urated and the cis unsaturated derivatives, as expected on the basis of conformational changes induced by introduction of the respective double bonds. Peaks 4, 13, 21, and 23 each represent a mixture of two fatty acid derivatives. Peak 23 can be resolved by omitting the solvent change to 97:3 acetonitrile-water and completing the separation using 80:20 acetonitrile-water. This is not done routinely because it prolongs the analysis time considerably. Should it be essential to resolve peaks 4, 13, and 21, the desired peak is collected from the chromatograph and re-injected in a separate analysis using the “recycle” mode of chromatograph. When applying this method to the analysis of fatty acids derived from a particular biological species, we often find it unnecessary to use the extensive separation illustrated in Figure 1. Consideration of the commonly occurring natural fatty acids (Peaks 1-6, 8, 12, 13, 16, 18, 19, 21, 23, and 24 in Figure 1) suggests that a faster separation with lower resolution might be adequate. The key elements in such an analysis are palmitoleic and arachidonic acids (peaks 5 and 6, Figure 1). Complete resolution of these two acids precludes use of a starting solvent system with acetonitrile composition >67%. If only one of these two acids is present, however, a rapid analysis is feasible. A separation of this type is illustrated in Figure 2. The time required for this analysis is 70 minutes as compared with four hours for the separation shown in Figure 1. Finally, the presence of the phenacyl moiety as the exclusive chromophore at 254 nm permits direct quantitation
of molar ratios of these fatty acid derivatives based on peak areas. The relationship of peak area to moles of fatty acids remains linear over the range 100 ng to 100 wg.
CONCLUSIONS The method described above affords a rapid and convenient method for the derivatization and subsequent analysis of fatty acid mixtures on the microgram scale with a high degree of resolution in most cases. This method has been applied extensively to the analysis of fatty acids composition in chick fibroblast phospholipids (10) and of both free fatty acids and phospholipids in platelets (11).Modification of this procedure for the analysis of prostaglandins will be reported separately.
LITERATURE CITED (1)A. Grunert and K. H. Bassler, Fresenius Z. Anal. Chem., 287, 342 (1973). (2)U. Hintze, H. Roper, and G. Gercken. J. Chromatogr., 87,481 (1973). (3)H. Ehrsson, Acta Pharm. Suecica, 8, 113 (1971). (4)E. 0.Umeh, J. Chrornatogr., 58, 29 (1971). (5) M. J. Cooper and M. W. Anders, J. Chromatogr. Sci., 13, 407-411 (1975). (6) I. R. Plitzer. G. W. Griffin, E. J. Dowty, and J. L. Laseter, Anal. Lett., 8, 539 (1973). (7)W. Morozowich, APhA Acad. Pharrn. Sci. Abstr., 69, (19723). (8)M. J. Cooper and M. W. Anders. Anal. Chem., 48, 1849 (1974) (9)W. T. Moreland. Jr., J. Org. Chem., 21, 820 (1956). (10)R. F. Borch and C. R . Moldow, unpublished results. (11) R. F. Borch and G. H. R . Rao, unpublishedresults.
RECEIVEDfor review May 27, 1975. Accepted August 20, 1975.
Separation and Detection of Ortho-, Pyro-, and Tripolyphosphate by Anion Exchange Thin Layer Chromatography R. A. Scott and G. P. Haight, Jr. School of Chemical Sciences, University of Illinois, Urbana, Urbana, Ill. 6 180 1
During work on the hydrolysis of various polyphosphates (in particular, pyrophosphate (PPi) and tripolyphosphate (PPPi)) as catalyzed by an inorganic redox system (HzOz oxidation of V02+) ( I ) , a simple, rapid technique for the separation and identification of Pi (orthophosphate), PPI, and PPPi was sought. Much work has been done in recent years toward finding such a technique for the separation of polyphosphates during which various types of chromatographic separations have been examined including paper, (2-6) and anion exchange using thin layers (7-11). In many of these separations, the detection and visualization of the polyphosphates was accomplished by hydrolysis to Pi, followed by formation of some type of phosphate-molybdenum complex which is subsequently reduced to form the mixed oxidation state (Mo(V,VI)) polymer, phosphomolybdenum blue (PMB) (3-6). Problems have arisen due to catalysis of polyphosphate hydrolysis by acid eluents (2), slow hydrolysis of polyphosphate spots after separation (4-6), and visualization of the spot as PMB (4-6). Anion exchange chromatography can be carried out on thin layers by the use of cellulose impregnated with an appropriate anion exchanger coated on glass plates or plastic sheets. Berger et al. (10) used thin layers of Bio-Rex 5 ion
exchanger to separate polyphosphates. Tanzer et al. (11) used polyethyleneimine (PE1)-impregnated cellulose-coated on glass plates and LiCl eluents to separate polyphosphates. For visualization, the chromatogram was sprayed with the reagent of Hanes and Isherwood ( 3 ) ,dried in an oven a t 150 OC (to hydrolyze all polyphosphates), irradiated with uv light, and exposed to NH3 vapors to develop the blue spots. Iida and Yamabe (12) used NHlCl and NaCl as the eluents and hydrolyzed the polyphosphates by spraying with an aqueous H N 0 3 solution before visualizing with molybdate and stannous chloride. The method presented here consists of the separation of polyphosphates (in particular, Pi, PPI, and PPPi) on PEIimpregnated cellulose-coated plastic sheets (thin layers) by use of LiCl eluents. The detection method used has the advantage of being simple and rapid, allowing visualization of polyphosphate spots in 5-10 minutes. This is accomplished by the simple expedient of using water to accelerate the hydrolysis process and PMB formation.
EXPERIMENTAL Thin Layers. Plastic sheets, 20 X 20 cm, coated w i t h 0.1-mm cellulose (MN 300) impregnated w i t h polyethyleneimine were obtained f r o m B r i n k m a n n Instruments, c u t i n t o 5 X 20 c m strips a n d stored a t -5 O C until used.
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Flgure 1. Chromatogram showing polyphosphate separation. (1) PPPI. (2) PPI. (3) Pi. (4) Pi and PPi. (5) PPi and PPP,
Rewashing. All thin layers were treated with B prewash using 1.0 M NaCl. The tops of the thin layers were placed in contact with an absorbent pad (several layers of Whatman No. 1 chromatography paper) and ascending wash was carried out for 12 hr. The thin layers were dried under a stream of cold air and then rinsed twice (5 hr each rinse) in the same manner with H20, drying between rinses. Reference Chemicals. Reagent grade NaH2P04.H*O(Pi), K4P20~3H20(PPi),.and NasPaOlo(PPPi) were used without purification. Solutions were made up 50 mM in phosphate and stored as frozen solutions to avoid any hydrolysis of the polyphosphates. It was found that spotting -0.2 fil of a 50 m M solution (-10 nmol of phosphate) on the origin of the thin layers gave an optimal amount of phosphate for detection. Apparatus. All elutions were carried out by ascending chromatography in an Eastman Chromagram Chamber Plate developing apparatus. This apparatus is known for the fast equilibration of the layer with the solvent vapor due to its small volume. Thus, no pre-equilibration of the layer was attempted. The spray reagents were applied by the use of Chromaflex glass sprayers ohtained from Kontes Glass Co. Normal spray bottles or atomizers did not deliver a fine enough spray to avoid diffusion of the phosphate spots and interference due to background coloration. Solvents and Elution. The phosphates were spotted on an origin 2 cm from the bottom of the 20-cm thin layer and eluted to a horizontal line scratched in the cellulose 2 crn below the top of the thin layer with 2.0 A4 LiC1. The elution takes -2 hr. The thin layer was then dried under a stream of cold air before visualization with spray reagents. Spray Reagents. The reagents used for visualization of the phosphate spots were adapted from the reagents used by Baginski et al. (13) for quantitative determination of Pi in solution. The molyhdate reagent consisted of B l% aqueous solution of (NH36Mo70zc4HzO (ammonium heptamolyhdate tetrahydrate) and the reducing agent was an aqueous solution of 2% ascorbic acid in 10%trichloroacetic acid (TCA). The molybdate reagent was sprayed on first, taking care not to saturate any portion of the thin layer. After the molybdate spraying, the thin layer was dried under B stream of hot air (to help promote the molybdate catalyzed hydrolysis of the polyphosphates) and sprayed with the reducing agent, taking the same precautions a8 with the molybdate spraying. While the thin layer was still wet with the reductant solution, it was placed in an oven at 100 'C. Within -5 minutes, the spots corresponding to Pi, PPi, and PPPi formed the desired PMB color. If saturation was avoided when spraying,hackground coloration was minimal.
RESULTS AND DISCUSSION Figure 1 is an example of the type of separation of Pi, PPi, and PPPi possible with this elution (2.0 M LiC1). Some tailing can he ohserved, hut this can he eliminated by 2440
reducing the amount of phosphate spotted. Approximately 5 nmol of phosphate was spotted at the origin of each spot on the chromatogram in Figure 1. For separation of mixtures of phosphates (numbers 4 and 5 in Figure l),50 nmol of each phosphate was spotted a t the origin. Reduction of the amount of phosphate spotted to 10 nmol per spot eliminates the observed tailing. Figure 1 also shows the R/ value for the three phosphates. Many concentrations of LiCl were attempted with 2.0 M LiCl giving the optimum separation. T h e R f values reported here (2 M LiCI) are in contradiction with those reported by Tanzer e t al. ( 1 1 ) who ohtained similar R/ values to ours (on PEI-cellulose thin layers) with an eluent of 0.2 M LiCl and very little separation a t higher concentration such as ours. Other than differences between the PEI-cellulose layers used, the source of this large deviation is not known. One of the advantages of the visualization method described here is the short amount of time required. Many previous methods suggest incubating the thin layer at high temperatures (-100 "C) to hydrolyze the polyphosphate spots only after first drying at room temperature. The results of this work indicate that, if the thin layer is dried and then heated to 100 "C, the hydrolysis requires -2 hours. However, if i t is placed in a 100 "C oven while still wet, the hydrolysis requires only -5 minutes. If the thin layer was allowed to dry completely after spraying with reducing agent, a light spray with H20 will again allow rapid (-5 minutes) visualization of the phosphate spots (if incubated a t 100 "C). Thus, i t appears that the reaction to form PMB requires a liquid medium to occur rapidly. Also, in the present procedure, the hydrolysis of the polyphosphates and the reduction of the molybdate to form PMB have been combined into one step. Other results (14, 15) have indicated the importance of the catalytic activity of redox processes toward the hydrolysis of various polyphosphates. The present work illustrates a similar catalysis of polyphosphate hydrolysis by another redox system: Mo(V1) -MOW). Preliminary results show that the above methods can also he adapted to the separation (and visualization) of organic phosphate esters. Rapid hydrolysis of such esters as monomethyl phosphate (Mep), diethylphosphate (Etzp), and dimethylpyrophosphate (MeppMe), by the visualization method described above has heen achieved. Thus, this method appears to he of general applicability in the separation of many types of phosphates and phosphate esters.
ACKNOWLEDGMENT The authors are indebted to R. I. Gumport for valuable and enlightening discussion.
LITERATURE CITED (1) G. M. Wonermann. R. A. Scott. and G. P. Haighl, Jr.. Abstracts 01 Papers, 169th National Meeting of the American Chemical Society. Philadelphia, Pa., 1975. (2) E. Karl-Kroupa. Anal. Chem., 28, 1091 (1956). (3) C. S. Hams and F. A. bherwood. Nature (London), 164, 1107 (1949). (4) D. N. Bernhan and W. B. Chess, Anal. Chem., 31, 1026 (1959). (5) R. H. Kolloff. Anal. Chem., 33, 373 (1961). (6) 0.Perengie, 2.Ansf. Chem., 158. 6 (1957). (7) D. T. Burns and J. D. Lee, Mikrochim Acta. 206 (1969). (8) T. iida and T. Yamaba, J. Chromtogr.,41, 163 (1969). (9) S. Ohashi and S.Takada, Bull. Chem. SOC.Jpn, 34, 1516 (1961). (10) J. A. Berger, G. Meyniel. and J. Petit, J. Chmmatogr., 29, 190(1967). (11) J. M. Tanzer. M. I. Krichevsky, and B. Chassy, J. Chromlogr.. 38. 526 (1968). (12) T. lida and T. Yamabe, J. Chromatogr.,56,373 (1971). (13) E. S. Baginski. P. P. Foa. and B. Zak, Clin. Chim. Acta. 15, 155 (1967). (14) 0.M. Woltermann. R. A. Scott. and G. P. Haight. Jr., J. Am. Chem. Soc.,96, 7569 (1974). (15) G. P. Haight, Jr., R. A. Scott, and G. M. Wollermann, Physbl. Chem. Phys.. 6, 375 (1974).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
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RECEIVEDfor review July 28, 1975. Accepted September 10, 1975. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American
Chemical Society, for partial support of this work, and to the National Science Foundation, the University of Illinois Foundation, and the Research Corporation.
Application of the Solvent Extraction Technique to the Investigation of the Kinetics of the Reaction of Nickel(l1) and Certain Bidentate Heterocyclic Nitrogen Ligands George COIOVOS,~ Akira Yokoyama,* and Henry Freiser Department of Chemistry, University of Arizona, Tucson, Ariz. 8572 1
Some years ago we initiated a simple extraction technique for the detailed examination of the kinetics and mechanisms of inherently very rapid metal complex formation reactions ( I , 2). The technique depends on the elementary principle that the rate of a reaction will decrease as the concentrations of one or more of the reacting species involved in the rate-determining step(s) is decreased. Such is the case when a chelating extractant is partitioned between an aqueous phase (where the reaction takes place) and an essentially immiscible organic solvent provided the extractant exhibits a sufficiently high distribution constant ( K D ~In ) . our laboratory, the technique was applied to the study of metal chelate systems (3-5) in which exceptionally low aqueous solubility precluded the application of any of the more conventional techniques, e.g., stopped flow, various relaxation methods, etc. (6). Despite the success and simplicity of the extraction technique to date, it has not been used for systems that have been studied by other means. In this report, we examine the kinetics of the wellstudied reactions of Ni2+ with phenanthroline and 5-nitrophenanthroline in order to compare our technique with more popular methods.
EXPERIMENTAL Apparatus. The extraction apparatus is essentially the same as that previously reported (2). Spectrophotometric measurements were carried out using a Gilford Model 2400 spectrophotometer. Atomic absorption measurements for the determination of Ni(I1) were made with a PerkinElmer Model 303 instrument. A Beckman Model G pH meter, equipped with glass-saturated calomel electrodes, and standardized vs. suitable Beckman buffer solutions, was used for all pH measurements. Reagents. Formate buffers a t pH 3.2 were prepared by adding suitable quantities of the appropriate acid to a 0.1 M solution of the sodium salt. The buffer solutions were freed from traces of metals by extraction with dithizone in CHC13 followed by a scrubbing step with CHC13. Chloroform was freed from alcohol by extraction with water. A stock solution of 0.100 M NiS04 was prepared by dissolving the proper amount of reagent grade N i S 0 ~ 6 H 2 )(Mallinckrodt) in water. It was standardized titrimetrically with EDTA. Stock solutions of M of 1,lO-phenanthroline (phen) and 5nitrophenanthroline (5-NO*-phen) were prepared by dissolving the proper amount of monohydrated phen (Mallinckrodt) and 5 NOz-phen (K & K Laboratories) in chloroform. Each stock solution was standardized spectrophotometrically after appropriate dilution using the following wavelengths and molar absorptivities: for phen, 266 nm (3.08 X lo4); for 5-NOz-phen, 267 nm (2.25 X 104). Present address, Science Center, Rockwell International, P.O. Box 1085, Thousand Oaks, Calif. 91360. Present address, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Kinetics of Formation of Ni(I1) Complexes with Phen and 5-NOz-phen. A series of approximately 10 identical samples containing appropriate concentrations of nickel, ligand, and buffer were prepared in 50-ml screw-cap vials in a manner that prevented phase mixing (careful pouring of upper phase along side of inclined vial). The contents of each vial was shaken for a definite time interval. The time of the reaction was taken from the time agitation was begun until it was stopped. Each sample was then allowed to stand briefly to ensure complete phase separation before removing an aliquot of the chloroform phase in which the concentration of the ligand was determined spectrophotometrically. In all systems, many runs with a wide range of concentrations of both nickel ion and ligand were carried out.
CALCULATION OF KINETIC PARAMETERS When a high total ligand to total nickel concentration ration (CBICNJ is used, and the spread of successive formation rate constants is sufficiently large, as was found with phen, then the rate of phen removal, plotted as -log [phen] vs. time, exhibits three distinct linear segments (Figure 1) from which the three stepwise rate constants can be obtained. Even when three distinct segments appear, the reaction steps are overlapping. Because these are pseudofirst-order reactions, these data can be treated in a manner analogous to that used in resolving concurrent first-order reactions such as mixtures of radioisotopes having different half lives. The linear segment corresponding to the reaction of slowest rate ( k 3 ) is extrapolated to shorter times and subtracted from the experimentally observed absorbances to obtain the true line corresponding to contribution from the second formation step. This line is in turn extrapolated to lower times and used in order to correct the values needed to obtain the first stepwise constant. Also, the first stepwise formation rate constant can be isolated using a high total nickel to total ligand concentration ratio (CNJCB). For the reaction of Ni2+
ki +B6 NiB2+ k-1
the rate of formation of NiB2+ can be expressed as d [NiB2+] = k1[Ni2+][B]- h-l[NiB2+] dt
(1)
Values of [B] and [NiB2+]follow from the ligand distribution equilibrium and mass balance equations:
[NiB2+]= Cp, -[Bl0
- [BH+]
[NiB2+]= Cp, -[B], where K D and K , are the ligand distribution and acid dis-
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