Actually, pure imidazole has very weak absorbance at 280 nm. The absorbance seen in solutions of imidazole is due to contaminants. Different imidazole products have different levels of UV absorbance at 280 nm. I faced the same problem and switched to a more expensive, higher-purity imidazole and nearly eliminated the background absorbance. Unfortunately, the low-absorbance products tend to be relatively expensive because of their high purity . I suggest you ask for samples from the chemical suppliers you use, make a 1 M solution of each, and measure their absorbances with a spectrophotometer. Either that, or buy the highest purity grade you can afford.

You can used to clean up imidazole by mixing it with activated carbon, shaking for 10 min, and then filtering through paper.

Exactly! But even the high purity - expensive ones - don't always check out as being very pure, in contrast to their claims.

Do you have a direct reference to a certain protocol to clean up imidazol preps via activated carbon?

Thanks already for this input!

I take a method for analysis of food :

https://fssai.gov.in/upload/uploadfiles/files/Manual_Food_Additives_25_05_2016(1).pdf

Prepare your chromatography buffers A and B.

At room temperature and mix until all water soluble substances are in solution (15 min).

For buffer with imidazole: For an example of weight 17g imidazole, add 60% or 10g activated carbon and mix thoroughly.

Let it stand for 30 min Filter under vacuum coarse fritted glass funnel with glass wool 1µm or 2µm, and after you use a second buffer filtration 0.45µm for chromatography.

We are currently having a similar problem. After forcefully switching to a new product after the old one being discontinued (both from Carl Roth: discontinued Cat. No. X998, new Cat. No. 2C4N), we ended up getting 140 mAU signal at 500 mM imidazole on Äkta (which translates to A280 = 0.17 for 0.1 M solution at 10 mm path). With the old product, we never got more than 20 mAU signal at the same imidazole concentration.

The fun fact is that the new product (Cat. No. 2C4N) should theoretically be PURER and has more stringent specs with regard to absorbance at 280 nm (A280 ≤0.1 for 2C4N vs ≤0.15 for X998, measured at 0.1 M solution). Thus, the new product 2C4N from Carl Roth does not even comply to its specification, which I of course reported to them and expect to get a refund.

In parallel, I am ordering imidazole from two other manufacturers (Serva 26081 and Sigma I2399) and will compare them for A280. I can post my results here if you wish. I will be also glad if you share the products you made good experience with.

Hi Mikhail and thanks for your answer, really helpful!

It would be great if you could post your experience with the other providers.

In the meantime we are still using 'what we have' so to not have to waste it: we simply collect the fractions during elution and check on SDS-PAGE, and do not rely on the peak area for protein concentration determination. In many cases we do downstream desalting or further purification steps, so the IZ is 'phased out' from interfering...

The thing is that, even from the same provider, some batches are good, some aren't...so our next purchase will also include an A280 background test, and if 'too high', we will return it and ask for either another batch or a full refund.

FYI: our best batch is from Carl Roth AND our worst batch is from Carl Roth...

Kevin De Muynck Which Cat. No. from Carl Roth are you currently using? Have you tried different ones? I also added Cat. No. 2X11 ('for biochemistry') into my comparison, although it should be less pure than 2C4N we are currently having problems with...

Mikhail Barkovskiy, yes we have different Carl Roth article numbers and tested these: X998.2 is fine and doesn't give background. X998.4 batch# 310299172 is also good... but then, X998.4 batch# 243331576 gives a lot of background. Acros organics (122025000) gives background, but lower than the contaminated Roth one... We also tested a Sigma batch from someone else: also some background, same level as Acros...

It's all super variable...

That's interesting - we've used X998 and never got that dirty batch. But X998 is now discontinued anyway, that is why we switched. Let's see what we get from our comparison. I wonder why there is so much variability in this production process - whether it comes from variation in the raw material quality or from insufficient process control. Actually, X998 and 2C4N are some of very few products that contain A280 in their specs at all - most don't even test for it (unless you want to pay 500 Eur for 100 g). So my hope is that Carl Roth will improve its batch quality and we stay with them...

Hi,

You may make an inquiry at Alfa Chemistry, they offer a wide range of high quality chemicals and laboratory equipment.

https://www.alfa-chemistry.com/inquiry

Mikhail Barkovskiy, do you have results yet of the imidazole from two other manufacturers (Serva 26081 and Sigma I2399)? Cheers!