Phenacyl Esters of Fatty Acids via Crown Ether Catalysts for Enhanced Ultraviolet Detection in Liquid Chromatography H. D. Durst, M. Milano, E. J. Kikta, Jr., S. A. Connelly, and Eli Grushka' Department of Chemistry, State University of New York at Buffalo, Buffalo, N. Y. 14214
Phenacyl esters of fatty acids have been formed In an essentially quantitative manner using crown ethers as catalysts. These esters absorb UV radiation strongly at 254 nm, allowing the detection of as small a quantity as 1 ng of C2 acid, and 50 ng of C20 acid. The method of synthesis is inexpensive and can be carried out in virtually any aprotic solvent system in a short period of time. Reaction conditions do not require total exclusion of water, thus simplifying the procedure. Liquid chromatographic Separations of these 8sters have been obtained on a 25-cm long column packed with Ce bonded phase. The Implications and uses of such a broad ranging synthesis are discussed.
Several reasons can be given for the use of derivatization in chromatography: e.g., to enhance solute volatility (GC), to enhance separation, and to enhance detectability. The selection of a derivative can often become much more difficult than the actual derivatization itself, In all areas of chromatography, the above effects have been successfully applied to difficult or otherwise impossible separations. Some recent examples of derivatization for, volatility enhancement are the study of Myher et al. ( 1 ) and of McMahon and Crowell (2). In liquid chromatography, Timko et al. ( 3 )utilized derivatives of amino acids for separation enhancement. In liquid chromatography, derivatization for detection enhancement is frequently needed, since no universal, sensitive, and simple-to-operate detector exists. The refractive index detector (RI) is incapable of detecting nanogram quantities, as was shown in recent studies of fatty acids analysis ( 4 , 5 ) . A UV detector can be quite sensitive but, in order to take advantage of this detector, derivatization of nonabsorbing species is frequently required. For example, Fitzpatrick and Siggia (6) have used benzoylation to form UV-absorbing compounds for the detection of hydroxysteroids. Fitzpatrick, Siggia, and Dingman ( 7 ) have also used 2,4-dinitrophenylhydrazinederivatives of 17-keto steroids to enhance detectability. Henry (8) et al. have detected 10 ng of steroids using 2,4-dinitrophenylhydrazinederivatives. Papa and Turner (9) have detected carbonyl compounds using 2,4-dinitrophenylhydrazonederivatives down to 5-ng levels, while Carey and Persinger (1.0) have detected similar compounds down to the 40-ppm level using dinitrophenyl derivatives. Nanogram level detection of hexachlorophene has been accomplished with p-methoxybenzoate derivatives by Porcaro and Shubiak (11). Recently, fluorigenic labeling of various classes of compounds has been applied in LC (12, 13) with detection limits down in the 1-to 10-ng range. One class of compounds which is of paramount importance, from a biological point of view, is the fatty acids. Recent papers have shown that all types of fatty acids, including both low molecular weight acids (14) and the higher molecular weight acids ( 1 5 ) , are more important in human To whom all correspondence should be addressed.
systems than previously believed. Since most fatty acid do not absorb UV radiation (at least not in the wavelength ranges of most commercial UV monitors), detection of quantities in the 1-ng range can be difficult (viz., 4 , 5 ) . Tagging the acids with a strongly UV absorbing species, by the deformation of suitable derivatives, is essential. The derivatization of fatty acids, and acidic substances in general, has been a problem in analytical-organic chemistry for many years. The classically used derivatives in qualitative organic chemistry have been amides and esters ( 1 6 ) .The main problem in using methyl esters of fatty acid in LC is that one cannot use the UV detector because of inadequate absorption. With diazo compounds, which have the proper absorption characteristics, the problem, in addition to their toxicity and instability is the poor reaction yields. Thus, a search for an alternate method of forming UV sensitive derivatives has been initiated in several laboratories. Politzer and Griffin ( 17) used 1-benzyl-3-p-tolutriazine as a diazo-like precursor which will react with free fatty acids to form benzyl esters. Regis Chemical Co. (18) has taken the same basic approach, except that the derivatizing agent was l-p-nitrobenzyl-3-tolutriazine, to give pnitrobenzyl esters of the fatty acids. These possess stronger UV absorption than the benzyl esters, and detection limits are reported to be in the 1- to 10-ng range. Disadvantages of this technique are the necessity to vent the nitrogen evolving during the reaction and that the derivatizing reagent is quite expensive and must be used in large excess. The reaction also produces by-products which may interfere with the subsequent chromatographic analysis. The classical synthesis of fatty acids esters from a nondiazo precursor has usually entailed the reaction of a carboxylate anion in a displacement reaction with a reactive alkylating agent ( 1 6 ) . One synthetic improvement over the classical procedure discussed above was developed by Hendrickson and Kandall ( 1 9 ) who performed the carboxylate displacement using dimethylformamide (DMF) as solvent. This basic synthetic procedure was used by Cooper and Anders (20) to derivatize fatty acids with 2-naphthacyl bromide to form esters which are suitable for UV detection in LC analysis. The in the lower detection limit was 4-90 ng of ester with A,, 247- to 248-nm region. The main disadvantage of this procedure lies in the usage of DMF, a highly polar and aprotic soivent. In addition, excess quantities of a strongly hindered nitrogen base (N,N-diisopropylethylamine) are introduced into the reaction. Depending on the fatty acid, this may effect the derivatization. Examination of current techniques indicates that a need exists for derivatizing agents which could react with fatty acids in a variety of solvents which are compatible with the chromatographic system, and yield compounds with large molar absorptivities at 254 nm (the wavelength most frequently used in current UV detectors). The present paper reports the formation of phenacyl esters using crown ether catalysts. Since the time of their discovery by Pederson (21-26), crown ethers have been shown to have a tremendous ability
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
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to complex metal salts (21-281, especially those of potassium, and aid by solvation of the cation the dissolution of these salts in nonpolar, aprotic solvents. Anions of these salts in solution have been shown by several groups to be unusually reactive (21-34), especially the carboxylate anions (35-37). One of the most interesting and important property of solid-liquid crown ether phase transfer of salts is that stoichiometric concentrations of the crown ethers are not necessary (35-37). Thus, one may use crown ethers in molar ratios of from 1:20 to 1:lOO to catalyze the phase transfer of carboxylate salts. Under these conditions, the nucleophilic properties of the carboxylate anion are enhanced and isolated yields of alkylated material of 92-98% (35-37) have been reported. The ability of the crown ether to aid in the dissolution or extraction of the fatty acid into an aprotic solvent, coupled with the unusually reactivity of the carboxylate ion under these conditions, forms the basis of the derivatization reaction such as shown in Scheme I, steps 1 and 2. SCHEME I step 1
@ Br-
+
RC02K
-
RC02
@
t
KBr 4
EXPERIMENTAL Apparatus. Liquid Chromatography. The liquid chromatograph has been described before (38). Data were collected on a Beckman Model 1005 and a OmniScribe Model 5110-T chart recorder. Detection was provided by an LDC UV detector with an 8-11 cell a t 264 nm. The column, 4-mm i.d. X 25 cm stainless steel, was dry packed with a reverse phase packing which consisted of a bonded nonyl group (CS)to Corasil 11. Injections were made with a Glenco 10-11 syringe through a homemade injection head fabricated from a V4-inch Swagelok Union-T specially modified to minimize dead volume. The column was thermostated with a water jacket. Water was circulated from a bath controlled to f0.02 "C by a Fisher proportional temperature controller. The temperature used in this study was 40 "C. The nonaqueous portions of the mobile phases used in this study were: n-heptane, chloroform, and methanol which were spectro grade chemicals obtained from Fisher Scientific. The water was distilled and then deionized through a Illinois Water Treatment Co. ion exchanger column. The detector attenuation was set a t 0.16 AFS, except when determining the detection a t which case 0.02 AFS range was used. T h e amount of acids injected varied between 0.25 to 1.0 fig. Ultraviolet S p e c t r a l Studies. Spectra of the acid derivatives were obtained with a Cary 14 spectrophotometer. Molar absorptivity measurements were made with a Cary 16 (12-nm bandwidth a t 254 nm) with a 1-cm quartz cell. Spectrophotometric grade methanol and reagent grade chloroform were used as solvents. The chloroform was passed through a column consisting of silica gel and molecular sieve (packed in alternate layers) for the removal of water and ethanol stabilizer. Reagents. All organic solvents (Fisher Scientific and J. T. Baker Chemical Co.) were commercial A.R. Solvents and were used without purification. apDibromoacetophenone was obtained from Aldrich Chemical Co. and was recrystallized from ethanol unless obtained as a white crystalline material, in which case it was used without further purification. 18-Crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) and dicyclohexyl-18-crown-6 (2,3,11,12-dicyclohexyl-1,4,7,1 0,13,16-hexaoxacyclooctadecane)were obtained from Aldrich 1798
Chemical Co. or P.C.R., Inc. or synthesized by the methods of Gokel and Liotta (391,Green ( 4 0 ) , or Pederson (21,22). Procedure. The pure crown is hydroscopic and is most conveniently stored as a standard solution in benzene or acetonitrile. The crown may be dried by azeotropic distillation from benzene. The alkylating solution was made in any aprotic solvent, such as benzene or acetonitrile, so that the molar ratio of alkylating agent/ crown was 20:l or 1O:l. The 20:l solution was most often used when alkylations of 0.5mM t o 20mM fatty acids were performed, while the 1O:l solution was used for amounts smaller than 0.5mM total acids. Potassium hydroxide (85%) was dissolved in MeOH and protected from the atmosphere. The derivatization was carried out as follows. Alkylation Method A (Total Acid Concentration 0.5mM t o 20mM). A sample of the organic acids, dissolved in either methanol or water, was neutralized to a phenolphthalein end point by the KOH/methanol solution. The solvent was removed under aspirator vacuum in a 50-ml pear-shaped flask. (Note: methanol is the most convenient solvent for the neutralization because it is easily removed under aspiriator pressure. The removal of water may be accomplished by a vacuum pump or lyophilization. If one is analyzing the acid product from a base hydrolysis, KOH in methanol should be used as the reaction medium and the solution neutralized to a phenolphthalein end point with HCl/methanol.) The salts are usually obtained as white or slightly pink solids. If the residue is oily or sticky, 10-15 ml of benzene can be added to the residue and removed under aspiration. Small amounts of water or methanol do not seem to affect the subsequent alkylation so complete drying of the salts is not necessary. If one standardizes the KOH/ methanol solution, the total number of milliequivalents of acid may be determined (in most cases this is not necessary). An excess solution of the alkylating agent/crown (20:l) is then added and the total volume of solution brought up to 10 ml with the appropriate solvent (most often acetonitrile). The solution was heated, with stirring, a t 80 "C for 15 minutes. It is then ready for the chromatographic analysis. If alkylated individually, the acids can be isolated by removal of the solvent under aspirator vacuum, taking the residue up in benzene, and filtering this solution through 5-15 grams of dry silica gel. The silica column is washed with benzene (10-15 ml) and the benzene removed under aspirator vacuum. Column filtration removes the crown ether and residual salts while allowing the alkylated derivatives to pass through. If necessary, the alkylated derivatives can be recrystallized from benzene/cyclohexane using the Craig tube technique (41). Alkylation Method B (Total Acid Concentration 0.001mM to 0.05mM). The potassium salts, generated as in Procedure A, were transferred to a 3-ml Reacti-Vial (Pierce Chemical Co., P.O. Box 117, Rockford, Ill. 61105) fitted with a Reacti-Vial stirring bar. An excess of alkylating agent/crown (lO:l), along with enough solvent (usually acetonitrile) to bring the total volume up to between 0.5 ml and 1.5 ml, was added to the vial. The Reacti-Vial was capped with a TUF-Bond septum disk and heated, with stirring, a t 80 OC for 15 minutes. The vial is then cooled and the solution chromatographed. I t should be noted that in either procedure A or B, excess quantities of potassium bromide or potassium chloride do not effect the alkylation. Alkylation Method C (Total Acid Concentration 0.001mM to 0.5mM). This procedure may be used when free acids are obtained and formation of the potassium salt is undesirable. Into a ReactiVial, fitted with a stirring bar, potassium bicarbonate in concentration of 3 to 5 times the total acids concentration was added. The rest of the reagents and their quantities were as in Method B. The heating period at 80 "C was extended from 15 to 30 minutes, depending on the acids involved (low-molecular weight acids tend to neutralize and react faster than high-molecular weight acids).
RESULTS AND DISCUSSIONS Derivatization Studies. The most important advantage in using crown ethers to catalyze the derivatizing of fatty acids is that very small amounts of the compounds are needed for the alkylation reaction. The two crown ethers used in this study are 18-crown-6 (I) and dicyclohexyl-18crown-6 (11). a,p-Dibromoacetophenone (111) was used as the alkylation agent because it is inexpensive, gives highly crystalline products (if isolation is necessary), and the resulting esters have very high molar absorptivity, as will be
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
-
i 10
I
I
I
i
I
l
l
I 1
l
I 1
30s rn < P O 6
-
0
o*
-
02
-
>
F
3 L
0 240
zm WAVELENQTH
320
(nm)
Figure 1. UV spectra of propionic acid derivative (a) in MeOH, ( b ) in chloroform Figure 3. Column, Corasii ll/C9, 4-mm i.d. X 25 cm. Mobile phase: methanol/50% water. T = 40 OC, 2.39 cm3/min
50%
(1) a,p-Dibrornoacetophenone, (2) acetic derivative, (3) propionic derivative, (4) butyric derivative
MINUTES Figure 2. Column, Corasil ll/C9, 4-mm i.d. X 25 cm. Mobile phase: 87.5% n-heptane/l2.5% CHC13. T = 40 'C, 1.77 cm3/min (1) apDibromoacetophenone, (2) heptanoic derivative, (3) butyric derivative, (4) propionic derivative, (5)acetic derivative
shown shortly. The crown ether solid-liquid phase transfer method may be used to generate any of the useful alkylated derivatives, Euch as benzyl esters, p-nitrobenzyl esters, pbromophenacyl esters, p-chlorophenacyl esters, p-phenylphenacyl esters, and 2-naphthacyl esters.
I 2
I
I
-
I
*
~
:
A
0
MiNUTES
Figure 4. Column, Corasil li/C9, 4-mm i.d. X 25 cm. Mobile phase: 62.5% methano1/37.5% water. T = 40 OC, 1.75 cm3/min (1) Acetic, propionic, and butyric acid derivatives, (2) heptanoic derivative
Another advantage of the crown ether process is that quantitative yields (>97%) of derivatives are obtained with no byproducts. Consequently, large excesses of alkylating reagent are not needed. A third advantage is that almost any solvent may be used in this process with no decrease in yield or product purity. T h e two most common solvents used in this study were benzene and acetonitrile, although cyclohexane, methylene chloride, or carbon tetrachloride were found to be interchangeable. Therefore, the solvent may be chosen so as to be ideal for the product solubility or subsequent LC analysis. Rigorous anhydrous conditions were not found to be necessary. The alkylated derivatives may be isolated and crystallized using Craig (41) tube techniques and the acids recovered by the method of Hendrickson and Kandall(19). Spectrometric Studies. Solutions of the p - bromophenacyl ester derivatives have a single absorption peak in their ultraviolet spectra, the position and intensity of the absorption being independent of the fatty acid derivatives,
but dependent on the solvent used (Figure 1).Absorption maxima occurred a t 257 nm in methanol solution and a t 261 nm in chloroform solution. The short chain C1, C2, Cs, Cq, Ci, C12 derivatives have a molar absorptivity of 18,700 f 126 1. mole-1 cm-l in methanol a t 254 nm. Chloroform was used as the solvent for the long chain (C16, CIS,C20) derivatives because of their low solubility in methanol. In chloroform a t 254 nm, the molar absorptivity of the derivatives is decreased to 17,200 f 102 1. mole-l cm-l, due to the shift in absorption peak maximum to 261 nm. Plots of absorbance vs. concentration in both solvents showed no significant deviations from linearity over the concentration range of 0.52 X 10-5M to 21 X 10-jM of the acid derivatives. The high molar absorptivities are to be noted since they determine the smallest amounts that can be chromatographed. Chromatographic Separations. All the separations were done a t 40 "C. The detector was set at 0.16 AFS and between 0.25 to 1 p g of each acid derivative (in the mixture) was injected. Figure 2 shows the separation of C7, Cq, Cs, and Cp saturated fatty acids using 87.5% n-heptanell2.5~0chloroform as a mobile phase. The compounds elute in a "normal phase" order of decreasing chain length or increasing polar-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
*
1799
L 0
U 5
IO
L 20
15
L 25
MINUTES
Figure 7. Column, Corasil ll/Cg 4-mm i.d. X 25 cm. Mobile phase: 7 5 % methanol/25% water. T = 40 'C, 0.58 cm3/min ~~
25
20
15 IO MINUTES
-
~
0
5
Figure 5. Column, Corasil ll/Cg, 4-mm 1.d. X 25 cm. Mobile phase: 7 5 % methanol/25% water. T = 40 OC, 0.63 cm3/min (1) a,pDibromoacetophenone, (2) lauric derivative, (3) palmitic derivative, (4) stearic derivative, (5) arachidic derivative
1
mobile phase change I
1:
!
2
,I 1
a 0
I
i
I
I
I
2
HOURS
Figure 6. Column, Corasil ll/Cg, 4-mm i.d. X 25 cm. Mobile phase: 62.5% methano1/37.5% water. Change at 2.2 hours (with a baseline shift) to 75% methanol/25% water. T = 40 OC, 0.30 cm3/rnin (1) a,pDibromoacetophenone, (2, 3) unknown impurities (from linoleic acid sample), (4) linolenic derivative, (5)linoleic derivative, (6) oleic derivative, (7) stearic derivative
ity. Since longer chain acids derivatives would tend to bunch up a t the dead volume peak, this normal phase separation is not very useful for acids longer than C7. Figure 3 shows the separation of C2, C3, and C4 acid derivatives using a reverse phase system where the mobile phase is 50% MeOH/5O% water. Using this mobile phase composition, the heptanoic acid derivative is retained so long that it could not be seen. Figure 4 shows that, upon changing the mobile phase to 62.5% MeOH/37.5% water, the Cz, CB, and C4 acid derivatives elute together and the C7 acid derivative is eluted in a short time. Hence, simple step or continuous gradient elution systems will have little 1800
( 1 ) a,pDibromoacetophenone, (2) lauric derivative, (3) linolenic derivative, (4) Oleic derivative, (5) stearic derivative, (6) arachidic derivative
difficulties in resolving the C1-C7 acids. Figure 5 further illustrates the effect of the mobile phase composition. Using 75% MeOH/25% water, aliphatic fatty acids derivatives of Cl2, CIS, CIS, and C20 acids were resolved. The above data illustrate that the C*-,C20 acid derivatives can be resolved on this column a t 40 OC starting a t 50% methanol/50% water and ending at 75% methanol/25% water. No attempts were made to optimize the system further for this separation, yet good resolution was obtained on a short, 25-cm, column with reasonable analysis times. Figure 6 shows the separation of four CIS acids derivatives. At -2.2 hours, the mobile phase was changed a step gradient, from 62.5% MeOH/37.5% H2O to 75% MeOH/25% H20. The separation of the C18 acid derivatives is comparable to that reported by Cooper and Anders (20). In fact, Figure 6 shows one more C18 acid (stearic) than Cooper and Anders' chromatogram. The impurity peaks (peaks 2 and 3) are from the linoleic acid sample used to prepare this synthetic mixture, and they are believed to be derivatives of lower fatty acids. No attempts were made to identify these two peaks since it was not crucial for this study. Mikes et al. ( 4 2 ) separated CIS acids methyl esters on a 50-cm X 2.1-mm i.d. Corasil I1 column coated with 0.8% AgN03/1.75% ethylene glycol stationary phase using 50% n-heptane/50% n-hexane as a mobile phase and a RI detector in 5 minutes. Even though this is a quicker separation, such a column is not broadly applicable to systems containing water because it would quickly degrade. For systems containing double bonds, the complexing feature of Ag+ is definitely advantageous and may be useful for resolving the phenacyl derivatives of elaidic and oleic acids. Finally Figure 7 shows a separation of the derivatives of C12, three (218, and C20 acids. Again no attempts were made to optimize this separation. The limit of detectability is an important criterion. For the Cz acid derivative, 1 ng could easily be detected by setting the detector attenuation at 0.02 AFS and expanding the recorder range to 1 mV. For the C20 acid derivative, 50 ng could be determined. These detectability limits were obtained without optimizing the chromatographic separation to get the best efficiencies. Since the peak height is a function of zone broadening, undoubtedly the lower limit of detectability could be improved substantially. It should be noted that samples were prepared in two ways. Mixtures of acids were made and derivatized. A sam-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
ple of the reaction mixture was then injected directly into the liquid chromatograph. On the other hand, pure derivatives of individual acids were made and isolated by crystallization. Synthetic mixtures were then made of these crystalline solids. A comparison of chromatograms of a real vs. a synthetic mixture of derivatives showed no significant deviations. This indicates that the derivatization reaction is quantitative with no significant interacid interference. This is especially important for the analysis of naturally occurring fatty acid mixtures. The a,p- dibromoacetophenone derivatives of fatty acids, catalyzed by crown ethers, show excellent promise in analytical biochemistry. The ease of preparation, using a range of solvents and quantitative yields, makes this derivatization technique useful in a broad variety of situations dealing with fatty acids and other organic acids. Work is now being performed to evaluate the method for dibasic acids and other biologically important compounds containing acidic functionality. LITERATURE C I T E D (1) J. J. Myher, L. Maras, and A. Kaksis, Anal. Biochem., 62, 188 (1974). (2) D. H. McMahon and E. P. Crowell, J. Am. Oil Chem. Soc., 51, 522 (1974). (3) J. M. Timko, R . C. Helgeson, M. Necomb, G. W. Gokel, and D. J. Dram, J. Am. Chem. Soc., 96, 7100 (1974). (4) G. A. E. Arvidson, J. Chromatogr., 103, 201 (1975). (5) C. R. Scholfield, J. Am. Oil Chem. Soc., 52, 36 (1975). (6) F. A. Fitzpatrick and S. Siggia. Anal. Chem., 45, 2310 (1973). (7) F. A. Fitzpatrick, S. Siggia, and J. Dingman, Sr., Anal. Chem., 44, 2211 ( 1972). (8) R . A. Henry, J. A. Schmit. and J. F. Diekman, J. Chromatogr. Sci., 9, 513 (1971). (9) L. J. Papa and L. P. Turner, J. Chromatogr. Sci., 10, 747 (1972). (10) M. A . Carey and A. F. Persinger, J. Chromatogr. Sci., 10, 537 (1972). (11) P. J. Porcaro and P. Shubiak, Anal. Chem., 44, 1865 (1972). (12) R . W. Frei and J. F. Lawrence, J. Chromatogr., 83, 321 (1973).
(13) R. W. Frei, J. F. Lawrence, J. Hope, and R . M. Cassidy. J. Chromatogr. Sci., 12, 40 (1974). (14) R. P. Michael, R. W. Bonsaii, and P. Warner, Science (London), 186, 1217 (1974). (15) N. Nicoiaides, Science(London), 186, 19 (1974). (16) N. D. Cheronis. J. B. Entrikins, and E. M. Hodnett, "Semimicro Qualitative Organic Analysis", Interscience. New York, N.Y., 1965. (17) I. R . Politzer, G. W. Griffin, B. J. Dowty. and J. L. Laseter, Anal. Lett., 6, 539 (1973). (18) Regis Lab Notes, No. 16, August 1974. (19) J. P. Hendrickson and C. Kandall, Tetrahedron Lett., 343 (1970). (20)M. J. Cooper and M. W. Anders, Anal. Chem., 46, 1849 (1974). (21) C. J. Pederson, J. Am. Chem. Soc., 89, 7017 (1967). (22) C. J. Pederson, J. Am. Chem. Soc., 92, 391 (1973). (23) C. J. Pederson, Fed. Proc., Fed. Am. SOC.Exp. Bioi., 27, 1305 (1968). (24) C. J. Pederson and H. K. Frensdorff, Angew. Chem., lnt. Ed. Engi., 11, 16 (1972). (25) J. J. Christenson, J. 0. Hill, and R. M. izatt, Science (London). 174, 459 (1971). (26) G. Gokel and H. D. Durst, Synthesis, in press (1975). (27) D. J. Sam and H. E. Simmons, J. Am. Chem. Soc., 94, 402 (1972); 96, 2252 (1974). (28) C. L. Liotta and H. P. Harris, J. Am. Chem. Soc., 96, 2256 (1974). (29) H. D. Durst, J. W. Zubrick, and G. R. Kieczykowski, Tetrahedron Leff., 1777 (1974). (30) H. D. Durst, J. W. Zubrick. and B. I. Dunbar, Tetrahedron Left., 71 (1975). (31) D. T. Sepp, K. V. Scherer, and W. P. Weber, Tetrahedron Leff., 2983 (1971). (32) D. Landin, F. Montanari. and F. M. Pirisi, Chem. Commun., 879 (1974). (33) C. W. Bowers and C. L. Liotta, J. Org. Chem., 39, 3416 (1974). (34) M. Makoza and M. Ludwikow, Angew. Chem., 86, 744 (1974). (35) H. D. Durst, TetrahedronLett., 2421 (1974). (36) H. D. Durst, M. M. Mark, D. Dehm, and R . Banden, Tetrahedron Lett., in press (1975). (37) C. L. Liotta, H. P. Harris, M. McDermott, T. Gonzalez, and K. Smith, Tetrahedron Lett., 2417 (1974). (38) E. Grushka and E. J. Kikta, Jr., Anal. Chem., 46, 1370 (1974). (39) G. W. Gokel, D. J. Cram, C. L. Liotta, H. P. Harris, and F. L. Cook, J. Org. Chem., 39, 2445 (1974). (40) R . N. Green, Tetrahedron Lett., 1793 (1972). (41) L. C. Craig, lnd. Eng. Chem., Anal. Ed., 12, 775 (1940). (42) F. Mikes, V. Schurig, and E. Gil-Av, J. Chromatogr., 83, 91 (1973).
RECEIVEDfor review March 17, 1975. Accepted June 11, 1975.
Novel Ion Exchange Chromatographic Method Using Conductimetric Detection Hamish Small Central Research, The Dow Chemical Company, Midland, MI 48640
Timothy S. Stevens Michigan Division Analytical Laboratories, The Dow Chemical Company, Midland, MI, 48640
William C. Bauman Inorganic Process Research, Texas Division, The Dow Chemical Company, Freeport, TX 7754 1
Ion exchange resins have a well known ability to provide excellent separation of ions, but the automated analysis of the eluted species is often frustrated by the presence of the background electrolyte used for elution. By using a novel combination of resins, we have succeeded in neutralizing or suppressing this background without significantly affecting the species being analyzed which in turn permits the use of a conductivity cell as a universal and very sensltive monitor of all ionic species either cationic or anionic. Using this technique, automated analytical schemes have been devised for Li', Na+, Kf, Rbf, Cs+, NH4+, Ca2+, Mg2+, F-, CI-, Br-, I-, NO3-, NOz-, Sod2-, S032-, Pod3- and many amines, quaternary ammonium compounds, and organic
acids. Elution time can take as little as 1.0 miniion and is typically 3 min/lon. Ions have been determined in a diversity of backgrounds, e.g., waste streams, various local surface waters, blood serum, urine, and fruit juices.
The demand for the determination of ionic species in a variety of aqueous environments is increasing rapidly and, as a result, there is an expanding need for automated or semiautomated analysis of chemical plant streams, environmentally important waters such as waste streams, rivers, and lakes, and fluids of biological interest such as blood, urine, etc. There are many examples where there is a continual need for routine analysis of common species such
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
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