酰胺键合色谱在分离小核酸异构体中的应用

gaoshan_lanjing

2012/09/27
食品检测

私聊

液相色谱（LC）

At the later structure modification, the nucleic acid fragment nucleus was to be produced position isomers frequently.

2’-BaG 3’-BaG Dimer-BaG

How to extract the target 2 '-BaG from these isomers, stationary phase to be selected correctly was important. If the silica gel was used, the Separation needs more than 10 hours, and the recovery was less than 40%; even the time packed column needed 5 hours and the time the stationary removed from column needed 3 hours; so the use-cost was very high. If the reverse phase HPLC was used to separate this mixture, the water in the solvent was removed which need high temperature(above 50℃), the sample may be metamorphic with this temperature. So the RP-HPLC is not the best choice. We chose a kind of propyl amide bonding silica gel for matrix, the results included loading sample amount、recovery、purity、separation time was satisfactory and the postprocessing could be carried out in a low temperature 40℃。

By RP-C18 analysis, we could detect the purities of the sample of each isomer. [ Fig.1]

Figure 1 the sample analysis chromatogram used column Venusil® XBP C18（L）(4.6×250 mm，5 μm，150Å)；Elution condition was the mixture of 0.03%TFA and CAN(25:75)；wavelength was 254 nm and flow rate was 1ml/min with 35℃ column temperature

Preparation HPLC condition：

With column Venusil® Hilic (30×250mm , 5μm, 100Å)，which with propyl amide bonding silica gel for matrix，and mobile phase used mixture of EA and PE(80:20), the separation cycle was less than 2 hours, and load sample amount was up to 2.5 gram. Three isomer was achieved a very good resolution.[Fig. 2]

Figure 2 The sample preparation chromatogram used column Venusil^® Hilic, and used Positive phase chromatographic mode, 3 isomers separated very well.

Purity detection of HPLC:

the sample fraction of the separation was enriched using rotation evaporator, we could get power 0.71 g , the recovery was more than 74%; and then purified by RP-C18 HPLC condition, the purity of 2’-BaG was up to 98%. [Fig. 3]

Figure 3 2’-BaG detection by reverse phase analysis chromatogram

Conclusions：

Nucleic acid fragment isomers such as 2’-BaG could be purified with propyl amide bonding silica gel by NP-HPLC, the results included loading sample amount、recovery、purity、separation time and postprocessing temperature had great advantages Compared with silica gel or reverse phase chromatography. So we may attempt to put this method into more separations.