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New Avenue for Mid-UV-Range Detection of Underivatized Carbohydrates and Amino Acids in Capillary Electrophoresis Cedric Sarazin,†,‡,§,^ Nathalie Delaunay,*,‡,§,^ Christine Costanza,† Veronique Eudes,† Jean-Maurice Mallet,§,||,# and Pierre Gareil‡,§,^ †
Central Laboratory of the Prefecture de Police, 39 bis, rue de Dantzig, 75015 Paris, France Chimie ParisTech, Laboratory of Physicochemistry of Electrolytes, Colloids and Analytical Sciences (PECSA), 75005 Paris, France § UPMC Univ Paris 06, 75005 Paris, France ^ CNRS, UMR 7195, 75005 Paris, France ENS, Laboratory of Biomolecules (LBM), 75005 Paris, France # CNRS, UMR 7203, 75005 Paris, France
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bS Supporting Information ABSTRACT: Capillary electrophoresis (CE) appeared as an interesting alternative to chromatographic methods for carbohydrate analysis, but it can be difficult to implement, because of the lack of easily ionizable functions and chromophore groups. Recently, a promising method was proposed by Rovio et al. for the CE separation under extremely high alkaline conditions of neutral carbohydrates under their alcoholate form and their direct UV detection [Rovio et al. Electrophoresis 2007, 28, 3129 3135; and Rovio et al. J. Chromatogr. A 2008, 1185, 139 144], which is claimed to be due to the absorption of enediolate at 270 nm. Even so, most of the detected compounds in Rovio’s paper (for example, sucrose) cannot give such enediolate, lacking a carbonyl group. In this work, a deeper insight was paid to the understanding of detection mechanism. In effect, unusual detection phenomena were observed in comparing reducing and nonreducing carbohydrate behaviors, which pointed to the existence of photochemical reactions in the detection window. A more systematic study of the influence of many parameters (carbohydrate nature, electrolyte pH, residence time in the detection window, and capillary diameter) was undertaken. In addition to this, most of this work was performed under cathodic (reversed) electro-osmotic flow conditions (using Polybrene-modified capillaries), to obtain much faster separations than under Rovio’s conditions. This study also opens up new avenues for the detection in mid-UV range of non-UV-absorbing compounds bearing reducing moieties, such as amino acids.
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arbohydrate analysis remains challenging, because of the great molecular diversity, the presence of several structural isomers, and the lack of easy-to-implement and sensitive detection methods. Currently, methods used for the separation of carbohydrates include thin-layer chromatography, 1 gas chromatography,2,3 and liquid chromatography (LC).4 6 Most often, LC methods used normal phase (HILIC),7 reversephase,8 or anion-exchange modes8,9 with direct low-UV-range or refractive index detections4 and most often mass spectrometry, ultraviolet (UV), or fluorescence detection after precolumn derivatization.10 12 More recently, important developments were carried out with the coupling between high performance anion-exchange chromatography and pulsed-amperometric detection.4,9 Currently, capillary electrophoresis (CE), with its high separation efficiency, low reagent consumption, and speed, appears to be an interesting alternative to chromatographic methods, r 2011 American Chemical Society
especially for sulfated and carboxylated carbohydrates,4,12 16 but the analysis of so-called neutral carbohydrates can be more difficult to implement, because of the absence of easily ionizable functions (other than anomeric hydroxyl group) and chromophore groups (see Table S-1 in the Supporting Information), and the high hydrophilic character of these compounds, which makes the use of micelles inoperable. Different strategies are available to circumvent these different drawbacks. Complexation with borate anions at pH comprised between 8.0 and 10.0 has long been used in carbohydrate analysis and exploited in CE with direct UV detection at 195 nm;17,18 however, this low wavelength does not favor selective detection, generates interferences, and suffers from poor sensitivity. As of today, the most widely used CE Received: May 27, 2011 Accepted: August 1, 2011 Published: August 01, 2011 7381
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Analytical Chemistry methods have involved the labeling of carbohydrates by a chromophore or fluorophore through a precolumn derivatization step.19 25 Limits of detection in the micromolar range were obtained; however, these methods suffer from complex and timeconsuming derivatization steps. Alternatively, indirect UV detection of underivatized carbohydrates can be carried out at extremely high pH (12 14) with the use of an anionic chromophore, such as 2,6-pyridine dicarboxylate26 28 (detection wavelength, 275 nm), 1-naphtyl acetate29 (detection wavelength, 220 nm), riboflavin30 (detection wavelength, 254 nm), tryptophan31 (detection wavelength, 280 nm), or sorbate32 34 (detection wavelength, 254 nm). Nevertheless, the limits of detections were observed to be within the range between 20 and 1000 μM, because of the deleterious presence of hydroxyl anion at high concentration. More recently, a new method described by Rovio et al. has allowed direct UV detection under extremely high alkaline conditions (130 mM NaOH, 36 mM Na2HPO4).35,36 According to the authors, carbohydrates were detected under their enediolate forms at 270 nm; however, this explanation cannot be valid for most of the detected compounds (for example, sucrose), which lack a carbonyl group. In this work, we revisited the experimental conditions of this approach, with respect to detection performances. In effect, unusual detection phenomena were observed in comparing reducing and nonreducing carbohydrate behaviors, which pointed to the existence of photochemical reactions in the detection window. A more systematic study of the influence of several parameters, including carbohydrate nature, background electrolyte (BGE) pH, residence time in the detection window, and capillary diameter, was undertaken. This study brings a deeper insight into the understanding of the physicochemical mechanisms involved in this new direct UV detection mode and opens up new avenues for detection in the mid-UV range of non-UV-absorbing compounds bearing reductive moieties, such as amino acids and peptides. In addition to this, most of this work was performed under cathodic (reversed) electro-osmotic flow conditions (using Polybrene-modified capillaries), to obtain much faster separation than under Rovio’s conditions.
’ EXPERIMENTAL SECTION Standards and Solutions. Polyols (glycerol, erythritol, xylitol, mannitol, sorbitol, and myo-inositol), cyclodextrins (R-CD, βCD, γ-CD, HP-R-CD, and heptakis(2,3,6-tri-O-methyl)-β-CD), amino acids (arginine, proline, glycine), naphthalene sulfonic acid (internal reference), and δ-gluconolactone were purchased from Sigma Aldrich (L’Isle-d’Abeau, France). Glucose, lactose, sucrose, fructose, and N-dimethylformamide (DMF) were obtained from VWR (Fontenay-sous-Bois, France). Individual solutions of each compounds were prepared weekly at concentrations of 1000 mg L 1 by dissolution in ultrapure water delivered by a Direct-Q3 UV system (Millipore, Molsheim, France). BGEs for CE were prepared from 1 M NaOH solution (Carlo Erba, Val-deReuil, France) and Na2HPO4 2H2O (Sigma Aldrich). The final BGE was composed of 130 mM NaOH (pH 13.1). Hexadimethrine bromide (HDMB), which was used as a coating agent, was purchased from Sigma Aldrich. HDMB at 0.1 g/100 mL was prepared by dissolving the appropriate amount in ultrapure water. Apparatus. Detection studies and separations were carried out with a Beckman Coulter P/ACE MDQ system (Villepinte, France) equipped with a diode array detector (DAD) set at 270 nm (measurement wavelength) and 350 nm (reference
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wavelength) and with bandwidths set at (10 nm and (40 nm, respectively. Instrument control and data acquisition were performed using Beckman 32 Karat software. Corrected areas were calculated as the peak-area-to-migration-time ratio (Ai/tMi). For comparison purposes, CE systems (Agilent Technologies, Models HP3DCE and HP 7100), equipped with DADs (Massy, France) were also used. A PHM210 Meterlab pH-meter, (Metrohm, Villebon Courtaboeuf, France), equipped with a high-alkalinity electrode was used to measure BGE pH. A Varian Cary 100 UV spectrometer, purchased from Bioserv (Thiais, France), was used to monitor the UV spectra. Electrophoretic Conditions. Electrophoretic separations were performed using 60-cm Polymicro bare fused-silica capillaries with internal diameters of 50, 75, or 100 μm purchased from Photonlines (Marly-Le-Roi, France). A window was created for the UV detection on the capillary at 10 cm from the anodic end, and detection windows of 200 and 800 μm aperture lengths were compared. Before first use, capillaries were conditioned by successive flushing with 1 M NaOH, 0.1 M NaOH, ultrapure water, HDMB solution, and finally BGE, each under 2.8 bar for 3 min (12 capillary volumes). Between each run, because of the high BGE pH, the HDMB coating was regenerated by percolating the solution under 2.8 bar for 1 min (4 capillary volumes) and, afterward, the capillary was re-equilibrated with BGE under 2.8 bar for 3 min (12 capillary volumes). Injections were performed hydrodynamically under 50 mbar for 5 s (0.7% of the capillary volume). Unless specified, separations were run at 20 C under 16 kV. BGE was renewed between each run.
’ RESULTS AND DISCUSSION Rovio et al. demonstrated that neutral carbohydrates can be separated in bare fused-silica capillaries within less than 30 min and detected in a new mode by direct UV absorbance at 270 nm, applying extremely high pH conditions. Better resolutions were obtained with a BGE composed of 130 mM sodium hydroxide and 36 mM Na2HPO4, as compared with a BGE composed of 130 mM sodium hydroxide only.35,36 We compared this (NaOH + phosphate)-based BGE with a BGE only composed of 130 mM NaOH and similar analytical performances were obtained in our case, even for resolutions (cf. the Supporting Information). Therefore, this NaOH-based BGE, which is simpler to prepare and provides slightly faster separations, was selected for this study. Interestingly, a decrease in peak corrected areas was observed with a decrease in separation voltage, which suggested the existence of a photochemical reaction in the detection window (see Figure S1-A in the Supporting Information). This assumption was corroborated by the fact that the sucrose UV spectrum in 130 mM NaOH, which was acquired with a classical grate spectrophotometer, did not exhibit any absorbance band at ∼270 nm (see Figure S1-B in the Supporting Information). No absorbance band (and no peak, either) was observed in our separation conditions (carbohydrate concentrations, capillary length, etc.) either with Agilent HP3DCE or HP 7100, which are equipped with a deuterium-lamp-based DAD detector. Moreover, when the Beckman Coulter P/ACE MDQ system was equipped with a fixed-wavelength UV detector (mercury lamp) at 280 nm, no signal was detected either. A particular feature of the Beckman DAD is that the incident UV light (from a deuterium lamp with a 160 400 nm emission continuum) does not go past a filter before reaching the sample, which means that 7382
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Analytical Chemistry
Figure 1. (A) Variation of absorbance front intensities as a function of residence time in the separation window for sucrose ((9) 800 μm aperture and (0) 200 μm aperture) and for DMF ((b) 800 μm aperture and (O) 200 μm aperture). (B) Variation of sucrose absorbance front intensities normalized by capillary inner diameter (25, 50, 75, and 100 μm) as a function of residence time in separation window. A bare fused-silica capillary, 50 μm id (A), 25, 50, 75, and 100 μm (B) 60 cm (UV detection at 10 cm). Capillary initially filled with 130 mM NaOH, then hydrodynamic short-end introduction of 0.05 mM sucrose + 0.01% (v/v) DMF in 130 mM NaOH solution. Direct UV detection at 270 nm (sucrose) and 210 nm (DMF).
the sample is irradiated by very short wavelength UV light (
