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Anal. Chem. 1986,58, 1380-1383
Liquid Chromatographic Determination of Guanidines with an Anion Exchange Column Used Simultaneously as Separator and Postcolumn Reagent Generator Hans Jansen, Elisabeth G . van der Velde, Udo A. Th. Brinkman, Roland W. Frei,* and Hans Veening*' Department of Analytical Chemistry, Free University, De Boelelaan 1083,1081 HV Amsterdam, The Netherlands
An anlon exchange llquld chromatographicprocedure for the determination of guanidine, methylguanidine, arglnlne, guanMinobutyric acld, guanidinopropionlcacid, and guanidlnoacetic acld wlth postcolumn derivatization and fluorescence detection ts reported. The anion exchange column is used in the hydroxlde form and serves simultaneously for the separation of the components and for the generation of hydroxide needed for postcolumn derivatization with phenanthrenequinone. This method was found to be reproducible with mlnlmum detection ilmlts ranglng from 3 to 40 ng and was successfully applied to spiked serum and hemodialysate samples. The potential for automated column regeneratlon wlth hydroxlde was demonstrated.
Elevated concentrations of guanidine compounds in blood serum and hemodialysate of uremic patients have been used for some time as an indicator of renal dysfunction (1-5).The availability of a rapid and sensitive method for the determination of these compounds in biofluids is, therefore, useful in clinical assay procedures. In past years, analytical methods for guanidine compounds have included classical column chromatography, paper chromatography, and gas chromatography as well as colorimetric and fluorometric procedures. The disadvantages of most of these methods are that relatively large sample sizes are needed and the detection limits are unacceptably high. More recently, high-performance liquid chromatography (HPLC) has become the method of choice for guanidine compounds. The first HPLC procedures using cation exchange and postcolumn derivatization with phenanthrenequinone (PQ) were published by Yamamoto et al. (6,7). Hiraga and Kinoshita used a cation exchange procedure to separate guanidine compounds with ninhydrin as a postcolumn reagent, followed by fluorescence detection (8).Baker et al. separated guanidine compounds by ion-pairing reversed-phase HPLC using on-line postcolumn derivatization with PQ and fluorescence detection (5). Kai et al. developed an HPLC method for guanidines using precolumn derivatization with benzoin followed by separation on a reversedphase column and fluorescence detection (9,lO).Hung et al. recently reported an automatic HPLC analyzer for guanidines using cation exchange and derivatization with benzoin (11). In this paper we report an ion exchange HPLC procedure for several guanidine compounds using postcolumn derivatization with PQ and fluorescencedetection. The novel feature of this method is the dual use of the anion exchange column, which serves simultaneously for the separation of the components and for the generation of one of the reagents (hydroxide ion) needed for successful postcolumn derivatization of guanidines with PQ. EXPERIMENTAL SECTION Apparatus. A diagram of the chromatographic system is shown in Figure 1. The mobile phase pump (Pl) was a homePermanent address: Department of Chemistry, Bucknell University, Lewisburg, PA 17837.
constructed syringe pump of 50 mL capacity; the column regeneration pump (P2) was an Altex Series 100 (Altex, Berkeley, CA) pump. Samples were injected into the column with either a 10 or 100-pL sample loop injector. The analytical column was constructed of stainless steel (100 X 4.6 mm i.d.) and was packed with the quaternary ammonium anion exchange resin, Aminex A-28 (particle diameter, 11pm; BioRad Laboratories, Richmond, CA) in the acetate form. The postcolumn reagent pump (P3) was a Model PCR-I (Varian Aerograph,Walnut Creek, CA) pump. The reaction coil was a PTFE knitted open tubular reactor (0.3 mm i.d.1 with an internal volume of 1 mL (12); the coil was heated to 70 "C using a constant-temperature water bath. The effluent from the reactor was passed through a Model 204 A fluorescence detector (Perkin-Elmer,Norwalk, CT) operated at, X = 375 nm and ,A = 460 nm. All chromatograms were recorded on a Kipp BD 40 recorder (Kipp & Zonen, Delft, The Netherlands). Preand postcolumn T-connectors were Valco 0.5- and 0.25-mm fittings, respectively. Reagents and Chemicals. Guanidine (G), methylguanidine (MG), arginine (ARG), guanidinobutyric acid (GBA), guanidinopropionic acid (GPA), guanidinoacetic acid (GAA),guanidinosuccinic acid (GSA), and "gold" label (99%) phenanthrenequinone (PQ) were obtained from Aldrich Chemicals (Milwaukee, WI). Taurocyamine (TC) was prepared as previously described (13). Reagent grade dimethylformamide(DMF)and HPLC grade methanol were obtained from J. T. Baker Chemicals (Deventer, The Netherlands). HPLC grade water was produced by a Milli-Q reagent water system (MilliporeCorp., Bedford, MA). All other chemicals were of reagent grade quality. Samples. Samples of normal serum, used in spiking experiments, were obtained from a healthy, male individual. Serum samples were used without any further treatment, except that during serum analysis, a homemade 10 X 2 mm i.d. precolumn, packed with Aminex A-28, was incorporated between the injector and the analytical column, in order to protect the latter. Hemodialysate solutions were collected from the overflow outlet of the dialyzer immediately after the first hour of dialysis treatment. All samples were stored at -30 "C until needed. Chromatographic Procedure. For packing the column, a slurry of ca. 2 g of Aminex A-28 in 10 mL of a mixture of 90% 25 mM aqueous acetic acid (adjusted to pH 7.0 with sodium hydroxide)and 10% methanol was prepared. The same solution was used as the packing solvent and as the mobile phase. During the packing procedure, the pressure slowly increased from 0 to 250 bar. Prior to use, the column was eluted at 1.0 mL/min with 10 mL of 0.5 M NaOH-methanol(9010,v/v) in order to convert the resin to the hydroxide form. This was followed by pumping 10 mL of mobile phase through the column at 1.0 mL/min. The column was regenerated twice a day using this procedure after 50 mL of mobile phase had been used during analyses. The regeneration procedure was carried out with pump 2 and with the waste outlet open (Figure 1). During chromatography, pump 1 was used to pump the mobile phase through the column at 0.26 mL/min with the waste outlet closed. Pump 3 (Figure 1) supplied the postcolumn derivatization reagent (2.5mM PQ in DMF) at a flow rate of 0.43 mL/min. The high pH (ca. 11)necessary for derivatization of the guanidines with PQ was achieved by the displaced hydroxide ions eluting from the column during the separation. The mixed column effluent and reagent was then passed through the reaction coil (70 OC). The delay time in the 1-mL coil was ca. 1.5 min.
0003-2700/86/0358-1380$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
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Y
0
52
s1
‘E -
-AEC
E 16-
Y
J
h
u
-
;1 4 0
0
8 -L
-WA
P3
-
12-
10-
53
0 20 40 60 80 column elution tirne(min)-
t
Flgure 1. Schematic diagram of the chromatographic system: S1, mobile phase supply; S2, column regeneration solvent supply; S3, postcolumn reagent supply; P1, moblie phase pump; P2, column regeneration pump; P3, postcolumn reagent pump; I V , injection valve; AEC, anion exchange column; WA, waste outlet (during regeneration); RC, reaction coil (70 OC); FD, fluorescence detector; R, recorder. Guanidines
-
A-2&MEOH
Figure 3. Decrease of total residence times for ARG, GBA, GPA, and GAA during column elution: t o = 3.5 min. Column elution time refers to the duration of column use after regeneration with scdlum hydroxide.
Table 11. Quantitative Reproducibility and Minimum Detection Limits for Several Guanidines min no. of av peak coeff of amt injected, samples height, variation, detection % limit, np“ analyzed cm compd ng
G MG ARG
365 361
GBA
240 196 380
GPA GAA
228
5
6 6 6 4 3
49.0 27.0 4.0 2.7 5.9 4.7
1.7 1.7 2.9 4.3 5.9 7.7
3 4 22 25 10
40
aBased on a signal-to-noise ratio of 3:l.
-
time (mln)
Flgure 2. Chromatogam for the separation of five guanidines: cdumn, 100 X 4.6 mm i.d. packed with Aminex A-28; mobile phase, 25 mM aqueous acetate/methanol (90: lo), pH 7.0; flow rate, 0.26 mL/min; sample size, 10 pL; detection, fluorescence, A,, = 375 nm, A,, = 460 nm.
Table I. Peak Broadening in the Chromatographic System ut, 8
compd
reaction coil
column
G MG ARG GBA GPA GAA
8
8 10 13 15
19 35
Wi a
total
totaf, s
11
24 34 36 38 48 84
13 15 17 21 36
RESULTS AND DISCUSSION Peak Broadening. The retention times of the guanidines on the ion exchange column varied from 1.5 min for G and MG to 23 min for GAA. Delay time due to the remainder of the chromatograpic system (reaction coil, T-connectors, capillary tubing, and detector cell) was ca. 2 min. The contributions to peak broadening (a3 due to the reaction coil and the column are reported in Table I. These values were found to be independent of the progress of change in the acetatehydroxide form of the column during elution. Also shown in Table I are peak broadening data (atand W,,,) due to the entire system. Separations. Figure 2 shows a chromatogram for the separation of G, ARG, GBA, GPA, and GAA. G and MG are unretained since both are positively charged under the con-
ditions used. For G and MG, the pK, values are 13.4 and 13.6, respectively (14).The other guanidines,however, are retained since they are negatively charged. The pK, values are 3.5, 3.8, and 2.86 for GPA, GBA, and GAA, respectively (5,15). During elution of the column with acetate eluent, the ion exchange resin gradually changes from the hydroxide to the acetate form. This process is accompanied by a constant decrease in retention times of the components, but peak broadening and peak heights were found to remain unchanged. This is surprising, since one would expect peak widths to decrease and peak heights to increase when retention times on the column decrease. It is possible that the column efficiency (plate number) decreases during the change from the hydroxide to the acetate form. The change in retention as a function of “column elution time” is proportionally equivalent for four guanidines tested (ARG, GBA, GPA, and GAA) and is shown in Figure 3 (“columnelution time” refers to the duration of column use after regeneration with sodium hydroxide). QuantitativeReproducibility, Linear Range, and Detection Limits. Table I1 lists the coefficients of variation for measured peak heights of successive standard mixtures of guanidines. These data were obtained during one “cycle”; Le., the column was not regenerated during the measurements. Peak heights were found to be randomly scattered around a mean value; i.e., no trends (constaht increase in peak height due to decreasing retention) were observed. Such trends would have been expected, however, as a result of conversion from hydroxide to acetate. It can be seen that the reproducibility tends to decrease as the retention times of the components increase. The linear dynamic ranges of the six guanidine compounds investigated were found to extend from the detection limit to l order of magnitude for GAA and 2.5 orders
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1988 GUANIDINES
MG
II
SPIKED SERUM ( E )
L SERUM ( A )
0
8
16
24
time (min)
Figure 4. (A) Chromatogram for 100 HL of normal serum. (B) Chromatogram for 100 pL of normal serum spiked with 288 ng of MG, 390 ng of GPA, 478 ng of GBA, and 352 ng of GAA. Conditions are the same as in Figure 2.
of magnitude for GPA, with regression coefficients of at least 0.995. The minimum detection limits ranged from 3 to 40 ng injected for G and GAA, respectively (Table 11). The calibration plot for GAA was found to have the smallest slope and, consequently,the highest minimum detection limit. The cause for this phenomenon is the high peak broadening value for GAA (see Table I). Other guanidine compounds (GSA and TC) could not be detected by this chromatographic system, apparently because of their permanent retention on the column under the condition used. This observation is consistent with the fact that the published carboxylic acid pKal and pK,, values for GSA are 2.65 and 4.26, respectively (16).Also, TC is a strong acid due to its sulfonate side chain; thus, both GSA and TC are negatively charged at pH levels above 3. Each of these two compounds was, however, easily detectable when the injector was placed after the column, the latter now serving solely as a source for hydroxide ion. Creatinine was very difficult to detect using the present system (the detection limit was ca. 1700 ng), although it was found to elute from the column, as supported by its pK, value of 4.87 (16). The cause of this problem is probably that a higher pH is necessary in order to achieve successful derivatization with PQ. Serum and Hemodialysate Samples. The method was applied to several normal serum and blank hemodialysate samples. In each case, a hemodialysate or serum sample (obtained from a healthy individual) was injected and a chromatogram was recorded. Known quantities of guanidine standards were then added to each sample prior to injection into the column. A typical set of results for a blank serum and a spiked serum sample are shown in Figure 4. It can be seen that ARG, which is always present in healthy serum, produces the only peak (as expected)in the blank sample. The standard addition of MG, GPA, GBA, and GAA to the serum sample resulted in peaks at the expected retention times. When injecting hemodialysate samples, no peaks were found in the chromatogram. The chromatogram obtained after spiking was identical with that obtained upon injection of a test mixture of guanidines (cf. Figure 2).
Potential for Automated Column Regeneration. It was found that the ion exchange column could be regenerated by injecting 2 mL of a solution consisting of 0.5 M NaOHmethanol (9O:lO) from a separate injection loop between analytical runs. Such a procedure eliminates the need for pump 2 (Figure 1)and has an obvious potential for automation. It would thus become possible to incorporate a flow switching device and two injection loops, one for the sample and one for the 2-mL NaOH plug. In an automated procedure, the latter would follow each separation, thus regenerating the column after each analytical run. This would result in constant retention times, since the time between regeneration and elution from the column would remain constant for each compound, and will also have a positive influence on the precision of the total analytical procedure. CONCLUSION It has been demonstrated that an anion exchange column can be used to separate guanidine compounds and simultaneously serve as a reagent (hydroxide) generator for postcolumn derivatization with PQ. The method is reproducible with minimum detection limits ranging from 3 to 40 ng. The order of elution of the guanidines is related to their pK, values; i.e., those components with the higher pKa values elute first. A disadvantage is the fact that negatively charged components, such as GSA and TC, cannot be determined under the conditions used, since they are apparently retained indefinitely on the anion exchange resin. Two components, G and MG, are not retained by the column because neither contains a carboxylic acid functional group. One of the advantages of the method is the elimination of the need for a sodium hydroxide postcolumn pump, as used previously (5, 111,since the column automatically generates this reagent. Thus, deterioration and damage to the pump due to concentrated caustic solutions is also eliminated. The pump that is needed for the regeneration procedure does not have to meet requirements such as pulse-free operation and high flow rate stability since it is not in use during analysis. Consequently, almost any high-pressure pump will be satisfactory. Additionally, the regeneration solvent is not as corrossive as the caustic solutions mentioned above, and flushing the pump with water after regeneration will further diminish the possibility of pump damage. As a result, the regeneration procedure in this method causes little if any wear and tear on the pumping mechanism. As a result of the shifting equilibrium retention conditions, the retention time of each of the solutes decreases with increasing column elution time. This can be seen as a disadvantage; however, surprisingly, peak heights and peak widths are not influenced so that quantitative determination of the guanidines remains possible. An alternative method of column regeneration could consist of repetitive injection of plugs of base between analytical runs. The regeneration pump would not be needed in this case, and retention times will be constant. This method has an obvious potential for automation and should be especially useful for routine analysis. The present method was found to yield satisfactory results with spiked serum and hemodialysate samples.
ACKNOWLEDGMENT Serum and hemodialysate samples were provided by the Free University Academic Hospital. Registry No. G, 113-00-8;MG, 471-29-4;ARG, 74-79-3;GBA, 463-00-3; GPA, 353-09-3; GAA, 352-97-6; PQ, 84-11-7. LITERATURE CITED (1) Sasakl, M.; Takahara, K.;Natelson, S. Ciin. Cbem. (Winston-Salem, N . C . ) 1973, 79, 315-321. (2) Baker, L. R. I.; Marshall, R. D. Ciin. Sci. 1071, 4 7 , 563-568.
Anal. Chem. 1986, 58, 1383-1389 (3) Sawynok, J.; Dawborn, J. K. Clin. Exp. Pharmacol. Physiol. 1975, 2 , 1-15. (4) Lazdlns, 1.; Dawborn. J. K. Clin. Exp. Pharmacol. Physlol. 1978, 5 , 75-80. ( 5 ) Baker. M. D.; Mohammed, H. Y.; Veening, H. Anal. Chem. 1981, 53, 1858- 1862. (6) Yamamoto, Y.; Saito, A.; Manjl, T.; Maeda, K.; Ohta, K. J . Chromatogr. 1979, 162, 23-29. (7) Yamamoto, Y.; ManJI, T.; Salto, A.; Maeda, K.; Ohta, K. J. Chromafogr. 1979, 162, 327-340. (8) Hlraga, Y.; Kinoshlta, T. J. Chromafogr. 1981, 226, 43-51. (9) Kai, M.; Mlyazaki. T.; Yamaguchi, M.; Ohkura, Y. J . Chromafogr. 1983, 268, 417-424. (10) Kai, M.; Miyazaki, T.; Ohkura, Y.; J. Chromafogr. 1984, 311, 257-266. (11) Hung, Y-L.; Kal, M.; Nohta, H.; Ohkura, Y. J. Chromatogr. 1984, 305, 28 1-289.
(12) (13) (14) (15) (16)
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Engelhardt, H.; Neue, U. D. Chromatographia 1982, 15, 403-408. Mori, A.; Hosotani, M.; Choong, T. L. Blochem. M e d . 1974, 10, 8-14. Angyal, S. J.; Warburton, W. K. J. Chem. SOC. 1951, 2492-2494. Failey, C. F.; Brand, E. J. Biol. Chem. 1933, 102, 767-771. Ratner, S.; Petrack, B.;Rochovansky, 0. J. Biol. Chem. 1953, 204, 95-113.
RECEIVED for review September 17,1985. Accepted January 15,1986. H.V. thanks the National Science Foundation (Grant INT-8317607)and Bucknell University for financial support while on sabbatical leave in The Netherlands. This work was supported by the Dutch Foundation for Technical Sciences, Grant VCH 33.0436.
Simultaneous Determination of Arsenic( I 11) and Arsenic(V) in Metallurgical Processing Media by Ion Chromatography with Electrochemical and Conductivity Detectors Liang K. Tan* and John E. Dutrizac
Mineral Sciences Laboratories, CANMET, Energy, Mines and Resources Canada, 555 Booth Street, Ottawa, Ontario K I A OG1, Canada
As( II I ) and As(V) In metallurglcai processlng solutions are determlned slmultaneously by Ion chromatography with electrochemlcal and conductivlty detectors, respectlvely. The effects of acld, base, or alkall salts on the ion chromatograms obtained from low concentratlons of As( I 1I ) are descrlbed. Cations such as Co(II), Cu(II), Fe(I1 and HI), and NI(I1) must be preseparated wlth an H+ cation exchanger to avoid precipltatlon of arsenlc wlth the metal hydroxides formed during dllutlon. Na+ or K+ In the sample must be preseparated slmllarly because of thelr effects on the current response of the arsenlte. The effect of H2S04on the current sensltlvlty of the arsenlte was determlned. The detectlon llmlts for arsenlte In 0 and 5 mM H,S04 are 0.005 and 0.025 mg/L As( III),respectlvely. The detectlon llmit for arsenate is 0.022 mg/L As(V). As(III)/As(V) ratlos as low as 0.025 can be determlned quantitatively.
The determination of As(II1) and As(V) in ferric chloride-hydrochloric acid leaching media (1)and in ferric sulfate-sulfuric acid processing solutions (2) by ion chromatography (IC) with conductivity detection was reported previously. Although arsenite and arsenate ions can be separated by the IC column, arsenite is not detected by the conductivity detector because of the low dissociation constant of arsenous acid (3). In the above studies, therefore, As(II1) was determined by difference following oxidation of the test solution with aqua regia ( I , 2). This method was useful for moderately high As(III)/As(V) ratios and had an accuracy of 5-10% at the 0.2-10 mg/L level. Although such accuracies are acceptable for most metallurgical applications, many processing solutions contain low As(II1) concentrations relative to the total arsenic concentration. In such solutions, it is impossible to determine As(II1) accurately by difference. Kinetic studies aimed a t examining the reduction of As(V) also require accurate As(II1) concentrations at low levels. A sensitive and 0003-2700/86/0358-1383$01 SO/O
direct method of measurement of As(III), such as by electrochemical detection, would be desirable and, ideally, such methods should also permit simultaneous measurement of As(V). The measurement of arsenic(II1) by polarography is wellknown, and the complex polarographic behavior of As(II1) in acidic, neutral, and alkaline media has been reviewed (4). A polarographic detector for flowing liquid has been investigated ( 5 , 6 ) . Although the dropping mercury electrode provides a constantly renewed surface, it presents several disadvantages as a detector for liquid chromatography that include oscillation of the measured current due to the growth and fall of the mercury drop and a complicated cell design. Recent studies have indicated that solid electrodes (Pt, Au, glassy carbon) for flowing liquid generally have greater sensitivity and simpler cell design than dropping mercury electrodes (5-9). Anodic detection of arsenic(II1) in a flow-through platinum-wire electrode for flow-injection analysis has also been reported (10, 11). The arsenic(V) concentration in the sample was, however, obtained by difference after the reduction of arsenic(V) to arsenic(II1) with hydrazine sulfate (11). The present study reports a method for the simultaneous determination of As(II1) and As(V) by IC with two detectors. After the separation of arsenite and arsenate by the IC separator column, the effluent containing the analytes passes through a flow-through electrochemical cell with a platinum working electrode which measures the arsenite concentration. It then flows through a suppressor column and finally passes through a conductivity cell which measures the arsenate concentration. The ion chromatograms resulting from the conductivity detection of arsenate-containing solutions have been documented previously (1-3). The ion chromatograms from the electrochemical detection of arsenite-containing solutions, however, have not been reported in the literature. Therefore, the purpose of the present work is to discuss the various ion chromatograms of arsenite in solutions containing acid, base or alkali salts, and to develop an analytical method
Published 1986 by the Amerlcan Chemical Society
