Original Paper Behavior of Humic Acid as a Micellar Phase in Micellar Electrokinetic Chromatography (MEKC)

Size: px

Start display at page:

Download "Original Paper Behavior of Humic Acid as a Micellar Phase in Micellar Electrokinetic Chromatography (MEKC)"

Emory Morris
5 years ago
Views:

1 Microchim Acta 151, (2005) DOI /s y Original Paper Behavior of Humic Acid as a Micellar Phase in Micellar Electrokinetic Chromatography (MEKC) Solange Leite de Moraes and Maria Olímpia Oliveira Rezende Universidade de S~ao Paulo, Instituto de Química de S~ao Carlos, C.P. 780, S~ao Carlos-SP, Brazil Received January 7, 2005; accepted May 12, 2005; published online July 18, 2005 # Springer-Verlag 2005 Abstract. The possibility of humic acids acting as micellar phase in micellar electrokinetic chromatography was evaluated. We investigated the separation of naphthalene in capillary electrophoresis using various samples of humic acids as micellar phase under different ph conditions, concentrations of humic acid, and temperature. The humic acid samples studied were from different origins including peat, vermicompost and a commercial sample acquired from Aldrich. Methanol was used as a marker for the electroosmotic flow. The results indicate that the formation of micelle depends on the number and nature of the hydrophobic association sites in an aqueous humic acid solution and on the origin and concentration of the humic acid at a defined ph. At lower ph values, the possibility of the humic acid molecule forming pseudomicelles increases due to a combination of neutralized and dissociated charged sites. Key words: Humic acids; pseudomicelle formation; capillary electrophoresis. Micellar electrokinetic chromatography (MEKC) is a mode of capillary electrophoresis (CE) that allows the separation of neutral analytes [1 5]. The separation mechanism by MEKC is based on the distribution of the analytes between a pseudo-stationary micellar phase and an aqueous mobile phase [6]. These two Author for correspondence. sollm@ig.com.br phases move at different rates according to electrokinetic phenomena. Therefore, the utilization of this mode in CE requires the addition of an ionic surfactant at a concentration above its critical micelle concentration (CMC) in buffer solution [7, 8]. The CMC is defined as the concentration below which the surfactant occurs as a monomer in solution and above which practically all additional surfactant forms micelles [9]. Knowledge of the CMC values of the surfactant is important mainly when novel surfactants are used. The surfactant properties of humic acids (HA) have been described in the literature based on the amphiphilic nature of these molecules which can be modeled as detergents presenting both hydrophobic and hydrophilic sites which are responsible, for example, for the enhancement of the solubility of organic solutes in water or the lowering of the water surface tension [10 12]. The ability of such solutes to partition into these hydrophobic domains depends on a range of solution conditions, including the concentration of HA and other solutes, and the nature of the cationic species present in solution [13 18]. These domains, which can be looked upon as intramolecular aggregates, are referred to as pseudomicelles because of their similarity to detergent micelles with regard to the structure and the polar character [19 21]. It was found that HA molecules consist of a long flexible polymer-like structure and coil creating regions that are like knots on a string

2 116 S. L. de Moraes and M. O. Oliveira Rezende [22]. The interior of this region is relatively hydrophobic, while the exterior is more hydrophilic because the HA molecule, which requires neutral or basic conditions for its solubilization, contains carboxylic and phenolic groups that are negatively charged in these solutions [21 24]. According to Piccolo and Conte, the polydispersity of HA is due to the self-assembly of different small molecules into supramolecular structures of different sizes. HAs behave like supramolecules that are able to form micelles and might form supramolecular ensembles with others compounds [25]. There are several methods for the determination of the CMC including electrical conductivity, surface tension, spectrophotometry, cyclic voltammetry and capillary electrophoresis [26 38]. By means of the capillary electrophoresis technique, Jacquier and Desbene [39, 40] demonstrated the evolution of effective electrophoretic mobility to be a function of surfactant concentration, since a sharp change in the slope was observed at the CMC. They observed that the electrophoretic mobility of naphthalene in borate buffer solution containing sodium dodecyl sulfate molecules (SDS) at a concentration below the CMC was very small, but the mobility increased markedly with the increase in surfactant concentration above the CMC. In this study, the micellar character of HA is investigated and thus the possibility of using it as pseudomicellar phase in MEKC. Experimental Humic Acids The HA samples evaluated in this study were HA extracted from peat (PHA), cm depth from Mogi-Guac u River bank, S~ao Paulo State, Brazil; HA obtained from vermicompost (VHA) originating from animal manure and treated with Eisenia foetida or Lumbriculus rubellus; and a commercial HA obtained from Aldrich (AHA), lot N KN. Extraction and purification of HA samples from peat and vermicompost were carried out according to a method adapted from the International Humic Substance Society (IHSS) [28, 29] based on acid and basic extractions followed by purification with successive elution through cationic and anionic resins. The nature and arrangement of the functional groups in the HA samples were discussed on the basis of FTIR spectroscopy. The infrared spectra were recorded from 4000 to 400 cm 1,usinga 4cm 1 resolution, on a Bomem MB-series 102 spectrometer. The samples were pressed with dried KBr pellets at a ratio of approximately 1:200 w=w. Chemicals and Solutions All reagents used were of analytical grade and acquired from Merck or Aldrich. The naphthalene was obtained from Aldrich. Deionized water obtained with a Millipore system was used to prepare all the solutions, standards and electrolytes. The electrolytes utilized were 20 mmol L 1 borate buffer of ph 9.3, 20 mmol L 1 carbonate buffer of ph 9.3, 20 mmol L 1 phosphate buffer of ph 7.0 and 20 mmol L 1 acetate buffer of ph 5. The standard solution of naphthalene had a concentration of 20 mg L 1 in methanol/water (50:50 v=v). The HA solutions had a concentration of 200 mg L 1 and were prepared directly in the running electrolyte. This stock solution was diluted to obtain the desired concentrations. An ultrasonic bath was applied for 3 5 minutes to enhance dispersion (Branson ultrasonic). Apparatus Separations were performed on a Hewlett Packard 3D Capillary Electrophoresis System equipped with a diode array detector (DAD). Uncoated fused silica capillary (50 mm i.d., 56 cm length to the detector, 64.5 cm total length) was obtained from Hewlett Packard. The samples were injected by means of pressure (50 mbar1 s), and a voltage of 20 kv was applied at a temperature of 25 C. The signals were recorded with UV-Vis detection at 214 nm. Prior to each analysis and in between determinations using different electrolytes, the capillary was flushed with 1 mol L 1 NaOH for 5 min, then with deionized water for 10 min and finally with the electrolyte for 15 min. Between each run, the capillary was flushed with 1 mol L 1 NaOH solution for 2 min, then with water for 2 min, and finally with the running electrolyte for 3 min. Changes in the analytical conditions are specified in the Figures. Results and Discussion The HAs chosen for this study were selected because of their differences in aromatic character. Figure 1 presents the FTIR spectra for all the HA samples. The FTIR spectra are similar, featuring small differences only. All spectra present a band in the range of 3500 to 3300 cm 1, which is assigned to OH stretching of the carboxylic groups, phenolic, alcohol or bonded water. The band in the region of cm 1 is attributed to the C H stretching of aliphatic groups. The band around 1720 cm 1 is attributed to the C¼O stretching of the COOH groups. For the 1720 cm 1 peak and that around 1650 cm 1, VHAs present bands of small intensities. This indicates that VHAs have less carboxyl groups than PHAs and AHAs. The band at 1650 cm 1 is assigned to the asymmetrical COO stretching of carboxylic groups and to aromatic C C stretching. The band at 1510 cm 1 is assigned to the stretching of the C¼C linkages of the aromatic groups. The band around 1460 cm 1 present in the VHA spectrum is due to C H angular aliphatic deformation. The band at 1400 cm 1 corresponds to the symmetrical COO stretching. The band at 1250 cm 1 is produced by CO stretching of phenolic groups and it is more intense for VHA, indicating that the acidity of this structure can be attributed to the phenolic compounds.

3 Humic Acid as a Micellar Phase in MEKC 117 Fig. 1. FTIR spectra of humic acid samples: humic acid from vermicompost (VHA), humic acid from peat (PHA) and humic acid from Aldrich (AHA) Table 1. Solubility of humic acids in different buffers Humic acid (50 mg L 1 ) Acetate ph 4.6 ph 5.0 Phosphate ph 7.0 Carbonate ph 9.3 AHA S S S S S PHA I I S S S VHA I RS S S S All buffer solutions have a concentration of 20 mmol L 1. S Soluble, I insoluble, RS relatively soluble. Borate ph 9.3 In order to investigate the HA behavior as pseudomicellar phase in MEKC, we first investigated the effect of the buffer solution on the solubility of the different HAs used (Table 1). The selection of these buffers was made on the basis that borate, phosphate and carbonate solutions are buffers commonly used in MECK. Because the HA structure contains a large number of carboxyl and phenolic groups, the degree of dissociation depends largely on the ph of the solution. It is noteworthy that only AHA shows solubility in ph<7. For ph values above 7, all HAs are soluble. At elevated ph, the mutual repulsion of the negatively charged sites causes a stretched configuration for certain HAs; this means that there is a lower tendency to form pseudomicelles. It implies that the hydrophobic domains in the HA molecules are relatively unstructured. At lower ph, the possibility of the HA molecules forming pseudomicelles increases due to a combination of neutralized and dissociated charged sites. Study of the Micellisation of Humic Acids by MEKC The separation of neutral compounds by MEKC occurs due to the partition of analyte between the micelle and the surrounding aqueous phase. Thus, if humic acid molecules form pseudomicelles in the buffer system, it is possible to visualize the separation of neutral compounds in the electropherogram. In this study, the micellisation process and the determination of CMC is a key parameter in the utilization of humic acids as a pseudomicellar phase in MEKC. However, it is important to choose the neutral compounds carefully. Naphthalene was chosen as a neutral compound because it can be incorporated into micelles and it is easily dissolved in buffer solution. These criteria are necessary to check the formation of micelle during the experiments in MEKC. The dissolution of the naphthalene in aqueous solution is possible in a mixture containing water and methanol (50:50 v=v). Figure 2 shows the mobility of naphthalene as a function of the concentration of PHA added to 20 mmol L 1 sodium carbonate, ph 9.3. Addition of PHA to the buffer solution did not promote the

4 118 S. L. de Moraes and M. O. Oliveira Rezende Fig. 2. Electrokinetic chromatograms of naphthalene as a function of the concentration of PHA in 20 mmol L 1 sodium carbonate, ph 9.3. Conditions: 20 kv, 1 s, 214 nm mobility of naphthalene. In this case, there was no formation of humic pseudomicelles or the concentration of the HA was below the CMC. This means that the neutral compounds moved along with the electroosmotic flow (EOF) and separation did not occur. The same electrolyte system (20 mmol L 1 sodium carbonate, ph 9.3) was used for VHA and AHA at a concentration of 50 mg L 1, cf. Fig. 3. The electropherogram shows that the naphthalene moved together with the EOF. It is assumed that pseudomicelles are not formed because at higher ph values all the carboxylic and phenolic groups of the HA are completely Fig. 3. Electropherogram of naphthalene in 50 mg L 1 AHA (a) and in 50 mg L 1 VHA (b) in 20 mmol L 1 sodium carbonate, ph 9.3. Conditions: 20 kv, 1 s, 214 nm Fig. 4. Electropherogram of naphthalene as a function of the concentration of PHA in 20 mmol L 1 sodium phosphate, ph 7.0. Conditions: 20 kv, 25 C, 1 s, 214 nm

5 Humic Acid as a Micellar Phase in MEKC 119 Fig. 5. Electropherograms of naphthalene in 20 mmol L 1 Conditions: 25 C, 25 kv, 1 s, 214 nm sodium phosphate buffer, ph 7, with different concentrations of VHA. dissociated, thus decreasing the tendency to form psedomicelles, interfering with the amphiphilic nature of HA and consequently compromising its ability of aggregation. Under these ph conditions, the HA could be totally hydrophilic. Therefore, the carbonate buffer solution is not suitable to promote the formation of humic psedomicelles due to the highly anionic environment. Then the other buffers and ph systems were tested. A decrease in the solution ph reflects in the gradual neutralization of the acidic sites, increasing the amphiphilic character of the HA molecule. It is not possible to reach the CMC of PHA at a concentration below 16 mg L 1 because there is no peseudomicelle formation, while above 60 mg L 1 bubbles begin to form, obscuring the visualization of the naphthalene peak (Fig. 4). Figure 5 shows a partial separation between naphthalene and EOF peaks. The results indicate that VHA is more suitable to promote micellisation than PHA. According to the FTIR data, VHAs present less carboxyl groups than PHA, which implies that VHAs present less negative charges under the experimental conditions. This characteristic improves the formation of micelles in solution. The influence of ph in the range of was evaluated (Fig. 6). Partial separation of naphthalene occurred at ph 7.0. Tiny variations in ph change the micellar environment, and consequently the retention of naphthalene in the humic pseudomicelle is also changed. The effect of temperature on the formation of humic pseudomicelles was examined. Temperature has a significant effect on the supramolecular organization of the surface-active compounds in aqueous solutions. Toerne et al. [24] showed that an increase in temperature can influence the tendency of HA aggregation, changing their detergent characteristics. However, in this study temperature values above 25 C did not influence the separation of naphthalene or the aggregation of HA, as shown in Fig. 7. VHA was not suitable for the intended purpose. At ph values above 5, carboxylic groups are deprotonated and strong negative charge repulsions between carboxylate groups can occur. It is not possible to study the VHA sample below ph 7 because the sample is not completely dispersed. Formation of Humic Pseudomicelle Figure 8 illustrates the mobility of naphthalene in 50 mg L 1 sodium acetate buffer and 50 mg L 1 AHA. Under these conditions the formation of humic pseudomicelle is possible because AHA has similar relative proportions between carboxylic and carboxylates groups. At lower ph values, the carboxylic groups present in HA are protonated, reducing the charge

6 120 S. L. de Moraes and M. O. Oliveira Rezende Fig. 6. Electropherograms of naphthalene in 20 mmol L 1 sodium phosphate buffer þ 50 mg L 1 VHA, different ph values. Conditions: 25 C, 25 kv, 1 s, 214 nm Fig. 7. Effect of temperature on naphthalene separation. Conditions: 20 mmol L 1 sodium phosphate buffer þ 50 mg L 1 VHA, ph 7, 25 kv, 1 s repulsion and increasing the tendency of HA to form psedomicelles and consequently the ability of adsorption for nonpolar molecules such as naphthalene. This indicates that AHA acts as a surfactant around ph 5.0 and could be utilized as pseudomicellar phase. The reproducibility was assessed by injecting 20 mg L 1 naphthalene over several days (three injections per day). The relative standard deviation was 6.8%. The migration time repeatability for naphthalene was 1.4%. The differences between the HA samples depend on the amount of acid groups in the HA that may be neutralized depending on the ph and on the amount of HA leading to the formation of humic pseudomicelles. AHA has a similar amount of hydrophobic and

7 Humic Acid as a Micellar Phase in MEKC 121 Fig. 8. Electropherogram of naphthalene in 50 mmol L 1 sodium acetate buffer, ph 5.0 þ 50 mg L 1 AHA, conditions: 20 kv, 25 C, 1 s hydrophilic sites compared to PHA or VHA, implying that AHA has better surfactant characteristics than PHA or VHA. Conclusions Capillary electrophoresis is an adequate technique for assessing the possibility of micelle formation by HA. The ph of the solution in which the HA is dissolved is an important parameter in the formation of humic pseudomicelles because their conformational behavior and aggregation characteristics are strongly influenced by acidic conditions. The parameters discussed in this paper show that, depending on molecular features such as aromaticity and aliphatic content, different HAs exhibit different microorganizations and that the micellar characteristics depend on this microorganization. Acknowledgements. The authors wish to thank the Brazilian financial agencies CNPq and FAPESP for financial support (fellowship to S.L.M., 00/ ). The CE equipment was acquired thanks to financial support from FAPESP. References [1] Tavares M F M (1997) Química Nova 20: 493 [2] Weinberger R (1993) Pratical capillary electrophoresis. Academic Press, New York, p 305 [3] Kuhn R, Hoffstetter K S (1993) Capillary electrophoresis: principles and practice. Springer, Berlin Heidelberg New York Tokyo, p 135 [4] Ewing G W (1997) Analytical instrumentation handbook. Marcel Dekker, New York, p 352 [5] Wendy M N, Tang Q, Harrata A K, Lee C S (1996) J Chromatogr A 749: 219 [6] Tagliaro F, Manetto G, Crivellente F, Smith F P (1998) Forensic Sci Int 92: 75 [7] Altria K D (1999) J Chromatogr A 856: 443 [8] Martinez R C, Gonzalo E R, Cordero B M, Pavon J L, Pinto C G, Lespada E F (2000) J Chromatogr A 902: 251 [9] Maniasso N (2001) Química Nova 24: 87 [10] Piccolo A (2001) Soil Sci 166: 810 [11] Hayes M H B, Clapp C E (2001) Soil Sci 166: 723 [12] Wershaw R (1998) Environmental Sci Technol 27: 14 [13] Fetsch D, Hradilová M, Mendez E M P, Havel J (1998) J Chromatogr A 817: 313 [14] Schmitt Ph, Freitag D, Garrison A W, Schiavon N, Kettrup A (1997) Chemosphere 35: 55 [15] Yates L M, Wandruszka R V (1999) J Soil Sci Soc Am 63: 1645 [16] Piccolo A, Nardi S, Concheri G (1996) Chemosphere 33: 595 [17] Wandruszka R V, Engebretson R (1997) Talanta 44: 805 [18] Ferreira J A, Nascimento O R, Martin L (2001) Env Sci Technol 35: 761 [19] Morra M L, Corapcioglu M O, Wandruszka RV, Marshall D B, Topper K (1990) J Soil Sci Soc Am 54: 1283 [20] Kopinke F D, Georgi A, Mackenzie K (1997) Anal Chim Acta 355: 101 [21] Engebretson R R, Wandruszka R V (1998) Environment Sci Technol 32: 488 [22] Engebretson R R, Wandruszka R V (1999) Environment Sci Technol 33: 4299 [23] Engebretson R R, Amos T, Wandruszka R V (1996) Environment Sci Technol 30: 990 [24] Toerne K, Rogers R, Wandruszka R V (2001) Langmuir 17: 6119 [25] Piccolo A, Conte P (2000) Adv Environment Res 3: 508 [26] Chen Z C, Lin J M, Uchiyama K, Hobo T (2000) Anal Chim Acta 403: 173

8 122 Humic Acid as a Micellar Phase in MEKC [27] Lin C E, Wang T Z, Chiu T C, Hsuch C C (1999) J Resolution Chromatograph 22: 265 [28] Lin C E, Chen M J, Huang H C, Chen H W (2001) J Chromatograph A 924: 83 [29] Lin C E, Lin K S (2000) J Chromatograph A 868: 313 [30] Tesarová E, Tuzar Z, Nesmerák K, Bosáková Z, Gás B (2001) Talanta 54: 643 [31] Moraes S L, Rezende M O O (2004) Química Nova 27: 701 [32] Benito I, García M A, Monge C, Saz J M, Marina M L (1997) Colloids Surf A: Physicochem Eng Aspect 125: 221 [33] Ysambertt F, Vejar F, Parede J, Salager J L (1998) Colloids Surf A: Physicochem Eng Aspect 137: 189 [34] Cimentes A, Bemal J L, Diez-Masa J C (1997) Anal Chem 69: 4271 [35] Ma G, Li G, Xu Y, Wang H, Ye X (1998) Colloids Surf A: Physicochem Eng Aspect 143: 89 [36] Mrestani Y, Neubert R, R uttinger H H (2000) J Chromatograph A 868: 317 [37] Lin C E, Huang H C, Chen H W (2001) J Chromatograph A 917: 297 [38] Gogová K, Maichel B, Gas B, Kenndler E (2001) J Chromatograph A 916: 79 [39] Jacquier J C, Desbene P L (1996) J Chromatograph A 743: 307 [40] Jacquier J C, Desbene P L (1995) J Chromatograph A 718: 167

Capillary electromigration techniques:

Capillary electromigration techniques: CE capillary electrophoresis CZE capillary zone electrophoresis EKC electrokinetic chromatography MEKC/MECC micellar electrokinetic chromatography MEEKC microemulsion electrokinetic chromatography CEC