Hi,
I have extracted and concentrated honey enzymes/proteins.
Most abundant ones are alpha glucosidases and alpha amylase. Beside of these, other enzymes and major royal jelly proteins are also available but at minor quantities. After spin colums concentration processes I have cleaned-up protein concentrates from its small molecules interferences like sugars, organic acids and phenolics using dialysis method. Final injection solution was insect saline (Tris.HCL buffered pH 7.4). Using Thermo Accela UHPLC system equipped with DAD detector and BioResolve RP mAb Polyphenyl Column ( 450Å, 2.7 µm solid core 150mm*4,6mm) I couldn't achieve to gain nice peak shapes. As you can see from the images of chromatograms, enzymes give fluctuated peak shapes. What could be the reason of this ?
As mobile phases, I have tested TFA or Formic acid modified (%0,1) water-ACN gradients applying diverse of linearity steepness...Due to pH limitation of the column, I couldn't increase pH of the mobile phases. Approximate pI values of the working enzymes were between 7 to 8.
I have evaluated the same protein solution at my downstream process based on enzyme purification. I have injected solution to Hydroxyapetite, Anion exchange and SEC flash columns as tandem purifying workflow using BioRad FPLC system under the flows of neutral buffers and all peak shapes were gauissian and resolution was good enough...
Any suggestion would be appreciated,
Thanks in advance...
peak fluctuation mainly occurs due to the comparatively high strength of the solvent than the mobile phase. You may gain fine peaks by just diluting the solvent.
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