950
Anal. Chem. 1982, 54, 950-953
orders of magnitude poorer than conventional fluorescence. This limits its current value in real analysis, but it is likely that state of the art pulsed laser and signal recovery equipment will make the sensitivity of the technique more competitive, especially when separations are performed on “microscale” columns.
2.5
LITERATURE CITED
z e
u u
2 1.5
45
.3’5
,215
L O G 11/f21
Flgure 5. Logarithmic plot of the two-photon excited fluorescence signal of a dilute PBD solution vs. the inverse square of the focusing lens focal length ( f ) . Flow cell dlmensions are (a) 1.0 mm i.d. by 2.0 mm o.d., (b) 0.50mm 1.d. by 1.0 mm o.d., and (c) 0.25 mm i.d. by 0.50 mm 0.d.
availability of lenses. In the case of SEF, thermal problems may also be a factor. Nevertheless, we predict that optical path lengths could be reduced by at least an order of magnitude, versus what is normally used in conventional fluorescence, without loss in signal, thereby making the sensitivity of SEF and TPEF more competitive with conventional fluorescence detection, when used with “microscale”columns. In summary we have presented a laser-based detection technique for liquid chromatography which tends to probe different excited states than conventional optical detectors, In addition, the technique has a higher degree of tunable selectivity. The demonstrated sensitivity of SEF is about 3
(1) Yeung, E. S.;Sepaniak, M. J. Anal. Chem. 1980, 52, 1465 A-1470 A. (2) Dieboid, G. J.; Zare, R. N. Science 1877, 796, 1439-1441. (3) Sepaniak, M. J.; Yeung, E. S. J . Chromatogr. 1980, 790, 377-363. (4) Folestad, S.; Johnson, L.; Josefsson, B. In “Proceedlngs of the Fourth Internatlonal Symposium on Capillary Chromatography”; Kaiser, R. E., Ed.; Instltute of Chromatography: Bad Durkheim, West Germanv. 1981; pp 405-427. (5) Hershberger, L. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1879, 57. 1444-1446. (6) Deutscher, S.B.; Richardson, J. H.; Clarkson, J. E.; Ondov, “Abstracts of Papers, 182nd National Meeting of the American Chemical Society, New York, NY, Aug, 1961; Amerlcan Chemical Society: Washington, DC, 1961; ANYL 48. (7) Carreira, L. A.; Rogers, L. B.; Gass, L. P.; Martin, G. W.; Irwin, R. M.; Von Wandruszka, R.; Berkowitz, D. A. Chem. Biomed. Envron. Instrum. 1980, 70, 249-271. (8) Sepaniak, M. J.; Yeung, E. S. Anal. Chem. 1977, 49, 1554-1556. (9) McClain, W. M. Acc. Chem. Res. 1974, 7 , 199-205. (10) Kasha, M. Faraday SOC.Discuss. 1850, 9 , 14-19. (11) Lln, H. 6.; Topp, M. R. Chem. Phys. Left. 1977, 48, 251-255. (12) Sepaniak, M. J. Ph.D. Dissertation, Iowa State University, 1960. (13) Sepaniak, M. J.; Yeung, E. S. J . Chromatogr. 1981, 277, 95-102. (14) Lin, H. B.; Topp, M. R. Chem. Phys. Lett. 1877, 47, 442-447. (15) Novotny, M. Anal. Chem. 1881, 53, 1294 A-1308 A. (16) Knox, J.; Gilbert, M. J . Chromatogr. 1979, 786, 405-418. (17) Swofford, R. L.; McCiain, W. M. Chem. Phys. Left. 1975. 34, 455-460.
RECEIVED for review December 14,1981. Accepted February 18, 1982. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research.
Agglomerated Pellicular Anion-Exchange Columns for Ion Chromatography Timothy S. Stevens” and Martln A. Langhorst Dow Chemical U.S.A., Michigan Division Analytical Laboratories, Midland, Michigan 48640
Slnce the conceptlon of ion chromatography, the bask technology for the anaiytlcal column packlng used for anion analysis has not changed. Mlcropartlcles of anlon exchanger (1000 to 50 000 A diameter) were agglomerated onto macropartlcles of cation exchanger (5-100 pm dlameter) to produce a low capacity pelilcuiar anion exchanger. The theoretical plate count for such columns Is low relative to modern practice, typlcaliy belng about 650. The use of smaller mlcropartlcles of anion exchanger (200-1 000 A dlameter) agglomerated onto efficlently packed beds of 15 pm cation exchanger resulted In Improved column performance wlth theoretlcai plate counts of about 2000. Separations that required 20 mln before, can now be obtalned In 10 min wlth better resolution.
Since the conception of ion chromatography the basic technology for the analytical column packing used for anion analysis has not changed. Microparticles of anion exchanger (1000 to 50 000 A diameter) were agglomerated onto macroparticles of cation exchanger (5-100 pm diameter) to produce
a low capacity “pellicular”anion exchanger (I). Improvements with this technology came with the use of monodisperse anion-exchange latex (2) rather than the previously used ground anion-exchange resin and by performing the agglomeration step in a polyvalent salt solution (3). The use of the monodisperse anion-exchange latex eliminated the need to refine the ground resin to obtain the optimum size range, and agglomerating in a polyvalent salt solution resulted in reproducible and dense agglomeration of the microparticles due to the resulting suppression of electrostatic repulsion forces between the microparticles. The performance of this type of exchanger is shown in Figure 1with a base line separation of fluoride, chloride, nitrite, phosphate, bromide, nitrate, and sulfate in 20 min using an eluent of 0.0024 M Na2C03,0.003 M NaHCO, at 138 mL/h with a 2.8 X 500 mm glass column filled with a packing composed of 3760 A diameter type 2 X 5 latex agglomerated onto 50 pm diameter surface sulfonated styrene divinylbenzene copolymer. Figure 1 is a reproduction of Figure 3 of reference 3. The theoretical plate count for the bromide ion peak in Figure 1is about 650. Theoretical place counts of about 650 for a 0.5 m long column are low by current liquid chroma-
0003-2700/82/0354-0950$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
951
0.7511 mho cm-’
T
I
0
,
10 TllvlE (MINI
15
20
Flgure 1. Performance of a 2.8 X 500 mm column containing 50-pm packing agglomerated with 3760
A type
2 X 5 latex.
t
tography standards, but am about as expected for a pellicular type of packing (4).Today, m a t liquid chromatography is practiced using the ”microparticulate” type of packing based on 5-10 pm diameter porous silica because of its superior efficiency, with plate counts of 2000-10000 per 0.25 m long column routinely observed, but silica based anion-exchange columns rapidly degrade in the basic eluents used with ion chromatography. However, there is a growing recognition that “...modern performance has not yet been incorporated into ion-chromatographs and is an area of research that may lead to fruitful results” (5). The subject of this contribution is the improved performance observed with agglomerated packings where the microparticle size is in the range of from about 200 to lo00A. Please note that the term “ion-chromatography” as herein used refers only to the technique of ref 1 and not to other chromatographic techniques for the analysis of ions. EXPERIMENTAL SECTION The ion chromatograph used was a preproduction prototype of the Model 10 unit available from Dionex Corp. Sunnyvale, CA. The following chromatographic conditions were used: eluent, 0.0024 M Na2C03,0.003 M NaHCO, at 138 mL/h; suppression column, 2.8 X 300 mm Dowex 5OW-X16, H+ ion form, 200-400 mesh; injection, 50 pL loop; detection, electrical conductivity. Analytical columns prepared were evaluated by their ability to separate an injection of seven anions prepared from reagent grade salts dissolved in deionized water: 0.8 ppm F, 1ppm Cl-, 2.5 ppm NOz-, 14 ppm PO>-, 2.5 ppm Br-, 9 ppm NO3-, and 13 ppm Column Pseparation. Three column packing techniques were used in this work: (a) slurry packing by gravity settling in 9 mm i.d. glass columns (LC-9-MA-13,available from Laboratory Data Control Co., Riviera Beach, FE); (b) conventional pressure packing (6) at 2000 psig using stainless steel columns employing Valco end fittings (Valco Instrument Co. Inc., Houston, TX), and (c) 8 X 100 mm Radial-PAK columns (custom packed by Water Associates, Milford, MA). The same macroparticle composition was used for all the column8 studied: fully sulfonated 10-20 pm diameter 65% styrene-35% divinylbenzene copolymer available from the Hamilton Co. or the Benson Co., both located in Reno, NE, and described herein as 15 pm Dowex 50W-X35. Anionexchange latex was then agglomerated onto the macroparticles packed into the columns by passing a 1%polymer solids suspension of the latex in a 10% aqueous solution of sodium carbonate through the column until an excess was seen to emerge. Then the column was rinsed with 20 bed volumes of eluent and considered ready for use. Ion-Exchange Latex. The ion-exchange latex used in this work was synt,hesized by first copolymerizing 5% divinylbenzene with 95% vinylbenzyl chloride using emulsion polymerization techniques and then quatemized with dimethylethanolamine. As such, the latex has the same anion functionality as the well-known
0
1
I
I
2
4 6 MINUTES
I
1 8
10
Flgure 2. Performance of a 9 X 110 mm column Containing 15-1m Dowex 50W-X35 agglomerated with 190 A type 2 X 5 latex. f
L
I
I
I
0
2
4
6
1 8
MINUTES
Flgure 3. Performance of a 9 X 76 mm column containing 15-pm Dowex 50W-X35 agglomerated with 220
A
type 2 X 5 latex.
Dowex 2 ion-exchange resin and is referred to herein as type 2 X 5 ion-exchange latex. RESULTS AND DISCUSSION When this work was begun, it was not realized that latex of a diameter less than 1000 A would prove optimum. However, it was postulated that a more efficient column would result if the agglomeration step occurred after column packing. Small size latex was thus required to hopefully eliminate the problem of column plugging previously experienced when larger latex was agglomerated in situ (3). The problem of column plugging caused previous workers to agglomerate before column packing (3), but we concluded that an efficiently packed column could not be obtained with preagglomerated packing because such packing tends to clump and not be free flowing. Thus, two type 2 x 5 latex sizes (535 and 910 A) were agglomerated onto 4.6 X 70 mm, 10-20 pm Dowex 50W-X35 columns. Neither column showed an increase in back-pressure after agglomeration and it was concluded that neither column was plugged with latex. At this time it was though that the optimum latex size was about 3000 A based on a theoretical treatment of pellicular ion exhangers (7). It was hoped that more would be gained by having an efficently packed column than would be lost from the use of a latex of a size smaller than optimum. However, the evalu-
952
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982 Y
I
so:
0.3811mho cm”
f
0.75~ mho cm-’ PO2
1
I 4
I 2
0
I 6
1
I
8
10
I 1
0
2
3
4
5
6
MINUTES
MINUTES
Figure 4. Performance of a 9 X 56 mm column containing 15-pm Dowex 50W-X35 agglomerated with 535 A type 2 X 5 latex.
Figure 6. Performance of a 4.6 X 140 mm column containing 15-pm Dowex 50W-X35 agglomerated with 535 A type 2 X 5 latex.
T
so:
3.380 mho cm”
Pop
A
i
I
I
I
0
1
4
6
1 8
1 10
~~
0
2
4
6
a
10
MINUTES
MINUTES
Figure 5. Performance of a 2.8 X 42 mm column containing 15-pm Dowex 50W-X35 agglomerated with 6250 A type 2 X 5 latex.
Figure 7. Performance of a Radial-PAK column containing 15-pm Dowex 50W-X35 agglomerated with 535 A type 2 X 5 latex.
ation of the above columns showed that the column made with 535-8 latex gave the same resolution of the seven ion standard as the one made with 910-8 latex but in about 40% less time. Smaller type 2 X 5 latex was then prepared in an attempt to find the optimum latex size. Chromatographic Performance as a Function of Latex Size. Figures 2-4 show chromatograms of the seven ion standard with gravity packed columns agglomerated with 190, 220, and 535 8 type 2 X 5 latex. For comparison, Figure 5 shows a chromatogram of the seven ion standard with a column prepared by first agglomerating 6250 type 2 X 5 latex with the 15 pm Dowex 50W-X35 followed by column packing as previously taught (3). The data in Figures 1-5 clearly demonstrate the improved performance seen with the use of relatively small latex as evidenced by better resolution in a shorter time. The use of latex smaller than 190 8 was not attempted because a relatively large column volume would be required. The effect of a large column volume is to increase the time between injection and the beginning of the chro-
matogram and to require relatively large amounts of mpensive macroparticle packing. The judgement was therefore made that the use of 535-8 latex provided improved performance with a minimum volume of the packing needed. The use of a macroparticle packing having a smaller diameter than 15 pm would result in more surface area per milliliter of column volume and would be appropriate for agglomeration with latex smaller than 535 8. Pressure packing of stainless steel columns resulted in better performance than the gravity packed columns, as illustrated in Figure 6 where resolution is about the same as Figure 4 but in two-thirds the time. Figure 7 shows the performance of a Radial-PAK 8 x 100 mm column, custom packed by Hubert Quinn of Waters Associates, Milford, MA, then agglomerated with 535 8 type 2 X 5 latex, and used with the Waters RCM-100 compression module. The separation in Figure 7 took the same time as Figure 4 but the resolution is better with the radial compression column. The theoretical plate count for the bromide ion peak in Figure 7 is about 2000. This
a
951
Anal. Chem. 1982, 5 4 , 953-957
level of efficiency is in the range of modern practice using microparticulate silica packings and provides ion-chromatographg - - - with a much needed performance improvement. ACKNQ WLEDGMENT The authors acknowledge the contribution of J. K. Solc, J. L. Pillepich, and 0. W. kindall regarding the preparation of the latex used in this work. LITERATURE CITED (1) Smali, H.; Stevens, T, inn1
s.; Baumann, w. c. Anal Chem. 1975, 4 7 ,
(2) SKaii, H.; Stevens, T. S. U S . Patent 4 101 460.
(3) Smith, F. C., Jr.; Chang, R. C. US. Patent 4 119580. (4) "Liquid Chromatography Product Guide"; Whatman Inc.. 1977; Bulletin
.Nn .-. 193 .--.
(5) Saunders, D. J. J. Chromatogr. Scl. 1979, 4 , 205. (6) Synder, L. R.; Klrkland, i. J. "Introduction to Modern Liquid Chromatography", 2nd ed.; Wiley: New York, 1979; p 210. (7) Hansen, L. C.; Gilbert, T. W. J. Chromatogr. Sci. 1974, 12, 464.
RECE~VED for review November 2,1981. Accepted Februrary 16, 1982. A United States Patent application has been filed on behalf of the authors covering the subject of this contribution and the technology has been licensed to the D~~~~ Corp.
Liquid Chromatography/Mass Spectrometry of Kepone Hydrate, Kelevan, and Mirex Thomas Calms," Em11 43. Slegmund, and Gregory M. Doose Department of Health and Human Services, Food and Drug Administration, Offlce of the Executive Director of Regional Operations, 1521 West Pic0 Boulevard, 1.0s Angeles, California 90015
The characterlzatlon of three related perchioro cage compounds, Kepone hydrate, Keievan, and Mlrex, Is reported by the use of liquid chromatography mass spectrometry with a moving beit interface system uslng methane and ammonla as reagent gases. Thermal degradatlon of Kelevan has been successfully avoided and a protonated molecular Ion cluster has been observed for the first time. Examination of a spiked banana pulp sample monltorlng only the protonated molecular Ion clusters has illustrated the advantageous selectivity provided by LCMS/MID In dMlngu1shlng the presence of Kelevan from Kepone hydrate. Mlrex has been postulated to undergo a novel nucieophliic substitution reaction Involving its major fragment Ion by collision with a neutral molecule of ammonla followed by subsequent ellmlnatlon of HCI.
During surveillance analysis of pesticide residues, a large number of possible compounds are frequently excluded from consideration since they cannot be identified and/or confiied by conventional gas chromatography mass spectrometry (GCMS). Three main reasons for this apparent failure in identification are thermal lability, low volatility, and high polarity. Wiith the recent advent of combined liquid chromatography mass spectrometry (LCMS), some of these difficulities can now be experimentally overcome ( I ) . Admittedly, the use of LC permits sepwation of thermally unstable compounds but the ionization of such molecules when introduced into the source of the mass spectrometer must avoid thermal degradation if molecular weight and fragmentation evidence are to be gained. The ionization process involving flash vaporization of an LC effluent deposited on a polyimide moving belt using chemical ionization has already proved to be successful in obtaining spectra containing molecular ions from sugars, glycosides, and glucuronides (2-4) as well as a variety of substituted phenols found in tannery effluents (5). It would seem that ionization conditions employing this moving belt interface allow enhanced volatility via flash vaporization. Additionally, the physical arrangement of the area of sample introduction being in very close proximity to the actual source itself has also assisted in providing protonated molecular ions in competition with thermal degradation. Similar charac-
teristics have been reported (6) in obtaining mass spectra of underivatized guanosine, deoxyguanosine, sucrose, and p nitrophenyl-6-D-glucuronide via direct exposure probe techniques. These initial successes have prompted investigation of the potential utility of LCMS as both an introduction mechanism and a novel ionization process to analyze the perchloro cage family of pesticides (7), namely, Kepone hydrate (I), Kelevan (11),and Mirex (111). Previous studies (8-12) have focused attention on the use of GCMS to identify and quantitate these closely related compounds. However, the thermal lability of Kepone hydrate and Kelevan upon introduction into the hot injection port of the gas chromatograph caused thermal degradation (49)to Kepone (IV).Hence both Kepone hydrate (I) and Kelevan (11) would coelute as the thermal degradation product, Kepone (IV). Although the 1975 concern of environmental contamination by Kepone hydrate and Kepone has diminished considerably, the report (13)that Kepone is the principal environmental degradation product of Mirex has stimulated renewed activity. Mirex has been used extensively as the most effective bait toxicant against the imported fire ant outbreak emanating from New Orleans.
po
@o: (I)
(11)
(111)
(IV)
In this paper, we report an LCMS analytical scheme to characterize and identify these three principal perchloro cage compounds using both methane and ammonia as reagent gases for chemical ionization. In addition, the observations recorded for Mirex under ammonia CI conditions have revealed that
This article not subject to US. Copyright. Published 1982 by the American Chemical Society
