INDIRECT DETECTION IN ION-EXCLUSION CHROMATOGRAPHY
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1 ACTA CHROMATOGRAPHICA, NO. 12, 2002 INDIRECT DETECTION IN ION-EXCLUSION CHROMATOGRAPHY B. K. Głód Medical Research Centre, Polish Academy of Sciences, Laoratory of Experimental Pharmacology, ul. Pawińskiego 5, Warszawa, Poland ABSTRACT Indirect detection is widely applied in ion-exchange chromatography and capillary electrophoresis. Equations are derived which predict its application in ion-exclusion chromatography is also possile. Two detection systems are discussed, photometric and conductometric. With the latter it was possile to present results from direct and indirect detection on one chromatogram, depending on the relative limiting conductivities (diffusion coefficients) of solute and ackground electrolyte. INTRODUCTION Ion-exclusion chromatography (IEC) finds application in the analysis of weak and medium-strength acids [1 3]. Detection techniques include direct UV asorance [3,4], refractive index (RI) [5,6], potentiometric [7,8], conductivity [9], and mass spectrometry [10]. Direct UV asorption at 210 nm is the detection method most frequently used for volatile fatty acids. The detection limit is rather high, ecause the solute lacks a chromophore. Because of the sensitivity of the detector to even trace amounts of contaminants [4], it cannot e used for complex samples without suitale sample pretreatment, owing to interference prolems. The conductometric detector, the detector most commonly used in IEC, is also characterized y high sensitivity ecause the dissociation of acids is small, and is also depressed y any uffer present. Although aliphatic acids can e separated in IEC with pure water as moile phase, resolution is poor ecause of the fronting peaks (leading tails) which result ecause the degree of dissociation (ionization) of the acids is not controlled, an effect which is increased y hydrophoic asorption of the undissociated forms of the acids [2]. Addition of modifiers to the moile phase has een proposed to overcome these prolems; such modifiers include:
2 (i) dilute strong acid, which acts as a uffer [4] (ii) ion-pairing reagent, which reduces the effective charge on the solute [8]; and (iii) low concentrations of polyalcohols and sugars, which increase the hydrophilicity of the cation-exchange resin [11]. These conditions are suitale for ulk property detectors, e.g. potentiometric [8], conductivity [11], or electrokinetic [12]. Indirect photometric detection has een already used successfully in ion chromatography [13] and in capillary electrophoresis [14]. For this mode of detection an asoring reagent with the same electric charge as the analysed solute and with a high molar asorption coefficient is added to the moile phase. In IEC indirect photometric detection has een applied to the analysis of volatile fatty acids [15]. The aim of this paper is to discuss theoretical prediction of indirect photometric and conductometric detection in IEC. EXPERIMENTAL Instrumentation Measurements were performed y means of a chromatograph comprising a GP40 gradient pump, AD20 asorance detector, ED40 electrochemical detector, LC30 chromatography oven, and PeakNet 5.1 chromatographic data acquisition and analysis software, all from Dionex (USA). Samples were separated on a 300 mm 7.8 mm i.d. TSKGel SCX(H + ) column from TosoHaas (Japan). Manual injection was performed with a 100 µl syringe (Scientific Glass Engineering, Ringwood, Australia). Reagents All reagents (BDH, Poole, UK; Fluka, Buchs, Switzerland; and Ajax Chemicals, Auurn, Australia) were of analytical-reagent grade and were used without further purification. Water was passed through Millipore (Bedford, USA) Milli-RO4 and Milli-Q water purification systems. Moile phases were filtered through a 0.22-µm memrane filter
3 RESULTS AND DISCUSSION Changes in the Concentration of the Background Electrolyte Indirect detection is ased on measurement of changes of the concentration of the ackground (proe) electrolyte, the electric charge of which should e the same as that of the solute. This means that for acid analysis the ackground electrolyte should also e acid a dilute solution of a strong, completely dissociated acid is usually used. Optimum conditions for detection are not always optimum for separation, however. Changes in the concentration of the ackground electrolyte can e calculated from a knowledge of the dissociation constant and the mass-conservation equation. For dilute solutes we can assume that the concentration of hydrogen ions is constant throughout the column, i.e. [H + ] = const. Under these conditions, outside the region of the solute (chromatographic peak) the electrical neutrality condition can e expressed as: [H + ] = [B ] (1) Injection of the test acidic solute, HR, on to the chromatographic column reduces the concentration of the dissociated form of ackground electrolyte. In accordance with the electrical neutrality condition at the peak maximum in the moile phase: [H + ] = [B ] max + [R ] (2) where B and R denote the dissociated forms of the ackground electrolyte and solute, respectively. Eqs (1) and (2) can e easily transformed to: [R ] = [B ] [B ] max (3) Conductometric Detection The conductivity, G, of the dilute electrolyte is a function of its limiting ionic conductivities, λ i, the stoichiometric coefficients, ν i, the valence, z i, the surface area of the electrodes, A, and the distance etween them, l: G = (ν + c + z + λ + + ν c z λ )A/l (4) For a monovalent monovalent acid eq. (4) can e rewritten: G = (λ B [B ] + λ H+ [H + ])A/l (5) At the peak maximum this conductivity depends also on the solute concentration:
4 G max = (λ B [B ] max + λ H+ [H + ] + λ R [R ])A/l (6) The chromatographic peak height can e calculated as the difference etween the conductivities otained y use of eqs (5) and (6): G = (λ B [B ] max + λ R [R ] λ B [B ])A/l = = (λ R [R ] λ B [R ])A/l (7) From eqs (3) and (7) we finally otain: G = (λ R λ B )[R ]A/l (8) From eq. (8) it is apparent that the height of the chromatographic peak should e directly proportional to the concentration of dissociated form of the analysed acid. It should also e possile to record results from oth direct and indirect conductometric detection on one chromatogram. Peak direction depends on the relative limiting ionic conductivity of the solute compared with that of the ackground electrolyte. Photometric Detection According to the Lamert Beer law, the ackground asorance, A, of the acid, HB, used as uffer can e expressed as: A = lε B [B ] + lε HB [HB] (9) where l denotes the length of detector cell and ε B and ε HB are, respectively, the molar asorption coefficients of the dissociated and undissociated forms of acid. Asorption at the peak maximum, on the asis of eqs (1) and (9) is descried y: A max = lε B [B ] max + lε HB [HB] max (10) The height of the chromatographic peak can e otained from eqs (9) and (10): A = lε B [B ] max + lε HB [HB] max lε B [B ] lε HB [HB] (11) By comining eq. (11) with eq. (3), using the definition of the dissociation constant, and assuming that the concentration of the undissociated form of the solute acid is constant throughout the chromatographic peak ([HB] max = [HB]), we otain: A = lε B [R ] (12)
5 Estimation of [R ] The mass conservation equation for the solute acid can e given as: c i V i = ([R ] + [HR])V P (13) where V P (= c i V i /c max ) denotes the volume of the peak maximum. From eq. (13) and the definition of the dissociation constant we otain: [R ] = c i V i /V P (1 + [H + ]/K a ) = c i V i K a /V P (K a + [H + ]) (14) After equiliration of the column the mass alance of the ackground electrolyte can e presented in the form: c = [B ] + [HB] (15) Because the concentration of the solute is usually small, and ecause the uffer suppresses its dissociation, it can e assumed that the concentration of hydrogen ions is constant throughout the column. Outside the chromatographic peak the concentration of the dissociated form of the ackground electrolyte is equal to the concentration of hydrogen ions, as descried y eq. (1). After resolving the quadratic equation otained from eqs (13) (15) we finally otain: [R ] = V P (2K a c V K K 2 a + 4K c where the peak volume is descried y: + i i K ) (16) V P = V R (2π/N) 1/2 (17) Eqs (8), (12) and (17) can e used to predict results from indirect conductometric and photometric detection. Chromatographic peak height should e directly proportional to the amount of the solute acid and should increase asymptotically with increasing dissociation constant. The largest peaks should e otained for completely dissociated acids (anions). Peak height, on the other hand, decreases with increasing uffered acid dissociation constant and concentration. Experimental Verification Ion-exclusion chromatography is used mainly for analysis of weak acids. Although pure water is frequently used as moile phase it has the disadvantages of poor resolution and fronting peaks [2]. To improve peak symmetry dilute, strong acids, e.g. sulphuric, are added to the moile phase. This stailizes the degree of ionization of the acid analysed. From
6 the discussion aove it ecame apparent that indirect photometric detection in ion-exclusion chromatography could e achieved y adding aromatic acids to the moile phase [15]. Preliminary results from indirect conductometric and photometric detection in IEC are presented elsewhere [16]. Aliphatic acids, including oxalic, malonic, formic, and acetic, were selected as test compounds. The IEC separation of these acids is presented in Fig. 1. A 1 mm solution of phthalic acid was used as moile phase. Two detectors, conductometric (A) and UV-300 nm (B) were serially connected to the column. Whereas indirect photometric detection was achieved for all the test solutes, indirect conductometric detection was achieved for acetic acid only. For the other acids analysed direct conductometric response was oserved. Finally, it should e also mentioned that phthalic acid affects the retention of the acid test solutes y competing for hydrophoic adsorption sites. Fig. 1 Ion-exclusion chromatograms otained from (1) oxalic, (2) malonic, (3) formic, and (7) acetic acids with conductometric (A) and UV-300 nm (B) detection. The moile phase was 1 mm phthalic acid, the flow rate 0.5 ml min 1, and the volume injected 20 µl
7 CONCLUSIONS The possiility of using indirect detection in ion-exclusion chromatography has een confirmed. Solutions of aromatic acids can e used as moile phases. Derived equations and experimental data show that indirect conductometric detection can e achieved for solutes for which the ionic conductivities (diffusion coefficients) are smaller than that of the ackground electrolyte. Direct detection (positive peaks) is oserved when the ionic conductivities are larger than that of the electrolyte. Quantitative correlation was otained etween derived equation and experimental results. Phthalic acid, used as indirect detection proe, led to reduced retention of aliphatic fatty acids ecause of competition for adsorption sites. REFERENCES [1] B. K. Głód, G. A. Czapski, and P. R. Haddad, Trends Anal. Chem., 19, 492 (2000) [2] B. K. Głód, Neurochem. Res., 22, 1237 (1997) [3] B. K. Głód, Acta Chromatogr., 7, 13 (1997) [4] E. Papp and P. Keresztes, J. Chromatogr., 506, 157 (1990) [5] C. W. Klampfl, W. Bucherger, G. Rider, and G. K. Bone, J. Chromatogr., 770, 23 (1997) [6] G. Iwinski and D. R. Jenke, J. Chromatogr., 392, 397 (1987) [7] K. Kihara, S. Rokushika, and H. Hotano, J. Chromatogr., 410, 103 (1987) [8] B. K. Głód, P. W. Alexander, P. R. Haddad, and Z. L. Chen., J. Chromatogr., 699, 31 (1995) [9] S. R. Bachman and M. E. Peden, Water Air Soil Pollut., 33, 129 (1987) [10] L. Yang, R. E. Sturgeon, and J. W. H. Lam, J. Anal. Atomic Spectr., 16, 1302 (2001) [11] K. Tanaka, K. Ohta, J. S. Fritz, Y. S. Lee, and S. B. Shim, J. Chromatogr., 706, 385 (1995) [12] B. K. Głód and W. Kemula, J. Chromatogr., 366, 211 (1986) [13] H. Small and T. E. Miller Jr, Anal. Chem., 54, 462 (1982) [14] E. Butler-Roerts and D. T. Eash, J. Liq. Chromatogr. Related Technol., 22, 2101 (2001) [15] Z. L. Chen, B. K. Głód, and M. A. Adams, J. Chromatogr. A, 818, 61 (1998) [16] B. K. Głód and P. R. Hadad, in preparation
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