Chromatography Forum

Hi,

Phosphate buffers with different pH values and 0.1%TFA are normally used reagents in mobile phase to improve peaks' separations and shapes.

In general, could any expert explain when we use which ?

Thanks.

The first difference here is that TFA is a good ion-pairing agent in water, whereas phosphate is not. So, I would view the TFA as having a primary role as an ion-pairing reagent, whereas the primary role of the phosphate is in pH control. Surely there are other issues in play here including UV absorption (TFA is not good at short wavelengths, but phosphate is), interference with electrospray ionization (TFA is tolerable at low levels, whereas phosphate is forbidden), and so on. The list of details is very long. If you have a specific question, please post it.

TFA is not good IP reagent, you need at least C4 carbon chain to have any IP properties. People call TFA ion-pairing reagent but it is a "urban myth" at least for small molecules. If you take a basic molecule and RP column you are not going to see much improvement in retention time by using TFA vs. phosphoric acid. The only difference might be is poor peak shape in case of phosphoric acid due to residual silanol interactions (because you create lower pH with TFA assuming same concentrations)

TFA is not good IP reagent, you need at least C4 carbon chain to have any IP properties. People call TFA ion-pairing reagent but it is a "urban myth" at least for small molecules. If you take a basic molecule and RP column you are not going to see much improvement in retention time by using TFA vs. phosphoric acid. The only difference might be is poor peak shape in case of phosphoric acid due to residual silanol interactions (because you create lower pH with TFA assuming same concentrations)

Thanks fro the reply.

I think TFA as an IP reagent helps to improve separation and peak shape especially for peptides and proteins. These components obviousely possess long carbon chains. The machanism is that the trifluoroacitates associate with the positively charged groups on peptite bonds leading to the polarity reduction of peptide/protein molecules, and resulting in better intereation with RP column stationary phase. Small molecules don't show much improvements are probably due to not having too many positively charged sites on target molecules. Is this correct?

TFA is not good IP reagent, you need at least C4 carbon chain to have any IP properties. People call TFA ion-pairing reagent but it is a "urban myth" at least for small molecules. If you take a basic molecule and RP column you are not going to see much improvement in retention time by using TFA vs. phosphoric acid. The only difference might be is poor peak shape in case of phosphoric acid due to residual silanol interactions (because you create lower pH with TFA assuming same concentrations)

Thanks fro the reply.

I think TFA as an IP reagent helps to improve separation and peak shape especially for peptides and proteins. These components obviousely possess long carbon chains. The machanism is that the trifluoroacitates associate with the positively charged groups on peptite bonds leading to the polarity reduction of peptide/protein molecules, and resulting in better intereation with RP column stationary phase. Small molecules don't show much improvements are probably due to not having too many positively charged sites on target molecules. Is this correct?

In my opinion TFA makes peptides and proteins more polar a it protonates amino groups, in some cases you might see slight increase in hydrophobicity if peptide has a lot of free carboxylic acids and you suppress ionization of these acids and make it less polar.

TFA is not good IP reagent, you need at least C4 carbon chain to have any IP properties. People call TFA ion-pairing reagent but it is a "urban myth" at least for small molecules. If you take a basic molecule and RP column you are not going to see much improvement in retention time by using TFA vs. phosphoric acid. The only difference might be is poor peak shape in case of phosphoric acid due to residual silanol interactions (because you create lower pH with TFA assuming same concentrations)

Thanks fro the reply.

I think TFA as an IP reagent helps to improve separation and peak shape especially for peptides and proteins. These components obviousely possess long carbon chains. The machanism is that the trifluoroacitates associate with the positively charged groups on peptite bonds leading to the polarity reduction of peptide/protein molecules, and resulting in better intereation with RP column stationary phase. Small molecules don't show much improvements are probably due to not having too many positively charged sites on target molecules. Is this correct?

In my opinion TFA makes peptides and proteins more polar a it protonates amino groups, in some cases you might see slight increase in hydrophobicity if peptide has a lot of free carboxylic acids and you suppress ionization of these acids and make it less polar.

Yes, all depend on the actual peptidre/protein targeted. The proton will combine with NH2-end group to for NH3+, while COOH remaining on neutral if an acidic enviroment is provided, thus the negative acetate, CF3COO- associates with NH3+ end of the target molecule to become an ion-pair. Therefore in overall its hydrophobicity indreased because of the ion-pair. Is this explanation reasonable?

Here it is an experiment example:

I am trying to separate two NH2 end capped dipeptide, Fmoc-D-Phe-L-Leu-OH and Fomc-L-phe-L-Leu-OH. Obviousely you can see the structural difference is very minimum, their palarities are very similar, they are not enantiomers as well. By using a 250x4.6 5u, c18 column and mobile phase A(0.1%TFA), B(MeOH+0.1%TFA) with gradient B 30-95% and 0.8mL/min flow rate, I got separaton factor of 1.6 at about 40 min for the two analytes which is too long time. The separation will lost if I shorten either gradient ratio or time length. So I would try to reduce the dipeptides' hydrophobicity by employing an approprate buffer to make the COOH end to become COO- in order to push peaks elute sooner. Of cause other options are available such as change column length, c18 to c8,.....ect. However, I would like to see how the mobile phase change, so that the analyte hydrophobicity change affects the separation in this case. Please common!

BTW, I don't have pka value informayion, but the pH of the analyes are 5-6. Do you have any buffer type recommandation please?

In you case I would try to stay with isocratic conditions. When you have compounds which are very similar you want to stay with isocratic. Your another alternative would be chiral chromatograpphy but it is more expensive. If are are willing to send me samples we can try to develop method for you free of charge on one of our mixed-mode columns. You can also try another IP reagent with longer chain.

In you case I would try to stay with isocratic conditions. When you have compounds which are very similar you want to stay with isocratic. Your another alternative would be chiral chromatograpphy but it is more expensive. If are are willing to send me samples we can try to develop method for you free of charge on one of our mixed-mode columns. You can also try another IP reagent with longer chain.

Hi Vlad
Thank you for your nice offerering. For some reasons I am not allowed to send out sample out. As you advised, I would change the mobile phase compositions including IP, pH and try running isocratic in stead.

In you case I would try to stay with isocratic conditions. When you have compounds which are very similar you want to stay with isocratic. Your another alternative would be chiral chromatograpphy but it is more expensive. If are are willing to send me samples we can try to develop method for you free of charge on one of our mixed-mode columns. You can also try another IP reagent with longer chain.

Two questions:
1. For a gradient example, the organic phase was 25-95% running 60 min with 0.8mL/min flow rate, and the peak eluted at 40min, how should I estiamte the organic concentration if ruunning isocratic in stead?
2. When running gradient, the IP should be put in the water or organic phase, or both phases?

In you case I would try to stay with isocratic conditions. When you have compounds which are very similar you want to stay with isocratic. Your another alternative would be chiral chromatograpphy but it is more expensive. If are are willing to send me samples we can try to develop method for you free of charge on one of our mixed-mode columns. You can also try another IP reagent with longer chain.

Two questions:
1. For a gradient example, the organic phase was 25-95% running 60 min with 0.8mL/min flow rate, and the peak eluted at 40min, how should I estiamte the organic concentration if ruunning isocratic in stead?
2. When running gradient, the IP should be put in the water or organic phase, or both phases?

1. The calculated organic con for isocratic elution is alout 72%.
2. The IP should only be added to the water as dissolve issues with organic phase.
3. In addition, why not give a try to ace? As in some cases, ace is complementary with met.

In you case I would try to stay with isocratic conditions. When you have compounds which are very similar you want to stay with isocratic. Your another alternative would be chiral chromatograpphy but it is more expensive. If are are willing to send me samples we can try to develop method for you free of charge on one of our mixed-mode columns. You can also try another IP reagent with longer chain.

Two questions:
1. For a gradient example, the organic phase was 25-95% running 60 min with 0.8mL/min flow rate, and the peak eluted at 40min, how should I estiamte the organic concentration if ruunning isocratic in stead?
2. When running gradient, the IP should be put in the water or organic phase, or both phases?

1. The calculated organic con for isocratic elution is alout 72%.
2. The IP should only be added to the water as dissolve issues with organic phase.
3. In addition, why not give a try to ace? As in some cases, ace is complementary with met.

1. 72%. Perfect!
2. I got it, thanks! However, what if 0.1% TFA is used which has no dissolve issues with either aqueous or organic (ACN, MeOH) phase? Only add it in water in this case please?
3. What does "ace" stand for please?