Anal. Chem. 1999, 71, 1111-1118
Strategies in Using Analytical Restricted Access Media Columns for the Removal of Humic Acid Interferences in the Trace Analysis of Acidic Herbicides in Water Samples by Coupled Column Liquid Chromatography with UV Detection Elbert A. Hogendoorn,† Ellen Dijkman, and Bert Baumann
National Institute of Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA, Bilthoven, The Netherlands Carmen Hidalgo, Juan-Vicente Sancho, and Felix Hernandez*
Analytical Chemistry, Department of Experimental Sciences, Universitat Jaume I, P.O. Box 224, 12080, Spain
In this comprehensive study, analytical restricted access media (RAM) columns were investigated to determine their performance in decreasing the humic acid interference encountered in the trace analysis of acidic herbicides in environmental water samples when employing reversedphase liquid chromatography with UV detection (RPLCUV). The selected analytical column RAM materials are 5-µm Semi-Permeable-Surface (SPS, Regis), 5-µm Internal Surface Reversed Phase (ISRP, Pinkerton, Regis), and 5-µm Hisep (Supelco); a precolumn containing 25-µm alkyl-diol-silica (ADS) RAM material (Merck) was also included in this study. Different LC configurations were investigated, including the single RAM column mode and column switching (LC/LC), employing one RAM column in combination with an analytical C18 column, or two RAM columns. Concerning methodology, both the singleresidue method (SRM) approach and the multiresidue method (MRM) approaches were tested by analyzing SPE C18 extracts of reference water samples spiked with the analyte(s) at the 0.5-1.0 µg/L level and containing dissolved organic carbon (DOC) between 3 and 12 mg/ L. Mecoprop (MCPP) was used as a model compound in SRM, and the more polar compounds metsulfuron methyl, bentazone, bromoxinyl, and MCPA were included to investigate the MRM approach. With regard to SRM using the LC/LC mode, the use of at least one analytical RAM column improved the resolution between the analyte and the humic interferences sufficiently to trace the analyte in extracts from water samples containing a high level of DOC (12 mg/L) without a preceding cleanup. In the MRM approach, ISRP/C18 was most favorable for the simultaneous analysis of the heterogeneous group of pesticides in water with a high DOC level. For samples with a medium DOC content (6 mg/L), C18/SPS using short columns (50 × 4.6 mm i.d.) and isocratic elution is an efficient alternative. The performance of the useful MRM procedures was tested by analyzing water samples con10.1021/ac980918x CCC: $18.00 Published on Web 02/10/1999
© 1999 American Chemical Society
taining 6 and 12 mg/L DOC and spiked with the analytes at the 0.5-1.0 µg/L level. Mean recoveries (n ) 5, each DOC level) ranged between 86 and 102%, with RSDs between 3 and 7.0%. The use of RAM columns in the different LC/LC configurations tested allows us to develop rapid, selective, and sensitive methods for the analysis of acidic herbicides, which are very adequate for screening purposes in environmental water samples. Acidic pesticides, e.g., chlorophenoxy and sulfonyl urea herbicides, can be efficiently separated with reversed-phase liquid chromatography (RPLC) on an analytical C18 column with a mobile phase at low pH (typically, 2.5-3.5). However, when analyzing environmental samples such as surface water, groundwater, and soil, and employing UV detection at low wavelength, chromatographic analysis is usually hampered by a broad hump originating from coextracted humic substances.1-8 This hump causes a severe baseline deviation and avoids, in many cases, identification and quantification at low levels. Humic substances have complex molecular structures and cover a wide range of molecular weights.9,10 In natural waters, the dissolved humic substances are operationally classified in * To whom correspondence should be addressed. E-mail:
[email protected]. † E-mail:
[email protected]. (1) Di Corcia, A.; Marchetti, M. Anal. Chem. 1991, 63, 580-585. (2) Di Corcia, A.; Marchese, S.; Samperi, R. J. Chromatogr. 1993, 642, 163174. (3) Pichon, V.; Cau Dit Coumes, C.; Chen, L.; Guenu, S.; Hennion, M.-C. J. Chromatogr. A 1996, 737, 25-33. (4) de Ruiter, C.; Minaard, W. A.; Lingeman, H.; Kirk, E. M.; Brinkman, U. A. Th.; Otten, R. R. Int. J. Environ. Anal. Chem. 1991, 43, 79-90. (5) Geerdink, R. B.; Tol-Wildenburg van, S.; Niessen, W. M. A.; Brinkman, U. A. Th. Analyst 1997, 122, 889-893. (6) Coquart, V.; Hennion, M.-C. Sci. Total Environ. 1993, 132, 349-360. (7) Masque, N.; Galia, M.; Marce, R. M.; Borrull, F. J. Chromatogr. A 1998, 803, 147-155. (8) Nilve, G.; Adnunson, G.; Jonsson, J. A. J. Chromatogr. 1989, 471, 151160. (9) Malcolm, R. L. Anal. Chim. Acta 1990, 232, 19-30. (10) Humic Substances in Soil, Sediment and Water; Aiken, G. R.; McKnight, D. M.; Wershaw, R. L.; MacCarthy, P., Eds.; Wiley: New York, 1985.
Analytical Chemistry, Vol. 71, No. 6, March 15, 1999 1111
humic and fulvic acids on the basis of their solubility. They are usually defined as the dissolved organic carbon (DOC) passing through a 0.45 µm filter.10 Fulvic acids are soluble in alkali and acidic aqueous solution, and humic acids are soluble in alkali but insoluble in acidic or neutral aqueous solutions. Due to the presence of phenolic and carboxylic groups, the ionization causes the presence of negative charges in the molecules.11 Because of this, upon lowering the pH, likewise the acidic target analytes’ ionization decreases, and obviously, coextraction with the analytes will take place when using standard extraction procedures, e.g., liquid-liquid extraction or solid-phase extraction (SPE). Several techniques, such as the use of selective sorbents for off-line,1,2 on-line3-7 or membrane trapping,8 have been developed to improve the selectivity concerning analytes and humic substances. Because of the large differences in molecular size between target analytes (small molecules) and humic substances (large molecules), the use of restricted access media (RAM) columns seems to be attractive.12,13 These columns have been successfully developed for the determination of low-molecularweight compounds in body fluids by direct injection.14,15 Recently, the various types of RAM columns,16 their mode of use, and their applications have been reviewed.16,17 Concerning the use of RAM columns in environmental analyses, only a few studies appeared. We first reported on the beneficial use of precolumns packed with 5-µm Internal Surface Reversed Phase (ISRP) from Pinkerton for improved RPLC separation between coextracted humic acid interferences and chlorophenoxy acid herbicides.18 Unfortunately, the reproducibility of these precolumns was poor, possibly due to the fact that these ISRP precolumns were not originally designed for this type of applications. In a more recent study, RAM precolumns packed with 25-µm alkyl-diol-silica (ADS) material, specially designed for the on-line processing of body fluids, showed high reproducibility and stability in trace analyses of triazines in water samples.19 However, as demonstrated, the analytical RPLC separation carried out with a mobile phase at pH 7 is not favorable to show the potential of the ADS precolumn concerning humic acid interferences. Based on the good results of a recent study on the use of analytical RAM columns for the determination of β-agonists in serum using coupled-column RPLC (LC/LC),20 we successfully developed efficient LC/LC methods for the determination of acidic herbicides in environmental samples.21,22 In these studies, the LC/ LC configurations of the RAM column and the 3-µm C18 column (50 × 4.6 mm i.d.) were different. For the analysis of mecoprop (11) Domenica, T.; Seeber, R.; Ciavatta, C.; Gessa, C. Fresenius J. Anal. Chem. 1997, 359, 555-560. (12) Hagestam, H.; Pinkerton, T. C. Anal. Chem. 1985, 57, 1757-1763. (13) Cook, S. E.; Pinkerton, T. C. J. Chromatogr. 1986, 368, 233-248. (14) Pinkerton, T. C. J. Chromatogr. 1991, 544, 13-23. (15) Perry, J. A. Prog. Pharm. Biomed. Anal. 1994, 1, 145-192. (16) Boos, K.-S.; Rudolphi, A. LC-GC Int. 1998, 11, 84-95. (17) Boos, K.-S.; Rudolphi, A. LC-GC Int. 1998, 11, 224-233. (18) Sancho-Llopis, J. V.; Hernandez-Hernandez, F.; Hogendoorn, E. A.; Zoonen van, P. Anal. Chim. Acta 1993, 283, 287-296. (19) Onnerfjord, P.; Barcelo, D.; Emneus, J.; Gorton, L.; Marko-Varga, G. J. Chromatogr. A 1996, 737, 35-45. (20) Hogendoorn, E. A.; Zoonen, van, P.; Polettini, A.; Marrubini, B.; Montagna, M. Anal. Chem. 1998, 70, 1362-1368. (21) Parrilla, P.; Kaim, P.; Baumann, R. A.; Hogendoorn, E. A. Mecoprop in soils. Submitted to Fresenius J. Anal. Chem., 1998. (22) Westhuis, K.; Dijkman, E.; Baumann, R. A.; Hogendoorn, E. A. Bentazone and Bromoxynil in surface water. Manuscript in preparation.
1112 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
Table 1. Information on Acidic Herbicides Used as Model Compounds
in soils, a 5-µm ISRP column (50 × 4.6 mm) was used as the first column (C-1),21 while for the analysis of bentazone and bromoxynil in surface waters, a 150- × 4.6-mm-i.d. column packed with 5-µm Semi-Permeable Surface (SPS) C18 was applied as the second column (C-2).22 The aim of this study is to investigate the beneficial use of different RAM columns in various LC/LC configurations in order to obtain a useful strategy in this field of analysis, which demands rapid, selective, and sensitive procedures that are suitable for performing screening analysis, less time-consuming and laborious than other methods based, for example, on GC/MS, and compatible with the environment due to minimal solvent comsuption. Based on their commercial availability and applicability,16,17,20-22 various RAM columns were tested in different LC modes. The performance of the different LC configurations was studied and evaluated by analyzing reference water samples containing 3-12 mg/L of DOC. Being in-line with previous work18 and our strategy concerning the screening of polar pesticides,23 an off-line solidphase extraction (SPE) of the samples was performed before instrumental analysis. Both the single-residue method (SRM) and the multiresidue method (MRM) approaches were considered. Providing differences in polarity and representing different chemical families, metsulfuron methyl, bentazone, bromoxynil, MCPA, and MCPP were selected as a heterogeneous group of acidic test compounds. Information on the selected compounds is given in Table 1. EXPERIMENTAL SECTION Chemicals. All the acidic herbicides (content >99%) were obtained from Dr. S. Ehrenstorfer (Promochem, Wesel, Germany). (23) Hogendoorn, E. A.; Hoogerbrugge, R.; Baumann, R. A.; Meiring, H. D.; de Jong, A. P. J. M.; Zoonen van, P. J. Chromatogr. A 1996, 754, 49-60.
Table 2. Information on Columns Used
a
code name
packing material (particle size/stationary phase)
Hisep ISRP SPS-50L SPS-150L C18-L50 C18-L100 C18-L30 PC-ADS
5 µm Hisep 5 µm Pinkerton ISRP GFF-II-S5-80 5 µm SPS-5PM-S5-100-ODS 5 µm SPS-5PM-S5-100-ODS 3 µm C18 Microspher 3 µm C18 Microspher 5 µm Hypersil ODS 25 µm LiChrospher RP-18 ADS
pore size (Å) 100 80 100 100
60
dimension (l × i.d. in mm) 50 × 4.6 50 × 4.6 50 × 4.6 150 × 4.6 50 × 4.6 100 × 4.6 30 × 4.0 25 × 4.0
Na (n/m)
used as
38 000 34 000 70 000 76 000 118 000 138 000 66 000
C-1, C-2 C-1 C-1, C-2 C-2 C-1, C-2 C-2 C-1 C-1
Plate number derived from the elution of fluoranthene in a mobile phase of acetonitrile-water (1 < k 99%), orthophosphoric acid (89% pure), and hydrochloric acid (37%) were bought from Merck. LC grade water was obtained by purifying demineralized water in a Milli-Q system (Millipore, Bedford, MA). Stock standard solutions (∼500 µg/mL) of the herbicides were prepared in acetonitrile. For the LC analysis, the stock solutions were diluted and mixed with LC grade water containing 0.05% (v/v) TFA. An aqueous stock solution of DOC (1.33 mg/mL) was prepared from a commercial humic acid material (Fluka; lot/product no. 35069 288/53680) under defined conditions.24 Mobile phases consisted of a mixture of methanol-0.05% TFA in water (pH 2.4) and were used at a flow rate of 1 mL/min. Disposable 3-mL SPE cartridges containing 500 or 200 mg of C18-bonded silica (40 µm) were obtained from J.T. Baker (Deventer, The Netherlands). The SPE cartridges were preconditioned with 3 mL of methanol, 3 mL of acetone, 3 mL of methanol, and 6 mL of 0.1% TFA in water, respectively. Columns. Analytical RAM Columns: 50-mm × 4.6-mm-i.d. column packed with 5-µm Pinkerton ISRP GFF-II-S5-80 (Regis, Morton Grove, IL); 50-mm × 4.6-mm-i.d. column and 150-mm × 4.6-mm-i.d. column, both packed with 5-µm SPS-5PM-S5-100-ODS from Regis; 50-mm × 4.6-mm-i.d. column packed with 5-µm Hisep (Supelco, Bellefonte, PA). When used as the only column or as the first column in LC/LC, a precolumn containing the same RAM material was always placed before the analytical column with dimensions of 10 mm × 3 mm i.d. (Pinkerton, SPS) or 20 mm × 4 mm i.d. (Hisep), respectively. The analytical column is always used in forward flush mode. RAM Precolumn: 25-mm × 4-mm-i.d. column packed with 25µm LiChrospher RP-18 ADS (Merck, Darmstadt, Germany). The precolumn is always used in the back-flush mode. Analytical C18 Columns: 50-mm × 4.6-mm-i.d. and 100-mm × 4.6-mm-i.d. cartridge columns packed with 3-µm C18 Microspher (Chrompack, Bergen op Zoom, The Netherlands), including a 10mm × 3-mm-i.d. RP guard column (Chrompack). Apparatus. A 24-place manifold from Varian (Harbor City, CA) was used to perform SPE. The LC system consisted of a model 233 XL autosampler from Gilson (Villiers-le-Bel, France) equipped with an auxiliary high-pressure valve for column switching, a (24) Hoop van den, M. A. G. T.; Leeuwen van, H. P.; Cleven, R. F. M. J. Anal. Chim. Acta 1990, 232, 141-148.
model 2150 isocratic LC pump from LKB (Bromma, Sweden) for the delivering of M-1, a model 9012 ternary gradient pump from Varian for the delivering of M-2, and a series 1100 UV diode array detector from Hewlett-Packard (Waldbron, Germany). Recording of chromatograms and quantitative measurement of peak areas were performed with an LC Chem Station (software version A.05.03) from Hewlett-Packard. A MicropH 2001 pH meter and Pipetmans (200, 1000, and 5000 µL) were obtained from Crisons Instruments (Barcelona, Spain) and Gilson, respectively. Water Samples. The reference water was prepared by adding a volume of the stock DOC solution to Milli Q water, providing water samples with DOC concentrations of 3, 6, and 12 mg/L. The natural water sample was from the river Kromme Rijn, Utrecht, The Netherlands, and was characterized by a DOC of 7.7 mg/L and a pH of 7.3. Sample Pretreatment. A 250-mL water sample was brought to pH 2.2 ((0.1) with hydrochloric acid and percolated through a preconditioned 500-mg C18 cartridge at a flow of approximately 4 mL/min. After sampling, the cartridge was dried by passing air for 30 min. The cartridge was transferred to the top of a calibrated tube, and by means of slight overpressure, 2 mL of acetone was passed through the cartridge and collected in the tube. After the volume was adjusted to 2 mL of acetone, 1 mL of extract was transferred to a glass tube and evaporated to dryness using a warm water bath and a gentle stream of nitrogen. The residue was dissolved by adding first 400 µL of methanol, followed by 1600 µL of 0.05% TFA in water. SRM and MRM Procedures. Information on columns and LC(/LC) conditions, including volumes for cleanup and transfer, are given in Tables 2 and 3, respectively. After the flows were set at 1 mL/min, a volume of 300 µL of the sample extract was injected on C-1. UV detection was carried out at a wavelength of 228 nm. In both SRM and MRM, quantification was done by external calibration with standard solutions in acetonitrile-0.05% TFA in water (20:80, v/v). RESULTS AND DISCUSSION General Aspects. The RPLC-UV trace analysis of acidic analytes in water samples is always severely hampered by coextracted humic (acid) substances causing a severe baseline deviation. Our recent studies21,22 showed that the use of an analytical RAM column in reversed-phase LC/LC significantly improves the baseline, allowing quantification at the required low levels in sample extracts without the use of additional cleanup. Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
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Table 3. Information on LC Configurations and Conditions
a
configuration in SRM/MRM
C-1;a M-1b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
C18-50L; (60:40) C18-50L; (60:40) C18-50L; (60:40) (C18-50L + SPS-150L); (60:40) Hisep; (60:40) C18-30L; (55:45) SPS-50L; (60:40) Hisep; (60:40) Hisep; (60:40) SPS-50L; (60:40) SPS-50L; (60:40) ISRP; (40:60) PC-ADS; (30:70) ISRP; (40:60) ISRP; (40:60) C18-50L; (50:50) C18-50L; (52.5:47.5) C18-50L; (50:50) SPS-50L; (52.5:47.5) Hisep; (40:60)
C-2; M-2b
cleanup/transfer volume (mL)
C18-50L; (60:40) SPS-150L; (60:40)
3.6/1.0 3.6/1.0
Hisep; (55:45) Hisep; (60:40) C18-100L; (60:40) SPS-50L; (60:40) C18-100L; (60:40) SPS-50L; (60:40) C18-100L; (60:40) C18-100L; (60:40) SPS-50L; (55:45) C18-50L; (50:50) SPS-50L; (50:50) SPS-50L; (52.5:47.5) SPS-50L; (55:45) C18-50L; (52.5:47.5) C18-50L; (40:60)
2.3/1.0 4.0/1.0 3.5/1.0 3.5/1.0 4.5/1.0 4.0/1.0 5.0/1.0 5.0 /3.0 2.2/3.6 2.3/2.9 2.3/7.7 1.9/5.9 2.3/4.7 2.0/7.3 2.8/0.80
Figure 1 1 1 1 2A 2B 2C 2D 3A 3B 3C 4 5 6A 6B 6C 7
Information on columns, see Table 2 and Experimental Section. b Ratio (v:v) of methanol-0.05% TFA in water.
These results prompted us to further investigate the potential of RAM columns in LC/LC in this field of analysis. Humic material is a complex and nonconsistent sample matrix. Depending on sample type and sampling time, variations in the composition and concentration of humic and fulvic acids can be expected. Therefore, to compare and evaluate results in a consistent way, reference water samples (see Experimental Section) were used containing DOC levels ranging from 3 to 12 mg/L, covering the levels between medium and high DOC content, respectively. The columns used in different LC configurations were selected on the basis of commercial availability, previous applications,19-22 recent comparative information,16,17 and their suitability for use in LC/LC. For example, we found that Biomatrix (Chrompack) and Ultrabiosep (Shandon, Runcorn, Cheshire, UK) RAM columns were not suitable for the determination of chlorophenoxy herbicides in serum and water samples (unpublished data). The low efficiency and relatively high retention capacity limit fast desorption kinetics, making them incompatible with the second analytical column. This study comprises several parts. After a presentation of the humic acid interferences encountered in RPLC, the use of the selected columns (see Table 2) in various LC configurations is discussed. In the next part, the LC configurations are investigated and evaluated for the single-residue method (SRM) approach. Finally, the feasibility of the most adequate LC configurations (SRM) is investigated to determine their applicability to the multiresidue method (MRM) approach. The most adequate LC configurations will be applied in testing the performance of the overall procedure involving the analysis of DOC-containing water samples spiked with the group of herbicides at the 0.5-1.0 µg/L level. Humic and Fulvic Acid Interferences in LC. The chromatograms displayed in Figure 1 illustrate the chromatographic interference of coextracted DOC material in different LC configurations. 1114 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
Figure 1 comprises four series of chromatograms (A-D) recorded with four different LC configurations (1-4) with columns and LC conditions as mentioned in Table 3. In the SRM approach, mecoprop was selected as a model compound. Series A shows the analysis of a standard of mecoprop, of which the concentration corresponds to the level of about 1 µg/L in a water sample. LC and LC/LC conditions such as mobile phase compositions, cleanup, and transfer volume were derived from previous work.18,25 To comply with a worse-case situation, a transfer volume of 1.0 mL was selected instead of the 400-µL peak elution volume of mecoprop. Series B and C correspond to the analysis of SPE extracts of water samples containing 3 and 12 mg/L DOC, respectively. These results clearly illustrate the effect of the humic and fulvic acids on the LC configuration. In the LC mode, mecoprop is not recovered from the humic substances in the first part of the chromatogram, and with LC/LC using two C18 columns, the analyte is on top of the hump, making quantification difficult. Obviously, LC/LC with C18 and SPS columns is more favorable. For samples with a DOC content of about 3 mg/L or lower, the use of a two-column system without column switching (see LC configuration 4) can be considered. Series D shows the LC analyses of a natural water sample taken from the river De Kromme Rijn having a relatively high DOC content of 7.7 mg/L. The LC baseline profile of the humic substances of the natural sample corresponds well with that of the reference water (DOC of 12 mg/L), indicating the suitability of the selected DOC reference material, which is one interesting finding of this work. Therefore, DOC reference material seemed to represent properly the behavior of humic and fulvic acids in real-world samples. In this way, this material was considered suitable for studying, from a chromatographic point of view, the humic acids interference in water samples. (25) Hogendoorn, E. A.; Zoonen van, P. J. Chromatogr. A 1995, 703, 149-166.
Figure 2. Performance of the Hisep column as C-1 or C-2 in LC/ LC in the SRM approach. SPE extract of reference water containing 12 mg/L DOC and spiked with mecoprop (MCPP) to a level of 1 µg/ L. For LC configurations and conditions, see Table 3.
Figure 1. Presentation of humic acid interferences in RPLC-UV (228 nm) for the trace analysis of mecoprop in SPE extracts of DOCcontaining water samples. Series A, standard of mecoprop (64 ng/ mL); series B, SPE extract (62.5 mL of sample/mL) of reference water containing 3 mg/L DOC; series C, SPE extract (62.5 mL of sample/ mL) of reference water containing 12 mg/L DOC; series D, SPE extract (62.5 mL of sample/mL) surface water from the river Kromme Rijn containing 7.7 mg/L DOC. Injection volume, 300 µL; for LC configurations and conditions of chromatograms 1-4, see Table 3.
Columns and Configurations. An overview of the tested columns is given in Table 2. The efficiency, N (number of plates per meter), of each analytical column was first tested by using fluoranthene as test compound and a mobile phase with an acetonitrile-water composition providing an adequate retention of the analyte (1 < k < 10). Because of the 25-µm packing material and its design to be used in back-flush mode, determination of the efficiency of the ADS precolumn has not been carried out. In residue analyses employing LC/LC, the total separation is divided over two columns, making selection of the dimensions of C-1 and C-2 an important aspect to be considered. From the financial, efficiency, and practical (pressure) points of view, one should avoid an overkill in separation power. Hence, short analytical columns, especially for C-1 to speed up cleanup, transfer, and conditioning time, are attractive for use in LC/LC. The degree of retention and efficient elution (band broadening) of the analyte(s) largely determines the position of the RAM column in LC/LC. For example, both efficiency and retention of
the ISRP column appeared to be relatively low in our previous applications,20,21 making it not suitable to perform a final separation. However, when used as C-1 in LC/LC, the distinctly higher eluotropic strength of M-2 will repair the band broadening of the analyte(s) by performing peak compression of them on C-2. The highly efficient SPS columns20,22 can be used as C-1 and/or C-2. Based on the considerations above, a large number of LC configurations and conditions listed in Table 3 were selected and tested to determine their performance in the analysis of mecoprop in extracts of water samples containing 12 mg/L DOC. Regarding the state-of-the-art in RAM technology and the commercially available material, and considering its suitability for LC/LC analysis, we have tested every possible configuration in order to test RAM column’s applicability for acidic herbicides analysis in environmental water samples. SRM Approach. Being the unexplored column, the potential of the Hisep column was first investigated. The degree of retention of mecoprop on the Hisep column is more or less similar to that on C18, providing a k of 7.0 with a mobile phase of methanol0.05% TFA in water (60:40, v/v). In the one-column configuration (LC configuration 5), Hisep provided an almost baseline separation between humic substances and mecoprop. Unfortunately, the baseline was not stable, and a broad and tailing system peak was present in the chromatogram coeluting with the last part of mecoprop. LC/LC with C18/Hisep (configuration 6) or SPS/Hisep (configuration 7) provided a very good recovery of mecoprop (Figure 2). However, as shown in Figure 2A,B, when using Hisep as C-2, the elution of mecoprop is not very efficient. Improved results were obtained when using Hisep as C-1 in combination with a C18 column (configuration 8) or an SPS column (configuration 9), as illustrated in Figure 2C,D, respectively. So, we can conclude that the best use for Hisep is as the first column, applying a good cleanup step. The performances of the other analytical RAM columns (LC/ LC configurations 10-12) are displayed in Figure 3. A short (50 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
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Figure 4. Performance of the ADS RAM precolumn in PC/LC in the SRM approach. SPE extract of reference water containing 12 mg/L DOC and spiked with mecoprop (MCPP) to a level of 1 µg/L. For LC configurations and conditions, see Table 3.
Figure 3. Performance of SPS and ISRP RAM columns in LC/LC in the SRM approach. SPE extract of reference water containing 12 mg/L DOC and spiked with mecoprop (MCPP) to a level of 1 µg/L. For LC configurations and conditions, see Table 3.
mm long) SPS column as C-1 (Figure 3A, configuration 10) and a C18 column as C-2 provides satisfactory resolution, more or less similar to that of the same columns used in the reverse order (see Figure 1, configuration 3). Figure 3B shows that LC/LC with two SPS columns is not favorable and produces a chromatogram with the analyte on top of the hump, comparable to that obtained with the use of two C18 columns (see Figure 1, configuration 2). The performance of the ISRP column as C-1 in combination with a C18 column as C-2 is displayed in Figure 3C (configuration 12). Because of the lower retention capacity of ISRP, the eluotropic strength of M-1 was lower than that of M-2. Taking into consideration that the baseline disturbance in the first part of the chromatogram is mainly caused by changes in TFA concentration, one can conclude that also the ISRP column provides an efficient separation between humic substances and mecoprop. Summarizing the results with Regis columns, one can establish the feasibility of the ISRP column for the cleanup step, while SPS can be used in both steps, depending on the humic acids load. Finally, the feasibility of the ADS precolumn was tested. The large particle size (25 µm) of this material is favorable for the sampling speed of various types of liquid samples. However, this RAM material requires the use of the back-flush mode in order to prevent excessive band broadening of the analytes.19,20 This means that, during sample loading and cleanup, the analyte has to be trapped and has to remain on top of the column. Experiments indicated that a mobile phase of methanol-0.05% TFA in water (30:70) was a good compromise between cleanup (5 mL) and elution prevention of the analyte. The complete desorption of mecoprop required 2-3 mL of the mobile phase of the analytical column. Under the selected conditions, the performance of the PC/LC analysis (configuration 13) is illustrated in Figure 4. It shows that the analysis of mecoprop is feasible but marginal. The remaining profile of humic acid interferences will distinctly hamper the quantification of more polar analytes. In conclusion, the best SRM approach for mecoprop taking into account the complete removal of interferences and good peak 1116 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
Table 4. LC Performance of Acidic Pesticides on Columns (50 × 4.6 mm i.d.)a 3-µm C18 compound
k
N
5-µm SPS 5-µm Hisep 5-µm ISRP k
N
k
N
k
N
metsulfuron methyl 4.1 7280 3.5 2060 3.0 2380 3.9 600 bentazone 4.7 8880 7.6 2030 9.5 300 11.7 2600 bromoxynil 6.0 4830 12.0 2680 7.9 310 4.5 330 MCPA 10.1 4830 13.6 2540 14.1 1440 9.3 1570 MCPP 17.8 5650 20.7 2940 7.9 1400 8.4 1820 a Mobile phase (methanol-0.05% TFA in water): C18 and SPS, (50: 50); Hisep, (55:45); ISRP, (40:60).
shape, used Hisep as C-1 in combination with a C18 column (configuration 8) as illustrated in Figure 2C. MRM Approach. In comparison to SRM, the LC/LC analysis of a group of pesticides with a wide polarity range requires larger transfer volumes; hence, selectivity will be less. Concerning humic substances, cleanup will become more difficult when more polar analytes than mecoprop are included because the available cleanup volume will be lower. Therefore, the MRM approach was investigated by including herbicides (more polar, see Table 1) with less C18 retention than mecoprop. Based on the SRM results, a LC/LC configuration employing Hisep as C-1 (see Figure 2C or D) was first selected. However, the data in Table 4 show that the elution order and efficiency for the analytes on the Hisep column deviated largely from those on the second column (SPS or C18). Apparently, the Hisep column provides a different separation mechanism for some of the analytes. Unfortunately, the reversed elution counteracts the second separation, and the excessive band broadening of bentazone and bromoxynil cannot be repaired with the second mobile phase. This makes Hisep incompatible with the second analytical column for the separation of this heterogeneous mixture of compounds. However, improved results can be expected when analyzing analytes belonging to one chemical family, e.g., sulfonyl ureas or chlorophenoxy acids. As shown in Table 4, the elution order on ISRP corresponds largely to the one on Hisep, and here a substantial increase in band broadening is obtained for metsulfuron methyl and bromoxynil. However, in this case the low retention capacity decreases the band broadening on C-2 with the higher eluotropic
Figure 5. Performance of LC/LC using ISRP/C18 columns in the MRM approach. SPE extract of reference water containing 12 mg/L DOC and spiked with metsulfuron methyl (1), bromoxynil (2), bentazone (3), MCPA (4), and MCPP (5) to a level of about 1 µg/L each analyte. Fort LC conditions, see Table 3.
strength of M-2. The performance of LC/LC using SPS or C18 (configurations 14 and 15) as C-2 was investigated. The latter combination provided a slightly improved baseline profile in the first part of the chromatogram. The MRM performance of ISRP/ C18 concerning the analysis of an extract of a DOC-containing water sample (12 mg/L) spiked with the herbicides at the 0.81.5 µg/L level is shown in Figure 5. It clearly illustrates the good resolution between humic interferences and analytes, allowing us to perform trace analysis. Considering distortion (see discussion above) and humic/fulvic acid elution behavior, the step size of the gradient elution should be kept as low as possible. The chromatographic performance of the analytes on ISRP/C18 decreased unacceptably when the difference between the eluotropic strengths of M-1 and M-2 was increased. The possibility of isocratic elution was studied with the C18/ SPS configuration by analyzing SPE extracts of water containing 6 mg/L DOC. A mobile phase with 50% methanol was first selected in order to maximize cleanup (selectivity) and a short (50 mm long) SPS column as C-2 to minimize the retention of the lasteluting analyte (sensitivity). However, as displayed in Figure 6A (LC configuration 16), the relatively large retention of mecoprop hampers sensitive analysis. Figure 6B shows that a small increase in eluotropic strength (LC configuration 17) provides a better compromise between selectivity and sensitivity. Figure 6C is recorded to demonstrate the less favorable baseline profile of the coeluted humic interferences when applying a step-gradient elution in order to improve the sensitivity of mecoprop by reducing its retention time. Finally, we investigated the feasible LC performance obtained in Figure 6B by using the columns in the reverse order, viz., SPS/ C18 (configuration 19). However, both the elution performance of the analytes and the baseline profile are worse than those with C18/SPS. In conclusion, from the various LC configurations investigated, it appears that the combinations of ISRP/C18 (LC configuration 15) and C18/SPS (configuration 17) are most adequate to perform rapid trace analysis of a heterogeneous group of acidic compounds in water samples using coupled-column RPLC-UV. In the MRM approach, ISRP/C18 was most favorable for the simultaneous analysis of the heterogeneous group of pesticides in water with a
Figure 6. Performance of LC/LC using C18/SPS columns and various mobile phase compositions in the MRM approach. SPE extract of reference water containing 6 mg/L DOC and spiked with metsulfuron methyl (1), bentazone (3), bromoxynil (2), MCPA (4), and MCPP (5) to a level of about 1 µg/L each analyte. For LC conditions, see Table 3. Table 5. Mean Recoveries and RSDs of the Herbicides Spiked to Water Samples Containing 6 and 12 mg/L DOC and Preconcentrated (100 mL) on 200-mg C18 SPE Cartridges (n ) 5) herbicide
spike level (µg/L)
mean recoverya (RSD) (%)
mean recoveryb (RSD) (%)
metsulfuron methyl bromoxynil bentazone MCPA MCPP
0.5 0.5 0.5 1.0 1.0
86 (4) 96 (5) 97 (5) 96 (5) 98 (4)
102 (7) 95 (5) 99 (5) 94 (6) 91 (3)
a DOC content, 12 mg/L; LC configuration 15 of Table 3. b DOC content, 6 mg/L; LC configuration 17 of Table 3.
high DOC level. For samples with a medium DOC content (6 mg/ L), C18/SPS using short columns (50 × 4.6 mm i.d.) and isocratic elution is an efficient alternative. To support this conclusion, the performance of the overall method was carried out by analyzing two series (n ) 5) of 100 mL of water samples spiked with the analytes at the 0.5-1.0 ppb level and containing a DOC level of 6 or 12 mg/L, respectively. The samples were processed with SPE on 200-mg C18 cartridges, and the extracts were, depending on the DOC content, analyzed by the LC/LC configuration mentioned above. Table 5 shows good results on both recovery and repeatability, emphasizing the good performance and efficiency of this approach. Only a small decrease in the recovery of metsulfuron methyl in the ISRP/C18 configuration for the high DOC level is observed, which can be explained by the need to perform a more accurate cleanup step in order to remove the major part of early-eluting interferences (which are higher at the 12 ppm DOC level), resulting in small losses of only the first analyte, but still maintaining very good reproducibility (RSD of 4%). CONCLUSIONS Strategies are presented concerning the advantageous use of analytical RAM columns in the analysis of acidic herbicides Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
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Figure 7. Performance of LC/LC using Hisep/C18 columns for the determination of the most polar acidic analyte. SPE extract of reference water containing 12 mg/L DOC and spiked with metsulfuron methyl to a level of 1 µg/L. For LC configurations and conditions, see Table 3.
pesticides in high and medium DOC-containing water samples using coupled-column RPLC (LC/LC) with UV detection. The use of RAM columns as first or/and second column in LC/LC and in the SRM or MRM approach depends on the efficiency, selectivity, and retention capacity of the RAM column. The single-residue method (SRM) approach, tested with mecoprop as a model compound, showed that always satisfactory results were obtained when using one or two different RAM columns in LC/LC. Evidently, the use of an ISRP or a Hisep column as C-1 in combination with a SPS (RAM) or a C18 column appeared to be most efficient. This LC/LC approach fully covers the wide polarity range of the involved analytes. This is clearly
1118 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
demonstrated in Figure 7, showing the LC/LC analysis of a highlevel DOC water sample spiked with the most polar analyte, metsulfuron methyl, at a level of 0.80 µg/L. In comparison to SRM, fewer LC/LC configurations are suitable in the MRM analysis of the heterogeneous group of acidic herbicides. The lower efficiency, high retention capacity, and different separation mechanisms for some of the analytes made Hisep incompatible with the second analytical column. The ISRP/ C18 configuration appeared to be most efficient in the removal of humic acid substances and allowed the determination of analytes to the 0.1 µg/L level in water samples containing up to 12 mg/L DOC. For samples with a medium DOC content (below 7 mg/L), LC/LC with C18/SPS appeared to be a suitable alternative in this field of analysis. The use of RAM columns in the LC/LC configuration is a powerful approach for the analysis of acidic compounds in water samples containing high levels of organic matter. In this way, a rapid, sensitive, and selective multiresidue procedure has been developed for the screening of a heterogeneous group of acidic herbicides, which cannot be done so easily by other techniques such as GC/MS, which would require the application of different derivatization steps, depending on the compounds to be analyzed. Besides, the potential of this approach seems to be very promising for coupling with MS in future applications (LC-LC/ MS or LC-LC/MS-MS), by removing the major part of interfering compounds and facilitating analysis at the ultratrace residue levels. Received for review August 13, 1998. Accepted December 10, 1998. AC980918X
