Hello!
column (SEC 10-400 кDа) https://www.waters.com/waters/partDetail.htm?partNumber=WAT011530
For calibration i chose standard protein:
- protein MW, kDa rt, min
- Aprotinin (5) 6,5 18,21
- Ribonuclease A (4) 13,7 16,06
- Carbonic Anhydrase (3) 29 15,3
- Ovalbumin (2) 44 14,62
- Conalbimin A (2) 75 14,31
- Aladalose (2) 158 13,84
- Thyroglobulin (1) 669 12,5
(1)-(5)- peak number (see photo)
Flow rate 0,6 ml/min isocratic - PBS (sigma) pH=7,4, column temperature- 25 0C
Sample volume-20 µl , sample - pool of all protein
I changed buffer ( discrease pH to 6,6 or another composition) and flow rate, but i didn t improve resolution (2)peak containes Ovalbumin,Conalbimin A and Aladalose
Do you have any suggestions?
Thanks for your time
Hi Antonina,
If my approach is right, I would like to know how pure were your samples after elution? Peak shouldering, I see, is an existing issue, though.
Navnit Kumar Ramamoorthy i used GE calibration kit with monomeric purity more 80% according to specification (often 90-100 % serificate of analysis)
And earlier when i worked on another column i could separate this proteins
After elution on this column i didn t reseach protein because i can t separate
Also standard proteins ( Aprotinin,Ribonuclease A, Carbonic Anhydrase, Ovalbumin, Conalbimin) were separated by electrophoresis SDS PAGE (one major band )
Hi Antonina,
SO is this chromatogram, a Calibration verification standard? If it's an actual sample, I would suggest you to head with Ion exchange- HIC- GPC combination. Addition of an appropriate quantity of NaCl, and maintaining the sample volume (1-3% lesser than the column volume), and maintaining a lesser flow rate than what you are using at present may help you solve issue, I modestly assume. Apart from this I am providing you with the links of some artciles, which may be of assistance to you.
https://www.waters.com/webassets/cms/library/docs/local_seminar_presentations/sp_sem_uplc_troubleshooting_inst_2014.pdf
https://www.agilent.com/cs/library/posters/Public/Final%20TIPS%20and%20Tricks%20HPLC%20Troubleshooting%20(2).pdf
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Supelco/Bulletin/4497.pdf
Navnit Kumar Ramamoorthy thanks!
But i can't use reverse phase chromatography on this type of column
Peak one is at your exclusion limit. Try adding a mixed bed column with a higher molecular weight range in series with your original.
Hi,
In many SEC cases, decreasing inj. volume or flow rate are good options to improve resolution. Since the comman SEC phases at protein purification applications demand mild salt concentrations at mobile phases for optimum performance. You may try to add approximately 100 mM NaCL to your buffer and if applicable, a bit of higher column temperatures may be tested for getting different protein diffusion rates to pores of SEC resin.
Good luck.
I agree with Ismail. Decrease the injection volume and change the flow rate and change the oven temperature!!