Chromatography Forum

Hello,

I'd like to know if someone could help me with something:

I'm currently working with a C18 cartridge, mobile phase Acetonitrile:water 40:60, isocratic. The products that I analyze are 5-methoxy-indole-3-acetamides.

The thing is that right now I'm working with two products. They have almost the same structure except for a piruvate substituent that one of them has (product 2). When I analyze them, the retention time of product 1 is higher that product 2. But, product 2 is more lipophilic than 1. Why does it run faster?

Thank you in advance,

V.-)

Hi Vandres

1. Is it possible the pyruvate substitiuent on (2) hasn't made it more lipophilic?

2. Is it possible that (2) is more lipophilic, but there are other factors affecting retention, such as solubility in the mobile phase or polar interactions?

3. Does elution order even matter, as long as you can achieve separation?

Thank you for your so interesting reply Ksharp,

Regarding point 1 I always thought that it was more lipophilic since people who synthesized it said so. They said that this substituent makes the structure more lipophilic and I just closed my eyes and believed it! but maybe I should think about it a little bit more... . I attached the image of the structure below.



2. I thought that these substances weren't so different but I should consider this too. I usually try to see things easier than they are... lack of experience! I'll learn...

3. No, the retention time doesn't really matter as I achieve the separation with no problems. I just wanted to understand why this was happening.

Thank you again for your time Ksharp! your reply has been very useful!!

V.-)

A couple of things to keep in mind about "reversed phase" chromatography:

While hydrophobicity (or lipophilicity, if you prefer) is a major factor in retention, it is not the *only* factor. "Secondary" interactions (for example, with residual silanols) can play a major role. So, while the "more hydrophobic = more retained" rule is general, it is not universal (that's why we do experiments!).

In the particular case of your molecules, I'm not sure that the addition of the pyruvate would make much difference in the hydrophobicity of the overall molecule. I'm not an organic chemist, but it looks to me like the relative number of hydrogen-bonding sites isn't all that different.

Several factors acting at the same time, that's the general rule in chromatography! an now I'll never forget it

Taking about hydrophobicity, ksharp comments woke up my curiosity. I found a web page that can give a prediction for any molecule (http://www.vcclab.org/lab/alogps/). Of course it's a prediction and not an experimental value but I think it's interesting. The result that I got was that the product with the substituent was more lipophilic than the one without it (logP=3.68 vs 2.33). So maybe I'll start to think again about polar interactions or different solubilities, perhaps the answer is there.

Thank you for your comments Tom,

V.-)

When I looked at the structure of the compound I got confused about what the piruvic acid substituent could be. To reassure myself I checked google, and sure enough, found only pyruvic acid. But: where is pyruvic acid (alpha-ketopropionic acid) in the drawing?
Incidentally, if there is really a tert-butyl group on one of the compounds it is not too surprising that it is more hydrophobic. On the other hand, even though I started out as a physical organic chemist, I would have tended to decline a polarity, etc., assignment of a molecule whose properties are foreign to me.

Eventhough there is t-butyl group, the pyruvic acid has two oxygen atoms, that makes the molecule more polar than its non derivative. As a rule of thumb in RP always more polar compounds elute first.

Hello again,

You are right HW Mueller, thank you, this substituent is called pivaloyloxymethyl but not piruvate. I memorized a wrong word to make it shorter, but a picture is worth a thousand words and fortunately everybody was talking about the same molecule :)

Thank you for your reply Umoshplc, what you say make sense and it's in accordance with previous comments so I think we're pretty close to the answer...

Thank you all for your help, you all make a great job in this forum and I'm really happy to have found it!