|Molecular Weight||103.122 g/mol|
Primesep C separates the isomers of aminobutyric acids by a combination of reversed-phase and ionic interaction mechanisms. alpha-Aminobutyric acid, beta-Aminobutryic acid, and gamma-Aminobutyric acid (GABA) are baseline resolved without ion-pair reagents. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) ammonium acetate with evaporative light scattering detection (ELSD).
|Column||Primesep 200, 4.6x150 mm, 5 µm, 100A|
|Flow Rate||1.0 ml/min|
|Class of Compounds||Drug, Acid, Hydrophilic, Ionizable, Hormone|
|Analyzing Compounds||Aspartic acid, Glutaric acid, Glycine, Hydroxytriptophan, GABA, Norepinephrine, Dopamine|
Amino acids are building blocks for peptides and proteins. Serine is not essential to human diet and it is synthesized in human body. Methionine is not synthesized in human body and needs to be ingested. GABA is used to enhance growth of specified plants, prevent development of powdery mildew on grapes, and suppress certain other plant diseases. In humans GABA helps to maintain normal brain function. Serine, methylserine, GABA and methionine are separated on Primesep 100 column by mixed-mode mechanism. Amino acids are retained by combination of reverse phase and cation-exchange mechanisms. At lower pH carboxylic acid fragment of amino acid is suppressed and not ionized, making amino acids more basic and slightly more hydrophobic. Amount of acetonitrile, buffer concentration and buffer pH can be used to adjust retention time. Underivatized amino acids are well retained and separated with perfect peak shape and symmetry. Fast method can be used for UV, ESLD and LC/MS quantitation of serine, methylserine, GABA and methionine.
Four underivatized amino acids (serine, methylserine, GABA and methionine) were separated on a Primesep 100 reversed-phase cation-exchange mixed-mode HPLC column. Two methods, one for UV and one for ELSD/LC/MS show good separation and peak shape for underivatized amino acids. This column and general HPLC approach can be used for analysis of underivatized amino acids. Primesep 100 is designed to replace reversed-phase HPLC column in combination with ion-pairing reagents.
GABA (neurotransmitter) and its isomers are polar zwitter-ionic compounds. Due to the position of amino-groups, all three compounds show different polar and basic properties. The isomers of aminobuturic acid are separated on an Obelisc N HILIC/cation-exchange column. Buffer concentration has a different effect on retention of alpha-, beta-, and gamma-aminobutyric acid. This general and robust method can be used for separation of other polar and ionizable compounds and isomers by mixed-mode chromatography.
Amino acids are building blocks for peptides and proteins; they are used in various supplement formulations and as starting reagents in chemistry to introduce chirality. GABA and GLU are used to enhance growth of specified plants, prevent development of powdery mildew on grapes, and suppress certain other plant diseases. L-Glutamic acid is one of the major amino acids naturally found in plant and animal proteins, and GABA helps to maintain normal brain function. Both amino acids behave as neurotransmitters. All amino acids have both basic and acidic groups. Depending on pH amino acids can be basic, acidic or zwitter-ionic. Due to the polar nature of underivatized amino acids, analysis is a very challenging task. Derivatization and ion-pairing reagents are used to provide retention of amino acids. Simple method is developed on Primesep 100 column using combination of acetonitrile/water with phosphoric acid as a mobile phase. Method uses UV detection. Amino acids are well retained without use of ion-pairing reagents. Fast reliable method can be developed for all underivatized natural and synthetic amino acids.
Amino acids are essential components of numerous formulation. Health supplements can contain various amino acids and vitamins and require quantitation of each ingredients. Amino acids are very polar compounds with limited or no retention in reversed-phase chromatography. The most common approaches are reversed-phase chromatography with ion-pairing reagent and hydrophilic interaction chromatography (HILIC). Underivatized amino acids can be retained by combination of reversed-phase and cation exchange mechanism on Primesep 100 mixed-mode. Retention time is controlled by amount of acetonitrile, buffer and buffer pH. Method does not require ion-pairing reagent. This method is for UV detection. LC/MS, ELSD or Corona CAD can be employed for analysis of amino acids with trifluoroacteic acid or ammonium formate in the mobile phase. This approach can be used for HPLC analysis of all underivatized amino acids.
|Column||Primesep 100, 4.6x150 mm, 5 µm, 100A|
|Mobile Phase||MeCN/H2O - 10/90%|
|Buffer||TFA - 0.1%|
|Flow Rate||1.0 ml/min|
|Detection||UV, 215 nm, ELSD|
|Class of Compounds||Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements|
|Analyzing Compounds||Glutamic acid, GABA|