Direct determination of tryptophan using high-performance anion-exchange chromatography with integrated pulsed amperometric detection.
Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.
Hanko
Valoran P
VP
Dionex Corporation, Sunnyvale, CA 94088-3603, USA. val.hanko@dionex.com
Rohrer
Jeffrey S
JS
D000821:Animal Feed; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012972:Sodium Hydroxide / Q000494:pharmacology; D014364:Tryptophan / Q000032:analysis*
D003470:Culture Media; D012710:Serum Albumin, Bovine; D012972:Sodium Hydroxide; D014364:Tryptophan; D005947:Glucose