Diacetyl Testing

[FireMang]

Hello everyone. I am in the process of getting my diacetyl testing perfected and had a couple questions on how other people are doing it.

My process now is I take a sample from a fermentor into a pyrex container with a lid. I make sure the lid is nice and tight so nothing leaks. It then sits in a <160F water bath for 1 hour. The sample is then cooled. We then taste it against a non-heated sample and see if butter flavor comes out. So far it has worked out well. My questions are as follows:

1: Does the heated sample need de-gassed before it is heated?
2: Anything I am missing?
3: I saw tests for VDK that can be sent to be done. We will be getting an actual lab soon. Does anyone do this at your in-brewery lab that this test is done? Where can I find info on setting this up?

We seem to be doing just fine with diacetyl NOT being present, but I was just curious what other people are doing to test for it.

[AT-JeffT]

I've read you only need to hold the sample @ 140F-160F for 10min. We hold it at temp for 10min and get meaningful results. You could probably shave some time off.

[mmussen]

There's quite a few breweries doing VDK tests in the lab - it does require a UV spectrophotometer. And that's not the cheapest piece of equipment. The test itself is relatively simple, I forget the exact ASBC method offhand but they do keep up to date methods and procedures handy.

[chronolite]

Beer-25

The method for UV Spectrophotometry for Diacetyl has been archived. Two methods, one for total VDKs and the other for a more precise measurement of diacetyl, both use much less inexpensive colorimeters. Ebay has UV Spectros for less than 400 on the regular, old analog Spectronic 21s can be had for less than 250 if you're patient. The deuterium bulbs are very hard to find though.

BEER - DIACETYL

MICRO DIMETHYL GLYOXIME METHOD
Reagents
(a) Dipotassium hydrogen phosphate. Prepare a solution containing 33 g K2HPO4 in 100 mL water. It is most conveniently kept in a plastic wash bottle.
(b) Hydroxylamine hydrochloride. Dissolve 11 g NH2OH·HCl in water and make to 250 mL.
(c) Sodium acetate. Dissolve 35 g NaC2H3O2 in water. Make to 100 mL total volume.
(d) Buffered hydroxylamine acetate. Prepare by mixing two volumes K2HPO4 + four volumes NH2OH·HCl + one volume NaC2H3O2 (reagents a, b, and c).
(e) Acetone-dipotassium hydrogen phosphate. Dissolve 29 g anhydrous K2HPO4 (or 38 g K2HPO4·3H2O) in water; add 40 mL pure acetone and make to 200 mL with water. Store in refrigerator.
(f) Tartrate ammonium hydroxide. Prepare a saturated solution of potassium sodium tartrate (Rochelle salt), ca. 270 g/150 mL. Mix 12 volumes of this solution with three volumes concentrated NH4OH.
(g) Ferrous sulfate. Dissolve 5 g FeSO4·7H2O in 100 mL 1% H2SO4. This solution should be discarded when it turns yellow (oxidation of ferrous iron).
(h) Diacetyl standard. Diacetyl of adequate purity used for the standard curve may be obtained from Polarome Manufacturing Co., Inc., 22 Ericsson Pl., New York, NY 10013; other suppliers also are listed.
(i) Diacetyl, stock solution. Prepare an aqueous solution containing 500 mg/L. The stock solution should be stored in an amber bottle in the refrigerator.

Apparatus
(a) Colorimeter or spectrophotometer (calibrated, 530nm).
(b) Generator trap assembly: The generator is a test tube, 250 × 25 mm, of 100-mL capacity. The trap is a glass-stoppered 10-mL footless graduated cylinder, Pyrex (Corning No. 2972). These cylinders take No. 00 rubber stoppers. The connection between the generator and the trap is a piece of bent tubing, 4 mm o.d., extending to bottom of the trap. The bends are smooth and gentle, with a slope toward the trap to minimize entrainment of condensing vapors.
(c) Tygon tubing of suitable size (1/8 in. i.d.) is used between the trap and the generator of the next sample.   Four units are connected in a train and contained in a water bath (a 2-L beaker serves well).
If more than a few samples are to be run, better replication can be obtained by use of a glass manifold designed for six samples such as is available from Pope Scientific Inc., N90 W 14337 Commerce Drive, Menomonee Falls, WI 53051.

Calibration
Prepare dilutions from stock of diacetyl (reagent i) to range from 0 to 20 mg/L. 

Develop colors as follows:
Pipet 1.0 mL diluted diacetyl solution into 10-mL cylinder containing 1 mL buffered hydroxylamine acetate (reagent d). Immerse for 10 min in a water bath maintained at 65–75°C. While it is still warm, add 0.5 mL acetone-K2HPO4 (reagent e), mix, and let cool. Proceed as described under Method, paragraph f, below. Plot absorbance (ordinate) against concentration (abscissa).

Method
Pipet 20 mL beer (which may be decarbonated at room temperature) into generator. Sample size may be increased to 30 mL or reduced to 10 mL, depending on diacetyl level.
Add 1 mL buffered hydroxylamine (reagent d) to trap and connect to sample tube. Then attach tube carrying CO2 gas into generator. Additional sets of tubes, up to 12, may be connected in series (not in parallel). Begin flow of gas at a rate of 100–150 mL per min (visually this rate is such that individual bubbles can be just distinguished).
The generators are immersed in a water bath maintained at 65°C (±5°C), but the traps must not be immersed in the bath; they should be left hanging outside, suspended by connected tubing.
Sweep for 1.5 hr (for 30-mL samples sweep for 2 hr; for 10-mL samples 1 hr is sufficient). When temperature and gas flow rate are properly adjusted, there is very little increase of volume in trap.
Disconnect traps (starting with last in train) while gas is still bubbling. Tips of trap inlets may be rinsed with a few drops of K2HPO4 solution (reagent a). If volume exceeds 3 mL, contents should be carefully evaporated down to about 2 mL (in water bath). This evaporation should be avoided, though, because of increased manipulation and risk of decomposition of dimethyl glyoxime by heat.

Color development
Prepare a reagent blank by placing 1 mL buffered hydroxylamine acetate (reagent d) in a 10-mL cylinder. Place all cylinders in a water bath at 70–80°C for 10 min. 
While the bath is still warm, add 0.5 mL acetone-K2HPO4 (reagent e) and mix by inverting. Allow to cool, and add 1.5 mL of tartrate-NH4OH (reagent f) and mix again. 
Finally add 0.1 mL FeSO4 (reagent g), mixing immediately after addition of this reagent. Adjust volume to 5.0 mL total with K2HPO4 solution (reagent a). 
Water must not be used, since it causes fading of color. A pink color will appear almost immediately after addition of the FeSO4. It reaches its maximum within 2– 3 min. 
Determine absorbance of solution in a 1-cm cell at 530 nm, or close to 530 nm, depending on apparatus available, within 15 min. 
Calculate concentration of diacetyl in sample from standard curve.

There's a HANNA Instruments Colorimeter for about $50 that runs at 525nm, it should work okay for this method, as long as you make your curve from it. There's also a ton of colorimeters on ebay for less than $100 and a DIY arduino one for $47 from http://www.iorodeo.com/store_new_2

PM me an email address for the complete Beer-25 method, careful with this one, the text layout is a bit wide.
If you want more than a few methods, the asbc membership is totally worth the price at <$1000 per year.

Hope this helps.

[aaron inkrott]

Yeast Book

Chonolite and AT-JeffT hit all the points Chris White discusses in his Yeast book. We currently do sensory where I work, but we're looking into VDKs.

[FireMang]

Thanks everyone. I found the 1 hour method from listening to a lecture on the BA website. Awesome info on the lab test. Hows about the degassing of the sample? are you taking the residual co2 out of solution before you heat it? Prost!

[DageraadBen]

Science is great and everything, but the point of not having diacetyl in your beer is so that drinkers won't smell/taste diacetyl. If you're sensitive to diacetyl and you can't smell it in a warm sample, nobody else should be able to smell it in cold beer. If you're not sensitive to diacetyl (every palate has its blind spots) find someone in your brewery who is.

I like science as much as the next guy, but we're making something that is meant to provide someone with a sensory experience. If you can't sense it, it doesn't matter what the UV spectrophotometer says.

[chronolite]

Diacetyl Testing

Thanks everyone. I found the 1 hour method from listening to a lecture on the BA website. Awesome info on the lab test. Hows about the degassing of the sample? are you taking the residual co2 out of solution before you heat it? Prost!

I degas tiny samples in the microwave, for more than 20ml or so, I degas in an erlenmeyer with a drilled stopper. If I have to degas more than 100ml then I use a tiny martini blender from target.

Too much shaking and it adds gas back in, so there's a bit of an art to it.

http://www.amazon.com/gp/aw/d/B00RM2C4SA/ref=mp_s_a_1_2?qid=1439396104&sr=8-2&pi=AC_SY200_QL40&keywords=battery+drink+blender&dpPl=1&dpID=31WQclYVliL&ref=plSrch

Asbc has a method for degassing beer, if you come up to Actual, we can go over the entire thing and play some Mario Kart

[MJMurphy]

Sensory v. measurement

The jump to using the ASBC method for diacetyl does come with a price tag: spectrophotometer, distillation apparatus, reagents, PPE, etc. That said, there is some comfort in being able to use a repeatable measurement process that you can validate. That said, you can also use the spectrophotometer for IBU testing, color checks, and chlorine testing on you water, among others. Rick Blankemeier from Stone Brewing has a great Article in the New Brewer about the various checks and analysis you can run with the spec.

We moved to the ASBC distillation method some years ago. Prior to that we used the sensory method. We would use a 20 minute 140 deg F bath, then chill the warmed sample and the "control" sample together in an ice bath so we could sample them at approximately the same temperature. We did not pay that close attention to the amount of degassing other than that we would obtain each sample the same way in a 500 ml nalgene container from a zwickle on the tank. Both samples would be taken at the same time and the container closed. One would go into the fridge, one into the warm bath. Then they would both go into the ice bath until they were at serving temperature and equalized with each other.

When we switched we validated our sensory method with the ASBC method. At our brewpub they still use the sensory method. We do regular (weekly to monthly) testing of their samples with the distillation method. Given our fermentation times and sampling practices this has worked out well.

A big caveat is that if there was a fluke or aberration in the fermentation, and your testers are not on their game that day (hosted a beer festival the day prior, or had hot wings for lunch), there is a greater chance that you will miss it in sensory.

We started with sensory, moved to analysis when we could, and validated our sensory method (to some extent). At this point we do both for different circumstances. We made these changes as we grew and the importance of the reliability of the results became more important. If you are sending it across the bar, a thumbs up can cover it. When you keg/bottle/can it and send it out into the world, having a number to point to is a good feeling.

MJM