Chen Y, Mehok A R, Mant C T, Hodges R S
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
J Chromatogr A. 2004 Jul 16;1043(1):9-18. doi: 10.1016/j.chroma.2004.03.070.
Trifluoroacetic acid (TFA) remains the dominant mobile phase additive for reversed-phase high-performance liquid chromatography (RP-HPLC) of peptides after more than two decades since its introduction to this field. Generally, TFA has been employed in a concentration range of 0.05-0.1% (6.5-13 mM) for the majority of peptide separations. In order to revisit the question as to whether such a concentration range is optimum for separations of peptide mixtures containing peptides of varying net positive charge, the present study examined the effect of varying TFA concentration on RP-HPLC at 25 and 70 degrees C of three groups of synthetic 10-residue synthetic peptides containing either one (+1) or multiple (+3, +5) positively charged groups. The results show that the traditional range of TFA concentrations employed for peptide studies is not optimum for many, perhaps the majority, of peptide applications. For efficient resolution of peptide mixtures, particularly those containing peptides with multiple positive charges, our results show that 0.2-0.25% TFA in the mobile phase will achieve optimum resolution. In addition, the use of high temperature as a complement to such TFA concentration levels is also effective in maximizing peptide resolution.
自三氟乙酸（TFA）被引入反相高效液相色谱（RP-HPLC）用于肽分析领域二十多年来，它一直是该领域主要的流动相添加剂。一般来说，在大多数肽分离中，TFA的使用浓度范围为0.05 - 0.1%（6.5 - 13 mM）。为了重新探讨这样的浓度范围对于分离含有不同净正电荷肽的肽混合物是否为最佳浓度范围这一问题，本研究考察了在25℃和70℃下，改变TFA浓度对三组含有一个（+1）或多个（+3、+5）带正电荷基团的10个残基合成肽进行RP-HPLC分析的影响。结果表明，肽研究中使用的传统TFA浓度范围对于许多（也许是大多数）肽应用并非最佳。为了有效分离肽混合物，特别是那些含有多个带正电荷肽的混合物，我们的结果表明，流动相中0.2 - 0.25%的TFA将实现最佳分离效果。此外，使用高温作为这种TFA浓度水平的补充，也能有效最大化肽的分离度。
