Chromatography Forum

I'm having trouble putting together an amino acid method using a UV detector. I'm trying to analyze raw amino acid material, specifically L-Arginine, L-Glutamine, Glycine and Lysine. I purchased PITC to try and derivate the amino acids so they can be detected in the UV range, however I'm not getting any results. Here is the sample prep and method I'm running.

Sample Prep: dissolve 5g of amino acid in 1L of H2O, dilute 10x500mL for a 0.10mg/mL concentration. Transfer 4mL of 0.10mg/mL Amino Acid sample into test tube and add 1mL EtOH:H20:TEA (2:2:1). Mix well. Add 500uL EtOH:H20:TEA:PITC(7:1:1:1) and incubate at room temperature for 30mn. Add 2mL Hexane to test tube, mix and extract Hexane layer into seperate test tube (perform in triplicate). Evaporate Hexane layer using air hose (it's all I have in the lab-no vacume set up). Reconstitute sample using 4mL H2O and filter into autosampler vial.

HPLC Specs:

mobile phase: A= 90/10 0.5% KH2PO4/ACN B= 60/40 ACN/H2O

Run on Gradient 20-100%B Convex in 60mn.

Column: 4um phenomenex synergi hydro pro RP C18

I'll try to paste a chromatogram of my results but I'm not sure how. Please help if you can, thank you.

Don't know why you do the hexane extraction step at the end. Try taking an aliquot of yur derivitized AA and drying it down. You can use a speed vac or lyophylizer. Bring the dried material back up in diluent. The diluent can be the same as the Waters Pico-tag method((PO4 buffer/ACN). See if you get peaks then.

Koby,

If you provide me with email address I will send you a method for the retention of underivatized aminoacids with UV detection (190-205 nm)
my email is mail@sielc.com.

lcguy1, thank you for the help. The reason I've been running a hexane extraction is to remove any by-product from the PITC AA reaction. Otherwise, I consistantly get a peak at around 20mn no matter what Amino Acid I'm running. I read some articles online and some have said to run a nitrogen vacum but if one is not available to run a heptane extraction. Well, I unfortunatley don't have a nitrogen vacum or any heptane in my lab at the moment, so I tried using the hexane. But you don't think the hexane extraction is needed? I wish I knew how to past a chromatogram here so I could show you the peak at 20mn I am talking about.

Kobe,

Check you email - I send you two links for amino acid analysis (it might be in your AOL bulk folder)