The aims of this study were to determine the effect of levels of various substances and reaction by-products, which are formed during hydrolysis of nucleic acids, on the derivatization and chromatography of nucleosides; and to investigate the silylation of mono- and dinucleotides. The effect of NaCl, KCl, MgCl 2, NH 4Cl, and (NH 4) 2SO 4 on silylation and chromatography of nucleosides was studied at various molar excesses of salt. The response values for all nucleosides were studied at various molar excesses of salt. The response values for all nucleosides were significantly affected at molar excess salt present values (MSP) between 1 and 10 for KCl, NaCl, NH 4Cl, (NH 4) 2SO 4 and between 0.1 and 1 for MgCl 2. It was noted that thymidine was more sensitive than other nucleosides if silylated in presence of these salts. Two chromatographic peaks at retention temperatures (RN) 240 and 251 were obtained for cytidine at MSP values of 10 −3 for NaCl, KCl, and MgCl 2, and 10 −4 for NH 4Cl and (NH 4) 2SO 4. In a mixture of nucleosides the RT = 251 peak was used for quantitative analysis of cytidine as the RT = 240 peak elutes with guanosine. Thus, these salts have a significant effect on the gas-liquid chromatography of trimethylsilyl (TMS) cytidine in a mixture of nucleosides, especially the RT = 241 peak. The effect of salts on derivatization can be explained in part as follows: (a) reduced derivatization of nucleotides due to a decreased solubility in the solvent system; (b) formation of TMS anion derivatives, e.g. TMS-SO 4, TMS-PO 4, with a reduced molar excess of BSTFA; (c) metal chelation by Mg ions or other divalent cations with nucleosides or BSTFA; and/or (d) an increased breakdown of TMS derivatives in presence of salt in the sample or on the top 3 in. of the column packing. Also, experiments were made on the effect of other substances such as Tris, phosphate, alkaline phosphatase, and KCl on completeness of silylation. The individual impurities showed no significant effect on the relative weight response (RWR) values of nucleosides; however, when a mixture was used, significantly lower RWR values were observed for all nucleosides except thymidine when using 1000 molar excess bis(trimethylsilyl)trifluoroacetamide (BSTFA). It was concluded that a molar excess of BSTFA greater than 1000 should be used for silylation and chromatography of nucleosides in an RNA hydrolysate. As reported earlier the best derivatization of nucleosides was achieved using closed tube silylation at 150° for 15 min with 225 molar excess BSTFA and chromatography on 4% OV-11 on Supercoport. In general, the presence of salts and other substances can be significant in quantitative work, thus it is suggested that they be removed using chromatographic cleanup methods. The stability of nucleosides as a function of concentration of HCl, at room temperature was studied and very low RWR values for nucleosides were obtained when stored for 48 h in > 0.001 N HCl. Trimethylsilylation of various nucleotides and dinucleotides were made at 15 min as a functin of temperature, and at 150° at different times. It was concluded that the optimal derivatization conditions were different for different nucleotides and dinucleotides. At 150° the best silylation time varied from 15 min to 4 h depending on the base attached. The multiple peaks obtained from dinucleotides corresponded to the TMS-nucleotide peaks, thus a breakdown of dinucleotides occurred.