超临界流体色谱法快速分离肽

由于超临界流体色谱（SFC）的应用能够减少分析时间，有机溶剂消耗和废物生产的兴趣正在增长。SFC已被广泛用于小分子药物手性纯化，并正在不断扩展到其他领域。对肽等水分子的探索最初被抑制，因为人们认为SFC不可能成功分离亲水和极性化合物。然而，这一应用说明说明了SFC分离多肽的能力。本应用说明中给出的结论：SFC成功的分离一种5肽混合物，说明了SFC不断扩大的能力。分析速度很明显，分析时间减少了五倍，环境友好性需要三分之二的溶剂，产生不到三分之一的有机废物。SFC APPLICATION NOTE 08-09 Rapid Peptide Separation by Supercritical Fluid Chromatograph y Interest in supercritical fluid chromatography (SFC)applications is growing due to its ability to reduce analysis times， organic solvent consumption and waste production. SFC is well established for small molecule pharmaceutical chiral purifications and is continually expanding into other areas. The exploration of aqueous molecules such as peptides was initially inhibited by beliefs that the successful separation of hydrophilic and polar compounds was unlikely by SFC. However， this application note illustrates the ability of SF to separate peptides. Experimental A JASCO SFC 2000series S VWas used d ffor the measurement.The system consisted of a PU-2080-CO2 l iquefied carbon dioxide delivery pump， PU-2080HPLC modifier delivery pump， an LV-2080-03 solvent selection valve， an AS-2059-SF autosampler， an HV-2080-06 column switching valve (2)， a CO-2060column oven， an MD-2010 diode array detector， a BP-2080 back-pressure regulator， and EZChrom Elite data system. The separation column Was a Princeton Chromatography 2-Ethylpyrindine column (4.6 mm ID x 250 mm L， 5 um， 60A). The standard 5 peptide HPLC mixture was purchased from Sigma-Aldrich . It contained G 3502 (GLY-TYR)， V 8376 (VAL-TYR-VAL)， M 6638 Methionine Enkephalin Acetate (TYR-GLY-GLY-PHE-MET)， L 9133 Leucine Enkephalin (TYR-GLY-GLY-PHE-LEU)， and A 9525 Angiotensin Ⅱ Acetate (ASP-ARG-VAL-TYR-ILE-HIS-PRO-PHE)all 0.5 mgs. The sample was dissolved in 1mL of methanol with 0.2% TFA. CO， with a modifier mobile phase of methanol with 0.2% TFA were used. Figure 1 shows a SFC chromatogram at 230nm of the standard 5 peptide HPLC mixture. The baseline drift is clearly evident due to the increased absorbance of the methanol/TFA modifier mobile increase over the gradient elution at 230nm. This baseline drift was not seen at 254nm. Figure 1. SFC chromatogram ofstandard peptide mixture. Peaks： 1=V 8376，2=G 3502，3=Leucine Enkephalin，4=Methionine Enkephalin， 5=Angiotension II. Gradient conditions： 30% MeOH 0.2% TFA to 40% MeOH 0.2% TFA over 10 minutes (1%/minute) at 2mLs /minute. 5uLs of 0.5mg/mL peptide mixture was injected. The separation of this 55 peptide mixture by HPLC required nearly 60 minutes (not including column re-equilibration). compared to less than 12 on SFC. The standard HPLC separation was conducted as a gradient from 5% acetonitrile with 0.1% TFA to 30% acetonitrile with 0.1%0TFA over 60 minutes at 1mL/min. These HPLC conditions required 60mLs of l iquid (Water-ACN)solvent producing 60mLs of waste compared to 40mLs of mobile phase (COz-Methanol) and 16mLsS of V waste (including column re-equilibration) for SFC. The resulting successful separation of a 5 peptide mixture i l lustrates the ever expanding ability of SFC. The analysis speed is clearly evident in the five fold decrease in time and 1analysis environmentally friendly nature requiring two-thirds the solvent and producing less than one-third of the organic waste.