Moritz R L, Simpson R J
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Victoria, Australia.
J Chromatogr. 1992 May 22;599(1-2):119-30. doi: 10.1016/0021-9673(92)85464-5.
A continuous gradient elution method for capillary column (less than 0.32 mm I.D.) liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2-3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which, in turn, was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-microns spherical, 300 A pore size, C8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins, peptides or phenylthiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (less than 2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6 microliters/min, proteins and peptides were recovered from the capillary columns in volumes of about 2-8 microliters. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tyrosine-containing peptides is discussed.
开发了一种用于毛细管柱（内径小于0.32mm）液相色谱的连续梯度洗脱方法。来自微径液相色谱仪的梯度洗脱液在进样器之前分流，以便泵输送的流动相中有准确比例（2 - 3%）流经毛细管柱。柱的出口连接到一段内径为0.075mm的熔融石英毛细管，该毛细管又连接到一个光程长为6mm的纵向毛细管流通池。内径为0.32mm的熔融石英毛细管柱用7微米球形、孔径300Å、C8键合相颗粒高效匀浆填充，并根据其分离蛋白质、肽或苯硫基乙内酰脲（PTH）-氨基酸衍生物混合物的能力进行评估。梯度洗脱曲线与使用微径（内径小于2.1mm）和较大内径柱获得的曲线一致。内径为0.32mm的毛细管柱上蛋白质和PTH -氨基酸的最低检测量分别为50pg和25fmol。在流速为每分钟3.6微升时，蛋白质和肽从毛细管柱中回收的体积约为2 - 8微升。讨论了使用多波长前向光学检测器鉴定含色氨酸和酪氨酸的肽。
