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Chapter 31 Other non - heterocyclic nitrogen compounds
Chapter 31
Other non- heterocyclic nitrogen compounds J . CHURACVEK
CONTENTS
...................................................................657 ............................................................... 657 ....................................................................... 659 ..................................................... 661 .................................................................... 664
Introduction Nitrocompounds Amides Cuanidineandureaderivatives References
INTRODUCTION In t h s chapter, the liquid chromatography of nitro compounds, urea and its derivatives, guanidines, nitrosamines, amides, azo compounds and aromatic hydrazo compounds is described. The chromatography of amides is mentioned only briefly because from the practical point of view the chromatographic separation of peptides and their mixtures is most important and therefore a special chapter is devoted to this aspect (Chapter 34). For the other compounds mentioned, the main aim of liquid column chromatography is their separation from other types of compounds. All modern chromatographic techniques have good prospects of being applied successfully in this field, but little attention has been devoted to them so far. An interesting technique was used for nitro compounds; some of them can be synthesized directly on the column on which they are then separated chromatographically from other components of the reaction mixture.
NITRO COMPOUNDS A typical example of the isolation of nitro compounds from industrial mixtures was described by Landram ef al. Chromatographic separation was used instead of the usual extraction procedures, which represents an important advance in work with explosives. This separation procedure was used satisfactorily for a number of double base propellants containing nitroglycerine, triacetin, 2-nitrodiphenylamine, resorcinol, ammonium perchlorate, aluminium, 2.4,6,8-cyclotetramethylenetriamine and nitrocellulose. The procedure is generally applicable to different types of propellants and it is not necessarily restricted to those which bear a large number of nitro groups. Both Chromosorb T (PTFE) and silica gel can be used for column packing in these instances. The principle of dry column chromatographic extraction should prove useful also with other non-propellant polymers. The main advantage of this procedure is the decrease in the operating time: in the example described above, the Soxhlet extraction took 1-4 days References p.664
657
658
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
while the chromatographic procedure took 1 h. In large-scale operations, the reduced consumption of solvents in chromatographic procedures is also important. I n the analytical field, ion-exchange chromatography is the prevailing method for the separation of nitro compounds. This method permits the separation of a series of nitroalkanes and nitro-aromatic compounds. Polarography is mainly used for detection (Kemula); a special chromatopolarographic apparatus was developed by Kcmula and Brzozowski for this purpose. The limiting diffusion current produced by organic substances is measured on a mercury drop electrode at a constant input potential (-1 V). Kemula and Brzozowski also described the separation of some nitroalcohols and nitrobenzoic acids by salting-out chromatography on a thermostatted cation-exchange column at 56,82,25 and 71°C. On Wofatit KPS-200, a mixture of four nitroalcohols was separated by elution with 1 M ammonium sulphate solution on a 37 X 0.7 cm column. The flow-rate was not an important factor in the separation. The sequence of the eluted compounds was 2-methyl-2-hydroxy-1,3-propanediol, 2-methyl-2-nitropropano1, 2-nitrobutanol and 2-hydroxymethyl-2-nitropentanol. Kemula and Brzozowski also separated a three-component mixture of nitroalkanes on a Dowex 50 column using ammonium sulphate solution as the mobile phase. The elution sequence observed was nitromethane, 2-nitropropane and 1-nitrobutane. The column dimensions were 1 1.5 X 0.7 cm and the flow-rate of the mobile phase did not exceed 9 ml/h. On the same ion exchanger, separations at various temperatures can also be effected: at 25°C with 1 Mammonium sulphate solution as eluent, nitromethane, 2-nitropropane and 1,3-dinitropropane are eluted first. After increasing the temperature in the column jacket to 71°C and using 0.5 M ammonium sulphate solution for elution, 1-nitrobutane and 1-nitropropane appear in the effluent. Under the same conditions, all three isomeric nitrobenzoic acids can also be separated, using 0.1 M ammonium sulphate solution in 0.019 M hydrochloric acid as the mobile phase. o-Nitrobenzoic acid is eluted at room temperature with a minimum retention time, p-nitrobenzoic acid is eluted at 56°C and m-nitrobenzoic acid at 82°C. The time required for the separation is about 5 h. During the analysis of the radiolytic products in the aqueous nitrate-ethylene system, ion-exchange chromatography on Dowex 50W-X8 was also employed. Ammonium sulphate solution of various concentrations served as the mobile phase. Nitromethane, hydrogen peroxide and sodium nitrite, present in the mixture, were detected polarographically. The curves were recorded a t a constant potential corresponding to the complete reduction of the dissolved oxygen and lower than the reduction potential of nitrate (Broszkiewicz and Przybylowicz). Kemula and Sybilska described a method for the separation and determination of 0.05-0.3 ing of a mixture of 0-,in- and p-nitroethylbenzene on a column packed with the clathrate nickel y-picoline thiocyanate. A solution of ammonium thiocyanate and y-picoline in acetone were used for elution. This separation, based on the formation of clathrates, may be considered to be a special case of ligand exchange. The specificity of the chromatography of nitro compounds consists in the possibility of preparing alkyl nitrates from the corresponding alkyl bromides on a silica gel column impregnated with silver nitrate, while the chromatographic separation of the mixture takes place simultaneously (Kuemmel). The reaction is unusual in that the breaking and forma-
659
AMIDES
tion of the covalent bonds occur on the adsorbent surface on reaction with the anion from the adsorbent, becoming covalently bound to the product. In addition, olefins are also produced from secondary bromides. The nitrates are readily separated from the olefins, which are strongly adsorbed on the column owing to the formation of silver complexes. A high ratio of adsorbent to sample is required so as to ensure the complete reaction of all the alkyl bromides.
AMIDES The possibility of applying liquid chromatography to the separation of coloured homologous N,N-dimethyl-p-aminobenzeneazobenzamides was demonstrated by ChuraEek and Jandera using an apparatus consisting of a pulse-free plunger feeding pump, a narrow bore column, a spectrophotometer with a flow-through measuring cell of their own design and a recorder. A strongly sulphonated styrene-divinylbenzene cation-exchange resin, Dowex 50W-X2 (H'), was used for the separation of the coloured compounds. Fig. 3 1.1 illustrates the chromatographic separation of some lower secondary amides. Their protonated forms are distributed between the external solution (mobile phase) and the solution in the resin particles in accordance with the basicities of the non-ionic forms. The equilibrium depends on the activity of H' in the external solution and in the resin particles.
0
4
8
12
16
20
Volume. rn 1
Fig. 3 1.1. Separation of secondary aliphatic amides of N,N-dimethyl-p-aminobenzeneazobenzoic acid (ChuriEek and Jandera). Column: 240 X 2.7 nim. Ion exchanger: Dowex 50W-X2 (H+) (200-400 mesh). Eluent: 0.925 M hydrochloric acid in 80.5%' ethanol. Flow-rate: 0.132 ml/min. Detection: optical density at 520 nm. Peaks: 1 = 1.5 p g of di-ti-butylamide; 2 = 1.5 p g of di+propylamide; 3 = 1.5 p g of diethylamide; 4 = 1.5 p g of dimethylamide; 5 = inert compound (I'onceau 6 R ) .
Because of the negligible solubility of these compounds in aqueous acid solutions, it is necessary to use mixed aqueous-organic media. The amount of the organic solvent present obviously affects the distribution equilibrium by its solubility and solvation effects, and also by the dielectric constant effect. References p.664
660
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
A cation-exchange resin with a low degree of cross-linking (Dowex 50W-X2) was used for the separation in order to improve the accessibility of the ion-exchange phase to the large molecules of the derivatives and to accelerate the diffusion rate in the resin. Coloured amides are sorbed quantitatively on Dowex 50W-X2 (H'), 200-400 mesh, from mixed aqueous-organic solutions (80%ethanol; 80%methanol). The sorbed compounds can be eluted with an aqueous ethanolic or aqueous methanolic solution of hydrochloric acid. The effect of the eluent composition on the chromatographic behaviour of some homologous amides was studied by ChurGCek and Jandera. Homologous amides are eluted in order of increasing basicities, i.e., in order of decreasing molecular weights. Amides with a higher basicity have a higher distribution coefficient than esters, the basicity of which is lower. Secondary amides are sorbed more strongly than the less basic primary amides. TABLE 31 .I VOLUME DISTRIBUTION COEFFICIENTS (DJOF SOME AMIDES OF N,N-DIMETHYL-pAMINOBENZENEAZOBENZOIC ACID ON THE CATION EXCHANGER DOJ'EX 50W-X2 IN 0.925 M HYDROCHLORIC ACID SOLUTION IN 80.5% ETHANOL (CHURACEK AND JANDERA)
D, is defined as the ratio of the amount of compound in unit volume of the ion-exchanger phase t o the same volume of external solution. Amide derivative
D,
Methylamide Ethylamide n-Propybdmide n-Bu tylamide n-Hexylamide Allylamide Dime thylamide Diethylamide Di-(n-propy1)amide Di-(n-buty1)arnide
8.6 7.4 6.5 5.6 4.7 6.4 9.9 6.6 4.7 3.5
TABLE 3 1 . 2 GEL CHROMATOGRAPHY OF AMIDES ON POLYACRYLAMIDES (STREULI) Column: 97 X 0.5 cm. Gels: 1 = Bio-Gel P-2, 100-200 mesh (polyamide gel),exclusion limit 2002600; 2 = Bio-Gel P d , 100-200 mesh, exclusion limit 1000-5000. Mobile phase: 0.01 Msodium chloride solution. Flow-rate: 1 ml/min. Detection: RI. Compound*
Urea Biuret Acetamide N-Me thylacetamide N,N-Dimethylacetamide Acrylamide N-revt. -Butylacrylamide N-Vinyl-2-p yrrolidone *Samples of 50 gl.
Kd
Gel 1
Gel 2
1.37 1.88 1.06 0.94 0.86 1.17 0.97 1.23
1.17 1.29 -
0.89 -
66 1
GUANIDINE AND UREA DERIVATIVES
The contribution of the CH2 group to the logarithm of the distribution coefficient proved t o be fairly constant for homologous primary aliphatic amides, increasing to some extent with decreasing hydrocarbon chain length. Secondary amides showed greater changes corresponding to the same molecular-weight contribution. Volume distribution coefficients (D,) of iso-derivatives are slightly lower in comparison with those of normal derivatives. The multiple bond contribution to D, does not seem to be significant (Table 3 1.1). The hydrochloric acid concentration in the mobile phase influences the equilibrium between the protonated and non-ionic forms of these compounds. Gel chromatography was also used for the separation of substituted amides. Bio-Gel P-2 and P-6 were used as the stationary phase and 0.01 M sodium chloride solution as the mobile phase. Distribution coefficients and other separation conditions are given in Table 3 1.2 (Streuli).
GUANIDINE AND UREA DERIVATIVES Kirkland separated urea derivatives, applied as antidiabetic agents, by means of highspeed liquid chromatography, using a 1 m X 2.1 mm column packed with Permaphase ETH and a 1% solution of dioxane in n-hexane as the mobile phase. Fig. 3 1.2 shows the
CI
0-
N H -CO -N ( CH, )2
Fenuron
Mon uron 4
Cl
Diuron
15
7.5 T I M E . MIN
References p.664
Fig. 31.2. Separation of substituted ureas (Kirkland). Column: 1000 x 2.1 mm. Sorbent: Permaphase ETH. Eluent: 1% solution of dioxane in n-hexane. Operating conditions: flowrate, 1 ml/min; temperature, 27" C; column pressure, 340 p.s.i.g. Detection: UV detector at 254 nm. Peaks: 1 = solvent; 2 = Neburon; 3 = Fenuron; 4 = Monuron; 5 = Diuron. Sample: 1.5 ~1 of a 0.25 mg/ml solution of each compound in methanol.
662
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
separation of a synthetic mixture of substituted aromatic ureas usinga relatively non-polar organic carrier. A similar mixture can also be resolved with the same packing using the reversed-phase technique with alcohol-containing water as the mobile phase. The order of elution of these substituted ureas is different in the two systems. Liquid chromatography was also used for the analysis of gaseous fluorinated organic nitrogen compounds of the guanidine type (Rebertus et al.). Silica gel is a satisfactory adsorbent for this type of compound. Alumina and molecular sieves tend to decompose the fluorinated unsaturated compounds. The separation of tris(difluoroamino)fluoromethane and pentafluoroguanidine is illustrated in Fig. 3 1.3. Either an inert fluoro-compound or a hydrocarbon such as n-heptane can be used as the solvent and mobile phase; however, the former is preferred because of its much greater stability toward oxidation. Tris(difluoroamino)fluoromethane is readily eluted with either of these solvents, but pentafluoroguanidine is removed completely only if large bed volumes are used. The separation of bis(difluoroamino)difluoromethane-tetrafluoroformamidine mixtures was also effected. The relative affinities of the compounds for silica gel increase in the order ( F ~ N ) J C F<(F2N)2CF2 <(FZN)ZC=NF< FzNCF=NF.
- 60 -
E .
NF II FZN-C-
F -
2 0
240
-
U I-
5 -
0 Z
0 O20-
F
0
2
4
6
8
NF2
VOLUME,ml
10
12
Fig. 3 1.3. Separation of tris(dif1uoroamino)fluoromethane and peiitafluoroguanidinc (Rebertus et al.). Column: 0.8 X 8 cni. Sorbent: silica gel (100-200 mesh). Eluent: n-heptane. Flow-rate: 1 ml/n~in. Detection: fractions were collected and elution with the solvent was continued until breakthrough of the trifluoroamidines was indicated by a positive test with 1 M potassium iodide solution. Sample: 3 ml of the fluoro-compound containing 1.5% (w/w) of tris(difluoroamino)fluoromethane and 2% (w/w) of penta fluoroguanidme,
663
GUANIDINE AND UREA DERIVATIVES
TABLE 3 I .3 ION-EXCHANGE CHROMATOGRAPHY 01: COMPOUNDS RELATED T O UREA (NOMURA ef a!.) Values listed are elution volumes (ml). Column: 15 X 0.8 cm. Flow-rate: 0.46 ml/min. Detection: Automatic thermal reaction energy detection. Temperature: 35°C. ~~
Conipound
Biuret Thiourea Dicyanodamide Nitroguanidine Urea Methylurea Ethylurea tert.-Butylurea
~
Mobile phase 0.1 N H a
Water
0.01 N HCI
Amberlite CG-I 20 (H') (200-400 mesh)
Dowex 50-X8 (Ht) (200-400 mesh)
Dowex 1-X8 (CI-) ( 2 0 0 4 0 0 mesh)
15.9 21.5 27.3 106.7 136.6 -
22.2 27.1 33.8 40.7 11 3.1 154.1 226
23.5 34.5 34.7
21.0 26.9 34.3 37.5 I 08 145.1 203
1.ON HCI
-
26.9 34.2 58
-
-
Water
-
18.4 18.9 20.2 30.4
TABLE 3 1.4 SURVEY O F DIFFERENT PROCEDURES APPLICABLE TO THE SEPARATION OF NITROGEN COMPOUNDS Compounds separated
Sorbent
Mobile phase
Reference
N-Halocyanamides and sulphonamides
Florisil
Dichloromethane, diethyl ether, n-hexane and other organic solvents
Neale and Marcus
Diphenylcarbazide and phenylsemicarbazide
Polyamide
Water-methanolacetic acid ( I :3:0.04)
Willenis et al.
Automatic quantitative analysis of guanidines (biochemical application)
PQ-28 resin; Dowex 50-X2
Durzan, Sodium citrate Carles and buffers of various compositions and pH Abravanel (amino acid analyzer)
Substituted acetanilides LFS pellicular and similar substances anion-exchange (drugs, analgesics) resin
References p . 664
Tris(hydroxymethy1) aminoethane ( I 21 g in 1000 ml aqueous solution) (pH = 9.0, adjusted with dilute HCI). Pressure 1000 p s i . at 60°C
Stevenson and Burtis
0.01 N HCl
23.7 34.7 34.5 18.2 18.4 19.6 29.9
664
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
lon-exchange chromatography of compounds related to urea was carried out on a 15 X 0.8 cm column packed with the cation-exchange resin Amberlite CG-120 or Dowex 50-X8 (H') or the anion-exchange resin Dowex 1-X8(Cl-), Solutions of hydrochloric acid of various concentrations (0.01-1 . O M ) and water were used as the mobile phase (Nomura et al.). Elution volumes found under these conditions on various ion exchangers and using hydrochloric acid solutions of various concentrations are given in Table 31.3. Other, less important, applications of the liquid chromatography of some nitrogencontaining compounds are listed in Table 31.4.
REFERENCES Broszkiewicz, R . K. and Przybylowicz, Z.,Anal. Chem.,41 (1969) 1121. Carles, J . and Abravanel, G., Bull. SOC.Chim. Biol., 52(1970) 453;C.A., 73 (1970) 8 4 4 5 4 ~ . Churitek, J. and Jandera, P., J. Chromatogr., 53 (1970) 69. Durzan, D. J., Can. J. Biochem.,47 (1969) 657. Kernula, W., Rocz. Chem., 29 (1955) 1153. Kernula, W . and Brzozowski, S., Rocz. Chem., 35 (1961)711. Kemula, W . and Sybilska, D., Anal. Chim. Acta, 38 (1967) 97. Kirkland, J . J.,Anal. Chem., 43 (1971)43A. Kuemmel, D . F.,Chem. Ind. (London), (1966) 1882;Anal. Abstr., 15 (1968) 815. Landrarn, G. K., Wickham, A. A . and DuBois, R. J., Anal. Chern., 4 2 (1970) 107. Neale, R. S . and Marcus, N. L.,J. Org. Chem., 34 (1969) 1808. Nornura, N., Shiho, D. I., Ohsuga, K. and Yamada, M., J. Chromatogr., 42 (1969) 226. Rebertus, L. R., Fiedler, K. R. and Kottong, C. W., Anal. Chem., 39 (1967) 1867. Stevenson, R. L. and Burtis, C. A,, J. Chromatogr.. 61 (1971) 253; Streuli, C. A., J. Chromatogr., 47 (1970) 355. Willerns, G. J., Lontie, R. A. and Seth-Paul, W. A., Anal. Chim. Acta, 51 (1970) 544.
Other non- heterocyclic nitrogen compounds J . CHURACVEK
CONTENTS
...................................................................657 ............................................................... 657 ....................................................................... 659 ..................................................... 661 .................................................................... 664
Introduction Nitrocompounds Amides Cuanidineandureaderivatives References
INTRODUCTION In t h s chapter, the liquid chromatography of nitro compounds, urea and its derivatives, guanidines, nitrosamines, amides, azo compounds and aromatic hydrazo compounds is described. The chromatography of amides is mentioned only briefly because from the practical point of view the chromatographic separation of peptides and their mixtures is most important and therefore a special chapter is devoted to this aspect (Chapter 34). For the other compounds mentioned, the main aim of liquid column chromatography is their separation from other types of compounds. All modern chromatographic techniques have good prospects of being applied successfully in this field, but little attention has been devoted to them so far. An interesting technique was used for nitro compounds; some of them can be synthesized directly on the column on which they are then separated chromatographically from other components of the reaction mixture.
NITRO COMPOUNDS A typical example of the isolation of nitro compounds from industrial mixtures was described by Landram ef al. Chromatographic separation was used instead of the usual extraction procedures, which represents an important advance in work with explosives. This separation procedure was used satisfactorily for a number of double base propellants containing nitroglycerine, triacetin, 2-nitrodiphenylamine, resorcinol, ammonium perchlorate, aluminium, 2.4,6,8-cyclotetramethylenetriamine and nitrocellulose. The procedure is generally applicable to different types of propellants and it is not necessarily restricted to those which bear a large number of nitro groups. Both Chromosorb T (PTFE) and silica gel can be used for column packing in these instances. The principle of dry column chromatographic extraction should prove useful also with other non-propellant polymers. The main advantage of this procedure is the decrease in the operating time: in the example described above, the Soxhlet extraction took 1-4 days References p.664
657
658
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
while the chromatographic procedure took 1 h. In large-scale operations, the reduced consumption of solvents in chromatographic procedures is also important. I n the analytical field, ion-exchange chromatography is the prevailing method for the separation of nitro compounds. This method permits the separation of a series of nitroalkanes and nitro-aromatic compounds. Polarography is mainly used for detection (Kemula); a special chromatopolarographic apparatus was developed by Kcmula and Brzozowski for this purpose. The limiting diffusion current produced by organic substances is measured on a mercury drop electrode at a constant input potential (-1 V). Kemula and Brzozowski also described the separation of some nitroalcohols and nitrobenzoic acids by salting-out chromatography on a thermostatted cation-exchange column at 56,82,25 and 71°C. On Wofatit KPS-200, a mixture of four nitroalcohols was separated by elution with 1 M ammonium sulphate solution on a 37 X 0.7 cm column. The flow-rate was not an important factor in the separation. The sequence of the eluted compounds was 2-methyl-2-hydroxy-1,3-propanediol, 2-methyl-2-nitropropano1, 2-nitrobutanol and 2-hydroxymethyl-2-nitropentanol. Kemula and Brzozowski also separated a three-component mixture of nitroalkanes on a Dowex 50 column using ammonium sulphate solution as the mobile phase. The elution sequence observed was nitromethane, 2-nitropropane and 1-nitrobutane. The column dimensions were 1 1.5 X 0.7 cm and the flow-rate of the mobile phase did not exceed 9 ml/h. On the same ion exchanger, separations at various temperatures can also be effected: at 25°C with 1 Mammonium sulphate solution as eluent, nitromethane, 2-nitropropane and 1,3-dinitropropane are eluted first. After increasing the temperature in the column jacket to 71°C and using 0.5 M ammonium sulphate solution for elution, 1-nitrobutane and 1-nitropropane appear in the effluent. Under the same conditions, all three isomeric nitrobenzoic acids can also be separated, using 0.1 M ammonium sulphate solution in 0.019 M hydrochloric acid as the mobile phase. o-Nitrobenzoic acid is eluted at room temperature with a minimum retention time, p-nitrobenzoic acid is eluted at 56°C and m-nitrobenzoic acid at 82°C. The time required for the separation is about 5 h. During the analysis of the radiolytic products in the aqueous nitrate-ethylene system, ion-exchange chromatography on Dowex 50W-X8 was also employed. Ammonium sulphate solution of various concentrations served as the mobile phase. Nitromethane, hydrogen peroxide and sodium nitrite, present in the mixture, were detected polarographically. The curves were recorded a t a constant potential corresponding to the complete reduction of the dissolved oxygen and lower than the reduction potential of nitrate (Broszkiewicz and Przybylowicz). Kemula and Sybilska described a method for the separation and determination of 0.05-0.3 ing of a mixture of 0-,in- and p-nitroethylbenzene on a column packed with the clathrate nickel y-picoline thiocyanate. A solution of ammonium thiocyanate and y-picoline in acetone were used for elution. This separation, based on the formation of clathrates, may be considered to be a special case of ligand exchange. The specificity of the chromatography of nitro compounds consists in the possibility of preparing alkyl nitrates from the corresponding alkyl bromides on a silica gel column impregnated with silver nitrate, while the chromatographic separation of the mixture takes place simultaneously (Kuemmel). The reaction is unusual in that the breaking and forma-
659
AMIDES
tion of the covalent bonds occur on the adsorbent surface on reaction with the anion from the adsorbent, becoming covalently bound to the product. In addition, olefins are also produced from secondary bromides. The nitrates are readily separated from the olefins, which are strongly adsorbed on the column owing to the formation of silver complexes. A high ratio of adsorbent to sample is required so as to ensure the complete reaction of all the alkyl bromides.
AMIDES The possibility of applying liquid chromatography to the separation of coloured homologous N,N-dimethyl-p-aminobenzeneazobenzamides was demonstrated by ChuraEek and Jandera using an apparatus consisting of a pulse-free plunger feeding pump, a narrow bore column, a spectrophotometer with a flow-through measuring cell of their own design and a recorder. A strongly sulphonated styrene-divinylbenzene cation-exchange resin, Dowex 50W-X2 (H'), was used for the separation of the coloured compounds. Fig. 3 1.1 illustrates the chromatographic separation of some lower secondary amides. Their protonated forms are distributed between the external solution (mobile phase) and the solution in the resin particles in accordance with the basicities of the non-ionic forms. The equilibrium depends on the activity of H' in the external solution and in the resin particles.
0
4
8
12
16
20
Volume. rn 1
Fig. 3 1.1. Separation of secondary aliphatic amides of N,N-dimethyl-p-aminobenzeneazobenzoic acid (ChuriEek and Jandera). Column: 240 X 2.7 nim. Ion exchanger: Dowex 50W-X2 (H+) (200-400 mesh). Eluent: 0.925 M hydrochloric acid in 80.5%' ethanol. Flow-rate: 0.132 ml/min. Detection: optical density at 520 nm. Peaks: 1 = 1.5 p g of di-ti-butylamide; 2 = 1.5 p g of di+propylamide; 3 = 1.5 p g of diethylamide; 4 = 1.5 p g of dimethylamide; 5 = inert compound (I'onceau 6 R ) .
Because of the negligible solubility of these compounds in aqueous acid solutions, it is necessary to use mixed aqueous-organic media. The amount of the organic solvent present obviously affects the distribution equilibrium by its solubility and solvation effects, and also by the dielectric constant effect. References p.664
660
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
A cation-exchange resin with a low degree of cross-linking (Dowex 50W-X2) was used for the separation in order to improve the accessibility of the ion-exchange phase to the large molecules of the derivatives and to accelerate the diffusion rate in the resin. Coloured amides are sorbed quantitatively on Dowex 50W-X2 (H'), 200-400 mesh, from mixed aqueous-organic solutions (80%ethanol; 80%methanol). The sorbed compounds can be eluted with an aqueous ethanolic or aqueous methanolic solution of hydrochloric acid. The effect of the eluent composition on the chromatographic behaviour of some homologous amides was studied by ChurGCek and Jandera. Homologous amides are eluted in order of increasing basicities, i.e., in order of decreasing molecular weights. Amides with a higher basicity have a higher distribution coefficient than esters, the basicity of which is lower. Secondary amides are sorbed more strongly than the less basic primary amides. TABLE 31 .I VOLUME DISTRIBUTION COEFFICIENTS (DJOF SOME AMIDES OF N,N-DIMETHYL-pAMINOBENZENEAZOBENZOIC ACID ON THE CATION EXCHANGER DOJ'EX 50W-X2 IN 0.925 M HYDROCHLORIC ACID SOLUTION IN 80.5% ETHANOL (CHURACEK AND JANDERA)
D, is defined as the ratio of the amount of compound in unit volume of the ion-exchanger phase t o the same volume of external solution. Amide derivative
D,
Methylamide Ethylamide n-Propybdmide n-Bu tylamide n-Hexylamide Allylamide Dime thylamide Diethylamide Di-(n-propy1)amide Di-(n-buty1)arnide
8.6 7.4 6.5 5.6 4.7 6.4 9.9 6.6 4.7 3.5
TABLE 3 1 . 2 GEL CHROMATOGRAPHY OF AMIDES ON POLYACRYLAMIDES (STREULI) Column: 97 X 0.5 cm. Gels: 1 = Bio-Gel P-2, 100-200 mesh (polyamide gel),exclusion limit 2002600; 2 = Bio-Gel P d , 100-200 mesh, exclusion limit 1000-5000. Mobile phase: 0.01 Msodium chloride solution. Flow-rate: 1 ml/min. Detection: RI. Compound*
Urea Biuret Acetamide N-Me thylacetamide N,N-Dimethylacetamide Acrylamide N-revt. -Butylacrylamide N-Vinyl-2-p yrrolidone *Samples of 50 gl.
Kd
Gel 1
Gel 2
1.37 1.88 1.06 0.94 0.86 1.17 0.97 1.23
1.17 1.29 -
0.89 -
66 1
GUANIDINE AND UREA DERIVATIVES
The contribution of the CH2 group to the logarithm of the distribution coefficient proved t o be fairly constant for homologous primary aliphatic amides, increasing to some extent with decreasing hydrocarbon chain length. Secondary amides showed greater changes corresponding to the same molecular-weight contribution. Volume distribution coefficients (D,) of iso-derivatives are slightly lower in comparison with those of normal derivatives. The multiple bond contribution to D, does not seem to be significant (Table 3 1.1). The hydrochloric acid concentration in the mobile phase influences the equilibrium between the protonated and non-ionic forms of these compounds. Gel chromatography was also used for the separation of substituted amides. Bio-Gel P-2 and P-6 were used as the stationary phase and 0.01 M sodium chloride solution as the mobile phase. Distribution coefficients and other separation conditions are given in Table 3 1.2 (Streuli).
GUANIDINE AND UREA DERIVATIVES Kirkland separated urea derivatives, applied as antidiabetic agents, by means of highspeed liquid chromatography, using a 1 m X 2.1 mm column packed with Permaphase ETH and a 1% solution of dioxane in n-hexane as the mobile phase. Fig. 3 1.2 shows the
CI
0-
N H -CO -N ( CH, )2
Fenuron
Mon uron 4
Cl
Diuron
15
7.5 T I M E . MIN
References p.664
Fig. 31.2. Separation of substituted ureas (Kirkland). Column: 1000 x 2.1 mm. Sorbent: Permaphase ETH. Eluent: 1% solution of dioxane in n-hexane. Operating conditions: flowrate, 1 ml/min; temperature, 27" C; column pressure, 340 p.s.i.g. Detection: UV detector at 254 nm. Peaks: 1 = solvent; 2 = Neburon; 3 = Fenuron; 4 = Monuron; 5 = Diuron. Sample: 1.5 ~1 of a 0.25 mg/ml solution of each compound in methanol.
662
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
separation of a synthetic mixture of substituted aromatic ureas usinga relatively non-polar organic carrier. A similar mixture can also be resolved with the same packing using the reversed-phase technique with alcohol-containing water as the mobile phase. The order of elution of these substituted ureas is different in the two systems. Liquid chromatography was also used for the analysis of gaseous fluorinated organic nitrogen compounds of the guanidine type (Rebertus et al.). Silica gel is a satisfactory adsorbent for this type of compound. Alumina and molecular sieves tend to decompose the fluorinated unsaturated compounds. The separation of tris(difluoroamino)fluoromethane and pentafluoroguanidine is illustrated in Fig. 3 1.3. Either an inert fluoro-compound or a hydrocarbon such as n-heptane can be used as the solvent and mobile phase; however, the former is preferred because of its much greater stability toward oxidation. Tris(difluoroamino)fluoromethane is readily eluted with either of these solvents, but pentafluoroguanidine is removed completely only if large bed volumes are used. The separation of bis(difluoroamino)difluoromethane-tetrafluoroformamidine mixtures was also effected. The relative affinities of the compounds for silica gel increase in the order ( F ~ N ) J C F<(F2N)2CF2 <(FZN)ZC=NF< FzNCF=NF.
- 60 -
E .
NF II FZN-C-
F -
2 0
240
-
U I-
5 -
0 Z
0 O20-
F
0
2
4
6
8
NF2
VOLUME,ml
10
12
Fig. 3 1.3. Separation of tris(dif1uoroamino)fluoromethane and peiitafluoroguanidinc (Rebertus et al.). Column: 0.8 X 8 cni. Sorbent: silica gel (100-200 mesh). Eluent: n-heptane. Flow-rate: 1 ml/n~in. Detection: fractions were collected and elution with the solvent was continued until breakthrough of the trifluoroamidines was indicated by a positive test with 1 M potassium iodide solution. Sample: 3 ml of the fluoro-compound containing 1.5% (w/w) of tris(difluoroamino)fluoromethane and 2% (w/w) of penta fluoroguanidme,
663
GUANIDINE AND UREA DERIVATIVES
TABLE 3 I .3 ION-EXCHANGE CHROMATOGRAPHY 01: COMPOUNDS RELATED T O UREA (NOMURA ef a!.) Values listed are elution volumes (ml). Column: 15 X 0.8 cm. Flow-rate: 0.46 ml/min. Detection: Automatic thermal reaction energy detection. Temperature: 35°C. ~~
Conipound
Biuret Thiourea Dicyanodamide Nitroguanidine Urea Methylurea Ethylurea tert.-Butylurea
~
Mobile phase 0.1 N H a
Water
0.01 N HCI
Amberlite CG-I 20 (H') (200-400 mesh)
Dowex 50-X8 (Ht) (200-400 mesh)
Dowex 1-X8 (CI-) ( 2 0 0 4 0 0 mesh)
15.9 21.5 27.3 106.7 136.6 -
22.2 27.1 33.8 40.7 11 3.1 154.1 226
23.5 34.5 34.7
21.0 26.9 34.3 37.5 I 08 145.1 203
1.ON HCI
-
26.9 34.2 58
-
-
Water
-
18.4 18.9 20.2 30.4
TABLE 3 1.4 SURVEY O F DIFFERENT PROCEDURES APPLICABLE TO THE SEPARATION OF NITROGEN COMPOUNDS Compounds separated
Sorbent
Mobile phase
Reference
N-Halocyanamides and sulphonamides
Florisil
Dichloromethane, diethyl ether, n-hexane and other organic solvents
Neale and Marcus
Diphenylcarbazide and phenylsemicarbazide
Polyamide
Water-methanolacetic acid ( I :3:0.04)
Willenis et al.
Automatic quantitative analysis of guanidines (biochemical application)
PQ-28 resin; Dowex 50-X2
Durzan, Sodium citrate Carles and buffers of various compositions and pH Abravanel (amino acid analyzer)
Substituted acetanilides LFS pellicular and similar substances anion-exchange (drugs, analgesics) resin
References p . 664
Tris(hydroxymethy1) aminoethane ( I 21 g in 1000 ml aqueous solution) (pH = 9.0, adjusted with dilute HCI). Pressure 1000 p s i . at 60°C
Stevenson and Burtis
0.01 N HCl
23.7 34.7 34.5 18.2 18.4 19.6 29.9
664
OTHER NON-HETEROCYCLIC NITROGEN COMPOUNDS
lon-exchange chromatography of compounds related to urea was carried out on a 15 X 0.8 cm column packed with the cation-exchange resin Amberlite CG-120 or Dowex 50-X8 (H') or the anion-exchange resin Dowex 1-X8(Cl-), Solutions of hydrochloric acid of various concentrations (0.01-1 . O M ) and water were used as the mobile phase (Nomura et al.). Elution volumes found under these conditions on various ion exchangers and using hydrochloric acid solutions of various concentrations are given in Table 31.3. Other, less important, applications of the liquid chromatography of some nitrogencontaining compounds are listed in Table 31.4.
REFERENCES Broszkiewicz, R . K. and Przybylowicz, Z.,Anal. Chem.,41 (1969) 1121. Carles, J . and Abravanel, G., Bull. SOC.Chim. Biol., 52(1970) 453;C.A., 73 (1970) 8 4 4 5 4 ~ . Churitek, J. and Jandera, P., J. Chromatogr., 53 (1970) 69. Durzan, D. J., Can. J. Biochem.,47 (1969) 657. Kernula, W., Rocz. Chem., 29 (1955) 1153. Kernula, W . and Brzozowski, S., Rocz. Chem., 35 (1961)711. Kemula, W . and Sybilska, D., Anal. Chim. Acta, 38 (1967) 97. Kirkland, J . J.,Anal. Chem., 43 (1971)43A. Kuemmel, D . F.,Chem. Ind. (London), (1966) 1882;Anal. Abstr., 15 (1968) 815. Landrarn, G. K., Wickham, A. A . and DuBois, R. J., Anal. Chern., 4 2 (1970) 107. Neale, R. S . and Marcus, N. L.,J. Org. Chem., 34 (1969) 1808. Nornura, N., Shiho, D. I., Ohsuga, K. and Yamada, M., J. Chromatogr., 42 (1969) 226. Rebertus, L. R., Fiedler, K. R. and Kottong, C. W., Anal. Chem., 39 (1967) 1867. Stevenson, R. L. and Burtis, C. A,, J. Chromatogr.. 61 (1971) 253; Streuli, C. A., J. Chromatogr., 47 (1970) 355. Willerns, G. J., Lontie, R. A. and Seth-Paul, W. A., Anal. Chim. Acta, 51 (1970) 544.