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Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Wed Feb 27, 2019 2:16 pm
by Fremouwski
Hello everyone,

I am experiencing issues with my HPLC analysis; Increasing pressure and loss of resolution, but replacing the guard-column resolved this. I expect issues with my sample preparation.

So a quick question:
* What would be good steps that can be undertaken to remove polysaccharides and proteins from water-based plant extracts?

I look forward to seeing your replies and suggestions.
Thanks in advance!

*Edited post to remove unnecessary details.

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Wed Feb 27, 2019 8:28 pm
by Zoraku
I don't have a lot of experience working with polysaccharides but if you are primarily interested in the amino acids themselves you might subject your extract to protein denaturation conditions such as 6M Urea, a very low or high pH aqueous solution or you can even attempt high salt to precipitate the proteins in hopes that it does not precipitate the amino acids (look up salting out for proteins). If you can precipitate the proteins in the extract you would just perform another centrifugation step and discard the pellet.

Do they make syringe filters that have a small enough pore size to catch proteins but let small molecules pass? Similar to the idea behind dialysis where the pore size allows molecules up to a certain size to pass through but larger molecules (such as proteins) cannot.

Just some ideas as I have never performed this kind of analysis...but hopefully this can provide insight or spark an idea from someone else.

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Thu Feb 28, 2019 7:49 am
by Fremouwski
Zoraku wrote:
I don't have a lot of experience working with polysaccharides but if you are primarily interested in the amino acids themselves you might subject your extract to protein denaturation conditions such as 6M Urea, a very low or high pH aqueous solution or you can even attempt high salt to precipitate the proteins in hopes that it does not precipitate the amino acids (look up salting out for proteins). If you can precipitate the proteins in the extract you would just perform another centrifugation step and discard the pellet.

Do they make syringe filters that have a small enough pore size to catch proteins but let small molecules pass? Similar to the idea behind dialysis where the pore size allows molecules up to a certain size to pass through but larger molecules (such as proteins) cannot.

Just some ideas as I have never performed this kind of analysis...but hopefully this can provide insight or spark an idea from someone else.


Thank you for your reply Zoraku. :)
Your suggestions give me leads to look for in literature, and I had not thought of looking into these size-exclusion filters myself, so thank you for the insights.

As you already remarked, these procedures might also influence the recovery of the AAs, but that is something that has to be tested out..

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Thu Feb 28, 2019 9:04 am
by Rndirk
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Thu Feb 28, 2019 9:18 am
by Fremouwski
Rndirk wrote:
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.


Hello Rndirk, thank you for the reply.
Just to clarify; Can acetonitrile or methanol be used for the simultaneous precipitation of both macromolecules?

Furthermore, I have so read elsewhere that acetone can be used for proteins precipitation. Do you know if this will also be effective to precipitate the polysaccharides?

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Thu Feb 28, 2019 9:37 am
by Rndirk
I think it depends what polysaccharides we are talking about. The larger (for example starch) the easier they will precipitate. But starch has a low solubility in water anyway..

There's a lot of literature on protein precipitation, as it is widely used as simple & quick sample preparation in clinical analysis (blood, urine).

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Thu Feb 28, 2019 3:51 pm
by Zoraku
Fremouwski wrote:
Rndirk wrote:
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.


Hello Rndirk, thank you for the reply.
Just to clarify; Can acetonitrile or methanol be used for the simultaneous precipitation of both macromolecules?

Furthermore, I have so read elsewhere that acetone can be used for proteins precipitation. Do you know if this will also be effective to precipitate the polysaccharides?


You can use acetone at a rough 3:1 ratio of acetone:sample solution but I have no idea how it will impact polysaccharides.

If you already know what sort of recovery you get with your normal extraction process, running a few of these precipitation reactions and comparing them to a normal extraction should help you gauge how much the recovery is affected by these other reactions.

I would also suggest trying to perform some of the precipitation reactions in a cold room, or with cold solvents (~4-8ºC) if you don't see success at room temperature.

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Fri Mar 01, 2019 10:14 am
by Fremouwski
Zoraku wrote:
Fremouwski wrote:
Rndirk wrote:
2 commonly used procedures come to mind to precipitate these macromolecules in aqueous extracts, followed by filtration or centrifugation:

- Add acetonitrile or MeOH, at least in a 1:1 ratio organic:water
- Use Carrez I & II, which are aqueous metal salt solutions

As Zoraku pointed out, there are more options.


Hello Rndirk, thank you for the reply.
Just to clarify; Can acetonitrile or methanol be used for the simultaneous precipitation of both macromolecules?

Furthermore, I have so read elsewhere that acetone can be used for proteins precipitation. Do you know if this will also be effective to precipitate the polysaccharides?


You can use acetone at a rough 3:1 ratio of acetone:sample solution but I have no idea how it will impact polysaccharides.

If you already know what sort of recovery you get with your normal extraction process, running a few of these precipitation reactions and comparing them to a normal extraction should help you gauge how much the recovery is affected by these other reactions.

I would also suggest trying to perform some of the precipitation reactions in a cold room, or with cold solvents (~4-8ºC) if you don't see success at room temperature.


Thank you, valuable suggestions!

Re: Sampleprep. for HPLC-FD analysis of watersoluble amino acids

Posted: Fri Mar 01, 2019 10:37 am
by Rndirk
Keep in mind that, if you're doing reversed-phase HPLC, there's a big chance peak shapes will suffer if you go from injecting aqueous extracts to organic extracts.

That's what I like about the carrez solutions; it doesn't add any solvent. But I can't tell if it works for amino acids. We use it for several HPLC sample preps on food extracts: antioxidants, sweeteners, ..