Hi, I'm doing amino acids of animals feed. I used Jajic et al. (2013) 's methods for the acid hydrolysis. Briefly, I will mix 0.5 gram of samples with 7 ml of 6 molars HCl-phenol solution, flush with N2 gas and heat in oven at 110 'c for 24 hours.
Then I will dry the acid to a small amount by rotary evaporator at 40 'c. The question is after this what kind of buffer/solvent should I used to dilute the solution to 50 ml?
AOAC suggested sodium citrate buffer (pH 2.2), however, that was mean for an amino acids analyzer. Meanwhile, Agilent recommended the Poroshell HPH-C18 column, rated for longer column lifespan under high pH operation due to derivatization with OPA/FMOC.
I want to know under my choices of instruments (column and derivatization reagents) and samples preparation, what kind of buffer/solvent would you recommend? In addition, which kind of materials of syringe filter will you used for that samples before injection into HPLC?
Thank you all.
I used following procedure--------------------
Samples were homogenised using a domestic blender to obtain equal portion. Then, weight 50 mg of sample into microwave vial and added 0.50 mL of Hydrochloric acid and 0.50 mL of propionic acid and freeze (-20 °C) into the domestic freezer for 1 hour. The frozen vials connected to the microwave accelerated unit (CEM, MARS 6, USA) and heated 160 °C (ramping time 10 min), and holding time was 15 min. After reaching the room temperature, hydrolysed solutions were completely dry using nitrogen gas and reconstitute it around pH 8 using, 983 µL 0.1N NaOH and 17 µL of 7.5 N NaOH. Then the following below mentioned optimized derivatization procedure.
In HPLC vial, 67.5 µL of MPA, 33 µL of OPA and 11.25 µL of sample (or standard) mixed together and wait 1 min prior to adding 15 µL of FMOC. Again, the vial was mixed and wait for 2 min and then added 0.1 N HCl 7.5 µL and injected to the HPLC.
A similar question was posed some years ago. I think the answers given then are still useful.
Check https://www.researchgate.net/post/HPLC_method_and_sample_preparation_for_amino_acids
Thank you all for your responds.
To Dr. B.K. Kolita Kamal Jinadasa,
It seems that you completely dried the HCl and used a neutralization step by NaOH to pH 8. I would like to know, how does formation of salts as a results of neutralization could interfere with HPLC and the column? Or salt has no effects on the analysis? Thanks.
To: Dr Andrei Blasko,
I checked the Regenerated Cellulose (RC), PES and PVDF syringe filters. They all have very low binding to proteinous samples. I want to go with RC, but it seems to be less suitable for acidic sample. Do you have more info to share about this?
To: Dr. Jos P M Wielders,
Thanks, I have go through the link you shared. The following buffers were found:
1. Borate buffer
2. 2:1 methanol/10mM ammonium formate solution
3. 20 mM HCl and Borate buffer
Would you share with me what are the criteria when selecting a buffer for sample? If it helps, these are my mobile phases:
Mobile Phase A: 10mM Na2HPO4, 10 mM Na2B4O7, pH 8.2: 5 mM NaN3. Adjusted to pH 8.4 with 1.2 ml conc. HCl.
Mobile Phase B: Acentonitrile:Methanol:Water (45:45:10, v:v:v).
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry