title: Far-ultraviolet absorbance detection of sugars and peptides by high-performance liquid chromatography.
abstract: A far-ultraviolet (FUV)-absorbance detector with a transmission flow cell was developed and applied to detect absorbance of sugars and peptides by HPLC. The main inherent limitation of FUV-absorbance detection is the strong absorptions of solvents and atmospheric oxygen in the optical system as well as dissolved oxygen in the solvent. High absorptivity of the solvent and oxygen decreases transmission-light intensity in the flow cell and hinders the absorbance measurement. To solve the above drawbacks, the transmission-light intensity in the flow cell was increased by introducing a new optical system and a nitrogen-purging unit to remove the atmospheric oxygen. The optical system has a photodiode for detecting the reference light at a position of the minus-first-order diffracted light. In addition, acetonitrile and water were selected as usable solvents because of their low absorptivity in the FUV region. As a result of these implementations, the detectable wavelength of the FUV-absorbance detector (with a flow cell having an effective optical path length of 0.5mm) can be extended down to 175nm. Three sugars (glucose, fructose, and sucrose) were successfully detected with the FUV-absorbance detector. These detection results reveal that the absorption peak of sugar in liquid phase lies at around 178nm. The detection limit (S/N=3) in absorbance with a 0.5-mm flow cell at 180nm was 21μAU, which corresponds to 33, 60 and 60μM (198, 360, and 360pmol) for fructose, glucose, and sucrose, respectively. Also, the peptide Met-enkephalin could be detected with a high sensitivity at 190nm. The estimated detection limit (S/N=3) for Met-enkephalin is 29nM (0.29pmol), which is eight times lower than that at 220nm.
authors.affiliation: Hitachi, Ltd., Research and Development Group, 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan. Electronic address: yuichi.uchiho.xs@hitachi.com.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Uchiho
authors.affiliation: Hitachi, Ltd., Research and Development Group, 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Hitachi, Ltd., Research and Development Group, 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Kamahori
authors.affiliation: Hitachi High-Tech Science Corporation, 1040 Ichige, Hitachinaka-shi, Ibaraki 312-0033, Japan.
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Aota
authors.affiliation: Hitachi High-Tech Science Corporation, 1040 Ichige, Hitachinaka-shi, Ibaraki 312-0033, Japan.
authors.forename: Atsuki
authors.initials: A
authors.lastname: Morisaki
authors.affiliation: Hitachi High-Tech Science Corporation, 1040 Ichige, Hitachinaka-shi, Ibaraki 312-0033, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hosen
authors.affiliation: Hitachi, Ltd., Research and Development Group, 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan.
authors.forename: Kimiyoshi
authors.initials: K
authors.lastname: Koda
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004151:Dipeptides / Q000032:analysis; D004744:Enkephalin, Methionine / Q000032:analysis; D005632:Fructose / Q000032:analysis*; D005947:Glucose / Q000032:analysis*; D057230:Limit of Detection; D010455:Peptides / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose / Q000032:analysis*
chemical_list: D004151:Dipeptides; D010455:Peptides; D005632:Fructose; D013395:Sucrose; D004744:Enkephalin, Methionine; C022013:glycyltyrosine; D005947:Glucose
keywords: Absorbance detector; Far-ultraviolet; High-performance liquid chromatography; Peptide; Sugar