Hops Spectra

I'm trying to figure out what I might be doing wrong when running a spectra analysis on known hops with an AA of 6.2. Following the ASBC 6A guidelines I get a spectra as shown below. Can anyone send me an example of what they get when they follow the 6A guidelines. They state that there should be peaks at 275, 325 and 355 nm however I don't get that? I believe that I have prepared the sample correctly to the ASBC. I'm getting something like 8.5 AA, and would think that if anything it would be less then what is published on the package because of the time degradation.

I've never done this one, but I've done IBUs which I think might be similar. Can you describe the method and your process?

Anatidaephobic said:

I've never done this one, but I've done IBUs which I think might be similar. Can you describe the method and your process?

Click to expand...

Anatidaephobic,
The ASBC 6A process is to take 2.5 g of hops, place in a Toluene solution, spin for a maximum of 40 minutes, let the liquid and solid separate. Take a aliquot (small quantity) and dilute with Methanol. This is solution A. This dilution is then diluted again with a 6M (corrected after 10/8 post) solution of NaOH Methanol that was made prior - this dilution is called dilution B.

The same thing happens for the baseline solution except you leave out the hops and proceed the same way. I call this dilution C.

Then dilution B and C are placed inside the quartz cuvette and spectra analysis from 400 nm wavelength down to 200 nm is carried out. The equipment is run in a double beam mode utilized solution C as the baseline and subtracts the result from B. That is the trace you see.

However, I don't get the peaks at 355, 325 and 275 nm that the standard says I should. But maybe I shouldn't have a peak here and just utilize the absorbance values as I did. However, when those calculation are done I get 8.2 AA which I think is to high as the value from the package is 6.5. It could be possible however ....

I guess what I was looking for was for someone to have run though this process (ASBC 6A) and send me an absorbance trace of what they get. This way I could say mine is correct or its not and learn a bit more how labs actually do this type of AA / BA analysis.

I'm thinking that a lab is doing an alpha acid analysis has some calibration standard and an output showing what the absorbances are at these (3) wavelengths. Then its just a plug and chug. Never having done this before and not having an organic chemistry background, leaves me in the dark about what might be happening as a result of this process.

Thanks for your interest. If you need further detail, PM me and I'll respond.
BB

So I looked up some articles on this method. The main thing that jumps out at me about your graph is that you have Abs <0 in the middle there, which would mean your sample cuvette is transmitting more than your reference cuvette at these wavelengths. It might just be that your cuvette was dirty or there were slight differences in the solvent preparation between Dilution B and C. For instance toluene absorbs strongly at 269nm, so a error in volume in the reference solution may account for the sub zero Abs around 270nm.

In any case, what equation are you using to calculate the AA%?

Using the equation provided here I get:
(2/3)*((-51.56*0.8)+(73.79*0.69)-(19.07*0.02))=6.19%
Which would suggest your spectrum is pretty close to the accepted value.

I found these graphs showing approximately what form of spectra you should expect.

Anatidaephobic,
I can't thank you enough for your support on my question. This forum is so great with the help that we give one another.

First off, I found my math error which you seem to have found too - used negative of a negative in the equation of the 19.07 coefficient. Hence, got a larger value than I was supposed to.

The reference graphs that you provided are really helpful. I wasn't sure if there should be a strong peak at the 355 / 325 nm. Maybe, someone who does these things on a routine bases will give some additional feed back however from the a couple of brewers that I have talked to, their equipment spits out the absorbances at the set frequencies, so they really don't see these type of plots over the entire range.

I'm going to do another sample of a commercially purchase hop and see what I get again. And then try some of my home grown stuff. I will post the results for others to see once I have completed that.

One other comment that I have pertaining to the ASBC 6A is the difficulty making the 6M solution of the NaOH. The 240 g / L (or 24 g / 100 ml what I made) does not go into solution easily and certainly seems to precipitate back out. Each time that I have run though this procedure, I've had to try and heat (30 C) and stir for 30 minutes prior to get it back into solution.

Thanks again,
BB

Anatidaephobic said:

...For instance toluene absorbs strongly at 269nm, ...

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Anatidaephobic,
Looking at your absorbance curves, it appears that there is a strong absorbance at 225 not at 269.

I did use my reference C again on the Amarillo and homegrown Centennial. It appears that I do get a negative value again. The next time I'll try and make the toluene C solution from scratch. I just didn't want to change more than one variable this time.

The cascade image has been updated and the Amarillo and homegrown Centennial are shown below.

BB

bbbrew said:

...

One other comment that I have pertaining to the ASBC 6A is the difficulty making the 6M solution of the NaOH. The 240 g / L (or 24 g / 100 ml what I made) does not go into solution easily and certainly seems to precipitate back out. Each time that I have run though this procedure, I've had to try and heat (30 C) and stir for 30 minutes prior to get it back into solution.

Thanks again,
BB

Click to expand...

240g/L is right above the solubility limit of NaOH in methanol (Wikipedia shows it as 238g/L)

A few posts above you wrote that you were using a 5M solution of NaOH. Is the 6M solution erroneously high?

Marc,
The ASBC 6A standard calls for a 6.0M solution of sodium hydroxide in methanol. My 9/26 post was incorrect in saying 5M. It has been corrected now. Thanks for that and the solubility insight.

BB