First figure out the net charge of your peptide

(K+R)-(D+E)= net charge

If the net charge is positive (ie basic) dissolve your peptide in something acidic to promote protonation of K + R. Use TFA 0.1%.

If the net charge is negative (ie acidic) dissolve your peptide in something basic but volatile like 50mM ammonium bicarb. or ammonium formate that you have pH'd to 9 or 8. You can also use TEA (Triethylamine) in small quantities.

If the peptide is neutral or highly hydrophobic use 0.1%TFA with increasing amounts of ACN like 50% ACN +0.1%TFA.

Good luck

Thanks Brian..but in PLRP column we can not use 1% TFA..i think it is not goood for column...!

I guess there are some advantages in silica!

I would suggest you to solubilize your peptide by adding also some Dimethylformamide (DMF) in place of Acetonitrile. Try for example 50% DMF/50 % water (plus 0.1-0.5% TFA). Another good solvent for peptides is Dimethylsulfoxide (DMSO) which is miscible with water in all ratios, like ACN or DMF.

Hello,

Hydrophobic peptides are always problematic. To help solubilizing, I use guanidine hydrochloryde in salt, directly in the solution. For very hydrophobic peptides, you can use pure DMSO ( up to 20 % of the solution), but beware of oxidation of Cys and Trp.

First figure out the net charge of your peptide

(K+R)-(D+E)= net charge

If the net charge is positive (ie basic) dissolve your peptide in something acidic to promote protonation of K + R. Use TFA 0.1%.

If the net charge is negative (ie acidic) dissolve your peptide in something basic but volatile like 50mM ammonium bicarb. or ammonium formate that you have pH'd to 9 or 8. You can also use TEA (Triethylamine) in small quantities.

If the peptide is neutral or highly hydrophobic use 0.1%TFA with increasing amounts of ACN like 50% ACN +0.1%TFA.

Good luck

Hi Shubhendu,

You could try the other way around and change your RP column to HILIC. Then the starting solvent is 95-98% ACN, 0.1 % TFA and you can decrease the ACN with a linear gradient. Of course your hydrophobic peptides will elute very early in this type of column and you should check that they are indeed resolved by the column.

Good luck

You haven't said what your initial conditions are. If this is an extremely hydrophobic peptide that elutes at 60% ACN, then you are fine to dissolve it in very high concentrations of ACN to match appropriately-increased ACN levels in initial loading conditions.

Yes we don't know enough of your situation to correctly help you. What is the pI of your peptide? What column are you using?

DMF is very bad for columns, most will dissolve.

DMSO might be ok, but you will not get ride of it.

Hi David... I have PLRP column. I am not sure if we can use DMSO in such column. Also we can not use more than 0.05% TFA in PLRP column. The theoritical pI of my peptide is 5.83. Also I have a N-terminal aminooxy group !

You can try adding some DMF instead of DMSO. Also, if your retention time is too high, try using a C4 column instead of the standard C18. Can you give us the sequence?

I only found data on a PLRP-s column. I will assume this is the same as yours. Very interesting. Its able to use the pH range from 1 to 14 and temperature up to 200C. The typical solvents are 0.1% TFA or 0.1% formic acid. I don't think you can use either DMF or DMSO from what I found.

But based on you pI and the pH range of the column, you really should try Ammonium Hydroxide instead of TFA. I found this resource great:

http://www.chem.agilent.com/Library/catalogs/Public/5991-1059EN%20LC_Columns.pdf

With aggregation prone peptides and proteins, I also have better luck in NH4OH at pH 10. Use 1% should be ok. The link above talks about using 1, 10 and 100mM, You can calculate this into % to see where 1% lies. Also they talked about using Methanol, but I wouldn't do that as it will precipitate most proteins. ACN and NH4OH, should work. Give it a try.

you could try to solubilize it in isopropanol instead of ACN and do the chromatography using isopropanol as solvent B as well. A colleague of mine has been successful with this years ago on surfactant protein C (sequence FGIPCCPVHLKRLLIVVVVVVLIVVVIVGALLMGL)

, even in the doubly palmitoylated form. She used a C4 column and around 40% isopropanol as starting concentration. With isopropanol you have to take care of the backpressure, usually you have to work at >40°C to reduce this to an acceptable limit.

Answer to David Bateman: 50-100 ul of DMF cannot damage/dissolve the column at all!

Hydrophobic peptides like formic acid not TFA, it should be possible to solubize them in 20% MeCN, 1% FA and separate on PLRP_S column at 40°C from 20-100% MeCN, 0.2% FA

To add to Marcus's answer, we've mixed acetonitrile and isopropanol 50/50 for solvent B to reduce backpressure to reasonable levels but retain better elution. You can also use acetone for solvent B for low backpressure, but I don't know what the chromatographic performance would be relative to acetonitrile.

Thank you all...I hope I will be able to purify my peptide with all your suggestions...It was great to know many things from this discussion.

....Dear Shubhendu and all...don't forget also the possibility of ethanol. It has more or less the same solvent power of isopropanol but without the backpressure problems of isopropanol.

Excellent suggestions above: IPA, ethanol, formic acid... You may also try TFE, 0.1-0.3 % in solvents A and B, a few % in your sample.

Depending on the sequence. You may use DMF and than dilute with you starting solution, also guanidine chloride, trifluoroethanol or DMSO. Do not used DMSO if you have Met residues in your sequence.

Ethanol can be a good choice as acetonitrile substitution to improve the solubility of hydrophobic peptides and furthermore is very transparent in UV quite as transparent as water so you can work at 210 nm with your UV detector with very good sensitivity. DMF and DMSO are not so good from transparency point of view.