Anal. Chem. 1995, 67, 1845-1852
Ribonucleotide Electrolytes for Capillary Electrophoresis of Polyphosphates and Polyphosphonates with indirect Photometric Detection Shahab A. Shamsi and Neil D. Danielson* Department of Chemistry, Miami Univetsily, Oxford, Ohio 45056
Ribonucleotide electrolytes, namely adenosine, cytidine, guanosine, and uridine monophosphates (AMP,CMP, GMP, and UMP),are investigated for capillary electrophoresis (CE) with indirect photometric detection (IPD) of polyphosphates (P,) and polyphosphonates ( R P n ) . A separation of 12 Pnand RP, componentsin about 30 min using 5 mM ribonucleotidewith 2 mM diethylenetriamine as electroosmoticflow suppressor with negative polarity CE is possible. Although all of the ribonucleotide monophosphates seem to provide reasonable selectivity,AMP is the best electrolyte because of its high molar absorptivity, large dynamic reserve, and favorable transfer ratio required for a sensitive IPD. However, the quality of separation for some P, (particularly P3- and Pcphosphates) can be dramatically improved by incorporating Mg2+in the running buffer with positive polarity. Detection limits for P, and RPn range from 0.84 to 11 pM, quite comparable to those o b w e d with commonly used HF'LC methods. The reproducibility of the migration time is between 0.5 and 1.0%RSD. One practical applicationis the determination of phosphates and phosphonates in soap and various brands of toothpaste. The quantitation of glyphosate and aminomethylphosphonic acid in a commmercial herbicide is performed with an average relative error of 1.6%.The developed CE method could be applied routinely for the analysis of samples having phosphorus-containingpolyvalent anions. Multifunctional polyphosphates and polyphosphonates are phosphoruscontaining polyvalent anions that find use in an array of industrial applications (e.g., food, beverage, and geochemical, biochemical, and environmental science^.^,^ Polyphosphates (P,) are linear polymers of phosphoric acid in an anhydrous linkage, whereas polyphosphonates (RP,) are organic derivatives of phosphorous acid. The structures of these anionic compounds are shown in Figure 1. Many analyticaltechniques for detecting P,, including indirect flow injection analysis using xylenol orange or methylthymol (1) Crapo, C. A; Crawford, D. L. J. Food Sci. 1991,56, 657-659. (2) Walsh, N. E.; Griftith, E. J.; Pany, R W.; Quin, L. D. Phosphorous Chemisty; ACS Symposium Series 486; American Chemical Society: New York, NY, 1991. (3) Griffith, E. J.; Spencer, J. M. Environmental Phosphorous Handbook; John Wiley and Sons, Inc.: New York, NY, 1973. (4) Francis, M. D.; Center, R. L. J Chem. Educ. 1978,55, 760-762. 0003-2700/95/0367-1845$9.0010 0 1995 American Chemical Society
b l ~ e ,polarography,i ~,~ and ion-selectiveelectrode methods,8have been developed. Since P, and RP, chelating agents do not absorb or fluoresce in the UV or visible spectral region, ion chromatography (IC) using postcolumn reaction with femc iong-" and gradient elution with suppressed condu~tivity~~J~ are the two HPLC methods that have been effectively used. We previously developed a simple ion chromatographic method for the determination of P, using a naphthalenetrisulfonate (NE)eluent with indirect photometric and nonsuppressed conductivity dete~ti0n.l~ Capillary electrophoresis (CE) with indirect photometric detection (IPD) has been successfully used to analyze a diverse array of small molecules, including anions,15-li c a t i ~ n s , ' ~organic J~ acids,1i$20 and surfactant^.*^^^^^^^ Despite the large volume of work describing small ion separations, there are relatively few reports concerning the CE of polyvalent anions. To date there is only one brief report on CE applied to the separation of PI-PY phosphates using a phthalate electrolyte at pH 4.0. However, the system was not fully optimized, and the method was reported to have marginal baseline stability and reprodu~ibility.~~ In this paper we provide an assessment of the suitability of a range of ribonucleotide monophosphates as electrolytes for the CE separation of P, and RP,. To the best of our knowledge, there have been no reports on the successful use of ribonucleotide (5) Yoza, N.; Kurokawa, Y.; Hirai, Y.; Ohashi, S. Anal. Chim. Acta 1980,121, 281-287. (6) Yoza, N.; Miyaji, T.; Hirai, Y.; Ohashi, S.J Chromatogr. 1984,283,89-98. (7) Al-Sulimany F.; Townshend, A Analyst 1973,98, 34. (8) Tanaka, T. Fresnius' 2.Anal. Chem. 1985,320, 278. (9) Dionex Corp. Determination of Sequesten'ng Agents; Application Note 44; Dionex Corp., Sunnyvale, CA, 1982. (10) Weiss, J.; Hagele, G. Fresenius'Z. Anal. Chem. 1987,328, 46-50. (11) Haddad: P. R; Jackson, P. E. Ion Chromatography,Principles and Applications; J. Chromatography Library 46. Elsevier Science Publishers, B.V.: Amsterdam, the Netherlands, 1990; pp 86, 371, 396. (12) Dionex Cop. Ion Chromatography Cookbook; Dionex Corp.: Sunnyvale, CA, 1987. (13) Kreling, J. R; Cowan, J. S.; Block, F.; Denvaan J. J Chromatogr. 1994, 671, 295-302. (14) Shamsi, S. A; Danielson, N. D.J Chromatogr. 1993,653, 153-160. (15) Romano, J.; Jandik, P.; Jones, W. R; Jackson, P. E.]. Chromatogr. 1991, 546, 411-421. (16) Fritz, J. S.; Benz, N. J. J. Chromatog. 1994,671, 437-443. (17) Shamsi, S. A; Danielson, N. D. Anal. Chem. 1994,66, 3757-3764. (18) Chen, M.; Cassidy, R M.J Chromatogr. 1993,640, 1425-1431. (19) Shi, Y.; Fritz, J. S. J Chromatogr. 1994,671, 429-435. (20) Devevre, 0.; Putra, D. P.; Bottom, B.; Garbaye, J. J Chromatogr. 1994, 679, 349-357. (21) Nielen, W. F.]. Chromatogr. 1991,588, 321-326. (22) Chen, S.; Pietrzyk, D. J. Anal. Chem. 1993,65, 2770-2775. (23) Stover, F. S.; Keffer, S. S. .] Chromatogr. 1993,657, 450-457.
Analyfical Chemistry, Vol. 67, No. 7 7 , June 7, 7995 1845
POLYPHOSPHONATES
POLYPHOSPHATES
HO-PC-P-OH
I 1 (Dequest 2010) m1 CH,a
[EDP 1-Hydroxyethylidene-1,l-diphosphonic acis 0
0 H a - I
n = 0 = PI-phosphate n = 1 = P2-phosphate n = 2 = P3-phosphate n = 3 = P4-phosphate
?
H-P-H
(Orthophosphate) (Pyrophosphate) (Tripolyphosphate) (Tetrapolyphosphate)
4TMP Aminotri(methy1enephosphonic acid)s
P
HO-74H
I
a3 EDTMP Ethylenediaminetetra(methy1enephosphonic acid) (Dequest 2041)
II
OH
IDTMP Hexamethylenediaminetetra(methy1enl Jhosphonic acid) (Dequest 2054)
Y
Cyclic-P,
(Trimetaphosphate) CyClic-Ps
I
HO-P-OH
(Hexametaphosphate)
I
OH
DETPMP Diethylenetriaminepenta(methy1enephosphonic acid) (Dequest 2060) Figure 1. Structures of polyphosphates (P,) and polyphosphonates (RP,) studied.
reagents as mobile phases for HPLC or CE with IPD. Our investigation indicated that the use of adenosine monophosphate in particular as a CE electrolyte with IPD is a valid alternative to the more traditional postcolumn IC method for the determination of both P, and RP, in a mixture. Parameters such as concentration and chain length of the electroosmotic flow suppresser (EOFS), pH, and use of an inorganic m o d ~ e are r varied to optimize the separation and maximize the peak capacity. The advantages of CE over IC for these compounds are large peak capacity, better efficiencies, and minimal sample and reagent requirements. The CE detection limits of P,, and RP, reported here are comparable to those obtained with HPLC methods. EXPERIMENTAL SECTION
Instrumentation. An Applied Biosystems (Foster City, CA) Model 270 A capillary electrophoresis instrument was employed to generate all electropherograms. The fused silica capillary (75 cm x 50 pm id., 320 pm 0.d.) with various effective lengths (Ld) ranging from 45 to 55 cm was also obtained from Applied Biosystems. A Hewlett Packard (Wilmiigton, DE) Model 3395 integrator with reversed polarity for IPD was used to record all the data. MacIntegrator I software purchased from the Rainin Instrument Co. (Wobum, MA) was used on a MacIntosh SE computer for quantitation purposes. Chemicals and Samples. The monosodium salts of adenosine, cytidine, guanosine, and uridine monophosphates (AMP, 1846 Analytical Chemistry, Vol. 67,No. 11, June 7, 7995
CMP, GMP, and UMP) were 5'-phosphorylated nucleotide isomers, with a purity range of 98-loo%, and were purchased from the Sigma Chemical Co. (St. Louis, MO). The disodium salt of 5'-adenosine diphosphate (ADP) was also bought from Sigma.The monosodium salt of naphthalenemonosulfonate(NMS) with 99.5% purity was from Eastman Kodak (Rochester, NY). The disodium salt of naphthalenedisulfonate (NDS) with 95%purity was obtained from the Aldrich Chemical Co. (Milwaukee, WI), whereas the trisodium salt of naphthalenetrisulfonate (NTS) with 97%purity was from American Tokyo Kasei (Portland, OR). Ethylenediamine (EDA), diethylenetriamine @ETA), and triethylenetetraamine ("A), all technical grade, sodium tetraborate decahydrate (99.5%),and boric acid (99.5%) were purchased from the Fisher Scientific Co. (Fair Lawn, NJ). Inorganic salts of P, were obtained from dserent manufacturers. The RP, (Dequest series) were provided free of cost from the Monsanto Co. (St. Louis, MO). N-(l'hosphonomethy1)glycine (glyphosate, GLYP) and aminomethylphosphonic acid (AMPA) were purchased from Sigma. The commercial herbicide solution containing GLYP and its major metabolite AMPA, marketed by Monsanto as Roundup, was bought locally. Crest toothpaste, Topol Plus toothpaste, and Lever 2000 soap were purchased from a local grocery store. Procedures. Preparation of Electrolyte Solutions. A 50 mM stock solution of each W-absorbing electrolyte (NMS, NDS, NTS,
AMP, CMP, GMP, UMP, and ADP) was prepared in triply distilled water and then used after subsequent dilutions. The running buffer contained 5 mM of the UV-absorbing electrolyte. The pH was varied as specified in the figures, being adjusted by adding 1 mM NaOH, LiOH, or KOH to the running buffer. The concentration of the organic modifer used as an EOFS @ETA or TETN varied from 0.25 to 4 mM (calculated from the density and the volume added). Other electrolyte compositions and operating conditions used in this study are presented in the figure captions. All of the final operating buffers were filtered using 0.2 pm IC syringe filters from Gelman Science (Ann Arbor, MI) by creating a vacuum inside the syringe. CE Procedures. Prior to first use, a new capillary was subjected to a standard wash cycle for 6 h using 1 M NaOH at 60 “C. As a daily routine procedure, the capillary was flushed with 1 M NaOH for 30 min and then equilibrated with the operating buffer for 10 min before any sample injections. The separation was initiated by applying a voltage (3~20-30 kV) between the two capillary ends which were immersed in vials containing the operating buffer. In between injections,the capillary was flushed with triply deionized water, 1M NaOH, and triply deionized water again for 2 min. The capillary was finally filled with operating buffer for 2 min. This procedure resulted in improved peak shapes, and the migration time reproducibility was 51.0%RSD for the analyte anions. RESULTS AND DISCUSSION
Indirect Photometric Reagent Evaluation. In a previous paper we reported the CE separation of a wide variety of inorganic anions, organic acids, and anionic surfactants using naphthalenesulfonate electrolyte^.'^ Although naphthalenemonosulfonate has been shown to be an excellent electrolyte for anionic sulfate and sulfonate surfactant^,'^ it was found unsuitable for these phosphorus compounds. The selectivity of the NDS and NTS reagents toward low molecular weight and more mobile phosphates and phosphonates (especially the PI type) is reasonably useful, but the separation for relatively higher molecular weight and less mobile P, and RF’, is poor. Based on the mobility data published for high-mobility electrolytes such as chromate and pyr0mellitate,2~it can be expected that neither of these electrolytes would provide a suitable match for the separation of medium to high molecular weight phosphoruscontaining anions. Theoretical considerations suggest that reagents that can be useful for indirect detection for these high molecular weight, multicharged, anionic compounds should have a low mobility, a relatively large molar absorptivity ( E ) , and a favorable dynamic reserve OR). From this point of view, we investigated the utility of ribonucleotide reagents as potential CE electrolytes. Table 1lists the ribonucleotides evaluated as electrolytes in this study, together with their measured migration times (mobility), measured E values (based on the capillary in the CE instrument), and DR numbers. It can be seen that all of the ribonucleotide monophosphates have a considerably low mobility, large 6,and favorable DR values. The one ribonucleotide diphosphate (ADP) has a large E , but its DR was difficult to measure due to baseline drift from reagent instability. As expected from the E data (Table l),the slope or the detector response (AU/mM) increases in the order AMP > GMP > UMP > CMP. The DR data predict that AMP should (24) Cousins, S. R; Haddad, P. R; Buchberger, W. J Chromutogr. 1994, 671, 397 -402.
Table I.Characteristics of Ribonucleotide Electrolytes
electrolyte 5’-UMP 5’-CMP 5’-AMP 5’-GMP 5’-ADP
relative charge“ migration timeb
effective -2.0 -1.9 -2.0 -1.0 -3.0
& (L mol-’ cm-1)
3.8 4.2 4.4 4.9 6.5
(Am3
DRd
7240 (261 nm) 5640 (271 nm) 9335 (259 nm) 8600 (254 nm) 9200 (259 nm)
503 426 612 429
Calculated using the equation in ref 11at pH 7.80. * Measured with respect to nitrate using 2 mM DETA, 100 mM H3B03, and 5mM NazB407 at pH 7.80. Obtained from slope of the calibration curve/ 5.0 x cm capillary path length. Calculated as ratio of background absorbance/background noise.
Table 2. Calibratlon Data for Detector Response vs Concentration of 5-AMP inside the Caplllary
C, of AMP ( x 103 mol/L) 0.5 1.0 2.0 4.0 5.0 10.0
background absorbance (AU)
0.025 0.050 0.099 0.195 0.240 0.469
background noisea (x 104 AU)
DR~
1.23 1.59 2.05 3.18 3.92 6.37
203 315 483 613 612 736
C /DRC
(x
13mol/L) 2.4 3.2 4.1 6.5 8.2 14
a Measured as peak-to-peak noise. Calculated as ratio of background absorbance/background noise. Clim = Cm/(DR x TR), a s suming TR = 1.0.
give the best detection limit. Table 2 shows the representative linearity data obtained with a standard solution of AMP. It can be seen that the background absorbance inside the fused silica capillary is linear up to -0.5 AU, a value dependent on both the type of electrolyte and the instrument. Similar results were also obtained with other ribonucleotide reagents (data not shown). Using linear regression statistical analysis, correlation coefficients of 0.999 or higher are calculated for all the ribonucleotidereagent plots. Table 2 also shows that because DR decreases with ,C, dramatic differences in CL,, the detection limit, are not observed as a function of .C, Even though the theoryz5predicts that at a k e d transfer ratio (TR), the lowest detection limits would be obtained by minimizing the (CJDR) ratio, in reality, electromigration dispersion at lower electrolyte concentrations should be taken into account. From both an electrophoretic separation and a detection point of view, a 5 mM ribonucleotide solution was found to be a good compromise. A comparison of five electropherograms obtained for the 12-componentphosphate and phosphonate mixture using different kinds but the same concentration and pH of the ribonucleotide reagents was made. The two best examples are shown in Figure 2. The order of migration of P, and RP, remains essentially the same in all the electropherograms. If the chargeto-mass ratio was the sole consideration for selectivity, the order of increasing migration time should have been HEDP < ATMP < EDTMP < HDTMP < DETPMP. However, other factors (e.g., hydration energy and ionic radius) must be contributing to this different CE separation mechanism, which is found to follow the order HEDP < HDTMP < ATMP < EDTMP < DETPMP. Three points of interest emerge from this study. Fist,PI-hypophosphite (25) Yeung, E. S. Acc. Chem. Res. 1989,22, 125-130.
Analytical Chemistry, Vol. 67, No. 1 1 , June 1, 1995
1847
10
6
I
! 6
AMP
I
t i i i i i i i i i i i i
6
8
10
12 14 16 18 20 22 24 26 28 32
Time (minutes) Figure 2. Comparison of UMP and AMP for the separation of a mixture of 12 Pn and RP,,. The electrolyte was composed of 5 mM UMP or AMP in 100 mM H3B03, 5 mM Na2B407,2 mM DETA, pH 7.80 buffer. IPD at 261 nm for UMP and 259 nm for AMP. Vacuum injection for 30 s, -30 kV applied for separation; current, 10-1 3 PA. Peak identification: 5-10 mg/L (pmollL) of 1, cyclic-P3 (21); 2, PIfluorophosphate (51); 3, P,-phosphite (62); 4, P?-phosphate(52); 5, PI-hypophosphite (78); 6, HEDP (49); 7, cyclic-PS (21); 8, Pzphosphate (57); 9, HDTMP (21); 10, ATMP (34); 11, EDTMP (23); and 12, DETPMP (18).
and cyclic-P6 (peaks 5 and 7) are fully resolved with AMP in comparison to the other ribonucleotide reagents such as UMP (Figure 2). Second, the prediction based on the data in Table 1 that AMP should give the best detectability was vedied. Even though the E of GMP is nearly the same as that of AMP (Table l), the Pz-phosphate, HDTMP, ATMP, EDTMP, and DETPMP peaks (solutes 8-12) show signals 2-3 times stronger when AMP is used as an electrolyte as compared to either GMP or CMP. The relatively lower DR values for these two electrolytes may be the reason for this behavior. Third, it is noteworthy that the 1848 Analytical Chemisfry, Vol. 67,No. 11, June 1, 1995
separation with ADP is not very effective, with poor baseline stability as expected (electropherogram not shown). In general, AMP possesses the highest E and DR It has a favorable TR and can be purchased at a relatively lower cost and higher purity. All these factors favor AMP as the best of the class of ribonucleotide reagents. For these reasons all further work was carried out with this electrolyte. Effect of Organic Modifier. Addition of organic modifiers to suppress the electroosmotic flow @OF) can lead to pronounced changes in migration time with occasional changes in migration order. A series of organic modfiers with increasing carbon number and amine functionalities, such as EDA, DETA, and 'IXTA, was investigated. The ability to suppress the EOF is found to follow the order EDA < DETA < TlX" Due to very weak adsorption of EDA, even at a concentration of 10 mM, little change in migration time of analyte anions is found (data not shown). The effect of DETA and TETA concentration on migration time is shown in Figure 3. Using negative polarity CE, the general trend toward shorter migration time with increasing concentration of DETA or TETA is based on the relative magnitude of electrophoretic and electroosmotic velocity vectors which oppose each other. At too high a concentration of organic modifer, poor resolution of P, and RP, (in particular, Prphosphate, HDTMP, ATMP, EDTMP, and DETPMP) is noted. The relative migration times are roughly 1.5 times higher for some RP, (HDTMP, ATMP, EDTMP, and DETPMP) when using the same concentration of DETA as compared to the TETA modifier. TETA provides shorter run times but at the expense of separating fewer RP,. It seemed that DETA is the best choice, maybe because of having three nitrogens instead of two or four, so the EOF inside the capillary is suppressed enough, but not excessively. A 2 mM DETA solution appears to provide optimum resolution with a reasonable migration time. Effect of pH on Detection Limits. Since both the electrophoretic mobility of phosphorus-containing anions and the EOF are influenced by pH, a pH range from 6.80 to 8.80 was studied. The four electropherograms shown in Figure 4 illustrate that both signal-to-noise (S/N) and migration time decrease as the pH is increased from 6.80 to 8.30. This modest decrease in migration time at higher electrolyte pH is due to an increase in EOF as well as in the electrophoretic mobility (due to the increase in the degree of ionization of P, and RP,,). Therefore, the overall mobility increase is not as much as one would have expected because the EOF and electrophoretic vectors oppose each other. The increase in the baseline noise at pH 8.30 may have been caused by the dilution of the AMP zones by the nonabsorbing hydroxide and borate ions. Excessive heating due to an increase in current from 6 pA at pH 6.80 to 13 pA at pH 8.30 results in greater mobility of the hydroxide ions, which may also contribute to an increase in baseline noise. At pH 28.80 (data not shown), the baseline becomes very unstable, and few analyte anions are detected. This is because the EOF which is in opposite direction to the electrophoretic velocity can no longer be suppressed at higher pH. Note that the cyclic-P,jand PI-fluorophosphate peaks (1and 2) are baseline resolved at pH 6.80. Increasing the pH to 7.30 decreases the resolution of this peak pair; however, the resolution of the ATMP and EDTMP peaks (10 and 11) is improved. DJ3TMP (peak 12) gives a stronger signal at pH 7.30 or 7.80 as compared to that at pH 6.80. Therefore, small changes in pH can affect the separation of closely migrating solutes as well as have
7
-+ Nitrate +- CYCLIC-PO
6 P,
+ P1-fluorophosphate
E5
+-
c 0 =4 Em
+ P1-phosphate
c
I
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Pl-hypophosphits
+ HEDP
3! >
-
--IC Cyclic-PG
L
+- P2-phosphate + HDThlP
$2
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+- ATMP
1
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4
0.25
EDTMP
4 DETPMP
mM DETA
II
0.5
I
I I I
1 mM TETA
I
I
2
I
4
Figure 3. Migration times relative to nitrate of 12 P, and RP, as a function of DETA and TETA concentration in a 5 mM AMP, 100 mM H3B03, 5 mM NazB407, pH 7.80 buffer.
10
-
pH = 7.30
;bH 6.80
5
,
4
9 1
I
I
,
,
,
6 8 10 12141S1820222426 283032 4
nrm (mlnutn)
f0 pH
-
6 8 10 1 2 1 4 1 6 1 8 2 0 2 2 2 4 1 2830 rime (mlnvhr)
pH = 8.30
7.80
Y
1 l 1 I I I l 1 1 1 1 1 1 4
6 8 10 1 2 1 4 1 6 1 8 0 2 2 2 4 2 6 28 nm* (minut-)
I l l 1 1 1 1 1 1 1 1 1 1 4
6 8 10 1 2 1 4 1 6 1 8 0 2 2 2 4 2 6 28 Time(mlnuc..)
Figure 4. Effect of electrolyte pH on the separation of 12 P, and RP,. The electrolyte was composed of 5 mM AMP, 2 mM DETA adjusted to various pH values with 1 mM NaOH. Vacuum injection for 15 s; other conditions as in Figure 2. Anion peak identification is the same as described in Figure 2.
some influence on detectability. The separation of P, and RP, was further studied by adjusting the pH of the AMP/H3B03 buffer with LiOH, NaOH, and KOH (data not shown). A comparison of these alkali metal hydroxides revealed that the lithium and sodium hydroxideadjusted buffer solutions gave equivalent resolution but that the potassium hydroxide-adjusted buffer led to markedly shorter migration times at the expense of very poor resolution and higher current. Table 3 compares the theoretical and experimentally determined detection limits of 12 P, and RPfianions at two different pH values. The AMP-analyte migration time differences ( A n
are compared with the transfer ratios W)expected on the basis of an equivalent-to-equivalentexchange between the electrolyte and the analyte. As discussed previously by Yeung, sensitive IPD requires a higher value of TRZ5However, the studies of NielenZ1 and Haddad et al.,24 and more recently the work in our laborahave presented more details about the factors influencing sensitivityin CE with IPD. One general consensus is that mobility matching between electrolyte and analyte is very crucial in obtaining a suitable equivalent-per-equivalentdisplacement and for maintaining a reasonable Gaussian peak shape. There can be situations in which a large TR value for some analyte is less important than a small AT value with respect to experimental detection limit. Several examples of this situation are shown in Table 3. At a pH of 7.80, Prphosphate, HDTMP, ATMP, EDTMP, and DETPMP have more favorable AT and TR values than PIphosphite, PI-phosphate,or PI-hypophosphite;the detection limits of the former group of analytes are 2-5 times better than the latter. However, at a lower pH of 6.80, the differencesin ATvalues and experimental detection limits between these two group of analytes are not great. A low pH decreases the contribution of hydroxide ion and improves the sensitivity but at the same time increases the ATvalues. Because of these two competing factors, the detection limits for PI-phosphite, PI-phosphate, and PIhypophosphite are improved, whereas the detection limit for DETPMP becomes worse. There is not much change in detectability of HDTMP, ATMP, and EDTMP at pH 6.80 compared to that at pH 7.80. HEDP has good detection limits at both pH values despite large AT differences. Our CE detection limits for P, and RP, of 0.84-11 pM (0.2-2 mg/L) are comparable to ion chromatographic methods. The H P X detection limits for RP, using FegII) or nitric acid eluents and direct UV or refractive index detection are reported to be in the 1.0-25 pM (2-15 mg/ L) range,10J1v26 whereas the detection limits of PI-P3-phosphates using direct UV detection range from 260 to 320 pM (45-80 mg/ L).l1vZ7Improved HPLC detection limits for P, of 0.42-5.8 pM (0.1-5.0 mg/L) using NTS with indirect UV or nonsuppressed conductivity detection have been recently published.14 However, (26) Wong, D.; Jandik, P.; Jones W. R; Hagenaars, A J. Chromatogr. 1987,389, 279-285. (27) Imnari, T.; Tanabe, S.;Toida, T.; Kawanishi, T. J. Chromatogr. 1982,250, 55-61.
Analytical Chemistry, Vol. 67,No. 1 7 , June 1, 1995
1849
Table 3. Comparison of Theoretical and Experimentally Determined Detection Limits at pH 6.80 and 7.80
effective charge Electrolyte Y-AMPR
6.80
7.80
-1.80
-2.0
APJ 6.80
7.80
+3.2 +3.0
TRd 6.80
7.80
calcd DL (x106 M)e 6.80
7.80
exptl DL (x lo6 M) 6.80
7.80
f2.8
2.0
+2.8 +2.6 +2.1 $1.8
0.84 2.4 2.5 3.1
10 4.2 8.3 8.4
Analyte
PI-fluorophosphate cyclic-P3 PI-phosphite PI-phosphate PI-hypophosphite HEDP (Dequest 2010) cyclic-Pc Pzphosphate HDTMP (Dequest 2054) ATMP (Dequest 2006) EDTMP (Dequest 2041) DETPMP (Dequest 2060)
-1.6 -1.3 -1.0 -2.4 -2.6 -3.3 -3.6 -2.6 -4.0
-1.9
+2.8
-1.8 -1.0
f2.3
-2.9 -3.0 -3.9 -3.9 -3.7 -5.1
$2.0 f1.2 +0.8 -0.5 -1.2 -1.6 -1.7 -3.0
0.86 0.70 0.55
+1.4 f1.4
1.3
-0.2
1.4
-0.1 -0.5 -0.9 -1.7
1.8 1.9 1.4
2.2
0.97 0.91 0.51 1.5
1.5 2.0 2.0 1.9 2.6
8.5
9.5 11 15 6.2
8.9 16 5.6 5.4 4.2 4.1 4.4 3.1
5.7 4.5 4.2 5.7 3.7
1.2 3.0 5.6 1.0 4.4 5.8
4.8
11 3.0 3.2 5.7
1.2 4.5 4.6 2.4
Calculated using the equation in ref 11. Measured with respect to nitrate. AT, relative migration time of AMP - relative migration time of analyte. dTR,effective charge analyte/effective charge AMP, ref 24. e Calculated using the Cjim equation in Table 2. the untreated fused silica capillary requires less maintenance than the polymeric HPLC columns required for the separation of these types of polyvalent compounds. Effect of Inorganic Modifier. The above separation schemes with negative polarity CE using a ribonucleotide/DETMH~B03 electrolyte system permit baseline separation of all RP,. Unfortunately, among the P, series, only PI- and Pzphosphates could be detected; all of the longer chain P, with lower charge-to-mass ratios move much more slowly toward the detector end (Le., anodic end used in negative polarity CE). For instance, elution of P3-phosphate takes at least 120 min with poor efficiency. To obviate the dif&iculties brought about by slow migration of longer chain P,, we explored an alternative separation scheme which employs positive polarity CE and Mget as an inorganic modifier in the AMP running buffer. Yoza et al. have demonstrated that Mgzc could be employed as a complexing agent in P,-Mg2+ binding studies; their indirect flow injection analysis method is based on the substitution reaction between P, and the Mg2+methylthymol blue complex.6 Chen and Pietrzyk reported the role of Mgz+in improving the CE resolution of both aliphatic and aromatic sulfonates and sulfates.22 For our study, developmental experiments were done to examine whether or not complexation occurs betweeen Mgz+ and AMP. If complexation occurs, one would expect a decrease in migration time of AMP because of charge neutralization between Mg2+and AMP. The increase in migration time of AMP from 6 to 22 min in the presence of 2 mM Mg2+suggested that no signiticant complexation between Mgz+ and AMP is taking place. The electrostatic attraction between the negatively charged silanol group and Mg2+may decrease the EOF, and therefore AMP migrates more slowly in the capillary. Figure 5a,b compares the separation of P1-P4-phosphates in the absence and presence of Mg2+. In the absence of Mgz+,only PI-phosphate could be swept by the EOF toward the detector. When the same P, mixture is injected into a Mgz+-AMP electrolyte system, selectivity of separation improves because P, reacts with Mg2+to form less anionic Mgz+-P, complexes, which results in a decrease in mobility of the P, toward the anode (Le., injection end used in positive polarity CE). However, the difference in migration times of Pp and P4-phosphate is narrow, and these could not be separated. Figure 5c represents the electropherogram obtained by addition of 0.2 mM DETA to the Mg2+-AMP 1850 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
WIi
.I 0
Tim
(minutes)
Figure 5. Separation of a standard mixture of P,. The electrolye was composed of 5 mM AMP, 100 mM H3603 adjusted to pH 7.10 with 1 mM NaOH, 35 "C:(a) no Mg", (b) 2 mM MgZc,and (c) 2 mM Mg2+with 0.2 mM DETA. Peak identification (mg/L, mmol/L): 1, PIphosphate (110, 1 . l ) ; 2, P2-phosphate (70, 0.40); 3, PS-phosphate (70, 0.28); and 4, P4-phosphate (70, 2.0). Vacuum injection for 1.5 s, +20 kV applied for separation; current, 11 PA. IPD at 259 nm.
electrolyte. The addition of DETA causes the separation window to become wider because the EOF is hindered through aminesilanol interactions. It is readily apparent that the addition of a small amount of DETA improves the resolution of the P3- and P4phosphates. The migration time of P, therefore increases in the order P2 < P3 < P4, which is consistent with an increase in their negative charge and stability constants with the Mg2+ cation.6 However, the migration order of PI-phosphate cannot be compared
.
Nl
2
4
6
I
10 12 14 16 18 Tlnw (minutes)
73
22
24
26
.
.
.
.
.
28 30
2
0.0
Nl
2
4
6
8
1012
14
1618
M
2
4
6
8
10
Time (minuter)
Figure 6. Analysis of (a) -0.20 9/50 mL of Crest toothpaste containing 1, sulfate;2, fluoride; 3, bicarbonate;and 4, P2-phosphate; (b) -0.4 9/50 mL of Lever 2000 soap containing 1, PI-phosphate;2, EDTA; and 3, HEDP; and (c) -0.15 9/50 mL of Topol Plus toothpaste containing 1, chloride; 2, PI-fluorophosphate; 3, fluoride; 4, PIphosphate. Vacuum injection for 1.O S for (a) and 0.5 S for (b) and (c),with -30 kV applied for separation; current, 6yA. The electrolyte was composed of 5 mM AMP, 2 mM DETA, 100 mM H3B03, pH 6.80. IPD at 259 nm.
with those of other P, (i.e., zPrphosphate) due to the low complexing ability of PI-phosphate, although the exact stability constant of the Mgz+- PI-phosphate complex is not known2*It was also found that increasing the Mg2+concentration from 0.5 to 2 mM decreases the migration time of P2-P4-phosphates and increases the migration time of PI-phosphate (data not shown). Applications. In view of the capabilities of the various buffer systems shown above for the CE separation and IPD of P, and RP,, it seemed desirable to complete this study by examining some realistic samples containing these polyvalent compounds. We selected two different analysis situations: the qualitative identification of some active P, and W, in soap and various brands of toothpaste, and the quantitation of glyphosate (GLYP) and aminomethylphosphonic acid (AMPA) in a herbicide. Figure 6a shows an electropherogramof Crest toothpaste. PZ Phosphate (peak 41, appearing around 24 min, is present as an active ingredient. Other anions noted, such as sulfate (peak l), fluoride (peak 2), and bicarbonate (peak 3), were confirmed by spiking the sample with their standard anion solutions. The peak at 28 min was not identified. Although a much faster separation of Pzphosphate can be obtained with positive polarity CE and AMP with Mga+,we chose to work with negative polarity CE conditions, ~~~~~~~~
(28) Sillen, L. G.; Martell, A. E. Stability Constants of Metal Ion Complexes; Special Publications 17 and 25; The Chemical Society: London, 1964 and 1971.
T i m (minutes)
10.0
Figure 7. Analysis of Roundup herbicide solution. The sample contained 1 mL of the commercial solution dissolved in few drops of methanol and diluted to 100 mL with triply deionized water. The electrolyte was composed of 5 mM AMP, 100 mM H3B03, pH adjusted to 7.10 with 1 mM NaOH. Vacuum injection for 1.5 s,f 3 0 kV applied for separation; current, 6yA. IPD at 259 nm. Inset: peaks for AMPA and GLYP at their limit of detection.
as this configuration can separate other mobile anions such as fluoride and sulfate along with Prphosphate in the same run. The analysis of PI-phosphate (peak l), EDTA (peak 2), and HEDP (peak 3) in a commercial soap (Lever 2000) is shown in Figure 6b. A slight shift in migration time of PI-phosphate and HEDP in Figure 6b as compared to their standard solutions (see Figure 4, separation at pH 6.80) is attributed to the high ionic strength of the sample. P#luorophosphate (monofluorophosphate) is widely used in commercial dentifrices as a fluorine donor.29 It is well established that in aqueous solution it can hydrolyze to fluoride and P~-phosphate.~~ Figure 6c shows the separation of the reactant (P1-fluorophosphate,peak 2) and products (fluoride, peak 3, and PI-phosphate, peak 4) in Topol Plus toothpaste. A sensitive detector setting was used in order to confirm the presence of minor components such as EDTA in Figure 6b and PI-phosphate in Figure 6c. No sample preparation other than dilution and filtration was required for any of the samples. Roundup, a well-known herbicide that has found widespread use in agriculture, is the trade name for a 0.96%GLYP solution prepared from the isopropylamine salt. AMPA is the major metabolite of GLYP in water, plants, and soil. Several separation methods have been employed for both GLYP and AMPA including gas chromatography,3l HPLC with various post-32s33or precolum1-1~~8~~ derivatizing reagents, and more recently CE after pred(29) Gron, P.; Encsson, Y. Curies Res. 1983,17 (Suppl. l ) , 1-136.
(30) Yoza, N.; Nakashima, S.; Nakazato, T.; Ueda, N.; Kodama, H. Anal. Chem. 1992,64, 1499-1501. (31) Deyrup, C. L.;Chang, S. M.; Weintraub, R A; Moye, H. A]. Agric. Food. Chem. 1985, 33, 944-947. (32) Cowell, J. E.; Kunstrnan, J. L.; Nord, P. J.; Steinmetz, J. R; Wilson, G. R]. Agric. Food. Chem. 1986,34, 955-960.
Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
1851
erivatization with p-toluenesulfonyl chloride.36 While this latter method provided a detection limit of 0.1 mg/L, analyte derivatization before separation will add to the total analysis time. CE with the AMP electrolyte and IPD can also provide comparable detection limits for these type of aliphatic herbicides with the added advantage of high sample throughput compared to the derivatization methods. The electropherogram of Roundup herbicide is shown in Figure 7 . The sample was diluted 1Wfold with water and analyzed with positive polarity CE. The less anionic AMPA has a lower mobility than GLYP toward the anode (injection end) and can be detected first at the cathode (detector end) due to EOF. The inset in Figure 7 shows the electropherogram of AMPA and GLYP at their detection limits of 0.2 and 0.1 mg/L, respectively, for S/N 2 2-3:l. Although negative polarity CE can provide even better detection limits due to oncolumn stacking of GLYP, the method cannot be used to determine both GLYP and AMPA due to the slow migration of AMPA toward the anode (detector end contiguration used in reverse polarity CE). The results of a study to assess the quantitation and regression parameters for GLYP and AMPA are shown in Table 4. Calibration curves for both compounds were linear over the lower (1(33) Cowell,J. E.; Oppenhuizen, M. E.J Assoc. Ojf Anal. Chem. 1991,74,317323. (34) Kawi, S.; Uno, B.: Tomita, M.J. Chromatogr. 1991,540, 411-415. (35) Tomita, M.; Okuyama, T.;Watanabe, S.; Uno, B.; Kawai, S. J Chromatogr. 1991,566, 239-243. (36) Tomita, M.; Okuyama, T.; Nigo,Y.: Uno, B.; Kawai, S. J. Chromatogr. 1991, 571, 324-330.
1852 Analytical Chemisfry, Vol. 67, No. 1 7 , June 1, 7995
Table 4. Quantitative Analysis of Glyphosate (GLYP) and Aminomethylphosphonic acid (AMPA)
AMPA
GLYP calibration params linearity range (mg/L) peak area RSD (%) slope (V-L/mg) intercept (V) corr coeff (12)
20-200
1-20
(n
= 5)
kl.2 8.8 x 10-3 4.4 10-3 0.9984
(n = 6)
1-20
(n
20-200
(n = 6) kO.9 51.7 f1.9 5.5 x 4.5 x 4.6 x 2.4 x 10-3 2.0 10-4 5.6 x 10-3 0.9994 0.9999 0.9996 = 5)
20 mg/L) and the higher (20-200 mg/L) concentration ranges and passed close to the origin, as evident from the y-intercepts. Peak area reproducibility (n = 3) for the calibration curves was between 0.9 and 1.9%. The value (68.7 mg/L, RSD = 1.4, n = 5) obtained for GLYP content in Roundup is in good agreement ( ~ 2 % relative error) with the certified value (69.8 mg/L) claimed on the label. ACKNOWLEDGMENT Purchase of the AB1 CE instrumentwas possible through funds granted by the Miami University Committee for Faculty Research and the Academic Challenge Program. Received for review November 23, 1994. Accepted March 21, 1995.@ AC941126F e Abstract published
in Advance ACS Abstracts, May 1, 1995.
