2215
Anal. Chem. 1985, 57, 2215-2219 (23) Burchill, P.; Herod, A. A,; Prichard, 271-295.
(19) Wright, E. W.; Peaden, P. A.; Lee, M. L.; Stark, T. J. Chromatogr. lQ82, 248, 17-34. 120) . , Later. D. W. I n "Handbook of Polvcvclic Aromatic Hydrocarbons": Bjorseth, A,, Ramdahi, T. Eds.; Mar% Dekker: New York, Vol. 2, in press. (21) M e r , D. W.; Lee, M. L.; Wilson, B. W. Anal. Chem. l Q W 5 4 , 117-123. (22) Later, D. W.; Andros, T. G.; Lee, M. L. Anal. Chem. IQ83, 55, 2126-2132.
E. J.
Chromatogr. lQ82, 246,
RECEIVED for review April 22, 1985. Accepted June 5, 1985. This work was supported by the Department of Energy, Office Research, 'Ontract No* DEOf and AC02-79EV10237.
Resolution of Ortho, Meta, and Para Isomers of Some Disubstituted Benzene Derivatives via CY- and P-Cyclodextrin Inclusion Complexes, Using Reversed-Phase High-Performance Liquid Chromatography J a n u s z Zukowski, D a n u t a Sybilska,* a n d J a n u s z J u r c z a k Institute of Physical Chemistry a n d Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44, 01-224 Warsaw, Poland
Separatlon of varlous dlsubstltuted benzene derlvatlves on a LiChrosorb RP18 column, wlth moblle phases contalnlng aor b-cyclodextrln, was systematlcally studled. The model compounds tested were cresols, fluoronitrobenzenes, chloronltrobenrenes, bromonltrobenzenes, lodonltrobenzenes, nltroanlllnes, nitrophenols, and dinitrobenzenes. Ethanol, 20 vol %, was used as an addltlonal component of the aqueous moblle-phase solutlon. The course of the relatlonshlp of the apparent capacity factor vs. cyclodextrln (CD) concentratlon was conslstent wlth that resultlng from slmple theoretlcal consideratlons only In the case of b-CD. This permitted evaluatlon of the capacity factors of P-CD complexes, as well as of thelr stablllty constants. I t was found that only 6-CD Imposes a dlstlnct selectlvlty toward ortho, meta, and para Isomers on RP systems, thus enabllng thelr complete separation In all cases studled.
Cyclodextrins (CDs) are torus-shaped cyclic oligosaccharides made up of six or more a-1,Clinked D-glucopyranose units. The nonpolar central cavity of CDs can selectively include various inorganic and/or organic species of neutral or ionic nature (1). These inclusion processes are influenced mainly by hydrophobicity and shape of guest molecules, Le., by the fit of the complexed molecule to the CD cavity. Hence CD complexation is a procedure of choice for separation of isomers. It has been used to advantage in classical liquid chromatography (2, 3 ) . However, the columns usually containing polymers with incorporated CD molecules are of very low efficiency, owing to the complex mechanism of sorption involving both gel permeation and molecular inclusion. CDs dissolved in the mobile phase have also been used in thin-layer chromatography with polyamide stationary phase ( 4 , 5 ) . The CD inclusion phenomena have recently been utilized for separation of disubstituted benzene isomers by highperformance liquid chromatography (HPLC) in two ways: by using chemically bonded a- and/or P-CD-silica stationary phases (6-10) and by applying CDs as the mobile phase components in reversed-phase systems (11, 12). Studies dealing with the latter method have concerned only three nitrobenzoic (11)and three nitrocinnamic acids (12). The present paper reports the results of further systematic 0003-2700/85/0357-2215$01.50/0
research on resolution of various disubstituted benzene derivatives of acidic, basic, or neutral nature. The work was aimed at establishing the general rules similar to those valid in clathrate chromatography (13,14)which relate the shape and size of molecules to their chromatographic behavior in reversed-phase systems containing a- or 6-CD in the mobile-phase solution. EXPERIMENTAL SECTION Reagents. a-and p-CD were supplied by Chinoin (Budapest, Hungary) and were purified by recrystallization from water. Methanol (MeOH), ethanol (EtOH), 1-propanol (1-PrOH), 2propanol (2-PrOH),and tetrahydrofuran (THF) were of p.a. grade. Water was purified by double distillation and ion exchange treatment. The following ortho-, meta-, and para-disubstituted benzene derivatives were used as solutes: cresols, fluoronitrobenzenes, chloronitrobenzenes, bromonitrobenzenes, iodonitrobenzenes, nitroanilines, nitrophenols, and dinitrobenzenes. All aromatic compounds purchased from various suppliers were of analytical or reagent grade and were used without further purification. Apparatus and Procedure. Chromatographic measurements were performed with a Type 302 apparatus (Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland) equipped with a 5-pL high-pressure injection valve and with a UV detector (254 nm) with Z-shaped passage (volume 8 pL). Stainless steel columns (100X 4.6 mrn i.d. and 150 X 4.6 mm i.d.) were packed with 10-pm LiChrosorb RP18 (E. Merck, Darmstadt, FRG) by the viscosity technique. All chromatographic experiments were carried out at constant flow rate of 2.4 mL/min and constant temperature of 25 f 0.1 "C (using a water jacket), unless specified otherwise. The mobile phases consisted of aqueous solutions containing 20 vol % organic solvents and a-CD at concentrations of 0.2 x lo-', 0.5 X lo-', 1.0 X lo-,, 1.5 X lo-', and 3.0 X lo-, M or P-CD at concentrations of 0.2 X 0.5 X lo-', 1.0 X lo-,, 1.5 X lo-', 2.0 X lo-', and 2.5 X lo-' M. The ethanolic solutions of solutes M) were injected onto the columns both separately (5.0 X and as mixtures. k'values were measured on a column (100 X 4.6 mm id.) whose dead volume, determined from NaNO, retention (15),was 0.72 mL. EQUILIBRIA AND EQUATIONS R P systems containing CD in mobile-phase solutions may involve many species of the solute: neutral, ionic, free, or bound to one or more CD molecules. Consequently the ad0 1985 American Chemical Society
2216
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
sorption and complexation equilibria are complicated. Supposing that only one species of the solute, i.e., neutral molecules (G), is present in the solution and takes part in the process of adsorption and complexation, 1:l stoichiometry complexes are exclusively formed, and CD does not influence the properties of the R P stationary phase, we then obtain the following simplified scheme for description of the equilibria: G,
+ CD,
2(G-CD), k'
RP-lSIGs RP-181(G.CD),
Table I. Capacity Factors of o-Iodonitrobenzene, m -Nitroadline, and p -Nitrotoluene, Determined on LiChrosorb RP-18Column in Various Aqueous Mobile Phases Containing 20 vol % Organic Solvent and p-CD
compounds
solvent MeOH
G,
1-PrOH
k b.cn
2-PrOH EtoH
I&
e(GCD),
where the subscripts s and m denote the stationary and mobile phase, respectively, KG is the stability constant of the G C D complex, and h'c, k'G.CD are capacity factors of a free guest molecule (G) and of its CD complex (GCD). For such a system the apparent capacity factor (k') can be expressed as follows:
yo2
~
4 CH3
Equation 1 describing a simple R P system (11)is analogous to that derived for the first time by Uekama et al. (16) for determination of the stability constant of CD complexes with various ionic species by ion-exchange chromatography. The analogous equation has been proposed by Horvath et al. for ion pair chromatography (17). Equation 2 arises from eq 1 by simple transformation for linearization:
where [solv],' is the initial molar concentration of organic solvent. Since in the experiments [solv],' >> [CD],', it is assumed in eq 3 that [ S O ~ V ]is, ~closely similar to the equilibrium concentration of organic solvent. KBolv is the stability constant of the 1:l CD inclusion complex with an organic solvent molecule.
RESULTS AND DISCUSSION Effect of Organic Solvent. Application of CDs in RPHPLC systems for separation of compounds that are slightly water soluble requires addition of an organic solvent to the aqueous mobile-phase solution. Unfortunately, only scarce data on the influence of various solvents on CD complexes stability have so far been published. This seems to result from the prevailing opinion that water is the only convenient matrix-medium for CD complexation. In this connection the present preliminary studies were performed in order to choose an appropriate solvent and to elucidate how various organic solvents influence the CD inclusion activity in R P systems. Table I presents, as examples, the values of capacity factors (k ') of o-iodonitrobenzene, m-nitroaniline, and p-nitrotoluene determined in the mobile phase containing p-CD and 20 vol % of MeOH, EtOH, 1-PrOH, 2-PrOH, or THF. Table I1
92 68 30 51 101 20 12 6 9 24 67 46 22 33 50
44 32 23 22 90 15 11 4 8 22 42 31 21 21 50
selectivi- 1O3[p-CD], M MeOH EtOH 1-PrOH 2-PrOH T H F ty factors meta)
(3)
143 90 30 57 106 24 14 6 ~ 10 23 82 51 21 34 52
M
[p-CD] = 10 X M
Table 11. Influence of p-CD Additions on Selectivity Factors a, and amiPof Isomeric Cresols upon Use of Various Eiuents
a(ortho/
The stability constants (KG) and capacity factors ( k 'G.CD) of the investigated compounds were evaluated in this work by the least-squares method using eq 2. The aqueous mobile-phase solutions used in this work contained an additional organic solvent whose molecules are also included in the CD cavities (18). The competitive influence of organic solvent on complexation equilibria may be expressed by eq 3 for the apparent CD molar concentration [CD], being smaller than the overall molar concentration [CDIm'
N
THF MeOH EtOH 1-PrOH 2-PrOH H THF MeOH EtOH 1-PrOH 2-PrOH THF
[p-CD] = 0M
k' [p-CD] = 2X
a(meta/ para)
0 2 10
1.02 1.06 1.14
1.03 1.05 1.10
1.01 1.03 1.03
1.03 1.08 1.03
1.25 1.23 1.22
0
0.99 1.07 1.24
0.99 1.03 1.18
0.97 0.99 1.00
0.97 0.97 1.02
1.01 1.01 1.02
2
10
illustrates the effect of the above solvents (20 vol %) on the selectivity of separation of isomeric cresols. A similar behavior was observed for other investigated benzene derivatives. From Tables I and I1 it is seen that aqueous 20 vol % solutions of 1-PrOH, 2-PrOH, and T H F almost completely destroyed the @-CDability to selectively determine disubstituted benzene derivatives. The effect of the preeence of 0-CD in these solutions was imperceptible in the chromatograms. Two solvents, i.e., MeOH and EtOH, seem to be appropriate as additional components of R P systems containing @-CD. These alcohols influenced p-CD complexation solutes to an extent depending on the stability of their own @-CDcomplexes (18). This effect is well explained by eq 3. Although 20 vol % methanol best preserved the selectivity generated by p-CD complexation, in further study ethanol was applied, since (3-CD is more soluble in an aqueous 20 vol % solution of EtOH than in MeOH. Influence of CD on Separation Processes. Figure 1 shows the plots of k'values vs. a-and p-CD concentrations in the mobile-phase solution, as exemplified by the abovementioned three cresols. A similar behavior was exhibited by the remaining compounds under study. Both a-or P-CD additions were followed by a decrease in the observed values of capacity factors ( k ') of solutes; for p-CD these effects were stronger. Moreover, only in the case of p-CD did the determined k'values satisfy the linear relationship k'vs. ( k ' ~k')/[CD] from eq 2. It means that an assumption of 1:l stoichiometry of the P-CD complexes is valid for our chromatographic system. This relationship enabled evaluation of the stability constants (KG) and capacity factors ( k b . 6 - c ~ ) for the complexes of all compounds studied; their values are recorded in Table 111.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
2217
Table 111. Capacity Factors of Solutes (kb)and Their /3-CD Complexes ( l ~ ' , . ~ and . ~Stability ~) Constants ( K , ) of p-CD Complexes of Disubstituted Benzenes meta
ortho Y
' G
b+CD
KG
19 54 67 90 51 21 15 16 14
3f1 2kl Of1 Of2 Of3 4f2 2fl 6f2 3f1
60 f 2 123 f 3 129 f 4 147 f 5 57 3 84 k 4 34 f 1 103 f 27 47 f 1
*
k'~ 26 69 93 142 60
15 15 13 8
para
~'G.B.CD
KG
5fl 7f5 8f2 11 f 4 If1 4f2 If1 3fl lf3
38 f 2 48 f 3 59 f 3 97 f 3 53 f 3 12 f 1 41 f 2 83 f 1 25 f 3
k'~ 19 51 69 122 54
KG
~'G.~-cD
77 f 11 56 f 4 89 f 4 142 f 8 110 f 2 9f6 69 f 6 89 f 5 117 f 2
10 f 1 614 12 f 3 9f4 3f2 Of1 If1 2fl 0+1
11 15 8 6
.P-
ic) m-
2
1
3x104M
JU
-
1111-1 15 10 5 10 5 0 [min] 10
c
5
Flgure 3. Separation of fluoronitrobenzenes (a) without CD, (b) with 3 X lo-' M a C D , and (c)wkh 2 X lo-* M P C D . Conditions are given in Figure 2. 1
2
xlO*M
Figure 1. Capacity factors (k') as a function of concentration of a C D (a) and P C D (b) for cresols (0, ortho; A, meta; 0,para). Solvent composition was 20 vol % EtOH in H,O.
0-, P-
m-
n
m0- m-,p-
n
0- m - p -
u 8
6
(iI(
uu 5
3 [min]
5
3
Figure 2. Separation of cresols (a) without CD, (b) with 3 X M a-Cb, and (c) with 2 X lo-' M PCD. Conditions were as follows: coiumn,'LiChrosorb RP 18 10 pm, 150 X 4.6 mm id.; solvent corn position, 20 vol YO EtOH in H,O; flow rate, 2.4 mL/min; temperature,
20 f 1 O C .
0-
u [min] 30
20
20
15
20
10
Figure 4. Separation of chloronitrobenzenes: (a) without CD, (b) with 3 X lo-' M a C D , and (c)w b 2 X lo-' M P C D . conditions are given in Figure 2.
The data given in Table I11 correspond to an aqueous 20 vol % ethanol solution. The course of the relationship k'vs. [a-CD] (exemplified in Figure la) indicates that the assumptions made in eq 1 are not true in the case of a-CD complexation processes in the RP sybtem. Chromatograms illustrating the separation of ortho, meta, and para isomers of cresol, nitrotoluene, fluoronitrobenzene, chloronitrobenzene, bromonitrobenzene, and iodonitrobenzene
2218
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985 P-
(ai
ibl
(ai 0- p-
[m,n] 40
1
D-
(CI
p-
ibi
m-O i
20
0
20
20
0
Figure 5. Separation of bromonitrobenzenes (a) without CD, (b) with 3 X lo-* M a C D , and (c)with 2 X lo-* M @-CD. Conditions are given in Figure 2. m-
0-
m-
Figure 8. Separation of iodonitrobenzenes (a) without CD, (b) with 3 X lo-* M a C D , and (c)with 2 X IO-' M @CD. Conditions are given in Figure 2. are shown in Figures 2-7. They enable a comparison of the chromatographic properties and selectivities generated by aand @-CDcomplexation between positional isomers of the above compounds. The present results (Table 111, Figures 2-7) lead to the following conclusions: 1. The adsorption of 0-CD inclusion complexes of disubstituted benzene derivatives on RP-18 stationary phase is much weaker, as compared with that of the corresponding free solutes: k'G >> k'G.p.CD. 2. Only p-CD complexation permits chromatographic separation of positional isomers of disubstituted benzenes. This chromatographic selectivity is due to the differences in the stability constants of inclusion complexes in mobile-phase solution, and the differences in the adsorption of these complexes on RP phase. These two factors can influence the separation in the same direction (enhancing effect) or in the opposite one (attenuating effect). The former situation is exemplified by the behavior of m- and p-nitrotoluene and the latter one by that of m- and p-iodonitrobenzene. As the capacity factors of P-CD complexes are comparatively low and their differentiation is also rather small, the final selectivity is mainly determined by the differences in the stability constants between @-CDcomplexes of isomeric disubstituted benzenes. 3. The sequence of elution of positional isomers of halogenonitro derivatives from an RP-18 column modified with
1 1 30
20
1
u15
10
15
[min]
5
Figure 7. Separation of nitrotoluenes (a)without CD, (b) with 3 X M aCD, and (c)with 2 X lo-' M PCD. Conditions are given in Figure n
L.
a p-CD solution is identical: ortho, para, meta. The present results concerning the @-CDcomplexing activity in mobilephase solutions, concerning positional isomers of disubstitubd benzene derivatives, are rather consistent with those obtained by other authors using @-CD-silica bonded phases (6-10). The recent results obtained with commericial @-cyclodextrin stationary phase demonstrate the great practical value of this new sorbent (19, 20). Nevertheless the CD complexation process in RP-HPLC proposed by us seems to be simple and comparatively cheap.
ACKNOWLEDGMENT The authors are deeply indebted to J. Szejtli (Chinoin, Budapest, Hungary) for kindly providing the a- and @-CDs. Registry No. a-CD, 10016-20-3; P-CD, 7585-39-9; P-CD.0fluoronitrobenzene, 97570-83-7; @-CD-m-fluoronitrobenzene, 97570-84-8; P-CDSp-fluoronitrobenzene, 97570-85-9; P-CD-ochloronitrobenzene, 97570-86-0; P-CD-m-chloronitrobenzene, 97570-87-1; P-CD-p-chloronitrobenzene, 97570-88-2; P-CD.0bromonitrobenzene, 97570-89-3; P-CDem-bromonitrobenzene, 97570-90-6; 0-CD-p-bromonitrobenzene, 97570-91-7; 6-CD-oiodonitrobenzene, 97570-92-8;P-CDSm-iodonitrobenzene,9757093-9; P-CD-p-iodonitrobenzene, 97570-94-0;p-CD.0-nitrotoluene, 97570-95-1;P-CDSm-nitrotoluene,97570-96-2;P-CD-p-nitrotoluene, 97570-97-3;p-CD-o-dinitrobenzene,97570-98-4;P-CDam-dinitrobenzene, 97570-99-5; P-CD-p-dinitrobenzene, 97571-00-1; PCD.0-cresol, 86563-45-3;P-CD-m-cresol,86563-46-4;P-CD.p-creso1, 86563-47-5;P-CD.0-nitrophenol, 97571-01-2;P-CD-m-nitrophenol, 80065-29-8;P-CD-p-nitrophenol,61955-24-6;P-CD-o-nitroaniline, 78153-70-5; P-CD-m-nitroaniline,78153-71-6; P-CD-p-nitroaniline, 78153-72-7;o-cresol, 95-48-7;m-cresol, 108-39-4;p-cresol, 106-44-5; o-fluoronitrobenzene, 1493-27-2;m-fluoronitrobenzene, 402-67-5; p-fluoronitrobenzene, 350-46-9; o-chloronitrobenzene, 88-73-3; m-chloronitrobenzene, 121-73-3;p-chloronitrobenzene, 100-00-5; o-bromonitrobenzene, 577-19-5; m-bromonitrobenzene, 585-79-5; p-bromonitrobenzene, 586-78-7; o-iodonitrobenzene, 609-73-4; m-iodonitrobenzene, 645-00-1;p-iodonitrobenzene, 636-98-6; onitroaniline, 88-74-4; m-nitroaniline, 99-09-2; p-nitroaniline, 100-01-6; o-nitrophenol, 88-75-5; m-nitrophenol, 554-84-7; pnitrophenol, 100-02-7; o-dinitrobenzene, 528-29-0; m-dinitrobenzene, 99-65-0; p-dinitrobenzene, 100-25-4; o-nitrotoluene, 88-72-2; m-nitrotoluene, 99-08-1; p-nitrotoluene, 99-99-0. LITERATURE CITED (1) Szejtii, J. "Cyclodextrins and Their Inclusion Complexes", Akadhial
KiadB: Budapest, 1982; English. (2) Hinze, W. L. Sep. Purif. Methods 1981, 10, 159-237. (3) SmolkovB-KulemansovB, E. J. Chromatogr. 1982, 251, 17-34. (4) Hinze, W. L.; Armstrong, D. W. Anal. Left. 1980, 13, 1093-1104.
Anal. Chem. 1985, 57, 2219-2222 (5) Armstrong, D. W.; Stine, G. Y. J . Am. Chem. SOC. 1983, 105, 2962-2984. (6) FuJimura, K.; Ueda, T.; Ando, T. Anal. Chem. 1983, 55, 446-450. (7) Kawaguchi, Y.; Tanaka, M.; Nakae, M.; Funazo, K.; Shono, T. Anal. Chem. 1983, 5 5 , 1852-1857. (8) Tanaka, M.; Kawaguchi, Y.; Nakae, M.; Mlzobuchi, Y.; Shono, T. J . Chromatogr. 1984, 299, 341-350. (9) Tanaka, M.; Kawaguchi, Y.; Shono, T.; Uebori, M.; Kuge, Y. J . Chromatogf. 1984, 301, 345-353. (10) Armstrong, D. W.; DeMond, W. J . Chromatogr. Scl. 1984, 2 2 , 411-415. (11) Sybilska, D.; Lipkowski, J.; WBycikowski, J. J . Chromatogr. 1982, 253, 95-100. (12) Sybilska, D.; Dgbowski, J.; Jurczak, J.; hkowski, J. J . Chfomatogf. 1984, 288, 163-170. (13) Sybilska, D. Proceedings of the VIth International Symposium on Chromatography and Electrophoresis: Presses Acad6miques Europ6en: Brussels, 1971; pp 212-221.
2219
(14) Sybilska, D.; SmolkovB-Kulemansovi, E. In "Inclusion Compounds"; Academic Press: London, 1984; Volume 3, pp 173-243. (15) Jinno, K. Chromatogfaphia 1983, 17, 367-369. (18) Uekama, K.; Hirayama, F.; Nasu, S.; Matsuo, N.; Irie, T. Chem. Pharm. Bull. 1978, 2 6 , 3477-3484. (17) HONath, C.; Meiander, W.; Nahum, A. J . Chromatogr. 1979, 186, 371-403. (18) Matsui, Y.; Mochida, K. Bull. Chem. SOC.Jpn. 1979, 52, 2808-2814. (19) Armstrong, D. W.; DeMond, W.; Alak, A,; Hinze, W. L.; Riehl, T. E.; Bui, K. H. Anal. Chem. 1985, 5 7 , 234-237. (20) Hinze, W. L.; Riehl, T. E.; Armstrong, D. W.; DeMond, W.; Ward, T. Anal. Chem. 1985, 5 7 , 237-242.
RECEIVED for review March 1,1985. Accepted June 3,1985. This study was supported within the Polish Academy of Sciences Project 03.10.
Simultaneous Determination of Nickel, Lead, Zinc, and Copper in Citrus Leaves and Rice Flour by Liquid Chromatography with Hexamethylenedithiocarbamate Extraction Susumu Ichinoki* and Mitsuru Yamazaki School of Pharmacy, Hokuriku University, Kanazawa 920-1 1, J a p a n
Reversed-phase llquld chromatography followed by solvent extraction with hexamethyleneammonlumhexamethylenedlthlocarbamate (HMA-HMDC) was carrkd out to determlne NI, Pb, Zn, and Cu In standard reference citrus leaves and rice flour. These samples (250 mg) were ashed wlth nltrlc acid and perchloric acid. The metals In the ash were extracted into chloroform as HMDC chelates which were then separated on a C 18 column and rnonltored at 260 nm. This method permitted Separation and determlnatlon of Cd-, NI-, Pb-, Zn-, Cu-, Hg-, Co-, and BI-HMDC chelates. Cd, Hg, Co, and BI could not be detected, but the microgram per gram levels of NI, Pb, Zn, and Cu In the standard blologlcal materials were simultaneously determined wlthln 25 min.
In recent years, high-performance liquid chromatography (HPLC) has been applied to the separation and determination of various metals. In principle, HPLC enables the simultaneous determination of several metals to an extent comparable to that of atomic absorption spectrometry (AAS) and spectrophotometry. Some reviews (1-3) and many papers on this subject have been published. A number of papers dealing with the separation of metal chelates have been published, but not many with the determination of metals in real samples, except for water. The following reports have been published: determination of Cd, Co, Cu, Pb, Hg, and Ni in zinc sulfate plant electrolyte (4); Mn, Fe, Co, Ni, Cu, Zn, and P b in steel ( 5 ) , Ni-Cr-Fe alloys, zirconium, and uranium (6);Cu, Ni, Pb, and Mn in a standard kale and fish meal (7). A common procedure is to separate the metals as metal chelates, and the most commonly used chelating agents are dithiocarbamates since they react with many heavy metals and the metal chelates have large molar absorptivity in the ultraviolet (UV) region. Consequently, the simultaneous and sensitive determination of heavy metals is possible with a 0003-2700/85/0357-2219$0 1.50/0
conventional HPLC equipped with a UV detector. On-column formation of dithiocarbamato chelates has been carried out by some workers (8-11). This method is simple, but the sensitivity is less than that by precolumn formation (10). Solvent extraction permits preconcentration of metals as chelates and provides relatively sharp peaks (good resolution). In previous work, we attempted the simultaneous determination of heavy metals by precolumn formation (solvent extraction) and reversed-phase HPLC with diethyldithiocarbamate (DDTC) (12), tetramethylenedithiocarbamate (13, 14), and HMA-HMDC (15,16) and published our results on the separation and determination of heavy metals (Cd, Ni, Pb, Zn, Cu, Hg, Co, Bi) in water (12,13,15), standard orchard leaves (14), and standard bovine liver and oyster tissue (16). The standard biological samples (14, 16) could be ashed by the dry ashing method (450-550 "C) without any loss of the metals to be determined. However, not all biological samples could be ashed successfully by dry ashing because of various matrices present in the samples. Actually, the standard citrus leaves and rice flour could not be ashed successfully with a muffle furnace. In this paper, wet ashing has been investigated for its application to the HPLC determination of heavy metals in plant samples. This method is based on a wet ashing method (HN03 + HC104) and precolumn formation, separation, and UV detection of the metal-HMDC chelates. A new eluent composition was employed so as to take advantage of a longer column.
EXPERIMENTAL SECTION Reagents. All reagents used were of analytical grade unless otherwise stated. Standard reference materials (citrus leaves and rice flour) were obtained from National Bureau of Standards (NBS, Washington DC). Nitric acid, perchloric acid, and ammonia water were of special grade; for example, the content of Zn was 10 ng/mL (ppb) and 0 1985 American Chemical Soclety
