0 Replies Latest reply on Sep 20, 2018 5:19 AM by cpt49

    Buffers choices for HPLC amino acids sample?

    cpt49

      Hi, I'm new to HPLC and amino acids analysis, therefore I have some questions that I need advise on.

      Forgive me for the long writing below, as I think it's better to clear out my methods before asking any questions.

       

      Background Information:

      My laboratory has an Agilent 1260 HPLC with quan pump vl, G1321B 1260 FLD, G1314B 1260 VWD VL and auto injector.

      I intend to run amino acids analysis (hereby refer as AAA) on fish feed and fish samples. The laboratory own a ZORBAX ECLIPSE PLUS C18 column (4.6 x 100 mm, 3.5 um) and is planning to acquire the HPH C18 (4.6 x 100 mm, 2.7 um; CAT#695975-702) soon.

       

      The current chosen references for HPLC setting are from Agilent application notes: #5990-4547EN, and #5991-5571EN.

      The digestion methods employed now are from both AOAC (2012), method 994.12, and Jajic et al. (2013) (link here).

      Our main objective is to be able to quantitatively determine the concentration of cysteine in our samples.

       

      Below are my questions:

      Q1: The app notes mentioned the uses of DAD for determination, since I'm not interested in any amino acids eluted after peak #20 lysine, can I just run the analysis based on one signal only using the VWD, which is Signal A: 338nm, 10nm bandwidth, and reference wavelength 390nm, 20nm bandwidth? Since this is based on the reasons that Signal B (262 nm, reference 324 nm) are meant for determination of Hydroxyproline, Sarcosine, and Proline which I'm not interested on.

      I understand FLD is possible but the particularly low peak area of cysteine in app notes seem to be unsuitable for concentration measurement.

       

      Q2: Based on Q1, since FMOC is means for hydroxyproline, sarcosine and proline, is it possible for me to omit FMOC from my purchases? When FMOC is not used, should I still follow the gradient programs for all 23 peaks, or should it be shortened earlier to 100% mobile phase B after peak #20 lysine?

       

      Q3: Based on my digestion method (Performic acid oxidation and HCl-Phenol hydrolysis), since cysteine is important for me, is the use of DTDPA compulsory? How would I benefits from the uses of DTDPA from Agilent (#5062-2479)? I need a clear instruction on how to used this during my digestion, since it wasn't mentioned in the app notes.

       

      Q4: Based on my digestion method, after HCl hydrolysis, I'm planning to dry the acids to near total dryness using rotary evaporator (40 degree Celsius). After that, what kind of buffer or solvent should I used? AOAC suggested sodium citrate buffer pH 2.2, while some papers used 0.1 M HCl or borate buffer, or even neutralization with NaOH.

      From my instrument and column choices, which buffer does Agilent recommends?

       

      Q5: For amino acids standard supplied by Agilent, is it possible to make dilution for a 3 points standard curve from only one 1 nmol stock solution into lower concentration?

       

      I thanks everyone first who took their times to look into my questions. Thank you.