laboratory have been internally consistent, this investigation affords the first opportunity to compare the method with those more widely used. In earlier studies, the major mass transfer was of the metal from the aqueous to the organic phase and the course of the reaction was monitored by (usually) radiochemical assay of the metal ion in the aqueous phase. In this study, involving water-soluble metal complexes, it is the ligand, which moves from the organic to the aqueous phase, that is monitored. Thus, the scope of the extraction technique has been enlarged to include water-soluble as well as extractable metal complexes. From the experiments under high C p h e n : C ~ iconditions, curves such as the one shown in Figure 1 were obtained, from which the values of the stepwise rate constants kl = 1.4 X l o 4 M-1 sec-I, k2 = 7.4 X 103 M-l sec-', and k 3 = 5.9 X lo2 M - l sec-I were calculated. These are in reasonable agreement with the value of kl obtained for high (Table Ia) and those for kz and k 3 obtained in runs using Cphen = 2 c N i and Cphen = 3 C ~ i respectively. , Our results are somewhat higher than those obtained by Holyer et al. (7) using the stopped-flow technique for the first stepwise constant (kl = 3.9 X lo3 M-l sec-l). It is interesting to observe that the determination of the second and third stepwise formation rate constants are accessible to the simpler extraction technique but apparently not, in this case a t least, to the more elegant and sophisticated stopped flow method.
The values of k l obtained under CB >> C N ~for , Ni-phen, also agreed reasonably well with that obtained for C N >> ~ CB. The Ni-5-nitrophenanthrolinesystem also proved to be well behaved. The corresponding kl = (5.0 f 0.5) X lo2 M-I value obtained (Table Ib) is in fair agreement with that reported by Holyer et al. (7). The drop in rate constant associated with the introduction of the electron-withdrawing group NOa, parallels the drop in the corresponding formation constant (log drops from 8.8 to 7.0) which implies that the rate of dissociation of the 1:l complexes is relatively independent of the substituent in the phenanthroline series of ligands. Finally, it can be noted that this study validates the use of the extraction technique for the examination of the kinetics of inherently fast reactions.
LITERATURE CITED C. B. Honaker and H. Freiser, J. Phys. Chem., 68, 127 (1962). B. E. McClellan and H. Freiser Anal. Chern., 36, 2262 (1964). J. S.Oh and H. Freiser, Anal. Chern., 39, 295 (1967). J. S.Oh and H. Freiser, Anal. Chem., 39, 1671 (1967). , P. R. Subbaraman, M. Cordes, and H. Freiser, Anal.'Chem., 41, 1878 (1969). (6) E. F. Caldin, "Fast Reactions in Solution", Blackwell Sci. Pub., Oxford, 1964. (7) R. H. Holyer, C. D. Hubbard, S. F. A. Kettle, and R. G. Wilkins, lnorg. Chem., 4, 929 (1965). ~
RECEIVEDfor review August 6, 1973. Accepted August 25, 1975.
Ligand-Exchange Chromatography of Diamines and Polyamines James D. Navratll and Harold F. Walton Deparfment of Chemistry, University of Colorado, Boulder, Colo. 80302
The first use of ligand-exchange chromatography was to recover a diamine by absorption on a copper-loaded ionexchange resin (1).In our laboratory, we found diamines to be held strongly by metal-loaded resins, and we were able to separate aliphatic diamines by chromatography on these resins (2). Interest in aliphatic diamines and polyamines was stimulated by the report of Russell in 1971 ( 3 ) that they were present a t elevated concentrations in the urine of cancer patients. The compounds in question were: putrescine, 1,4-diaminobutane; cadaverine, 1,Ei-diaminopentane; spermine, H~N(CH~)~NH(CH~)~NH(CH~)~NHZ; spermidine, H2N(CH2)3NH(CH2)4"2. They are widely distributed in animal and plant tissues (4, 5). Tabor and Tabor, in their pioneer work, used ion exchange to absorb these compounds and separate them as a group from accompanying substances. Later, they were separated from one another by ion-exchange chromatography and determined quantitatively by their reaction with ninhydrin (6-11) or fluorescamine (12). Amino-acid analyzers were used. The diamines are retained more strongly than amino acids and are eluted by concentrated buffers, usually a t high pH. Liquid chromatography of dansyl derivatives on alumina, with UV detection has recently been used (13). Dansyl derivatives were separated by thin-layer chromatography (14). We have studied the separation of diamines and polyamines by ligand-exchange chromatography, exploring different combinations of cation exchangers and coordinating metal ions. Aqueous ammonia solutions were used as eluents, with refractometric monitoring.
EXPERIMENTAL Materials. These cation exchangers were used: Cellex-CM, modified cellulose with functional carboxyl groups; Cellex-SE, with functional sulfonate groups; Bio-Rex 70, 37-44 p , irregular particles of a methacrylate polymer with functional carboxyl groups; Aminex ($150 S,20-35 p, polystyrene beads with 8%crosslinking and functional sulfonate groups; Aminex A-7, 7-11 p , polystyrene beads chemically similar to Aminex Q-150 S.All were supplied by Bio-Rad Laboratories, Richmond, Calif. Before being placed in the columns, they were converted to the desired metal form by stirring with the metal-ammonia salt solution, then washed with aqueous ammonia. Chemicals were obtained from several commercial sources. Spermine, spermidine, putrescine, cadaverine, histamine, and histidine hydrochlorides came from ICN Pharmaceuticals, Inc., Cleveland, Ohio. For some of the tests, solutions of spermine and spermidine bases were prepared from solutions of the hydrochlorides by passing through a short column of anion-exchange resin in the OHform. Except for an ammonium chloride peak a t the void volume, it made no difference whether the amines were introduced as salts or as the free bases. The diamines 1,2-diaminopropane, l,g-diaminopropane, l,&diaminobutane (putrescine), and 1,5-diaminopentane (cadaverine) were purified by redistillation, but 1,6-diaminohexane and n-butylamine were used without redistillation. Equipment. Glass columns of 6.3-mm and 9.0-mm internal diameter, fitted with PTFE plungers, were obtained from the Chromatronix Division of Spectra-Physics, Inc., Santa Clara, Calif., and Glenco Scientific, Inc., Houston, Texas. Chromatronix sample-introduction valves with 0.1-cm3 loops and a Model 3100 liquid chromatograph were used; a Model 6000 solvent delivery system and Model R401 differential refractometer were obtained from Waters Associates, Milford, Mass. Metal concentrations in influents and effluents were measured by atomic absorption (Model 360, PerkinElmer Corporation, Norwalk, Conn.). . .
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2443
Table I. Elution Volumes (as Multiples of the Bulk Column Volume) Ce Ilex -CM
Aminex Q-150s
Amine
cu
Zn
Ni
1,3-Diaminopropane 1,4-Diaminobutane 1,5-Diaminopentane 1,6-Diaminohexane Spermidine Spermine Histamine Lysine Histidine Arginine n-Butylamine Ammonia, M
14.3 2.9 3.4 4.4 4.2 8.9 4.4
2.0 2.9 4.4 5.6 3.6 4.2 1.4 0.8
5.4 1.7 2.3 2.4 1.9 2.0
1.1
2.0 3.1 1.9
...
1.6 1.5
... ...
4.0
1.2 1.9 1.6
2.1
1.3 2.0 1.7
1.1
"4
1.9 3.3 4.8 6.4 4.3 5.5 1.2
1.4 1.1
~-
Table 11. Ligand Replacement Ratios Cellex CIM Amine
1,3-Diaminopropane 1,4-Diaminobutane 1,5-Diaminopentane Spermidine Spermine
--_
cu
Zn
Ni
Aminex, cu
2.0 1.2
1.0
...
.o
1.o
2.0 2.0
1.6 1.6 1.6 1.6
-___
1.0 0.5 1.5 1.5
12.2
2.1 3.6 7.4 14.4 4.6 5.1
...
...
8.4 3.9 6.4 18.6 < 1.0 < 1.0 5.8
Bio-Rex 7 0
*..
... ...
7.1
Zn
5.6 2.6 3.3 3.5 2.3 1.9 4.9
1.5 2.3 2.5 2.8 1.7 2.0
1.5 6.0
1.2
1.3
...
< 1.0
1.3 4.3 7.6
...
cu
...
1.2
1.0
3.8
4.9
I
u
En 2-
2.5 2.5
X W
0
z -
__--
RESULTS Table I gives an overview of the elution volumes and elution sequences found with various metal-exchanger combinations. I t does not show band widths or theoretical-plate heights; these are discussed below, along with other factors. General features of Table I are: (a) of the diamines, Cu binds 1,3 most strongly, while Zn binds 1,6 most strongly; (b) Cu has a much stronger affinity for histamine than Zn; (c) Cu discriminates better between spermine and spermidine than does Zn; (d) Bio-Rex 70 favors absorption of spermidine over spermine, whereas spermine is more strongly held than spermidine in all other cases. Table I1 gives "ligand replacement ratios", the slopes of the lines found by plotting the logarithm of the corrected elution volume vs. the logarithm of the ammonia concentration. A ratio of 2.0, for example, implies that 1 molecule of amine replaces 2 molecules of ammonia in the resin. These numbers may be used to predict elution volumes a t ammonia concentrations different from those quoted. Cellulosic Exchangers. A few tests were made with Cellex-SE in the copper and zinc forms. They were abandoned because the ammonia eluents stripped inordinate amounts of metal from the exchanger and, moreover, the theoretical-plate heights were large (1-4 mm). However, the copper form gave excellent separation of spermine from spermidine. The carboxylic exchanger Cellex-CM retained metal ions more strongly, and was tested with copper, nickel, zinc, cadmium, and ammonium as counterions. The cadmium form, not included in Table I, discriminated poorly between spermine and spermidine, though the plate height was good (0.5 mm). An elution curve with Cellex-CM-Zn is
Zn
vj
... ...
The experimental technique was like that used with amino sugars (15).The resin Bio-Rex 70, being soft, will stand only a moderate pressure drop; the cellulosic exchangers tolerate even less. Soft column packings collapse and choke the flow when the total pressure across the column (rather than the pressure gradient) exceeds a certain amount. It is better, therefore, to use two short columns in series than to use a single column having the same total length. We could double the total pressure drop in a column of cellulosic exchanger by sealing a coarse glass frit across the middle of the tube, effectively making two columns.
2444
-
cu
W
2
c 0 a
r LL w
a
0
20
40
1
1
1
60
EO
IO0
I
l2Oml
Figure 1. Elution on Cellex CM-Zn Column, 0.9 cm X 13 cm: solvent, 0.97M NH3, 10-4M ZnS04; flow rate, 20 mllhr. Amounts injected: 1,3-diaminopropaneand l.Cdlaminobutane,0.5 mg each: 1,6diaminohexane. 1.0 mg; spermidine. 3 HCI, 1.1 mg: spermine. 4 HCI, 1.7 mg
shown in Figure 1. The peak separation is good, but (a) the spermine elution took 5 hours, (b) cadaverine and spermine eluted together. If only the five diamines, 1,2 through 1,6, were present, a good chromatogram was obtained in 3 hours. Copper counterions gave larger plate heights than zinc, and nickel gave even larger plate heights. The same relation was found with the Aminex resins. Polystyrene-Sulfonate resins. Most work was done with Aminex Q-150 S a t room temperature. Metal sulfates, 1 X 10-4M, were added to the influents. The copper-loaded resin showed large differences in elution volumes (see Table I), but plate heights were 2-4 times those found with zinc-loaded resin and, moreover, arginine eluted close to spermine. Adding more copper salt to the influent did not greatly change the elution volumes, and it appeared to give broader peaks. A column was therefore packed with zinc-loaded Aminex A-7, a resin similar to Q-150 S but having smaller particles (7-11 microns, compared to 20-35 microns). Chromatograms obtained with this column are shown in Figure 2, and elution volumes are given in Table 111. Raising the temperature to 55 OC gave sharper bands; the spermidine peaks in Figure 2 correspond to plate heights near 0.3 mm, compared to 0.7 mm a t 20 "C. An important variable is the zinc-ion concentration in the solution. Raising the temperature increased considerably the stripping of zinc from the column, and more zinc
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
ZINC I N OUT
i 2
Z I N C I N : 110 p p m OUT = 8 0 p p m
4 6 ppnr 59 p p m
'3
W
n
I
cs
w IV
a a L.
W
I
P
0
IO
20
30
ML 5.5
40 0 IO 5 AMMONIA
20
30
40
Table 111. Elution from Temp, C Ammonia, M Zn, influent, ppm effluent, ppm 1,2-Diaminopropane 1,3-Diaminopropane 1,4-Diaminobutane 1,5-Diaminopentane 1,6-Diaminohexane Spermidine Spermine Histamine aColumn, 0.63 cm uncorrected.
Aminex A-7-Zna 20 5.1 50 40 60 16.5 21 40
55 5.4 46 60 45 16.6 19.3 33.7 60 26.3 32.5
*..
24 30
55 5.5 110 80
55 5.6 115 112
15.0 15.7 27.7
...
...
...
...
16.5 27.7
...
21.0 20.2 24.5 23.1 ... ... 35.5 ... X 24 cm; elution volumes in ml,
Figure 2. Elution on Aminex A7-Zn Column, 0.63 cm X 24 cm; solvent, 5 . 5 M "3, Zn concn as shown; flow rate, 20 mllhr; temp. 55 OC. Amounts injected: curve A, 0.65mg of each diamine, 0.46 mg spermine, 0.47 mg spermidine (as bases): curve 8, 1.0 mg of each diamine, 0.43 mg spermine, 0.25 mg spermidine. Refractive index scale: before vertical dashed lines, 1 ordinate unit = 40 ppm change in Ri: after vertical dashed lines, 1 unit = 20 ppm (curve A ) , 10 ppm (curve B)
sulfate had to be added to the influent. Raising the zinc concentration reduced the elution volumes and decreased the separation between 1,3-diaminopropane and 1,4-diaminobutane, while increasing the separation between spermine and cadaverine; see Figure 2 and Table 111. Good base-line separation of small concentrations of putrescine, spermidine, spermine, and cadaverine was found after the column had come to equilibrium with an influent containing 115 ppm of zinc (last column of Table 111); see Figure 3. Curves B and C, Figure 3, recorded at high sensitivity, show shifting base lines, a common problem with refractometric recording. The ammonia concentration influences the refractive index, and a slow loss of ammonia from the solvent reservoir causes the base line to fall. Ideally, this solvent should be dispensed from a large-volume syringe pump. The amino acids arginine, histidine, and lysine eluted between 1 and 3 void volumes, and did not interfere with the diamines and polyamines. Carboxylate Resin (Bio-Rex 70). This resin retains metal ions very strongly, and an influent metal concentration of 1 x 10-4M was quite adequate to prevent loss. Because the association between the metal ions and the fixed carboxyl ions is strong, retention volumes of amines are smaller than with sulfonate resin and the ammonia concentration of the eluent could be reduced. Copper-loaded resin gave narrower bands than the zincloaded resin. In both the copper and the zinc forms, the polyamines were eluted before putrescine and cadaverine, and spermine appeared before spermidine, a contrast to the behavior of the polystyrene sulfonate resin; see Table I. The early appearance of spermine and spermidine means a short analysis time. A typical run with copper-loaded resin is shown in Figure 4. Resolution of spermine and spermidine was improved b:y lengthening the column and decreasing the flow rate. In view of our observation with amino sugars on this resin ( 1 5 ) that the elution can be followed by the ultraviolet absorption of the copper complexes eluted from the resin, we tried to detect the diamines and polyamines by ultraviolet absorption of the effluent from the copper-loaded column. Peaks in the ultraviolet absorption were obtained, but the sensitivity was about ten times poorer than with refractometric detection. The polyamines do, indeed, form intensely colored copper complexes that absorb
I hour
VOLUME A N D TiME
Figure 3. Elution of low concentrations Same column and conditions as Figure 2, except Zn concn = 115 ppm influent, 112 ppm effluent (see Table Ill). Putrescine, spermidine, spermine, cadaverine were eluted in this order. Quantities injected (micrograms, as free bases): A, 180, 150, 150, 180; 8, 18. 15, 15, 18: C, 32, 11, 15, 14. Refractive index, full-scale deflection: A, 20 ppm; Band C, 5 ppm
W _I
a u
v)
J -I
3 LL
En 0
(D X W
0
z
W
I Iu a LL
LL W
e
\ 0
50
90ml
Figure 4. Elution o n Bio-Rex 70-Cu Column, 0.63 cm X 36 cm; solvent, 5.3MNH3, 1 X 1 0 - 4 M C ~flow ; rate, 50 ml/hr; room temp. Quantities injected: 0.5 mg of each diamine, 0.5 mg spermine. 4 HCi, 0.8 mg spermidine. 3 HCI
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2445
strongly in the ultraviolet, but the complexes are not sufficiently stable to be visible a t the ammonia concentrations used. Sensitivity. The lower limit of detection was explored with the Bio-Rex 70-Cu and the Aminex A-7-Zn columns. With the latter, injection of 10-15 micrograms of spermine or spermidine gave peaks with signal-to-noise ratios about 10, with the refractometer at full sensitivity. The refractive index response per unit mass was 1.5 times as great with cadaverine, and twice as great with putrescine as with the polyamines. For spermine and spermidine, therefore, a quantity of 5-10 micrograms is the smallest that can be measured with any degree of accuracy, unless a more sensitive detector can be used. Ninhydrin and fluorescamine cannot be used in the presence of much ammonia. For clinical purposes, one should be able to measure 1 microgram. The concentrations of putrescine and spermidine in normal urine are about 1-2 mghiter (3, 10, 11) and in cancerous urine may be five times as great. Two ways suggest themselves to make our method useful for clinical analysis: one, to use it simply as a method of separation, to collect the appropriate effluent fraction, evaporate it to remove the ammonia, and measure the polyamine by another means; the other, to take a relatively large urine sample, say 10-100 ml, and concentrate the polyamines on a small column of metalloaded resin before injecting them into the analytical column. Preliminary attempts to concentrate polyamines from urine are promising. In one such test, 1.0 mg each of spermine and spermidine hydrochlorides were added to 10 ml of urine; the urine was passed through a short column, 4 cm X 0.63 cm, of Bio-Rex ~O-CU,after which the column was washed with 10 ml of 0.25M ammonia, then connected to the inlet of a second column of Bio-Rex ~O-CU,61 cm X 0.63 cm (actually, two columns in series, to avoid having too great a pressure drop in one column). This was eluted with 5.9M ammonia. Well-separated, well-defined peaks for spermine and spermidine were obtained, and their areas indicated about 80% recovery. The ammonium form of Bio-Rex 70 was also used to concentrate diamines and polyamines from urine following hydrolysis with hydrochloric acid. Most of the excess acid was removed by evaporation, and the remainder neutralized to pH 9 by ammonia before passing into the column. After hydrolysis and before removing the ammonia, the solution was spiked by adding spermine and spermidine hydrochlorides. After absorption, the short column was washed with 10 ml of 0.25M ammonia, then connected to the analytical column as before. One hundred micrograms of each polyamine were added, and 80-90% were recovered.
2446
CONCLUSIONS Ligand-exchange chromatography with refractometric detection provides a simple means of analyzing mixtures of diamines and polyamines with minimum interference from accompanying amino acids. By varying the combination of metal ion and exchanger, a great variety of elution orders can be realized. The best separations were obtained with a zinc-loaded sulfonated polystyrene resin, Aminex A-7, and with a copper-loaded acrylic-type resin, Bio-Rex 70. Data are not available for the formation constants of the various metal-amine complexes in solution, but it is evident that Cu(I1) forms much more stable complexes with 1,2- and 1,3-diamines than does Zn(II), compared to the complexes with other diamines, and it is also clear that the nature of the exchanger influences the binding of the amines. The inversion of the elution order of spermine and spermidine with Bio-Rex 70, compared to Cellex-CM and the Aminex polystyrene-type resins, is surprising. Refractometric detection is simple and easy, but it does not provide the sensitivity that would be needed for clinical analysis unless a preliminary concentration step is performed.
ACKNOWLEDGMENT We are grateful to Jerry W. Harder for help in the laboratory.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
F. Helfferich, J. Am. Chem. SOC.,84, 3242 (1962). J. J. Latterell and H. F. Walton, Anal. Chim. Acta, 32, 101 (1965). D. H. Russell, Nature (London), New Blol., 233, 144 (1971). H. Tabor and C. W. Tabor, Phermacol. Rev., 16, 245 (1964). C. W. Tabor and S. M. Rosenthal, Methods fnzyml., 8, 615 (1963). R. A. Wall, J. Chromatogr., 37,549 (1968). H. Hatano, K. Sumizu, S. Rokushika, and F. Murakami, Anal. Biochem., 35, 377 (1970). H. J. Bremer and E. Kohne, Clin. Chlm. Acta, 32,407 (1971). H. Tabor, C. W. Tabor, and F. Irreverre, Anal. Blochem., 55,457 (1973). L. J. Marton, D. H. Russell, and C. C. Levy, Clin. Chem., 19, 923 (1973). C. W. Oehrke, K. C. Kuo, R. W. Zumwalt, and T. P. Waalkes, J. Chromatogr., 89, 231 (1974). H.Veenlng, W. W. Pitt, andG. Jones, J. Chromatogr., 80, 129(1974). M. M. AbdeCMonem and K. Ohno, J. Chromatogr., 107, 416 (1975). 0 . Dreyfuss, R. Dviv, A. Harell, and R. Chayen, Clln. Chim. Acta, 48, 65 (1973). J. D. Navratll, E. Murgia, and H. F. Walton, Anal. Chem., 47, 122 (1975).
RECEIVEDfor review June 26, 1975. Accepted August 21, 1975. This work is taken from the Ph.D. thesis of J.D.N. (University of Colorado, 1975), who received financial support from Dow Chemical Co., Rocky Flats Division. Support is also acknowledged from the National Science Foundation grant GP-37779X, and from the National Institutes of Health Biomedical Sciences Support Grant to the University of Colorado, which assisted in purchasing equipment.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
