Solvent Effect

Since the mobility is strongly affected by the viscosity of the solvent and the dissociation constants depend on the dielectric constant of the solvent, the use of a nonaqueous solvent or a mixed solvent may improve electrophoretic separations (the solvent effect). The other advantage of the use of a nonaqueous systems is that it enables the analysis of substances with low water solubility. Many solvents have been successfully applied for isotachophoresis, such as methanol, ethanol, dioxane, acetone, propanols, di-methylformamide, etc. The migration behaviour in a nonaqueous solvent is very different from that in an aqueous solvent. The use of a mixed solvent

Figure 6 Simulated and observed isotachopherograms of 19 anions, AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, IMP, IDP, ITP, UMP, UDP, UTP, pyrophosphoric acid (P2O7), triphosphoric acid (P3O10), tetraphosphoric acid (P4O12) and propionic acid (Prop) at pHL = 4.7 (creatinine buffer). The terminator was pelargonic acid (Pel). Current, 75 mA. Sample amounts, 1.2-3 nmol. The leading electrolyte contained 0.2% hydroxypropylmethylcellulose (HPMC). Buffer, creatinine.C'L, concentration of leading ion.

Figure 6 Simulated and observed isotachopherograms of 19 anions, AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, IMP, IDP, ITP, UMP, UDP, UTP, pyrophosphoric acid (P2O7), triphosphoric acid (P3O10), tetraphosphoric acid (P4O12) and propionic acid (Prop) at pHL = 4.7 (creatinine buffer). The terminator was pelargonic acid (Pel). Current, 75 mA. Sample amounts, 1.2-3 nmol. The leading electrolyte contained 0.2% hydroxypropylmethylcellulose (HPMC). Buffer, creatinine.C'L, concentration of leading ion.

(aqueous-nonaqueous) provides further possibilities for mobility control to improve separations.

Figure 7(A) and (B) show the simulated and observed isotachopherograms at pH 8 using triethanol-amine buffer when methanol is used as solvent. The leading ion is perchlorate. Obviously, the separation is complete. On the other hand, insufficient separation was predicted for the aqueous system where the leading ion was chloride when pHL = 3.6 (^-alanine buffer), as shown in Figure 7(C). Apparently the step heights of 2-naphthalenesulfonate, picrate and o-chlorobenzoate were similar and they may form a mixed zone in the actual analysis. No satisfactory separation was estimated when pH effects in the aqueous system were used.

Thus it is evident that the separation behaviour in isotachophoresis is strongly affected by the solvent used. As demonstrated above, high separability can be expected by the use of methanol solvent for particular samples but it should be noted that carboxylic acids are esterified gradually on standing in methanol solution, although the production of esterified compounds during analysis is negligible.

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