, the retention time of N-n-hexadecylmethanesulfonamide observed with F-60-Z is nearly sixteen times that of the parent amine. Nevertheless, these derivatives exhibit good gas chromatographic properties, as illustrated in Figure 3 with an F-60-Z phase. A variety of suitable derivatives are available for work with amines. The free amine is eluted more rapidly than any of its derivatives; the order of elution of derivatives is the same for the three phases used in this work, although the relative retention times are different. Figure 4 shows the elution pattern of four derivatives with a CXSi phase. The data in Tables I to I11 indicate that the elution order for ordinary amides is acetamide < propionamide < butyramide. The corresponding fluoro derivatives show a remarkable difference VOL. 36,

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F-60-2 n- C g N W Y s

n

tl

n-Cl4N H Ma

I\

A

Figure 3.


The methanerulfonamides of n-dodecylamine (n-ClzNHMr), cyclododecylamine (cyclo-CnNHMs), n-tetradecylamine (n-ClaNHMr), and n-hexadecylamine ( n C16NHMr) with

F-60-Z

Table I.

Relative Retention Times Observed with Phase SE-52

Relative retention timen Derivativeb

CycloCIZNH,

Free amine TFAc PFPr HFBu Ac Pr Bu llethyl carbamate Methyl thiolcarbamate AIethanesulfonamide Acetone-SB T F Acetone-SB Cyclopent-SB Cy clohex-SB Cyclohept-SB

0.097 0 14 0 13 0 15 0 37 0 46 0.63 0.33 0.73 1.05 0.16 0.11 0.49 0.67 1. O O

n-Ciz1;Hz 0.07 0 12 0 12 0 14 0 33 0 42 0.55 0.27 0.61 0.88 0.14 0.089 0.42 0.60 0.89

ni-Ci4NHz

n-CieNHz

0.13 0 27 0 25 0 26 0 68 0 90 1.18 0.59 1.33 1.91 0.28 0.19 0.91 1.28 1.89

0.32 0 58 0 55 0 62 1 49 1 90 2.51 1.26 2.83 4.15 0.63 0.41 1.93 2.73 3.99

a Column conditions were as given in the Experimental Section. All retention times are relative t o that of n-docosane (retention time, 23.5 minutes). b Abbreviations: TFAc, trifluoroacetyl; PFPr, pentafluoropropionyl; HFBu, heptafluorobutyryl; Ac, acetyl; Pr, propionyl; Bu, butyryl; SB, eneamine or Schiff base; TF Acetone, l,l,l-Trifluoroacetone; Cyclopent, cyclopentanone; Cyclohex, cyclohexanone; Cyclohept, cycloheptanone.

Table II.

Relative Retention Times Observed with Phase F-60-Z

Relative retention timen Derivativeb Free amine TFAc PFPr HFBu AC

Pr

Bu

Methyl carbamate 3Iethyl thiolcarbamate ~lethanesulfonamide Acetone-SB TF Acetone-SB Cyclopen t-SB Cyclohex-SB Cyclohept-SB

CycloClzNHz 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0

084 14 13 14 39 49 72 34 82 05 14 10 46 65 98

n-C,,SHz 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

052 13 11 12 32 42 62 27 66 85

11

08 39 57 8i

n-Ci4rL"z 0 0 0 0 0

12 28 25 30 76 00 37 60 49 88 25

1 1 0 1 1 0 0 18 0 88 1 27 1 95

ni-Cie,NH, 0 0 0 0 1 2 3 1 3 4 0 0

26 59 56 64 59 16

02 33 29 11 56 39 1 96 2 83 4 33

Column conditions were as given in the Experimental Section. All retention times are relative to that of n-docosane (retention time, 28.5 minutes). b ilbbreviations are given in Table I. 0

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in behavior in that no large increase in retention time is observed as one proceeds from the trifluoroacetamide to the heptafluorobutyramide [similar effects have been observed with sterol esters (15)l. Indeed, with C S S i the heptafluorobutyramides are eluted slightly more rapidly than the t,rifluoroacet'amides, and the elution order with all three phases is trifluoroacetamide 3 pentafluoropropionamide 6 heptafluorobutyramide. The shortest retent,ion time observed within each group was that of the pentafluoro derivative. Amine derivatives may also be prepared by condensation with ketones to form eneamines or Schiff bases ( 1 , 2, 6). The gas chromatographic properties of a number of these compounds were studied. .Icetone, trifluoroacetone, cyclopentanone, cyclohexanone, and cycloheptanone were used as reagents. The retention data in Tables I to 111 indicate that t,hese derivatives provide a sat'isfactory separat'ion of n-dodecylamine from cyclododecylamine, and that for a given amine, in general, the greater the molecular weight of the derivative, the greater the retention time. The only exception, as might be expected, was the eneamine from trifluoroacetone. Ai comparison of separation patterns for cyclopentanone and cycloheptanone derivatives with CNSi is shown in Figure 5. .is indicated in Tables I to 111, t,he retention of a long chain amine-cycloheptanone derivative is very similar to that found for t'he cyclopentanone derivat,ive of the amine with two additional methylene units in the chain. An advantage in the use of these derivatives is that the reagent is also the solvent; the mixture is injected directly into the gas chromatographic system. Retention Time Behavior and Structure. The steroid number concept (3,14) was advanced by VandenHeuvel and Horning in order to describe

n-C14NHPFPr n-CI

CNSi

n-C14NHC:0 0 CH,

I

I

15

30

45

MINUTES

Figure 4.

Gas chromatographic separation of n-tetradecylamine and several of its derivatives with CNSi

The compounds a r e n-tetradecylamine (n-CldNHg), N-n-tetradecylpentafuoropropionamide (n-ClrNHPFPr), N-n.tetradecylmethy1carbomote (n-C14NHCOOCH3), N-n-tetradecylpropionamide (n-ClrNHPr), and N-n-tetradecylmethylthiolcorbamate (n-C14NHCOSCH )

CNSi

I

n-C14N=C@

Figure 5.

A,


The cyclopentanone-condensation products (eneomines or Schiff bases) ( N = C 5 ) (panel A ) and cycloheptanone-condensation products ( N = C 7 ) (panel n-dodecylamine (n-Clp), cyclododecylamine (cycIo-C12), n-tetradecylamine (n-C14), and n-hexadecylamine (n-Cle) (panel A only) with CNSi

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relationships between the structure of steroids and their retention times observed in gas chromatography. By defining the steroid number in experimental terms as an additive (logarithmic) value determined from retention times relative to two reference steroids rather than one, it was possible to obtain a set of values independent of

Table 111.

temperature over a relatively wide range. Furthermore, it was possible to obtain values characteristic of the functional groups present in steroids, and these values may be used to study structure-retention time relationships. A similar approach to the correlation of structure and gas chromatographic behavior has been studied for

5.00200-

Relative Retention Times Observed with Phase CNSi

Relative retention timea Cyclo-

Derivative* CdHz n-CizNHz n-C14"2 n-ClsNHz Free amine 0.11 0.071 0.16 0.36 TFAc 0.38 0.38 0.85 1.93 PFPr 0.30 0.32 0.74 1.65 HFBu 0.34 0.36 0.81 1.82 Ac 0.97 0.89 1.96 4.47 Pr 1.13 1.07 2.37 5.38 Bu 1.52 1.45 3.23 7.21 Methyl carbamate 0.56 0.48 1.07 2.39 Methyl thiolcarbamate 1.48 1.35 3.01 6.69 Methanesulfonamide 3.23 3.38 8.10 18.7 Acetone-SB 0.17 0.14 0.31 0.67 TF Acetone-SB 0.14 0.11 0.26 0.56 Cyclopent-SB 0.59 0.50 1.11 2.43 Cyclohex-SB 0.79 0.70 1.56 3.43 Cyclohept-SB 1.20 1.07 2.33 5.17 a Column conditions given in the Experimental Section. All retention times are relative to that of n-docosane (absolute time, 19.0 minutes). * Abbreviations are given in Table I. Table IV.

Methylene Unit Values Observed with Phase

SE-52

Methylene Unit (189" C.) Derivative5 Free amine TFAc PFPr HFBu

CycloCizNHz 15.8 16.8 16.6 16.9 19.3 19.95 20.7~ -. 19.05 21.15 22.15 17.1 16.15 20.05 20.9 22.0

AC

Pr

Bu

Methyl carbamate Methyl thiolcarbamate LMethanesulfonamide Acetone-SB TF Acetone-SB Cyclo ent SB Cyclogex-SB Cyclohept-SB Abbreviations are given in Table I.

n-CizNH2

n-Ci4NHz

n-Cis?;HZ

14.75 16.4 16.25 16.65 19.05 19.7 21)_ 4 18.55 20.75 21.65 16.75 15.6 19.7 20.6 21.65

16.75 18.5 18.3 18.65 20.95 21.7

18.85 20.5 20.35 20.7 23.05 23.7 _24_ 422 6 24 8 25.75 20.75 19.6 23.75 24.65 25.7

~

22 4

io 6

22.75 23.7 18.55 17.6 21.75 22.65 23.7

Q

Table V.

Methylene Unit Values Observed with Phase F-60-Z

Methylene Unit (190' C . ) Derivative" Free amine TFAc PFPr

CycloCizNHz

n-CizNHz

15.8 17.05 16.8 17.15 19.65 20.2 21.15 19.3 21.5 22.1 17.0 16.15 20.1 20.9 21.95

14.6 16.8 16.5 16.85 19.1 19.8 20.75 18.7 21.0 21.6 16.5 15.6 19 65 20.6 21.7

HFBu Ac Pr Bu Methyl carbamate Methyl thiolcarbamate Methanesulfonamide Acetone-SB TF Acetone-SB Cyclopent-SB Cyclohex-SB Cyclohept-SB a Abbreviations are given in Table I.

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n-CiaNHz 16.7 18.8 18.5 18.95 21.2 22.0 22.8 20.7 23.0 23.55 18.55 17.65 21.7 22.6 23.7

I 001'

'

16

I

I

18,65 20.7 20.55 20.9 23.15 23,9 24.8 22.7 25 0 25.55 20.55 19.6 23.65 24.6 25 65

I

I

26

Figure 6. Methylene unit chart showing the relationship between temperature and the slope of the n-docosanen-tetracosane line used to determine methylene unit values from relative retention times The mode of determination of the methylene unit value (20.71 of N.n-hexadecyltrifluoroocetamide (relative retention time, 0.59 at 190') is shown

long chain amines and their derivatives. Two long chain hydrocarbons, n-docosane and n-tetracosane, were used as reference compounds; retention times were calculated relative to n-docosane. The methylene unit value (so called because retention times are transformed into values based on methylene units) for a compound may be found by comparing its retention time with that observed for long chain hydrocarbons on a logarithmic basis. The methylene unit value of n-docosane is 22, and that of n-tetracosane is 24 (by definition); Figure 6 shows the method of determining the methylene unit value for LV-n-hexadecyltrifluoroacetamide A ith F-60-Z at different temperatures. hlethylene unit values for a number of amines and their derivatives are in Tables IV to VI. The expression

n-CisNHz

I

18 20 22 24 METHYLENE UNITS

Mllc

=

JICS

+

Mc'F1

+...MCFn

may be used to describe an additive relationship, where MC' is the observed methylene unit value, JIG'S is the methvlene unit value for the carbon skeleton, and JI c'F1 . . . .li L'pn are values characteristic of the functional groups. Table VI1 contains JICF values for a variety of functional groups obtained from the X c ' values found for the long chain compounds listed in Tables IV to VI through the use of th!s equation. Relative retention times are temperature dependent, and th s fact can be observed both from Figure 6 and Table

Table VI.

Methylene Unit Values Observed with CNSi

Methylene Unit (195' C.) Derivative" Free amine TFAc

PFPr HFBu Ac

Pr Bu Methyl carbamate Methyl thiolcarbamate Methanesulfonamide Acetone-SB T F Acetone-SB Cyclopent-SB Cyclohex-SB Cyclohept-SB

CycloCIzYHz

n-CizNHz

16.4 19.5 18.95 19.2 21.9 22.3 23.05 20.5 23.0 24.8 17.45 17.0 20.6 21.4 22.45

15.3 19.55 19.1 19.4 21.7 22.15 22.95 20. I 22.75 24.85 16.95 16.45 20.2 21.05 22.15

n-Ci&Hz

ni-Ci&Hz

17.3 21.6 21.2 21.4 23.7 24.15 24.95 22.15 24.8 26.95 19.0 18.55 22.25 23. I 24.15

19.4 23,65 23.25 23.5 25.8 26.25 27.0 24.2 26.85 28.95 21.0 20.5 24.25 25.15 26.2

.4bbreviations are given in Table I.

Table VII. Methylene Unit Values for Functional Groups (MUF)"

Functional group NHz KHTFAc NHPFPr NHHFBu NHAc SHPr NHBu YHCOOCH, XHCOSCH, NHSOpCH, K=C(CHa)z N=CCH,CF, N=Ca N=C6 s=c7

SE-52 F-60-2 2 . 7 5 2.65 4.7 4.5 4.5 4.3 4.65 4 . 9 7.15 7 0 7.9 7 7 8.8 8 4 6 6 6.7 8 75 9 . 0 9.6 9 7 4.55 4 7 3 6 3.6 7 75 7 . 7 o c o u 8 65 9 7 9 7

CKSi 3.3 7.65 7.2 7.4 9.7 10.2 11.0 8. 10.8 12.95 5.0 4.5 8n . 2?5 3 .I 10.15

Determined with the columns and conditions described in Tables IV to VI. a

TableVIII. Variation in MU and MUF Values with Temperature (F-60-Z Phase)

Rel. ret. time

Compound n-Docosane

1.ooa

MU 22.0

MUF

...

180" C. 2.34 0.24 0.54 0.57 1.35 1.62

24.0 18.65 20.55 20.7 22.7 23 15

2.65 4.55 4.7 6.7 7.15

190" C. 2.23 0.26 0.56 0.59 1 33 159

24.0 18.65 20.55 20.7 22.7 23,l5

2.65 4.55 4.7 6.7 7.15

n-Tetracosane n-Cls"Z n-CisY\i=C(CH3)2 n-CiJVHTFAc n-CisNHCOOCHa n-Ci6XHAc

n-Tetracosane n-ClsXH2 ~ - C ~ B N =CH3)2 C( n-CishHTFAc n-Ci6KHCOOCH3 TZ-C~GSHAC

200"

c.

n-Tetracosane 2 12 n-Cis>-Hn 0 28 ~ - C , ~ X = C I C H I ) ~0 58

24 0 18 65 2 65 20 55 4 55 n-Ci6SHTFAc 0 61 20 7 4 7 ?%-CI~SKCOOCH~ 1 31 22 7 6 7 n-CioSHAc 1 55 23 15 7 15 a Retention times: a t 180", 50.0 minutes; at 190", 28.0 minutes; a t 200°, 17.1 minutes.

VIII. The change in the slope of the n-docosane/n-tetracosane plot with change in temperature indicates that a temperature dependency exists, and serves to emphasize the point that M C values should be determined from relative retention times obtained under the same conditions used for the determination of the slope of the n-docosanelntetracosane line. Table VI11 gives the relative (to A-docosane) retention times found for n-hexadecylamine and several of its derivatives a t three different temperatures, and these values are ternperature dependent. The corresponding MIU values, calculated with the use of the appropriate slope, however, are not affected by the changes in temperature. The M C T F values, charact,eristic of the various funct'ional groups, are also temperature independent. The gas chromatographic behavior of several derivatives of two biologically important' amines, putrescine (1,4diaminobutane) and cadaverine (1,5diaminopentane) is summarized in Table IX. With CSSi, in the case of the acyl derivatives, the fluoro-substituted compounds are eluted much more rapidly than the corresponding ordinary derivatives. The elution order is di-acetamide < di-propionamide < dibutyramide, and for the fluoro compounds the order is di-trifluoroacetamide > di-pentafluoropropionamide < di-heptafluorobutyramide. X striking difference in volatility b e h e e n the diheptafluorobutyramide of cadaverine and the corresponding di-butyramide is evident from the data. The former compound, although possessing a molecular weight t,wice that of the di-butyramide, is eluted in approximately one tenth the retention time. The usefulness of the ML' concept in predicting retent,ion behavior is illustrated in Table X, which compares the calculated or predicted M L ' values for the diamides of putrescine and cadaverine with those observed Kith CSSi. ~

Ketones can condense with both ends of a diamine to form di-eneamines. Table X compzres the predicted JIC' values for the di-eneamines of putrescine and cadaverine with t,hose found with CNSi and SE-52. The possibility of forming a mixture of eneamiries should exist when a diamine is dissolved in a mixture of ketones. For the three cyclic ketones used in this study, the general question of the elution order of the six theoretically possible derivatives arides, and a more specific question is whether the cyclopentyl-cycloheptyl or the di-cyclohexyl di-eneamine would be elut,ed more slowly, since both contain the same number of carbon at,oms. I t should be possible to identify the peaks from the chromatogram of such a mixture through the use of the M C concept, and Table XI list,s the predicted and observed ML' values for t,he mixed enearnines from putrescine and cyclopentanone, cyclohexanone, and cycloheptanone. The retention behavior of the di-cyclopent,anone, di-cyclohexa-

Table IX. Relative Retention ~i~~~ for Derivatives of 1 , 4 - ~ i ~ ~ and i ~ ~ b ~ t ~ 1,5-Diaminopentane

Relative retention time* Derivative"

1,4-DA4B 1,5-DAP SE-52

ACZ

Prz Buz Cy clopent-SBn Cyclohex-SBz Cyclohept-SBp

0.27 0.34 0.61 0.39 0.76 1.69

0.31 0.50 0.91 0.55 1.12 2.44

F-60-Z Acz Prz

Bu. Cyclopent-SBZ Cy clohex-SBZ Cyclohept-SBz

0.42 0.56 0.95 0.38 0.78 1.86

0.64 0.89 1.46 0.55 1.15 2.72

CXSi TFAcz PFPrz HFBup ACZ Prz BUZ Cyclopen t-SB2 Cy clo hex-SBZ Cyclohep t-SBz

0.66 0.50 0.62 1.74 2.49 4.58 0.59 1.14 2.66

0.90 0.70 0.83 2.75 4.02 7.48 0.82 1.64 3.84

a Abbreviations: 1,4-DAB, 1,4-diaminobutane; 1,5-DAP, l,5-diaminopentane; ACZ, di-acetyl; Prz, di-propionyl; B u ~ ,dibutyryl; Cyclopent-SBs, di-eneamine from cyclopentanone; CyclohexSBp, di-eneamine from cyclohexanone; Cyclohept-SBz, di-eneamine from cycloheptanone; TFAc2, di - trifluoroacetyl; PFPrz, di-pentafluoropropionyl; HFBup, di-heptafluorobutyryl. Same conditions as Tables I, 11, and 111. Retention times relative t o that of n-docosane.

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F-60-Z

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Figure 7. Gas chromatographic separation of the six theoretically possible condensation products from putrescine and a mixture of cyclopentanone, cyclohexanone, and cycloheptanone with F-60-Z The compounds are the di-cyclopentanone product [5C=N(CH&N = CS], the cyclopentanone-cyclohexanone product [5C=N(CH2)4N = C 6 ] , the di-cyclohexanone product [6C=N(CH& N = C 6 ] , the cyclopentanone-cycloheptanone product [SC=N(CHz)dN = C7],the cyclohexanone-cycloheptanone product [6C=N(CH2)4N = C 7 ] , and the di-cycloheptanone product [7C=N(CHz)rN = C 7 ]

none, and di-cycloheptanone condensation products was determined separately to establish reference points. The predicted result is borne out by the actual elution order; Figure 7 illustrates the separation of the mixture of eneamines. Quantitative Relationships. In order to ascertain whether satisfactory quantitative relationships could be obtained with the hydrogen flame ionization detection system two mixtures of reference compounds were chromatographed with a F-60-Z column. Eminently satisfactory results were obtained both with a mixture of fatty acid methyl esters (methyl palmitate, methyl stearate, methyl arachidate) and a mixture of long chain hydrocarbons (n-eicosane, n-docosane, n-tet-

racosane) ; typical analytical results are given in Table XII. To ascertain whether satisfactory quantitative results could also be obtained with amine derivatives, two test mixtures were used. I n the first, a mixture of n-dodecylamine, n-tetradecylamine, and nhexadecylamine was allowed to react with trifluoroacetic anhydride (pyridine catalyst) in ethyl acetate, and aliquots of the reaction mixture were analyzed; a typical result is in Table XII. I n another test, a mixture of n-dodecylamine, n-tetradecylamine, and n-hexadecylamine was dissolved in cyclohexanone, and aliquots were chromatographed; again, the result observed in the table was highly encouraging. These data indicate that no correction factors

Table XI. Calculated and Found MU Values for Eneamines from 1,4-Diaminobutane and Cyclopentanone, Cyclohexanone, and Cycloheptanone

Compound*

Cyclopen t-SB2 19 4 19 Cyclopent, Cvclohex-SB2 2 0 . 3 20 Cyclohex-SB221 2 21 Cyclopent, Cy~lohept-SB~21 4 21 Cyclohex, Cyclohept-SBn 22 3 22 Cyclohep t-SB2 23 4 23 a Abbreviations given in Table IX

Methylene Unit Values for Derivatives of 1,4-Diaminobutane 1 ,5-Diaminopentane

CNSi Derivativea

1,5-DAP

1,4-DAB Calcd. Found 23.4 24.4 26.0

23.4 24.3 25.85

Calcd.

Found

24.4 25.4 27.0

24.55 25.5 27.1

21 5 23 2 25 3

21 45 23 25 25 4

C?jSl Cyclopent-SB2 Cyclohex-SBp Cyclohept-SBp

20 5 22 2 24 3

20 6 22 3 24 45

and

Methyl palmitate Methyl stearate bfethyl arachidate n-Eicosane n-Docosane n-Tetracosane n-Dodocvl NHTFAr n-Tetradkcyl SHTFrlc n-Hexadecyl SHTFAc n-Dodecylamine Cy clohex-SB n-Tetradecylamine Cyclohex-SB n-Hexadecylamine Cyclohex-SB

SE-52

1556

19 5 21 3 23 4

T a b l ~IX


19 5 21 2 23 4

20 5 22 3 24 4

20 4 22 3 24 4

IYeigh t % , Known Found 27 36 36 14 32 53 15 34 50

2 0 8 6 3 1 0

2 8

27 36 36 14 32 53 17 33 49

1 2 6 6 1

3 1

2 7

18 0

18 2

32 6

33 3

49 4

48 5

Measurements of area were height x width at half-height: these values are from typical runs. In separate experiments the precision was found to be about 0.5-1.05;. Column conditions were as given in the Experimental Section. Abbreviations given in Table I. a

Cyclopent-SBz Cyclohex-SBp Cyclohept-SBa a Abbreviations given in

5 4 3 45 4 5

Table XII. Analysis of Reference Mixtures with F-60-Z"

Compoundb Table X.

1,4-DAB ______

Calcd. Found

are necessary for work with mixtures of long chain compounds which contain the same functional group but, which differ in chain length. Members of closely related groups of compounds usually show the same response (ion yield) rharacteristics (see above), but it s,hould not be assumed that compounds of similar carbon skelet,on but differing in functional groups will possess similar response characteristics. Inderd, as has been pointed out by Slveeley and Chang (II),the detector response for closely relat’ed steroids differing in functional groups is not uniform. T o investigate the effect of varying the functional group present on a long hydrocarbon chain, a number of quantitative niistures were prepared, each cont,aining 2.5 pg. of n-docosane per wl. of solution. The detector response for a known amount of long chain amine or amine derivative was then compared to that for the hydrocarbon, both on a weight basis, and on a mole basis; the results are given in Table X I I I . The low response observed for n-hexadecylamine suggest’ed that some irreversible adsorption of the free amine might be occurring, and this was further suggested by the trailing exhibited by these compounds during chromatographic separation (see Figure 1). If loss of solute occurs on a column, it is often less apparent a t increased load levels, and so a sample with a threefold increase of amine concentration was analyzed; as can be seen in Table X I I I , the relative response increased, but the values are still far below those for the derivatives. The relative response with a flame ionization detector for the trifluoroacetamide is much greater than that for the amine, but significantly lower than that found for the acetamide on a weight basis. On a molar basis, however, the two amides display similar response values. Both the methyl carbamate and methanesulfonamide exhibit relative responses slightly less than t,he acetamide on a weight basis (probably due to a n increase in oxygen content), but on a molar basis the values for the carbamate and sulfonamide are reversed. When the trifluoroacetamide was prepared from n-hexadecylamine and trifluoroacetic anhydride (pyridine catalyst) in the n-docosane-containing solution, rather than added in solution as a pure compound, the relative response was somewhat reduced. With the acetone-condensation product the reverse is true, in that a lower relative response is found for t,he sample prepared with the isolated eneamine. These compounds are quite labile, especially in a solution which does not contain the reactive ketone, and there was evidence for a small amount of reversal to the free amine, which would account for the lowered relative re-

4

Figure 8.


A mixture of the pentafluoropropionamider (PFP) (upper chromatogram) and propionamides (Pr) (lower chromatogram) of benzylmethylamine ( 1 1, a-methylbenzylomine (21, and 4-methylbenzylamine ( 3 ) with

F-60-Z

sponse. If the amine and n-docosane are dissolved in the ketone, which serves as both solvent and reagent, however, gas chromatographic evidence indicates complete conversion to the Schiff base, and this is reflected by the higher relative response factor observed. With trifluoroacetone the relative response for the Schiff base is much lower than for acetone, both on a weight and a molar basis.

Table XIII.

Aromatic Amines. Separation patterns for aromatic amines may be altered markedly through the use of different stationary phases and derivatives. Three isomeric methyl-substituted benzylamines, benzylmethylamine, a-methylbenzylamine and 4-methylbenzylamine, cannot be resolved with either F-60-Z or SE-52 columns, and a CNSi column of normal efficiency, 2500 to 3000 theoretical

Determination of Response (Ion Yield) for Amine Derivatives Compared with Response (Ion Yield) for the Hydrocarbon n-Docosane

Mixturea n-CieNHz

Weight Molar ratio ratio Composition

Areab ratio Found

0.67 2.03 0.92 2.19 2.08 1.18 0.92 1.10 1.28

0.11 0.55 0.59 1.45 1.32 0.70 0.51 0.62 0.77

0.25 1.58 1.00 2.00 2.00 1 .oo 1.00 1.00 1.16

Area ratio Weight ratio

Area ratio Molar ratio

21 35 59 73 66 70 51 62 67

17 27 64 66 64 59 55 56 60

x 100

x 100

n-CiJHTFAc n-CieXHAc n-CieNHCOOCH? n-CizSHSOtCHs n-Ci6NHz TFAA n-CisS=C( CHa)z n-Ci6NHt Acetone n-Ci6NHz TF Acetone 1.39 1.29 0.39 28 30 The a The second component of each mixture was a known amount of n-docosane. ratios are calculated with respect to the hydrocarbon. b Measurements of area were height X width a t half-height; precision was 0.5-1.0%. Column conditions same as for Table XII.

+ + +

VOL. 3 6 , NO. 8, JULY 1964

1557

I. lo

2. F-60-Z PF P

1

F-60-2

CN Si PFP

I5

MINUTES Figure

9. Gas chromatographic

A mixture of the pentafluaraprapionomider (PFP) of benzylamine ( 1 mixture of the methyl carbamates with F-60-Z (center panel)

"1

separation

and benzylmethylamiae (2") with F-60-Z (left panel) and CNSi (right panel), and of

Smphot

Amphri

F- 60-Z Amphrt

Amphet+PFPA

11

*o Amphet t CIC,scH

A

+ Pr20

3

30 45 60 MINUTES Gas chromatographic separation of a mixture of amphetamine and several of its derivatives with F-60-Z IS

Figure 10.

+

The eompoundr are amphetamine (Amphct), amphetamine pentofluarapropionamide (Amphet PFPA), amphetamine methyl carbamate (Amphet CICOOCHs), amphetamine propianamide (Amphet PrzO), and amphetamine methyl thiolcarbamate (Amphet CICOSCHII

+

1558


+

+

plates, would just effect a base line separation of a-methylbenzylamine from benzylmethylamine. The three compounds may be readily separated as the pentafluoropropionamides with SE-52. Wit,h F-60-Z, t,he propionamides of benzylmethylamine and a-methylbenzylamine do not separate, but a very clear cut separation is effected as the pentafluoropropionamides, as illustrated in Figure 8; the greater volatility of the fluoro compounds is clearly evident,. The three methyl-substituted amines may also be separated as the methyl carbamates and methyl thiolcarbamates; the elution order for these derivatives of benzylmethylamine and a-methylbenzylamine with F-6CZ is the reverse of that found for the pent'afluoropropionamides. Excellent separations were also achieved through the use of the pentafluoropropionamides, propionamides, methyl carbamates, and methyl thiolcarbamates with a CNSi column. The acetone-condensation products of a-methylbenzylamine and 4-methylbenzylamine were readily separated with all three stationary phases used in this investigation, and the same effect was found for the cyclohexanone condensation products. The close proximity of the methyl group to the functional group in a-methylbenzylamine may reduce the extent of interaction between the functional group and the stationary phase and account for the lower retention times found for this compound when compared to the 4-methyl isomer; a similar behavior was observed for acyl derivatives. Benzylmethylamine, a secondary amine, does not yield a Schiff base when dissolved in acetone, and no new peak was observed during gas chromatography. When a sample of benzylmethylamine was dissolved in cyclohexanone; however, a new compound was produced which was eluted with all t,hree columns a t a rate intermediate to those of the cyclohexanone Schiff bases of a-methylbenzylamine and 4-met h ylbenzylamine ; the benzylmethylamine condensation product is presumably an eneamine, possibly benzylc yclohexenylmethylamine. The gas chromatographic separation of a primary amine from the corresponding secondary methyl amine may be illustrated by the separation of benzylamine from benzylmethylamine. Separation as the free amines does not occur with SE-52 or CNSi, but a satisfactory separation factor was observed wit'h F-60-Z (see Table XIV). An SE-52 column will readily separate the pentafluoropropionamides, and these derivatives were also well separated with F-60-Z; with CNSi their order of rlut,ion i3 reversed (see Figure 9). The order of elution may also be reversed with F-60-Z by forming the methyl carbamates. This effect is shown in Figure 9. The fact' that elu-

Table XIV.

Relative Retention Times" Observed for Aromatic Amines and Their Derivatives

Derivativeb

Benzylamine

Benzyla-Methylmethylamine benzylamine

Amine PFPr Pr COOCH, COSCHa Acetone-SB Cyclohex-SB

0.062 0.13 0.68 0.38 1.08 0.17 1.22

0.065 0.18 0.75 0.38 1.11

Amine PFPr Pr COOCHB COSCHa Acetone-SB Cyclohex-SB

0.052 0 15 0 98 0 50 1 73 0.17 1.37

0.060 0 19 0 92 0 43 1 40

4-MethylBenzylbenzylamine dimethylamine

SE-52

...

1.30

0.062 0.13 0.69 0.42 1.16 0.15 1.00

0,095 0.22 1.22 0.69 2.01 0.29 2.08

0.062

0.057 0 14 0 94 0 53 1 69 0.15 1.07

0.089 0 24 1 64 0 84 2 85 0.31 2.43

0.050

F-60-Z

...

1.46

CXSi Amine 0.075 0,076 0.071 0.12 0.052 PFPr 0.71 0.41 0.59 1.07 Pr 2.88 2.08 3.01 4.88 COOCHa 1.08 0.81 1.10 1.78 COSCH, 3.96 2.45 3.84 6.33 Acetone-SB 0.29 0.22 0.51 Cyclohex-SB 2.17 1.81 1.50 3.65 Column conditions were as given in the Experimental Section. All retention times are relative to that of n-hexadecane (times: SE-52, 29.2 minutes; F-60-Z, 24.0 minutes; CSSi, 9 . 0 minutes). Abbreviations are as given in Table I ; COOCH,, methyl carbamate; COSCH,, methyl thiolcarbamate.

tion orders may be readily altered indicates the high degree to which gas chromatography is sensitive to small differences in structure and to changes in functional groups. The gas chromatographic characterization of amphetamine, which may be considered to be a typical drug of the aromatic amine type, is given in Table XV. The fluoroamides, with a given column, were found to have very similar retention times. The relative retention times for the acyl, carbamate, and sulfonamide derivatives are of the same order of magnitude with SE-52 and F-60-Z. The latter phase showed somewhat enhanced selectivity; for example, the relative retention time of the carbamate was 0.60 with SE-52, but 0.74 with F-6Ck-Z. The separation of a mixture of amphetamine and several of its derivatives is illustrated in Figure 10. The selective retention shown by a CXSi phase for these derivatives is significantly greater, however, as evidenced by the large relative retention times in Table XV; the methanesulfonamide was retained to a particularly great extent. Amphetamine may also be characterized by using various reactive ketones to form eneamines. LITERATURE CITED

(1) Brochmann-Hanssen, E., J . Pharm. Sci. 51, 1071 (1962).

Table XV. Gas Chromatographic Behavior of Amphetamine and Amphetamine Derivatives with Three Stationary Phases ~

Compound Amphetamine TFAc PFPr HFBu Ac Pr Bu Methyl carbamate Methyl thiolcarhamate Me thanesulfonamide Ace tone-SB TF Acetone-SB Cyclopent-SB Cyclohex-SB Cyclohept-SB

Relat,ive retention timea--__ SE-52 F-60-Z CKSi 0 10 o 22 0 21 0 25 0 68 0.96 1.44 0.60

0 09

0 16

23 0 22 0 27 0 91 1.24 1.87 0.74

0 94 1 10 3 01 3.75 5.56 1.54

1.66

2.23

5.09

2.06 0.21 0.12 0 76 1 18 2 10

3 . 9 7 13.9 0.18 0 . 2 3 0.12 0 26 0 77 1 08 1 21 1 68 2 18 2 83

o

i io

a For column conditions, see Experimental Section and Table XIV. All retention times are relative to n-hexadecane. * Abbreviations are given in Table I.

(2) Fales, H. M., Pisano, J. J., Anal. Biochem. 3 , 337 (1962). (3) Hamilton, R. J., Vanden Heuvel, W. J. A., Horning, E. C., Biochini. Biophys. Acta 70, 679 (1963). ( 4 ) Holmstedt. B.. Vanden Heuvel. W. J. . A., Gardine;, W. L., Horning, E . C.,

Anal. Biochem., in press. E. C., Moscatelli,

(5) Horning,

VOL. 36, NO. 8, JULY 1964

a

E.,

1559

Pweeley, C. C., Chem. Ind. (London) 1959, 751. (6) Horniny, E. C., Vanden Heuvel, W . J . A , , .4nn. Rev. Biochem. 32, 700 ( 1963).

( 7 ) Horniny, E. C., Vanden Heuvel, W . J. h.,Creech, B. G., in “Methods of Biochemical Analysis,’’ D. Glick, ed., Vol, XI, Interscience, Kew York, 1963. f X 1 Link. IT. E.. Morrissette. R . A.. J . -1m. 011 Chem.’Soc. 37, 364 il960). ’ ( 9 ) Metcalfe, L. D., Schmitz, A. A., J . Gas Chromatog. 2, 15 (1964).

(10) Selson, J., Chem. lnd. (London) 1960, 663. (11) Sen, 1.P., McGreer, P. L., Biochem. Biophys. Res. C o m m . 13, 390 (1963):

E. C., in “Biochemical ,Applications H. A. Szymanski, ed Plenum Press, Sew York, in press.

(12) Sweeley, C. C., Chang, T-C. L., ANAL.CHEM.33, 1860 (1961).

RECEIVEDfor review March 26, 1964. Accepted May 14, 1964. Presented at 2nd International Symposium on Advances in Gas Chromatography, University of Houston, Houston, Texas, March 23-26, 1964. This work was supported in part by Grant HE-05435 of the National Institutes of Health.

Ibid., 14, 227 (1964).

(13) Sze, Y . L., Borke, M. L., Ottenstein, D. M., I b i d . , 35, 240 (1963). 114) Vanden Heuvel. W. J. A.. Hornine. E, C., Biochem. ’ Biophys. ‘ d c t a &, 416 (1962). (15) Vanden Heuvel, W. J. A., Horning,

of Gas Chromatography, ~

Analysis of Sulfur Compounds with Electron Capture/ Hydrogen Flame Dual Channel Gas Chromatography DUDLEY M. OAKS, HAROLD HARTMANN, and KEENE P. DlMlCK Wilkens lnstrurnent & Research, lnc., P.O. Box 3 7 3, Walnut Creek, Calif.

b A dual channel gas chromatograph, equipped with both an electron capture and a hydrogen flame detector, has been used to study organic sulfide compounds and the sulfur components present in garlic. The ratio of electron capture response to hydrogen flame response for a given peak is defined and designated as 4. A series of standard sulfur compounds was obtained and chromatographed. Both relative retention times and 4 values were calculated. These values were then used in identifying peaks found in chromatograms of garlic headspace gas and hexane extracts. Cases are shown where identification is possible only because of the availability of the 4 values. Components have been found in garlic which were hitherto unknown, and many of these have been identified.

D

gas chromatography refers to a technique using a single column with two different detectors. The column effluent is divided by a splitter and the two streams are fed to two detectors. The signal from each detector is then separately amplified to drive a two-pen recorder system (Figure 1). The detector combination employed for this u-ork consists of electron capture and hydrogen flame ionization detectors. Each detector is entirely different in operation and response to a compound. This combination was suggested by Lovelock ( 7 ) , and was discussed with applications by Amy and Dimick ( I ) and Hartmann, Oaks, and Dimick (6). The flame detector is quite sensitive and responds nearly equally to all organic compounds. The electron capture detector is sensitive to only certain organic comUAL CHANNEL

1560


pounds such as conjugated carbonyls, nitro compounds, halogenated compounds, and sulfur-containing compounds. Furthermore, sensitivity will vary markedly between types of compounds. For this reason the ratio of electron capture response to flame response becomes an important value for component identification. This response ratio (EC/FL), which we have designated as $, is an empirical value based on 1X attenuation for both electrometers and assumes a 1: 1 split in the column effluent. This study was undertaken to show the versatility of the dual channel system for the detection and identification of sulfides in general, and of the volatile sulfides in garlic in particular. Three parameters were employed in peak identification: relative retention times (RRT) on a polar column, R R T on a nonpolar column, and 4 values. To form a basis for peak identification, 31 organic sulfide standards were purchased or synthesized. These standards were chromatographed on a Carbowax

20M column. Their retention times relative to diallyl disulfide and their response ratios, $, were tabulated. The garlic samples were chromatographed on both a Carbowax 20h4 and a silicone oil column. The retention times relative to diallyl disulfide for each column were displayed as a cross plot. From these data it was possible to tentatively identify 11 of the 21 peaks. Some of the components, most notably diallyl sulfide and diallyl disulfide, have been previously reported by Kertheim ( 2 1 ) and Semmler (9) as being present in garlic. Carson and Wong ( 2 ) , in work of a similar nature, reported the presence of methyl disulfide, methyl trisulfide, methyl n-propyl disulfide, methyl n-propyl trisulfide, npropyl disulfide, and n-propyl trisulfide in onions. EXPERIMENTAL

Apparatus. T h e gas chromatograph used was a Rilkens Aerograph twochannel instrument equipped with a standard flame detector and a concentric tube electron capture detector.

0

Figure 1.

Schematic diagram of a dual channel chromatograph and recorder A. B. C.

D.

Injector Column Splitter Flame detector

E. F1 Fp G.

Electron capture detector Electrometer, flame Electrometer, EC Two-pen recorder

Characterization and Separation of Amines by Gas Chromatography

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