Huber C G, Krajete A
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
J Chromatogr A. 2000 Feb 18;870(1-2):413-24. doi: 10.1016/s0021-9673(99)00953-x.
Fused-silica capillary columns of 200 microm inner diameter were packed with micropellicular, octadecylated, 2.3 microm poly(styrene-divinylbenzene) particles and applied to the separation of oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography. Oligonucleotides were eluted at 50 degrees C with gradients of 3-13% acetonitrile in 50 mM triethylammonium bicarbonate. Addition of sheath liquid to the column effluent allowed the detection of oligonucleotides by electrospray ionization mass spectrometry using full-scan data acquisition with a detectability comparable to that obtained with UV detection. The signal-to-noise ratios with different sheath liquids increased in the order isopropanol<methanol<acetonitrile. The incorporation of volatile acids or bases such as triethylamine or hexafluoroisopropanol into the sheath liquid was found to influence the charge state distribution of oligonucleotides longer than 20 nucleotide units whereas no significant effect was observed with shorter oligonucleotides. Organic acids and bases in the sheath liquid generally deteriorated the signal-to-noise ratios in the chromatograms and mass spectra mainly because of increased background noise. Only a few charge states were observed in the mass spectra of oligonucleotides because of charge state reduction due to the presence of carbonic acid in the eluent. With triethylammonium hydrogencarbonate as chromatographic eluent and acetonitrile as sheath liquid, very few cation adducts of oligonucleotides were observed in the mass spectra. However, the presence of small amounts of monopotassium adducts enabled the calculation of the charge state of multiply charged ions. With acetonitrile as sheath liquid, 710 amol of a 16-mer oligonucleotide were detected using selected ion monitoring data acquisition with a signal-to-noise ratio of 3:1. Finally, capillary ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was applied to the separation and characterization of heterooligonucleotide primers.
内径为200微米的熔融石英毛细管柱填充有微球型、十八烷基化、2.3微米的聚（苯乙烯 - 二乙烯基苯）颗粒，并应用于通过离子对反相高效液相色谱法分离寡核苷酸。寡核苷酸在50℃下，用50mM碳酸氢三乙铵中3 - 13%乙腈的梯度进行洗脱。向柱流出物中添加鞘液，可通过电喷雾电离质谱法检测寡核苷酸，采用全扫描数据采集，其检测限与紫外检测相当。不同鞘液的信噪比按异丙醇＜甲醇＜乙腈的顺序增加。发现将挥发性酸或碱如三乙胺或六氟异丙醇加入鞘液中会影响长度超过20个核苷酸单元的寡核苷酸的电荷态分布，而较短的寡核苷酸则未观察到显著影响。鞘液中的有机酸和碱通常会使色谱图和质谱图中的信噪比变差，主要是因为背景噪声增加。由于洗脱液中存在碳酸导致电荷态降低，寡核苷酸的质谱图中仅观察到少数电荷态。以碳酸氢三乙铵作为色谱洗脱液，乙腈作为鞘液时，质谱图中寡核苷酸的阳离子加合物很少。然而，少量单钾加合物的存在使得能够计算多电荷离子的电荷态。以乙腈作为鞘液，使用选择离子监测数据采集，在信噪比为3:1的情况下，检测到710 amol的16聚体寡核苷酸。最后，毛细管离子对反相高效液相色谱 - 电喷雾电离质谱法应用于异源寡核苷酸引物的分离和表征。
