核酸水解产物嘌呤、嘧啶碱基在BDS柱上的分离及测定

Separation and Determination of Purine Bases and Pyrimidine Bases from Nucleic Acid Hydrolysis by HPLC on BDS Column

用高效液相色谱法测定了核酸水解的中间产物及最终产物 6种嘌呤、嘧啶碱基 ,探讨了色谱柱、流动相等对其分离的影响 ,确定了最佳色谱条件为 :HypersilBDS C18柱 ,乙腈 0 1mol/LKH2 PO4 (H3 PO4 调节 pH至4 0 5 ) (体积比为 2∶98)作流动相 ,紫外检测器在 2 6 0nm波长下检测。方法的精密度在 3%以内 ,回收率在 82 %～ 114%。方法应用于酵母核酸样品的测定中 。

The hydrolysates of nucleic acid, six purine bases and pyrimidine bases(cytosine,uracil,guanine,hypoxanthine,adenine and thymine) were separated and determined by using HPLC. It is discussed how the column and mobile phase affect the separation. The peaks of cytosine and adenine are tailed on ordinary C 18 column, and they are very good on BDS C 18 column. The KH\-2PO\-4 H\-3PO\-4 buffer can be used in separation the hydrolysates of RNA and DNA,and the NaAc HAc buffer is only used in DNA. In addition,pH value is a very important factor for separation.With pH value of mobile phase increasing, the retention times of guanine, hypoxanthine and thymine were first increased and then decreased, adenine was increased, and cytosine and uracil were almost constant. The chosen mobile phase was 0 1 mol/L KH\-2PO\-4 H\-3PO\-4 buffer, with a pH value of 4 05. It was detected at UV 260 nm. The determination was completed within 10 min. The RSDs were all less than 3% and the recoveries were in the range of 82% 114%. The method has been applied to the detection of yeast hydrolysates.