G a s 4 quid Chromatography Separation and Microestimation of Volatile Aromatic Amines A.
T. JAMES
National lnstitute for M e d i c a l Research, M i l l
Hill, London, N.W. 7, England
The development of gas-liquid chromatography as an analytical technique will be facilitated by the publication of information on the relationship between structures and chromatographic behavior. .4 study has been made of the behavior of a variety of substituted anilines on three chemically distinct types of gasliquid chromatogram. The information presented will be of use both in the analysis of aniline bases and in the elucidation of the effect of structural change on retention volume.
Figure 1.
Separation of a series of aromatic amines at 205' C.
Column length 4 feet, total load 0.6 mg., sensitivity 0.1N, nitrogen pressure 12 cm. Hg., nitrogen flow rate 11.3 ml. N per minute. Recording of xones by gas-density meter. Stationary phase high boiling lubricating oil
I
N T H E initial papers describing the gas-liquid chromatogram (1, 2, 6) the method employed for the detection of the vapors emerging in the gas stream from the column was a continuous automatic titration in aqueous media. This technique limited the substances that could be separated to those acids and bases possessing suitable pK values-Le., aliphatic acids ( 8 ) , aliphatic bases (1, 6) and pyridine bases (1). Titration in nonaqueous media would allow detection and estimation of weaker acids and bases. The technique has now been applied to the separation of aromatic amines; the titration is carried out in glacial acetic acid with perchloric acid by means of the automatic recording buret described by James and Martin ( 2 ) . The sensitivity of the titration technique is such that as little as 0.1 mg. of aniline is detectable (corresponding to a step height of 1 mm.). The later development of the gas-density meter by Martin (7) has allowed the detection limit to be lowered even further. This device, unlike the automatic titrator, records the instantaneous concentration of the material emerging from the chromatogram, so that the more familiar Gaussian-shaped zones are recorded automatically by the apparatus. S n example of its use is shown in Figure 1. Quantitative estimation of the amount of material in the zone necessitates measurement of area under the peak, a slower process than measurement of step height in the titrator record.
tains a 0.04,V solution of perchloric acid in glacial acetic acid. From this point on the whole process is autopatic. I t is important that the amines contain not more than a few per cent of water; otherwise the separations are impaired. The stationary phases used were not chosen in order to show the best separations but to show singularity of type of solution force-viz., in liquid paraffin only van der Waals forces are involved, in Lubrol M O van der Waals forces plus hydrogen bonding are involved, and in benzyldiphenyl van der Waals forces plus interaction of a polar type with the benzene ring are involved. I t is hoped that the results presented here will aid in the choice of a suitable stationary phase for any particular separation. RESULTS
APPARATUS
The columns consist of 4-foot lengths of glass tubing 3 to 4 mm. in internal diameter, drawn down a t one end to form a short length of capillary over which is fitted a small rubber sleeve. A short length of glass yarn (Fibreglas Corp.) is pushed down into the narrowed end of the column to support the packing. This consists of a mixture of kieselguhr (Celite 545), size graded as described by James and Martin ( 2 ) and treated with alcoholic alkali ( I ) , and the stationary phase chosen (40y0 by weight). The columns are packed by vibration as previously described (2).
The stationary phases are: paraffin wax, melting point C., or liquid paraffin (low boiling point). Lubrol h I 0 (Imperial Chemical Industries), a polyethylene oxide condensate; benzyldiphenyl; and an aromatic extract of a lubricating oil, high boiling point ( 3 ) . The column is placed in the vapor jacket of the apparatus and loaded by inserting into the end of the column a micropipet containing the sample to be analyzed (melted if necessary, or a solution of the amines in alcohol may be used). The nitrogen supply from the manostat ( 2 ) is connected, the clock driving the recording drum is switched on, and the titration cell. of the automatic recording buret is filled with a solution of crystal violet (or metanil yellow) in dry glacial aretic acid. The buret con49'
1564
In Figure 2,A, is shown a typical separation of aniline, N methylaniline, N-dimethylaniline, 2,4-dimethylaniline, PmethylN-dirnethylaniline, and AT-diethylaniline using a '&foot column a t 137' C. with liquid paraffin or paraffin wax as the stationary phase ( 4 ) . Each step denotes a separate substance and the horizontal line between steps denotes a period in which no titrable material is emerging from the columns. It has been indicated (1, 3, 6) that the order of emergence of a series of substances from columns having paraffin hydrocarbon stationary phase is determined by their relative van der Waals interactions with the stationary phase. To a first approximation these forces can be considered to increase with molecular weight, with certain main exceptions (see below). In Table I, column 1, is given a list of retention volumes (the volume of nitrogen passing through the column before the center of a zone emerges) of a number of aromatic amines relative to the retention volume of aniline for a column with liquid paraffin as the stationary phase. The amines are arranged in order of emergence from the column. Relative retention volumes rather than corrected retention volumes, V i . are given, as they are more easily reproduced by other workers. With the exception of fluoro compounds, certain ortho-substituted tertiary amines, and the phenylenediamines, molecular weight is the main factor controlling position on the chromatogram. Many of the amines of similar molecular weight cannot be separated on paraffin columns. A difference of roughly 20% in relative retention volumes is necessary for complete separation,
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
1565
a difference of 10 t o 15% Rill show two united peaks, and differences of less than 7% will barely show up. In most cases a pair of substances that overlap in position on the paraffin column will separate completely on one of the other types of column, as can be seen by reference to Table I. The Lubrol MO columns, having a polyether stationary phase, allow hydrogen bonding by primary and secondary amines but not by tertiary amines (see Figure 2 3 ) . An effect smaller in magnitude but qualitatively similar is given with the aromatic hydrocarbon, benzyldiphenyl, as stationary phase. This is particularly well illustrated by the series aniline, N-methylaniline, N-dimethylaniline. In the paraffin column the relative retention volumes are aniline 1.0, A'-methylaniline 2.05, and N-dimethylaniline 2.6; in Lubrol MO, 1.0, 1.28, and 1.05; and in benzyldiphenyl, 1.0, 1.73, and 1.68. The inability of the N-dimethylaniline to show donor hj-drogen bonding is responsible for its relative speeding up in
both Lubrol MO and benzyldiphenyl. This effect is shown by all tertiary amines [James ( I ) discusses similar effects in aliphatic amines]. Effect of Substituents on Retention Volume. ALKYLGROUPS In the monosubstituted compounds very little difference in retention volume is shown by the 0-, m-, and p- isomers. In the paraffin stationary phase the order of emergence is p-, followed by m-, followed by 0-, the differences being insufficient to show separation on a 4foot column. Complete separation could be attained only by the use of a longer column. The order is changed in the Lubrol and aromatic hydrocarbon columns, but the differences are still insufficient to allow complete separation. Where the nitrogen atom possesses one or trro alkyl groups, the 0- isomer runs fastest, and the Nz- and p - isomers run together. This ortho- effect is common to all three types of column and increases in magnitude with chain length of the N-alkyl group.
Table 1. Retention Volumes of Aromatic Amines Relative to Aniline in Three Types of Column at 137" C.
/A2 S H 2
5\/3 4
Substituents in Position N Atom
... ... ... ...
2
FI'
...
CH8 (CHIL' (CH3)Z
... CH3-'
...
'
CH8
c1-
C2Ha-
'
...
EEJ. . . ... ... ...
...
CHiOCH3 ... ...
... CH'3
. .
(CHIjZ'
...
(CHsjZ CzHr (CHdz (CzHdz n-CaH7 CzHsCzHs-
...
...
CH3
CHI c1Br ... ... CHI .. ...
... , . .
...
... ...
...
...
CHI'"
Nti2-' C1-
c1I-
... ...
...
...
ISO-c~H,,
(CHd2
C Ha
...
... c1... ...
...
"%'2
CHiOCHs-
... ...
...
...
...
CHI
...
...
...
...
...
cH3... ... ... ... ... ...
cHJ'-'
cl:"
...
...
...
CHI-
... ...
FF-
-
2"
, . .
... ...
,..
...
...
...
F...
FF-
CHaOCHgO,
.
...
CHs iyH2'-'
... ... Br ... Br -
...
C1-
... ...
... ... ...
CHa
IC1:"
...
,,.
... ... ...
...
... ... . . ...
... '
-'
'
...
... ...
...
CH;"
...
... CHsO-
CHI1
...
... c€i8'-' ... ...
...
.. ... ...
...
...
...
, . .
...
..
...
CHsOC H3 ,..
...
... ...
CHI
CZHIO Br-
-
... ...
...
CI CHI-
... , . .
,..
. .
...
. .
c1-
CH;" ... ... I-
... . . . .
...
... ...
CHaO
...
"2'-
c1,..
, . .
... c1
...
Br-
,..
... ...
... ... ... ... ... ... ...
... ,,,
Column Stationary Phase Paraffin Lubrol Benaylwax RIO diphenyl 0.72 0.74 0.79 0.96
...
CHI-
-
6
, . .
C Ii3
. I .
CH;"
...
CHI -' ' CHs-
CHJ
... ...
...
...
...
... ...
F-
5
4
3
CHa
1.88 1.95 2.0 2.0 2.05 2.05 2.56 2.60 2.8 3.18 3.4 3.4 3.4 3.7 3.76 3.8 3.84 3.92 4.0 4.1 4.2 4.25 4.27 4.3 4.4 4.6 4.8 4.9 4.9 5.1 5,21 5.4 5.45 5.82 5.9 6.0 6.2 6.2 6.4 6.4 7.35 8.8 9.8 10.0 10.0 10.1 10.5 10.8 11.0 13.0 13.1 14.0 14.6 16.4 16.8 17.3
...
0.75 1.08 1.28 0 99 2 28 1.63 2.28 1.56 1.28 1.53 0.71 1.05 2.34 1.66 2.7
...
...
5.25
0.63
...
...
1.1 1.82 ..,
1.75
...
1.86
1.73 1.8 1.07 1.68 2.6 2.33 3.7 3.05 3.28 4.4
...
...
5.35 2.60 7.95
4.4 3.36 6.6 3.14
. . 3.5
10.0 3.22 3.8 3.7 6.6 4.0
'j:;
3,27
...
5 8
3.34
...
4.5
...
1.7 2.34 1.8
1.9 2.7 3.02 2 9
... ...
10.0 10.0
... ...
... ... ...
...
...
5 0 5.7 2.74 3.66 2.9 3.0 4.0 4.4 4.4 12.4 8.8 8.8 9.0 6.9
...
... ...
...
,..
3'2 3 1 4.7
22.4
...
... ...
6.4 6.8
5.0
5,l
7.0 23.2 23.2
... ... ... ...
13.3
A N A L Y T I C A L CHEMISTRY
1566 Table 11.
Effect of Position of Substituent Alkyl Groups on Retention of Xylidines NHY
"1
Structure NHz
CHa Stationary 'phase Paraffin Lubrol M O Benzyldiphenyl
NHi
"1
CHa Retention Tolume Relative to -4niline 4.0 4.26 4.27 2.7 2.5 3.27 3.4 3.22 3.80
3.84 2.6 3.36
4.6 3.34 4.0
With disubstituted compounds (the xylidines) the maximum retention volume in all three columns is given by the 2,3 compound. Where, however, the two alkyl groups are arranged on either side of the amino group, the compound shows a lower retention volume (see Table 11). The results suggest that adjacent arrangement of the groups in the nucleus allows maximum van der Waals interaction, but that where the largest group is in the middle, both van der Waals forces and hydrogen bonding are decreased, presumably because of steric factors. GROUPS CAPABLE OF ACTINGAS HYDROQEN BONDACCEPTORSIn all types of column only E.G., F, C1, Br, I, OCHs, OC2Hs,"2. very small differences in retention volume between the m- and p isomers are found, except with the phenylenediamines. In all cases, however, the 0- isomers run much faster than the p- or mretention volume of m- or p- isomer isomers; indeed, the ratio retention volume of o- isomer is constant throughout the series (again except for the phenylenediamines) for each type of column (see Table 111).
The fact that the ratio is greater in the case of the Lubrol and benzyldiphenyl columns suggests that not only the van der Waale forces with the solvent but also the hydrogen bonding forces are decreased. The only conclusion that can be drawn is that internal hydrogen bonding is taking place as has been postulated for o-substituted phenols, causing a constant difference in free energy of solution between the o- and p- isomers, irrespective of the nature of the substituent group. That the same effect le not shown by an o-alkyl group or by an o-amino group can be adduced as additional evidence for this hypothesis, since in the first case no hydrogen bonding is possible and, in the second, the 0- group can manifest both donor and acceptor functions in hydrogen bonding. In the case of the substituted tertiary amines, the decrease in retention volume of the 0- isomer compared with the p- and misomers cannot be due to hydrogen bonding and must, thcrefore, be ascribed to steric interference between the -~ groups. From these results certain generalizations on the behavior of aromatic amines can be drawn, that should be useful in defining the conditions Curve 0 necessary for any given separations. I t is likely, moreover, that these generalizations would also apply to phenols and many other aromatic compounds.
I
3228. 2420.
16-
8- 24
u
2 4- 2 0
0
'I
N-methylaniline
12. 28
Aniline t N-dimethylaniline
8
I0
20
30
40
50
TIME I N MINUTES
Figure 2.
Separation of aromatic amines at 137' C. uce 47.2 cm. Hg, nitrogen
In general, m- and p - isomers are inseparable on short gas-liquid chromatograms. For such separations either long gas-liquid columns should be used or advantage should be taken of the difference in pK value of the isomers by using either a suitably buffered liquid-liquid column or an ion exchange column. W h e r e s u b s t i t u e n t groups are capable of hydrogen bonding with either a primary or secondary amino group, the 0- isomer runs faster than the m- and p - isomers and is easily separable from them. With the tertiary aromatic amines all types of osubstituted compounds show lower retention volumes than the m- and p compounds. In non olar solvents unable to hydrogen-Bond the order of emergence is determined largely by the molecular weight and number of groups in the molecule. I n solvents capable of showing polar jnteractions such as hydrogen bonding, a tertiary amine will show a lower retention volume than the corresponding secondary amine. In hydrogen bonding solvents an N-alkylaniline runs faster than the isomeric nuclear alkylaniline and can be easily separated from it.
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6 Bearing these generalizations in mind, it should be possible to identify almost any volatile aromatic amine by its behavior on two or more types of chromatogram. CONCLUSIONS
The advantages of the gas-liquid chromatogram in the analysis of aromatic amines may be summarized as: The method is highly sensitive and quantitative results are easily obtained. Information on the structure of an unknown amine can be obtained by studying its behavior on two or more types of column. Relative retention volumes are put forward as physical constants as useful in identification as melting points, boiling points, etc. LITERATURE CITED
(1) J a m e s , A. T., Biochem. J . 52,242 (1952). (2) J a m e s , A. T.. M a r t i n , A. J. P., Ibid., 50, 679 (1952). (3) Ihid.. 6 3 , 144 (1956).
1567
Table 111. Constancy of Ortho Effect in Primary Amines Replaced by Group Capable of Acting as Hydrogen Bond Acceptor (Evidenced by ratio of retention volume of p - isomer t o 0 - isomer) Benzyldiphenyl Substituent Group Liquid Paraffin Lubrol MO -F -c1 -Br
-1
...
-0CHs -0CzHh --SHn
1.78 1.54 1.59
... ...
1.26 1.48
(1) J a m e s , -4. T., Martin, -1.J. P., Brit. M e d . Bull. 10, 170 (1954). (5) J a m e s , d.T., M a r t i n , A. J. P.. J . A p p l . Chem. 6 , 105 (1956). (6) James, -4.T., M a r t i n , A. J . P., S m i t h . H. G., B b c h e n . J 52, 238 (1952). (7) Martin, A. J . P., J a m e s , -4. T., Ibid., 6 3 , 138 (1956). RECEIVED for review Iiovember 23, 1955. Accepted June 2, I956
Colorimetric Determination of Fluoride in Water by Meteropoly Blue System ROBERT
P. CURRY1 and M. G. MELLON
Department of Chemistry, Pordoe University, Lafayette, Ind.
A new method for the determination of fluoride in waters utilizes the distillation of fluoride as silicon tetrafluoride from a sulfuric acid medium. The silicon tetrafluoride is carried by a nitrogen gas stream into a sodium borate-boric acid buffer and hydrolyzed, after which the soluble silicate is determined by formation of molybdosilicic acid and subsequent reduction to the corresponding heteropoly blue. This method affords accurate and precise determination of fluoride in the range from 0.1 to 2.0 mg. The effects of the following variables were investigated : the presence of 25 diverse ions, distillation time, and water concentration on the distillation system. Beer's law is followed from 0.1 to 2.0 mg. of fluoride with a standard deviation of 0.024 mg. The sensitivity of the method is 0.18 absorbance unit per 0.1 mg. of fluoride.
and Shuey (14) improved the distillation. The procedure and apparatus described here are modifications of those of Adolph, Shuey, and Wagner and Ross. The heteropoly system involving molybdosilicic acid and its blue reduction product are well knovn, and this heteropoly blue method is recommended for determining silicate in water, The recommended reagent for reducing molybdosilicic acid to the blue product is a solution of sodium hydrogen sulfite, disodium sulfite, and l-amino-2-naphthol-4-sulfonic acid. 1 survey of the literature revealed no attempt to establish a method for fluoride in water based on the formation of molybdosilicic acid from the silicate formed by hydrolysis of silicon tetrafluoride after the removal of the fluoride from the sample by volatilization as silicon tetrafluoride. The following reactions are assumed to be involved:
+ SiOnHzSOc SiF, + 3H20 + SO3 heat SiF4 + HzB03- + 2 OHSi03-- + BF-a + 2 H 2 0 4HF
T
recent ext'erision of fluoridation of water supplies eniphasizes the need for a better method of determining fluoride in 110th treated and untreated waters. This study &-as undertaken to investigate the possibility of making such det,erminationa nsing reactions of heteropoly systems in conjunction with the distillation of fluoride as silicon tetrafluoride from concentrated riilfuric acid. In st,rongly acidic solution fluoride reacts with sources of silicon t,o form volatile silicon tetrafluoride. When this gas is passed into an aqueous solution, hydrolysis yields silicic and fl(iosi1icic :i.cids. Bein and Wohler (2, 18) xwre among the first t,o establish analytical methods for fluoride based on this volat,ility. -1dolph (1) distilled silicon tetrafluoride from concentrated sulfuric acid and titrated the liberated fluosilicic acid. Penfield (11) collected the distillate in an alcoholic solution of potassium chloride and weighed the insoluble potassium fluosilicate. Wagner and Ross (16) used distillation for separating fluorides quantitatively, HE
1
Present address, Ethyl Corp., Baton Rouge, La.
---+
+
Sios--
+ 12 hIoOa-- + 22 H + + S i ( ? * 1 0 ~ 0 ~ ~+) ~HS03-4
+
S ~ ( M O ~ O ~ , , ) 11 ~ - H20 ~ -+
heteropoly blue
Feigl and Krumholz (4)and Feigl and Leitmeier (5) used this 3equence of reactions for the qualitative identification of fluoride. Peregud and Boikina (12) describe the determination of fluoride in gas streams of fluororganic compounds by decomposing the compounds at 900" C. in a oxygen stream in the presence of quartz and platinum and hydrolyzing the silicon tetrafluoride formed. Subsequent formation of molybdosilicic acid and determination of the silicate by visual comparison complete the determination. GENERAL EXPERIMENTAL WORK
Apparatus. Photometric measurements were made in 1-cm. matched cells with a Cary Model 10-llM recording spectrophotometer. All pH measurements were made with a Beckman Model H-2 glass electrode pH meter.
