It is a cationic peptide of 40 aa. I used solid phase extraction as a concentration method, after a cation exchange that yields more than 97% purity, determined by RP-HPLC.
I would assume the peptide is not binding to matrix. Check the absorbance at 210 nm of the wash.
If I´m checking the purity by RP-HPLC and the peptide eluted with 40 % of AcN:H2O 0.1 %TFA, why did not bind to the matrix in Sec-Pack C18 Merck cartridge?
It's possible like Toufic says the peptide is aggregating before entering the sec-pack column. Does the crude peptide have low solubility? If it does, this may be an indication of aggregation. I had this problem before and got around it by dissolving the crude peptide in 6M guandinine hydrocholoride before solvent exchange and purification.
I am attaching the spectra of the sample called FII and the spectrum of unbounded or whatever it did not stick to the matrix while I'm loading the secpack cartridges with the sample derived from cationic exchange. Please tell me if you get the information or if you can read properly, it is the first time I am using this way of exchanging information.
I am surprising with the lack of absorption maximum at 280 nm, although the peptide has no tryptophan. However, note that the sample which is not retained in the matrix (unbound) exhibits a maximum at 280, but is small.
These spectra are superimposed after performing scanning each separately, each one made with the solvent that appears in the description of the sample in the legend. That is, the sample that eluted from sec pack FII with 80% ACN 0.1% TFA, was used the same solvent as blank. The sample came without adhering to the column while loading the column was performed with a blank TFA0.1%. I attached the spectrum in the .pdf document. It may be possible that AcN deformed the absorption profile? I ask given the absence of the maximum at 280 nm!!
Let's see if I understand:
Blue line: Sample eluting from the SecPak C18 column with binding?
Green Line: Sample eluting from the C18 column without binding?
My first guess is the capacity of the SecPack C18 column used to concentrate the peptide is exceeded. All of the binding sites on the column are occupied so the remainder of the peptide washes out without binding.
Does your peptide have a tyrosine? Tyrosine also absorbs at 280 nm (1200 vs 5500 molar absorbtivity).
Yes, you have understood. The peptide has 5 Y and 2 F. I supposed that these aa. are also contributing to the Abs at 280 nm. But still intrigue me the absortion profile of the sample FII. I know that some solvents has strong shift effects over the absortion spectra, therefore I am not sure if I can take this Abs measure at 280 nm without maximun to determine the concentration of my sample using the molar absorptivity of this peptide.
On the other hand, the SecPack columm has 3 mL capacity and the sample had approximatelly 0.2 mg/mL in 18 mL of total volume. The column could be old, and its binding capacity lesser??
How can I know that the peptide is aggregated?? I have no signal of instability!!
OK, false alarm. The Sep-Pack cartridges were renovated and just now is that I realized that the pore size of the current cartridge is 60 A, so I deduct that is the reason for the loss of ability to bind to the matrix. This definitely seems to be the problem! Thank you very much to the contributors, your comments were very helpful!
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