|Molecular Weight||131.175 g/mol|
Glutamine, valine, isoleucine, and leucine are baseline resolved in a dietary supplement with a mobile phase of acetonitrile, water, and sulfuric acid with ultraviolet (UV) detection at 210 nm on a Primesep 100 column. Increasing the sulfuric acid concentration allows elution of arginine. Primesep 100 separates underivatized amino acids and amines by combining reversed-phase and ion-exchange mechanisms. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN), and sulfuric acid with ultraviolet (UV) detection at 210 nm.
Essential and non-essential amino acids can be retained and separated in zero-organic mode on Primesep mixed-mode HPLC columns. Zero-organic mode is required to monitor isotopes of carbon. Amino acids are retained by combination of reversed-phase and cation-exchange mechanisms. At lower pH, some of the amino acids are more hydrophobic. Buffer pH will affect ionization state of amino acids, and at higher pH (above 2.5), the amino acids will be less hydrophobic and retentive in zero-organic mode. Amino acids can be monitored by low UV. Method can be used in archeological research for analysis of various molecules where presence of organic component of the mobile phase interferes with analysis.
Application Notes: Amino acids are polar ionic compounds which are not retained on reversed-phase column without ion-pairing reagent. In our application, lysine and arginine can be separated from other amino acids. Amino acids with a pH between 3 and 5 and with one basic and one acidic group become very polar. Therefore these amino acids don’t have strong ion-exchange interaction with Primesep C stationary phase. Amino acids with two amino groups still carry positive net charge and can interact with stationary phase by cations-exchange mechanism. pH variation of the mobile phase can be an effective tool to adjust selectivity of separation for zwitter-ionic, basic and acidic compounds. This method can be used for separation of mono-charged compounds from compounds having an extra charge.
Application Columns: Primesep C
Application compounds: Aspartic acid, Glutamic acid, Aspargine, Glycine, Proline, Alanine, Phenylalanine, Tyrosine, Leucine, Lysine, Arginine
Detection technique: UV, LC/MS, ELSD/CAD
|Column||Primesep 100, 4.6x250 mm, 5 µm, 100A|
|Mobile Phase||MeCN/H2O - 35/65%|
|Buffer||H2SO4 0.05% 12 min hold, gradient 0.05-0.20, 13 min|
|Flow Rate||1.0 ml/min|
|Detection||UV, 200 nm|
|Class of Compounds||Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid|
|Analyzing Compounds||Amino acids|
Both β-hydroxy-β-methylbutyric acid (HMB) and 2-hydroxyisocaproic acid (HICA) are metabolites of the essential dietary branched chain amino acid leucine.
|Column||Primesep 100, 4.6x150 mm, 5 µm, 100A|
|Mobile Phase||MeCN - 5%, 3 min hold, Gradient MeCN 5-30% 12 min|
|Buffer||H2SO4 - 0.05 % 3 min hold, GradientH2SO4 0.05-3% 12 min|
|Flow Rate||1.0 ml/min|
|Detection||UV, 210 nm|
|Class of Compounds||Acid, Hydrophilic, Ionizable, Normal human metabolite, Dietary supplement, Essential amino acid, Branched-chain amino acid (BCAA)|
|Analyzing Compounds||β‑Hydroxy-β-methylbutyric (HMB), 2-Hydroxyisocaproic Acid (HICA), Leucine|