Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection.
Anion-exchange chromatography with integrated pulsed amperometric detection (AE-IPAD) separates and directly detects amino acids, carbohydrates, alditols, and glycols in the same injection without pre- or post-column derivatization. These separations use a combination of NaOH and NaOH/sodium acetate eluents. We previously published the successful use of this technique, also known as AAA-Direct, to determine free amino acids in cell culture and fermentation broth media. We showed that retention of carbohydrates varies with eluent NaOH concentration differently than amino acids, and thus separations can be optimized by varying the initial NaOH concentration and its duration. Unfortunately, some amino acids eluting in the acetate gradient portion of the method were not completely resolved from system-related peaks and from unknown peaks in complex cell culture and fermentation media. In this article, we present changes in method that improve amino acid resolution and system ruggedness. The success of these changes and their compatibility with the separations previously designed for fermentation and cell culture are demonstrated with yeast extract-peptone-dextrose broth, M199, Dulbecco's modified Eagle's (with F-12), L-15 (Leibovitz), and McCoy's 5A cell culture media.
Hanko
Valoran P
VP
Dionex Corporation, Sunnyvale, CA 94088-3603, USA. val.hanko@dionex.com
Heckenberg
Andrea
A
Rohrer
Jeffrey S
JS
D000085:Acetates; D000596:Amino Acids / Q000032:analysis* / Q000302:isolation & purification; D002241:Carbohydrates / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D003470:Culture Media / Q000378:metabolism*; D005285:Fermentation; D008239:Lysine / Q000302:isolation & purification; D013402:Sugar Alcohols / Q000302:isolation & purification; D013997:Time Factors
D000085:Acetates; D000596:Amino Acids; D002241:Carbohydrates; D003470:Culture Media; D013402:Sugar Alcohols; D008239:Lysine