• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>other >Improving the Sensitivity Resolution and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing
【2h】

Improving the Sensitivity Resolution and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

机译：通过使用温度辅助柱上溶质聚焦技术提高大体积进样的毛细管液相色谱中梯度洗脱的灵敏度分辨率和峰容量

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Capillary HPLC (cLC) with gradient elution is the separation method of choice for the fields of proteomics and metabolomics. This is due to the complementary nature of cLC flow rates and electrospray or nanospray ionization mass spectrometry (ESI-MS). The small column diameters result in good mass sensitivity. Good concentration sensitivity is also possible by injection of relatively large volumes of solution and relying on solvent-based solute focusing. However, if the injection volume is too large or solutes are poorly retained during injection, volume overload occurs which leads to altered peak shapes, decreased sensitivity, and lower peak capacity. Solutes that elute early even with the use of a solvent gradient are especially vulnerable to this problem. In this paper, we describe a simple, automated instrumental method, temperature-assisted on-column solute focusing (TASF), that is capable of focusing large volume injections of small molecules and peptides under gradient conditions. By injecting a large sample volume while cooling a short segment of the column inlet at subambient temperatures, solutes are concentrated into narrow bands at the head of the column. Rapidly raising the temperature of this segment of the column leads to separations with less peak broadening in comparison to solvent focusing alone. For large volume injections of both mixtures of small molecules and a bovine serum albumin tryptic digest, TASF improved the peak shape and resolution in chromatograms. TASF showed the most dramatic improvements with shallow gradients, which is particularly useful for biological applications. Results demonstrate the ability of TASF with gradient elution to improve the sensitivity, resolution, and peak capacity of volume overloaded samples beyond gradient compression alone. Additionally, we have developed and validated a double extrapolation method for predicting retention factors at extremes of temperature and mobile phase composition. Using this method, the effects of TASF can be predicted, allowing determination of the usefulness of this technique for a particular application.
机译：带有梯度洗脱的毛细管HPLC（cLC）是蛋白质组学和代谢组学领域的首选分离方法。这是由于cLC流速和电喷雾或纳喷雾电离质谱（ESI-MS）的互补性质。较小的色谱柱直径可实现良好的质量灵敏度。通过注入相对大量的溶液并依靠基于溶剂的溶质聚焦，也可以实现良好的浓度灵敏度。但是，如果进样量太大或在进样过程中保留的溶质差，则会发生体积过载，从而导致峰形改变，灵敏度降低和峰容量降低。即使使用溶剂梯度也能提早洗脱的溶质特别容易受到此问题的影响。在本文中，我们描述了一种简单的自动化仪器方法，即温度辅助的柱上溶质聚焦（TASF），它能够在梯度条件下聚焦小分子和多肽的大体积进样。通过注入大量样品，同时在低于室温的温度下冷却一小部分色谱柱入口，溶质在色谱柱顶部浓缩成窄带。与仅聚焦溶剂相比，快速升高该塔段的温度可导致分离，峰宽减小。对于小分子混合物和牛血清白蛋白胰蛋白酶消化物的大量进样，TASF改善了色谱图中的峰形和分离度。 TASF的浅梯度显示出最显着的改进，这对于生物学应用特别有用。结果表明，采用梯度洗脱的TASF能够提高超载样品的灵敏度，分辨率和峰容量，而不仅仅是梯度压缩。此外，我们已经开发并验证了双重外推法，用于预测极端温度和流动相组成下的保留因子。使用这种方法，可以预测TASF的效果，从而确定该技术对特定应用的有用性。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号