was almost complete when 4 ml of fluid was the maximum amount applied to each column. Note that as the volume of fluid added to a column of this height increased, accuracy decreased presumably because of a build-up of interfering substances from the fluid. Iodide is a competing ion in the dye reaction and care must be taken to avoid collecting the copper iodide precipitate which is formed in a pellet on the bottom of the centrifuge tube. Other Ions. The inadequacy of the Collinson-Boltz method ( 9 ) for micro perchlorate determination is that diverse ions compete with perchlorate in formation of the dye complex. The introduction of ion exchange from the selective binding of perchlorate to the resin has eliminated this problem. Perchlorate Concentration. The standard graph was obtained by following the procedure of Collinson and Boltz (9). Duplicability was satisfactory. Conformity to Beer's law was observed between 5 and 50 pg of perchlorate in 25 ml of aqueous solution or in 10 ml of ethyl acetate. A slight negative deviation was observed above 100 pg.
Recovery of different amounts of perchlorate i n 2-1111 urint. samples was measured. Full recovery (96-100 %) was obtained when 40, 80, and 100 pg C10,- were added to each sample and then measured independently. A precision determination on a series of 5 serum samples each containing the same amount of CIOa- (80 pg per 2-ml sample) yielded a mean recovery of 97 + 1 %. As little as 5 pg/ml of perchlorate in urine or serum can be accurately determined in 2 ml of fluid. Copper Concentration. Hydroxylamine reduces copper(I1) to copper(1). Sufficient copper(1) is required to precipitate out all of the iodide as well as t o ensure complete formation of the perchlorate complex. pH. A p H in the neutral range was used for all experi, ments. Collinson and Boltz point out that the perchlorateneocuproine complex is stable in the pH range 3-10 (9). RECEiVED for review July 22, 1971. Accepted September 29, 1971. Supported in part by United States Public Health Grant No. A M 10992.
Proton Magnetic Resonance Studies of Adsorption of Salts on Chromatographic Gels Joseph J. Pesek' and Robert L. Pecsok2 Department
OJ
Chemistry, Unifiersity oj California, Los Angeles, Calif 90024
NUCLEAR MAGNETIC RESONANCE (NMR) has proved to be a valuable tool for investigating the properties of ion-exchange resins ( / - / 3 ) . In most systems the N M R spectrum will consist of two sets of peaks. One set represents the bulk solvent [exterior peak(s)] and the other represents solvent in the hydration sphere of the counter-ion inside the resin [interior peak(s)]. The solid resin does not show any peaks in the spectrum as a result of its restricted molecular motion. It has been reported that certain chromatographic gels are capable of separating inorganic salts (/4-18). Previously, Present address, Department of Chemistry, Northern Illinois University, DeKalb, Ill. 601 15. Author to whom ccrrespondence should be sent. Present address, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822 (1) J. E. Gordon, J. Phys. Cliem., 66, 1150 (1962). (2) J. E. Gordon, Cliem. Ind. (London), 1962, 267. (3) R. H. Dinius and G. R. Choppin, J. Phys. Clzem., 66, 268 (1962). (4) R. H. Dinius, M. T. Emerson, and G. R. Choppin, ibid., 67, 1178 (1962). (5) R. H. Dinius and G. R. Choppin, ibid., 68, 425 (1963). (6) J. P. DeVilliers and J. R. Parrish, J . Polym. Sci., A2, 1331 (1964). (7) D. Reichenberg and I. J. Lawrenson, T r a m Faraday SOC.,59, 141 (1963). (8) R. W. Creekmore and C. N. Reilley, ANAL.CHEM.,42, 570 (1970). (9) Ibid., p 725. (IO) L. S. Frankel, ibid., p 1638. (1 1) D. G. Howery and B. H. Kohn, Anal. Lett., 3,89 (1970). (12) D. G. Howery and M. J. Kittay, J . Mucromol. Sci., Part A , A(4), 1003 (1970). (13) T. E. Cough, H. D. Sharma, and N. Subrarnanian, Cam J . Cliem., 48, 917 (1970). 620
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
these gels have been used mainly to separate macromolecules and to estimate their molecular weights (19-21). Because chromatographic gels have a n affinity for inorganic salts, N M R should be as useful a technique for studying these systems as it is for studying ion-exchange resins. The mechanism for separation of inorganic salts by chromatographic gels is unclear. The most likely mechanisms involve the selective adsorption of ions, the exclusion of ions from the pores of the gel, especially in high per cent polymer gels, or some combination of these two. Because the pore size of most chromatographic gels is large compared to radii of even the most hydrated ions, a mechanism based on selective adsorption would seem the most reasonable. Another N M R technique should prove valuable in studying the adsorption of ions by chromatographic gels (22-24). Frozen aqueous solutions of proteins and polypeptides show broad proton magnetic resonances which have been ascribed to water of hydration. Similar resonances should be ob(14) B. Gelotte, J. Chromatogr., 3, 330 (1960). (15) B. Lindqvist, Acta Cltem. Scand.. 16, 1794 (1962). (16) N. V. B. Marsden and H. R. Ulfendahl, Acta. Physiol. Scatid., 59, Supp. 213, 100 (1963). (17) S. Ohashi, N. Yoza, and Y. Ueno, J. Cl~romatogr.,24, 300 ( 1966). (18) D. L. Saunders and R. L. Pecsok, ANAL.CHEW, 40, 44 (1968). (19) R. L. Pecsok and D. L. Saunders, Sepnr. Sci., 1, 613 (1966). (20) D. M. W. Anderson and J. F. Stoddard, A d . Cliim. Acta, 34, 406 (1966). (21) H. Determann and W. Michael, J . Clirornatogr., 25, 303 (1966). (22) I. D. Kuntz, T. S. Brassfield, G. Law, and G. Purcell, Scierice, 163, 1329 (1969). (23) I. D. Kuntz,J. Amer. Cliem. Soc.,93, 514(1971). (24) Ibid., p 516.
served for gels having bound water. This could be water adsorbed directly by the gel or it may be water in the hydration sphere of adsorbed ions. Because salt solutions d o not exhibit any resonances below their freezing point, resonances in salt-gel solutions different from that of a pure gel solution may be evidence for adsorption of ions.
A
A
EXPERIMENTAL Chromatographic gels, 100-200 mesh, (Bio-Gel P 2-10, Bio-Rad) were placed in 1M salt solutions prepared with deionized water. This mixture was transferred by capillary pipet to standard N M R tubes so that upon settling the gel height a t the bottom of the tube was about 5 cm. The sample was allowed to stand for a t least 24 hours before the spectra were taken. An identical procedure was used to prepare samples containing 100--200 mesh ion-retardation resins (AG l l A 8 , Bio-Rad) or 100-200 mesh glass beads (Bio-Glas, Bio-Rad). The N M R spectra were obtained o n a Varian A-60D spectrometer equipped with a variable temperature unit. F o r low temperature experiments (all a t - 30 “C), the samples were first placed in small plastic tubes sealed o n the bottom with epoxy glue. The plastic tube containing the sample was then placed inside a standard N M R tube. This technique was used t o prevent cracking of the sample tube and probe insert as the frozen solutions warmed. The samples were frozen quickly by placing the N M R tube in dry ice-acetone and then they were allowed to warm up t o -30 O C in the probe.
B
C
Figure 1. NMR spectra of salt-gel solutions A . 1M NaCl and Bio-Gel P-2 B. 1M CaBr? and Bio-Gel P-2 C. 1M .4Ir(S0& and Bio-Gel P-2 Each division represents 10 Hz
A
RESULTS AND DISCUSSION The distribution coefficient, KD, in gel chromatography is given by 250 where V , = elution volume, V, = column void (interstitial) volume, and Vi = volume of solvent in the interior of the gel beads. Differences in Kn values account for the separation of various substances using chromatographic gels. KD values for a large number of inorganic salts o n Bio-Gel P-2 have been previously reported (18). Figure 1 shows typical N M R spectra obtained for 1M solutions of various salts containing Bio-Gel P-2. Figure 1A is a typical spectrum of a n aqueous suspension of gel without a salt or for those salts with relatively low KD values ( 51.5). The line width is about 4 Hz. Salts in this category are NaC1, K F , and KCl. Figure 1B is a typical spectrum for salts of somewhat larger KD values (1.5 5 KO 5 2.5). The line width is between 4 and 20 Hz. Salts in this category are CaBrs, MgBrz, and Ba(NO&. Figure 1C is a typical spectrum of salts with very large KD values ( 2 2 . 5 ) , such as A1(N0J3 and Al,(SO&. I n this case, two separate resonances are observed. One represents the bulk water an:i the other the hydrated water as in ion-exchange resins which have firmly bound ions. By analogy to the ion-exchange system ( I ) , peak CI should be the interior water and peak b the bulk water which is broadened because of field inhomogenieties in the vicinity of particles of diamagnetic susceptibility different from that of the surrounding liquid. Although the peaks in Figure 1 are centered near the normal water resonance, exact chemical shifts are not reported because of small variations in these values which depend o n the particular salt in solution. I n order t o compare the above results with a system that is known to adsorb ions, spectra of various 1M salt solutions containing a n ion-retardation resin were obtained. This
Hz
560
-250
0
A
B
A
C
250
0
-250
Figure 2. Low temperature spectra of Bio-Gel P-2 and Salt-Gel Solutions A . Bio-Gel P-2 B. 1M NaCl and Bio-Gel P-2 C. 1M Al2(SO4)3and Bio-Gel P-2 All spectra were recorded at -30 “C
system contains paired cation (carboxyl groups) and anion (quaternary ammonium groups) exchange sites. The spectra are similar t o those obtained for chromatographic gels. The salts known to be held most strongly by the resin (25) (NaCI, KC1, and NHdCI) gave two separate peaks, salts which are retained t o a moderate degree (CaBra, MgC12, and BaC12)produced a single broad peak, and salts that are held very weakly ( 2 5 ) W. Rieman and H. F. Walton, “Ion Exchange in Analytical Chemistry,” Pergamon Press. Oxford, 1970, p 204.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
621
A
B
very broad peak is attributed to water adsorbed by the gel. The second sharper peak (-40 Hz) is centered near the maximum of the broad peak. Using solutions of salts with increasingly larger K D values decreases the amplitude of the broad peak and increases the amplitude of the sharp peak until a spectrum such as Figure 2C is obtained for salts with the highest KO values [Al(NO& and Al2(SO,),]. Apparently water adsorbed by the gel and the salts are competing for the same sites. As more of the salt is adsorbed by the gels (salts with higher KD values), the water adsorbed by the gel is displaced. The sharpness of the hydrated water is probably due to the relatively high salt concentration of the environment. This water does not freeze and its increased mobility leads to a somewhat sharper peak. The NMR spectra of frozen solutions containing high salt concentrations have been reported previously (22). None of the above solutions showed any water resonance at -30 "C when the gel was not present. I n addition, spectra of samples containing silanized Bio-Glas were also obtained. Silanized Bio-Glas has no adsorptive properties and does not separate any inorganic salts. Neither the sample containing Bio-Glas in deionized water nor those containing 1 M salt solutions and Bio.Glas showed any water resonance a t - 30 OC. Figure 3 shows the N M R spectra of solutions containing various Bio-Gels. As can be seen, the amount of water adsorbed decreases from P-2 to P-6 and cannot be detected by this technique when P-10 is reached. From P-2 to P-10, the pore size increases and the total amount of polymer decreases. This decrease in the total amount of material means there are fewer sites for adsorption which accounts for the decreasing ability to separate salts from P-2 to P-10. O n Bio-Gel P-10, all salts have virtually the same KD value. From a summary of all the data presented, it should be possible t o make a reasonable choice concerning the mechanism of separating inorganic salts by chromatographic gels, Salts with high KD values give N M R spectra similar to salts which are highly adsorbed on ion-retardation resins and vice versa. Low temperature studies show that salts with high KD values displace more water adsorbed by the gel and have a larger peak which is characteristic of solutions with high salt concentrations than those with low KD values. The greater amount of water adsorbed by P-2 indicates it has more adsorption sites and therefore is more effective in separating salts than gels which adsorb less water. It is apparent that the mechanism for separation is selective adsorption.
A, 500
Hz
250
0
-250
C
7+ -2 5 0
Hz
500
250
D - ..
4
Hz
50'0
250
0
-250
Figure 3. Low temperature spectra of various Bio-Gels Bio-Gel P-2 B. Bio-Gel P-4 C . Bio-Gel P-6 D. Bio-Gel P-10 All spectra were recorded at -30 "C A.
[AI(NO& and AI,(SO,),] gave a single sharp peak. It should be noted that the relative order of adsorption by the ion-retardation resin is characteristic of the strong field carboxyl group and opposite to that of the weak field sulfonate cation exchange resins (26). O n the other hand, the chromatographic gel has the same relative order of adsorption as the sulfonate cation exchange resin. These results support the assumption that a n increasing KD value for a salt on a chromatographic gel means greater adsorption of the salt by the gel. Further evidence for adsorption of salts by chromatographic gels was provided by the N M R spectra of frozen salt-gel solutions. Figure 2A shows the spectrum of a frozen solution containing Bio-Gel P-2. The broad peak is similar to that obtained in previous work (22-24) and has a line width of -300 + 20 Hz. The resonance is centered a t the normal water chemical shift and is apparently due to water adsorbed by the gel. Figure 2B shows the spectrum of frozen 1 M salt (relatively low KO value such as NaC1) solutions containing Bio-Gel P-2. Two peaks are observed in the spectrum. The (26) W. Rieman and H. F. Walton, "Ion Exchange in Analytical Chemistry," Pergamon Press, Oxford, 1970, p 47.
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ANALYTICAL CHEMISTRY, VOL. 44,
NO. 3, MARCH 1972
RECEIVED for review July 19, 1971. Accepted September 29, 1971. This research was supported by the Office of Saline Water Grant No. 14-01-0001-1319.
