使用表面带电杂化(CSH)C18色谱柱提高反相肽分离的峰容量3
粒径和UPLC/HPLC的放大性
许多肽分离操作仍在传统的HPLC仪器上进行。因其背压过高，使用亚2 μm填充的色谱柱通常不适合与HPLC系统联用。而2.5 μm色谱柱因其背压较低可以在任何LC仪器上使用。为确定CSH130 C18,1.7μm色谱柱得到的峰容量是否可以成功转移到CSH130 C18 XP,2.5 μm色谱柱上，针对2.5 μm XP色谱柱设置了一种从一根1.7μm色谱柱转移出来的HPLC兼容的方法。这种转移如下所述：按照ACQUITY UPLC色谱柱计算器的建议18，将流速降低，增加梯度时间，降低和增加的系数均为1.5。图6显示，使用1.7 μm色谱柱进行的分离成功地转移到了2.5μm XP色谱柱上。选择性系数的一致性强调了这一观察结果，如表2所示。与1.7颗粒的色谱柱相比，使用2.5μm XP颗粒进行分离时背压出现极大地降低(~3000psi比 ~8000 psi)，从而表明了CSH技术可以轻松应用于UPLC或HPLC进行的高峰容量肽分离中。
结论
由于创新性的施加了低水平的正电荷，CSH 130 C18色谱柱已经被证明是一种能够实现肽分离的技术。根据对9种肽混合物进行分析，我们发现，与非表面带电的C18色谱柱相比，CSH 130 C18显示出更高的峰容量和独一无二的选择性。经过观察，CSH 130 C18色谱柱的性能对强离子对试剂(例如TFA，可以抑制电喷雾离子化)的依赖性显著降低。此外，在背压较小的HPLC条件下，使用CSH 130 C18,1.7 μm色谱柱得到的分离结果可以转移到CSH 130 C18,2.5 μm XP色谱柱上——尽管以牺牲分析时间为代价。CSH 130 C18可以使用不同的粒径，便于采用UPLC作为常规使用(以往这些实验室的日常使用被限制在HPLC仪器上)，也可以拓展到压力上限为9000~12000psi的UHPLC上。
参考文献
1. Yates JR, Ruse CI, Nakorchevsky A. Proteomics by mass
spectrometry: approaches, advances, and applications. Annu Rev Biomed
Eng. 2009; 11: 49-79.
2. Richardson J, Shah B, Xiao G, Bondarenko
PV, Zhang Z. Automated in-solution protein digestion using a commonly
available high-performance liquid chromatography autosampler. Anal
Biochem. 2011; 411(2): 284-91.
3. Neue UD. Theory of peak capacity in gradient elution. J Chromatogr A. 2005; 1079(1-2): 153-61.
4.
Wyndham KD, O’Gara JE, Walter TH, Glose KH, Lawrence NL, Alden BA, Izzo
GS, Hudalla CJ, Iraneta PC. Characterization and evaluation of C18 HPLC stationary phases based on ethyl-bridged hybrid organic/inorganic particles. Anal Chem. 2003; 75(24): 6781-8.
5.
Iraneta PC, Wyndham KD, McCabe DR, Walter TH. Charged Surface Hybrid
(CSH) Technology and Its Use in Liquid Chromatography. Waters White
Paper 720003929EN. 2011.
6. Berthelette KD, Summers M, Fountain KJ.
Modernization of the USP Assay for Clarithromycin Using eXtended
Performance (XP) Column Technology. Waters Application Note 720004461EN.
2012.
7. Fountain KJ, Hewitson HB, Iraneta PC, Morrison D.
Practical Applications of Charged Surface Hybrid (CSH) Technology.
Waters Application Note 720003720EN. 2010.
8. Summers M, Fountain
KJ. A Quality by Design (QbD) Based Method Development for the
Determination of Impurities in a Peroxide Degraded Sample of
Ziprasidone. Waters Application Note 720004072EN. 2011.
9. Summers M, Fountain KJ. Rapid Method Development through Proper Column Selection. Waters Application Note 720004353EN. 2012.
10. ACQUITY UPLC CSH Columns. Waters Applications Notebook 720003537EN. 2010.
11. Lauber MA, Koza SM, Fountain KJ. Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases. Waters Application Note. 720004571EN. 2013.
12.
Yan B, Yates Z, Balland A, Kleemann GR. Human IgG1 hinge fragmentation
as the result of H2O2-mediated radical cleavage. J Biol Chem. 2009;
284(51): 35390-402.
13. Yu XC, Joe K, Zhang Y, Adriano A, Wang Y,
Gazzano-Santoro H, Keck RG, Deperalta G, Ling V. Accurate determination
of succinimide degradation products using high fidelity trypsin
digestion peptide map analysis. Anal Chem. 2011; 83(15):5912-9.
14. Annesley TM. Ion suppression in mass spectrometry. Clin Chem. 2003; 49(7): 1041-4.
15. Temesi D, Law B. LC-GC Int. 1999;17: 626-32.
16.
Apffel A, Fischer S, Goldberg G, Goodley PC, Kuhlmann FE. Enhanced
sensitivity for peptide mapping with electrospray liquid
chromatography-mass spectrometry in the presence of signal suppression
due to trifluoroacetic acid-containing mobile phases. J Chromatogr A.
1995;712(1): 177-90.
17. Kuhlmann FE, Apffel A, Fischer S, Goldberg G, Goodley PC. J Am Soc Mass Spectrom. 1995; 6 (12): 1221-1225.
18. Jones M D, Alden P Fountain KJ, Aubin A. Waters Application Note 720003721EN. 2010.
-
焦点事件
