|Molecular Weight||146.190 g/mol|
11 underivatized amino acids (aspartic acid, glutamic acid, alanine, valine, methionine, isoleucine, cysteine, phenylalanine, histidine, lysine, and arginine) are separated by a Primesep 100 HPLC column by reversed-phase and ion-exchange mechanisms with LC/MS compatible conditions without the use of ion-pair reagents. The HPLC separation uses a TFA (trifluoroacetic acid) gradient in a mobile phase of water acetonitrile (MeCN, ACN with evaporative light scattering detection (ELSD).
Polar amino acids are very often used as components of vitamin and supplement composition. Analysis of such complex composition is a challenging task. In this application, 5 amino acids (asparagine, glutamic acid, proline and arginine) and two preservatives (methyl paraben and propyl paraben) are separated on a Primesep 100 reversed-phase cation-exchange column with LC/MS compatible mobile phase. Method does not require ion-pairing reagent in the mobile phase. Compounds are monitored by ELSD and UV. Method is validated for quantitation of underivatized amino acids in complex mixtures. The method is simple and robust and can be used for analysis of various vitamin formulations.
Short-chain peptides are usually very polar compounds. If the peptide contains lysine moiety it also become very basic in nature. Analysis in traditional reversed-phase of HILIC mode can produce poor peak shape. The method was developed for lysine-based peptide on Primesep AP column in HILIC/cation-exclusion mode. Column can be used for analysis of small peptides with multiple basic groups. Compounds can be monitored by UV, LC/MS or ELSD.
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 S, 4.6x150 mm, 5 µm, 100A|
|Mobile Phase||Gradient MeCN - 85-60%, 10 min 2 min hold|
|Buffer||Gradient AmFm pH 3.0- 10-80 mM, 10 min 2 min hold|
|Flow Rate||1.0 ml/min|
|Detection||CAD (Corona) MS- compatible mobile phase|
|Class of Compounds||Sugar, Amino Acid, Hydrophilic, Ionizable, Vitamin, Supplements|
|Analyzing Compounds||D-Glucose, L-Lysine|
|Column||Primesep 200, 4.6x150 mm, 5 µm, 100A|
|Mobile Phase||MeCN/H2O - 10/90%|
|Buffer||AmFM pH 3.0 - 10 mM|
|Flow Rate||1.0 ml/min|
|Detection||CAD (Corona), MS- compatible phase|
|Class of Compounds||Acid, Hydrophilic, Ionizable, Carboxylic acid, Drug, Amino Acid|