Hop Extraction and Characterizing the Extracted Compounds

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Trouble-Brewing

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I am doing a special project in my organic chemistry class and am wondering if there is a fairly simple way to characterize the compounds I extract. I plan on boiling hop pellets in a ratio of 3g of hops in 25ml of ethanol for an hour, then cooling and filtering and drying under vacuum. I did the extraction at home using some gin I distilled down to somewhere near 80% ethanol and it definitely extracts the bittering compounds I just need a way to separate the acids from the essential oils and prove that they have been extracted. All of the papers I have found use high performance liquid chromatography or centrifugal partition chromatography neither of which I have access to. I do have access to NMR, mass spec, gas and column chromatography. Any help would be great, thanks
 
This is supposed to be a mass-spectrograph and I believe the six sharp spikes are the bittering compounds. The article was rather vague.

gc-ms-Beer.jpg
 
Using the logic of boil additions:
Short extractions will leave you with mostly flavor compounds, and long extractions will leave you with more of the bitter compounds.

I would also assume that the aroma compounds or more hydrophobic, and the bitter compounds more hydrophilic.

Putting these together, aromas should come out in a quick extraction with cold EtOH. After that boil the crap out of the hops in water. The volitile aromatics will vaporize, and you will be left with the bitters.

Another approach would be to destroy the aromatics through oxidation.

Also, be sure to choose different hops. Some are much higher on aromatics, while others are almost strictly bittering compounds.
 
These might contain some useful information.

ADVANCES IN THE LIQUID-CHROMATOGRAPHY OF HOP BITTER COMPOUNDS
VERZELE M, STEENBEKE G, DEWAELE C, VERHAGEN LC, STRATING J
HRC-JOURNAL OF HIGH RESOLUTION CHROMATOGRAPHY

also

Hop (Humulus lupulus L.) proanthocyanidins characterized by mass spectrometry, acid catalysis, andgel permeation chromatography
Taylor AW, Barofsky E, Kennedy JA, Deinzer ML
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

Hope this helps. :mug:

~andrew
 
NMR won't do you any good until you separate the compounds from one another. HPLC really is the way to go. I'm not sure the compounds are volatile enough to run through a GC without having to derivitize them first - which is probably why people use HPLC - saves a step.

You might be able to go old school and do this by thin layer chromatography. I bet there are some papers from the 70's or 80's that have a method. Hmmmm, that might be a way for the home hop grower to get an idea of the AA content of their hops.
 
With some NMR skill, it should be easy to tell the components apart, but I don't know how much they'd pile up in a mixture. I'd just extract the hops with a non-polar solvent such as hexane, and run a liquid chromatography column on it. This should at least yield a crude separation.

The hydrogens on the carbon directly next to the carbonyl is where I'd look. I remember looking at some of the compounds in my NMR class. Are you looking at acids in raw hops, or the iso-alpha acids you get from boiling?
 
Thanks for the responses. I forgot to mention I had access to solid and liquid IR too, not that it will really help without separating the compounds. Thanks for the articles I will have to check them out at school tomorrow all I can get right now is abstracts. When I was looking yesterday I couldn't seem to find any old school stuff, thin layer would be perfect though.
 
CHansen6 said:
With some NMR skill, it should be easy to tell the components apart, but I don't know how much they'd pile up in a mixture. I'd just extract the hops with a non-polar solvent such as hexane, and run a liquid chromatography column on it. This should at least yield a crude separation.

The hydrogens on the carbon directly next to the carbonyl is where I'd look. I remember looking at some of the compounds in my NMR class. Are you looking at acids in raw hops, or the iso-alpha acids you get from boiling?
Click to expand...

Dont really have much NMR skill, just learning all that right now. I have been reading that you can get more out with a polar solvent which is why I am leaning towards ethanol. Not sure about the iso or raw, though after an hour boiling in ethanol, if that is how I end up extracting, then iso. Guess it really comes down to what I can find for papers on separation. My teacher wants something that has already been done, though I might be able to convince him to let me do a crude separation as you described
 
Check the boiling temps of the things you want to separate you might be able to get away with a rotovap.
 
Yeastie said:
Check the boiling temps of the things you want to separate you might be able to get away with a rotovap.
Click to expand...

The a acids are all very similar, really doubt that boiling points are very far off. Most of the molecule is the same just a different R group comming off the ring
 
Author(s): Kac J, Mlinaric A, Umek A
Source: JPC-JOURNAL OF PLANAR CHROMATOGRAPHY-MODERN TLC Volume: 19 Issue: 107 Pages: 58-61 Published: JAN-FEB 2006
Times Cited: 3 References: 10 Citation MapCitation Map
Abstract: A sensitive thin-layer chromatographic method has been established for quantification of xanthohumol in hops (Humulus lupulus L.) and hop products. Chromatographic separation was performed on silica gel 60 HPTLC plates with toluene-dioxane-acetic acid, 77 + 20 + 3 (v/v), as mobile phase. The plates were scanned densito-metrically at lambda = 368 nm. The method was validated for precision, repeatability, and accuracy. It was found to be precise - intra-day and inter-day RSD were 1.7% and 2.3%, respectively. Instrumental precision and repeatability for the method were found to be 0.38 and 1.5 (%CV), respectively. The accuracy was checked by measuring the recovery at three levels; average recovery was 103.9%. The method was used for the estimation of xanthohumol in hop extracts and xanthohumol-enriched beer and was confirmed to be suitable for this purpose.
Click to expand...

Here's a start. Its for an oil though, not an AA. I did find some perfect articles from '68 and '74, but the journal archives online don't go back that far - no abstracts either
 
pjj2ba said:
Here's a start. Its for an oil though, not an AA. I did find some perfect articles from '68 and '74, but the journal archives online don't go back that far - no abstracts either
Click to expand...

Thats the problem I have been running into, seems like all the articles old enough to have simple technology are not available to me
 
a few options you might have are: 1) using the American Chemical Soceity website. They have a ton of good stuff that goes back ages. 2) if your school has SciFinder Scholar, you might want to try that. Again, great search engine. 3) your library might have an interlibrary loan program which allows you to get articles either via email or hard copy. I'll check my library tomorrow to see what we have access to.
 
If you do try NMR, I can help you out a bit. The hydrogens directly next to the carbonyl on the relevent sidechain are the ones I'd look at.

Humulone will exhibit a ddqq pattern, with a big geminal splitting (16-18Hz). One of the doublets may go to an AB quartet depending on how much the chiral center affects it.

Cohumulone should have a qq pattern and integrate to 1H

Adhumulone is present in small percentages, but if isolated or concentrated will show a tq pattern.

I don't think you'd be able to discern cohumulone from colupulone, or adhumulone from adlupulone by this method. However Lupulone should lose one of the doublets from humulone because the chiral center is lost.

Below is an old scientific american article I came across while looking up the various molecules.

The Bitter Substance Of Hops
 
celts said:
a few options you might have are: 1) using the American Chemical Soceity website. They have a ton of good stuff that goes back ages. 2) if your school has SciFinder Scholar, you might want to try that. Again, great search engine. 3) your library might have an interlibrary loan program which allows you to get articles either via email or hard copy. I'll check my library tomorrow to see what we have access to.
Click to expand...

I have not tried the chemical society website yet but my school has something similar to scifinder as well as the interlibrary loan program. Its just a matter of searching the right terms which apparently havent yet.

Well what have you tried? Did you try column chromatography or TLC?
Click to expand...
Nothing yet, I need to show my teacher an article before I can start

If you do try NMR, I can help you out a bit.
Click to expand...
Thanks. I am going to check that article out right now
 
Yeastie said:
hope this helps. I find it stupid you need an article first, only being allowed to repeat something someone else did doesn't sound very groundbreaking or in the spirit of science.
Click to expand...

Yeah its not all that ground breaking but at least its not another experiment out of the boring lab book. The need for a paper is needed to prove to my teacher that I did isolate what I claim to

I did brew a small 2.5 gallon batch with some hop extract that I extracted from some pacific gems. Distilled 2 cups of cheap gin down to a cup of what should have been 80% ethanol and then boiled an ounce of crushed pac gems then filtered off the hops and washed them with warm water. To distill the gin I put a small bowl in the bottom of a pot on top of a metal measuring cup so it was off the heat a little. Then I put the gin in the bottom and the lid on upside down with ice on the top. Ethanol boiled up hit the cold lid and dropped into the cup. I put it in a simple pale ale, and added the extract at the beginning of the boil then added a half ounce of cascade in the last 5 min. I just tried it today and it came out pretty good, needs some more time to condition but definitely damn bitter.
 

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