|Molecular Weight||147.131 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).
Primesep 100 separates the components of a Swiss cheese extract by HPLC using cation exchange and reversed phase as retention mechanisms. The amino acids, glutamic acid and proline, as well as glycine betaine were resolved in less than 10 minutes. A mobile phase gradient of water, acetonitrile (MeCN, ACN,) and phosphoric acid (H3PO4) and ultraviolet (UV) detection was used.
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.
Glutamic acid and GABA are neurotransmitters. Glutamic acid and GABA are non-essential amino acids. They are hydrophilic and zwitter-ionic in nature . At lower pH, carboxylic acid groups of amino acids are not ionized, making them more hydrophobic and basic. Underivatized glutamic acid and GABA were retained and separated on a Primesep 100 column using ACN/water/TFA mobile phase. Amino acids can be monitored by low UV or ELSD/CAD. Retention is provided by reversed-phase and cation-exchange mechanism. Method can be used for analysis of underivatized amino acids in various matrices including supplements, vitamin and other complex mixtures. various mobile phase can be used with corresponding detection techniques.
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 - 20/80%|
|Buffer||H3PO4 - 0.1%|
|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|