Hi, I need to analyze amount of cysteine spray-coated on animal feed. I read the AOAC (2000) which briefly stated that:
(1) Mix 100 mg feed with 5 ml performic acid and oxidized on ice for 16 hours.
(2) Add 50 ml 6M HCl-phenol and hydrolyze under reflux for 24 H at 110'c.
(3) Add 20 ml norleucine internal standard. Dry with rotary evaporator, and rinse with water.
(4) Filter the hydrolysate and adjust the pH with NaOH and sodium citrate buffer.
Here are my questions:
Q1: Many methods stated that hydrolysis need to be under vacuum but AOAC didn't mentioned it. (i) Does performic acid oxidation required vacuum? (ii) Does HCl hydrolysis required vacuum?
Q2: If vacuum is required, can you share with me your protocol for vaccuming? Which specific pump model you used. Is that a video for this step? This step has been bothering me much as I'm not sure if I can do it in my lab.
Q3: If I used this hydrolysis tube, which volume should I go with? 1/6/18 ml? Link to product: https://goo.gl/bs99Tt
Q4: The AOAC method uses rather a large amount of chemicals, is this common or are there any newer methods I can use?
Q5: There are many derivatization reagents. Which derivatization agent will you recommend? Fyi, I will be using pre-column derivatization technqiue with Agilent 1260 infinity and Agilent ZORBAX Eclipse Plus C18 (4.6 x 100 mm x 3.5-Micron).
Sorry for my rather long post, as this is my first time doing this analysis. Thank you all first for your time and effort in responding to these questions.
As an alternative for the atmospheric hydrolysis, you could try performing the hydrolysis in concentrated HCl (19- 26 v%) in a microwave oven at 150C for approx. 4 hours. Conversions are usually >90%.
Hi Dr. Stjepan Krešimir Kračun Thank you so much for your input.
Here are some of my further questions:
Q1: So you are suggesting for me to use the pump that comes with a rotavap to vacuum my test tube? That sound feasible but how would I determine if my hydrolysis tube is sufficiently vacuum (without over-doing that it crack). Is this a process of mere seconds to few minutes?
Q2: In this paper that I just found (http://www.doiserbia.nb.rs/img/doi/0352-5139/2013/0352-51391200144j.pdf), the authors compared a non-vacuum to vacuum method, and found that a non-vacuum method is non-practical for HPLC to produce reasonably peak. What's your opinion on this?
Q3: Between rotavap drying and nitrogen gas drying of the solution after hydrolysis, is it okay if I prefer the nitrogen method? I actually have both in the lab but thinking that N2 blow can handle multiple samples at once.
Q4: For derivatization agent, I'm considering WATERS AccQ-Tag method. DO you have any comment about this over the Agilent OPA/FMOC?
That's all. Thanks.
Hi Dr. Ronald Paulissen. Thank you too for your reply.
Your method is interesting too. Is that a citable reference to this? I would like to read more about it. Also, does this method require the tube to be vacuum?
By microwave, just to clarify, are you referring to a regular microwave or advanced microwave-assisted digester such as this? https://www.milestonesrl.com/en/microwavedigestion/ethos-up/21-publications.html?
I am quite in accord with Stjepan Krešimir Kračun. However I wouldn't expect to be an easy task to fully dry with nitrogen 50 ml of 6 M HCl. Better to handle only a smaller aliquot (1-5 ml or even less) at once after having taken to a known volume the total hydrolysate. That even when using a rotavapor where in case successive 2-3 evaporation cycles after successive small washing water addition to the residue are taking the pH near to neutrality thus making easier or even unnecessary the addition of NaOH.
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