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NEMO-Chemistry
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[*] posted on 8-6-2016 at 14:13
How to measure fermentable sugar in grass?


I know grass contains reducing sugars etc, and I know these can be extracted with 70% Ethanol. How would you go about measuring the amount of fermentable sugar in a given weight of grass?

I apologize for the slightly odd question! But I would like to measure the ferementable sugar content in grases from different areas, I am unsure if you titrate for all sugars or just the reducing ones? I am also unsure which would be the best method to use.
Cheers Nick
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[*] posted on 8-6-2016 at 16:57


Why not collect grass samples and ferment them then determine the alcohol content ?
This I think would give the most usable measurements.

I know that sucrose, a minimally-reducing sugar can be fermented so I guess you should measure all sugars.
In grass, sugar is mostly fructans, conditions that affect concentration;

. Higher in stressed pastures than in lush grass
. Higher when night-time temperatures drop below 40 degrees (because the grasses do not grow, so the excess remains stored in the stems)
. Lower in new spring grass (first 3-6 inches), but also lower in fiber
. High in mature grass (8-10 inches), but also higher in fiber
. Lower in the morning when days are sunny and nights warm
. Higher in the afternoon/evening on a sunny day
. Lower in rainy, wet weather
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[*] posted on 8-6-2016 at 19:42


BRIX prism or meter?
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[*] posted on 9-6-2016 at 04:13


Quote: Originally posted by Sulaiman  
Why not collect grass samples and ferment them then determine the alcohol content ?
This I think would give the most usable measurements.

I know that sucrose, a minimally-reducing sugar can be fermented so I guess you should measure all sugars.
In grass, sugar is mostly fructans, conditions that affect concentration;

. Higher in stressed pastures than in lush grass
. Higher when night-time temperatures drop below 40 degrees (because the grasses do not grow, so the excess remains stored in the stems)
. Lower in new spring grass (first 3-6 inches), but also lower in fiber
. High in mature grass (8-10 inches), but also higher in fiber
. Lower in the morning when days are sunny and nights warm
. Higher in the afternoon/evening on a sunny day
. Lower in rainy, wet weather


Thank you for the information, although i now kind of have no reason to do the experiment seeing as you have told me what i was experimenting about :D.

I will still do it just for the fun and experience, in a way at least my results should match your information, so i guess thats a good thing :D.

Interesting it is higher is stressed grass! that would seem counter intuitive, my assumption would have been stressed grass would be low in almost mot things.

[Edited on 9-6-2016 by NEMO-Chemistry]
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[*] posted on 9-6-2016 at 04:17


Quote: Originally posted by feacetech  
BRIX prism or meter?


Partly i was interested in doing some kind of titration, its a basic skill i could do with more practice in. I havnt managed to get many of titrations to give reliable results.
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[*] posted on 9-6-2016 at 15:41


For titrations you need
good calibrated glass ware
good quality reagents
accurate calibrated scales for primary standards
and oven for drying primary standards (a calibrated thermometer to verify your oven)
good quality distilled or DI water
good technique

try this

Standardisation of Sodium Hydroxide

Apparatus
25 mL 'A grade' burette
250 mL stoppered conical flasks

Reagents For Standardisation
Carbon Dioxide - Free Water
Boil 1000 - 1500 mL distilled water for 20 minutes in a 2000 mL conical flask. Cool in a stoppered container. This must be prepared fresh each day.

Potassium Hydrogen Phthalate
Dry a quantity of AR grade potassium hydrogen phthalate (KHC8H4O4) in a weighing bottle for at least two hours, but preferably overnight at 100 oC (+/- 1 oC). Cool in a desiccator.

Phenolphthalein Indicator (1%)
Dissolve 2.000 g phenolphthalein in 200 mL ethanol

Procedure
Weigh to the nearest 0.1 mg, an aliquot of potassium hydrogen phthalate into a 250 mL stoppered conical flask.

[NaOH] : Aliquot size (g KHC8H4O4)
1.0 : 3.5-4.5
0.25 : 0.9-1.0
0.1 : 0.35-0.45
0.01 : 0.035-0.045

Add 50 mL warm carbon dioxide-free water. Stopper the flask and swirl gently until the salt dissolves. Titrate against the unknown sodium hydroxide solution using three drops of phenolphthalein indicator.

Repeat as necessary to give satisfactory replication.

Keep in mind this titration is just so you can standardise NaOH for other titrations

[Edited on 9-6-2016 by feacetech]
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[*] posted on 9-6-2016 at 23:08


Thanks i will give that a go!
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[*] posted on 21-6-2016 at 15:57


did you manage to try this standardisation titration using a primary standard. What were your results if you did?
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[*] posted on 21-6-2016 at 16:41


Quote: Originally posted by feacetech  
did you manage to try this standardisation titration using a primary standard. What were your results if you did?


Thanks i had forgotten about the post!! To answer your question, YES and no!

As mentioned in another post i broke the tip off my burrete (sp), it was a class B, although i am not too sure of the difference between A&B.

I am trying to use it as a chromatography column seeing as i broke it while cleaning it to use for the first time.

I have ordered another burette and this time went for a class A (Ouch!), i also ordered a couple of flasks (forgotten the bloody name!! ahhhh) for measuring out accurate solutions.

Bloody hell no idea whats is going on with my memory! They are the flasks with a single line on and you add the solid then fill to the line and have a stopper, as soon as my brain re engages i will edit and put the right name :D.

Anyway i have the flasks arrived a couple of days ago and the burrete should be here Friday, I ordered Potassium Hydrogen Phthalate from APC in the UK and they sent something else in error.

I should have the Potassium Hydrogen Phthalate Tomorrow (fingers crossed), they did get the phenolphthalein right but its a solution, is this going to matter?

If it does then i can get some from school (if i ask nicely and part some cash) also on Friday when i am next in.

No point doing the water until the other bits arrive but i have been double distilling water ready.

I also downloaded a couple of papers related to titration for reducing sugars.


Thanks for taking the time to write out the procedure i appreciate it. I have also now got a book :D! My scales are not the best but i got what i could afford, i am hunting ebay daily for a decent (half decent) used lab scales to come up at a reasonable price.

Reading up on a couple of things, i noticed that sodium carbonate dried and kept in a desiccator can also be used as a primary.

So i have some sitting in my home made desiccator, hopefully when my burette and stand get here i can take then for a spin!

On my hit list is sugar's and starch to begin with. I have been looking around the area where i live, interesting how grass suddenly dosnt look all the same when you pay attention.

I do have an end plan but it's likely to be a while! I am interested in collecting some grass seed and planting it in pots of coir. I want to see what components play major roles in producing sugar in grass.

So magnesium etc will tested out in ferts (withdrawn) and the sugar content tested at various stages.

I also have a small list of other plants i want to test for sugar. I will post (with pics) all my attempts!

It's taken a while to get everything but i am on a limited budget.

Thanks again for the help

NK

EDIT

BRAIN finally slipped into gear! The word i was looking for is volumetric flask! No idea why i went completely blank but there you go :D. I have 5 of different sizes, ranging from 15ml to 250 ml

[Edited on 22-6-2016 by NEMO-Chemistry]
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[*] posted on 21-6-2016 at 16:54


One question i forgot to ask was about N and M. I see in some papers solutions are measured as x amount of a Mol (M), and in other it says something like use 0.005N of x solution. What is the difference between them? I dont understand what N is, i can work out Mols (mostly) but had a bit of a struggle working out the difference between the two.

The following paper is one example

edit

For example to quote the paper "Dilute sulfuric acid (0.005 N) was less
suitable for starch digestion than enzymatic hydrolysis"

Attachment: Tree Physiol-2004-Chow-1129-36.pdf (107kB)
This file has been downloaded 304 times

[Edited on 22-6-2016 by NEMO-Chemistry]
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[*] posted on 21-6-2016 at 16:54


Sucrose is not a reducing sugar, at all. To detect it via any conventional REDOX assay (e.g. DNS (dinitrosalicylic acid and Rochelle salt) you would need to invert it, first (cleave into one mole equivalent each of glucose and fructose). This can be done with invertase enzyme (yeast make their own in-situ) or by heating with acid (see Clerget procedure). Fructose isn't a reducing sugar, either--but, most assays are alkaline which isomerizes it to glucose/mannose, which are reducing and, hence, detectable.

"Fermentable" is a vague term. The capacity of a given microorganism to consume a particular carbohydrate (or, glycerol, lactate, mannitol, for that matter) depends on its genetic coding for carbohydrate transporters/metabolism. For example, some bugs can eat xylose, some cannot. Ergo, the definition of "fermentable" depends on the bug you want to use.

So, simple maceration with water, filter, measure the brix (refractometer). Weigh an aliquot, invert with acid or invertase, and titrate for reducing sugar (Lane-Eynon boiling titration, Fehling's reagent) or run a DNS assay which can be compared with standards visually or, better, with a vis-spectrophotometer. The original method, for urine, works very well. I use it all the time for quick enzyme kinetics (there have been some improvements, but the old assay is simple and effective): http://www.jbc.org/content/47/1/5.full.pdf

Because many higher carbohydrate polymers and oligos also have a reducing end, it might be a good idea to first ppt your aqueous extract with 70% EtOH. The EtOH should be removed prior to assay: http://www.rroij.com/open-access/effect-of-ethyl-alcohol-on-...

I think you may have a hard time getting a decent S/N with the straight extract (most lawn-type grasses don't make very much sugar), so you might need to concentrate the extract (avoid heat and alkaline pH).

I run HPAEC-PAD (high pressure anion exchange chromatography with pulsed amperometric detection) for this kind of thing, so sensitivity is not an issue.

Hmm, I might need to extract some grass and see if I get anything, now.

O3



[Edited on 22-6-2016 by Ozone]




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[*] posted on 21-6-2016 at 16:58


Quote: Originally posted by Ozone  
Sucrose is not a reducing sugar, at all. To detect it via any conventional REDOX assay (e.g. DNS (dinitrosalicylic acid and Rochelle salt) you would need to invert it, first (cleave into one mole equivalent each of glucose and fructose). This can be done with invertase enzyme (yeast make their own in-situ) or by heating with acid (see Clerget procedure). Fructose isn't a reducing sugar, either--but, most assays are alkaline which isomerizes it to glucose/mannose, which are reducing and, hence, detectable.

"Fermentable" is a vague term. The capacity of a given microorganism to consume a particular carbohydrate (or, glycerol, lactate, mannitol, for that matter) depends on its genetic coding for carbohydrate transporters/metabolism. Some bugs can eat xylose, some cannot, for example. Ergo, the definition of "fermentable" depends on the bug you want to use.

So, simple maceration with water, filter, measure the brix (refractometer). Weigh an aliquot, invert with acid or invertase, and titrate for reducing sugar (Lane-Eynon boiling titration, Fehling's reagent) or run a DNS assay which can be compared with standards visually or, better, with a vis-spectrophotometer, the original method, for urine, works very well. I use it all the time for quick enzyme kinetics (there have been some improvements, but the old assay is simple and effective): http://www.jbc.org/content/47/1/5.full.pdf

Because many higher carbohydrate polymers and oligos also have a reducing end, it might be a good idea to first ppt your aqueous extract with 70% EtOH. The EtOH should be removed prior to assay: http://www.rroij.com/open-access/effect-of-ethyl-alcohol-on-...

I think you may have a hard time getting a decent S/N with the straight extract (most lawn-type grasses don't make very much sugar), so you might need to concentrate the extract (avoid heat and alkaline pH).

I run HPAEC-PAD (high pressure anion exchange chromatography with pulsed amperometric detection) for this kind of thing, so sensitivity is not an issue.

Hmm, I might need to extract some grass and see if I get anything, now.

O3



We posted at the same time sorry.

Not sure if it makes a difference but the grass i am looking at is pasture grasses, also some wild meadow flowers. I will also try different lawn grass now you mention it :D, as well as some of the much coarser grasses we have around here.
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[*] posted on 21-6-2016 at 17:02


Thanks for the papers, i will give them a study.

I only have fairly basic equipment, i dont study chemistry at school and we only have a week and a bit before the summer holidays. When i get back after the break i will see if i can join the after school chemistry club, it might give me access to more equipment but that is speculation!! To be fair the chemistry technician has been pretty good at letting me have old glass ware etc.
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[*] posted on 21-6-2016 at 17:12


Also, I wouldn't recommend fermentation to ethanol as a method of assay--ethanol is but one of several metabolites you may get, and unless you have an HPLC and a good understanding of the Biochemistry of the bug you are working with, you're probably screwed. For example, one organism I work with produces 2 equivalents each of lactate, formate, acetate, and ethanol per glucose consumed.

Also, unless there is a lot of sugar in there, e.g. sugar cane (Saccharum grass), you would need to ferment an obscenely large amount in order to distill any recoverable amount. If you can't get at least 5%/ferment ethanol, don't even bother (even so, losses during handling/distillation would kill your measurement). Otherwise, you'll end up with a soup containing a small amount of ethanol and a bunch of other complex junk. Teasing it out, chemically, without an LC/GC is practically impossible.

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[*] posted on 21-6-2016 at 17:22


Quote: Originally posted by Ozone  
Also, I wouldn't recommend fermentation to ethanol as a method of assay--ethanol is but one of several metabolites you may get, and unless you have an HPLC and a good understanding of the Biochemistry of the bug you are working with, you're probably screwed. For example, one organism I work with produces 2 equivalents each of lactate, formate, acetate, and ethanol per glucose consumed.

Also, unless there is a lot of sugar in there, e.g. sugar cane (Saccharum grass), you would need to ferment an obscenely large amount in order to distill any recoverable amount. If you can't get at least 5%/ferment ethanol, don't even bother (even so, losses during handling/distillation would kill your measurement). Otherwise, you'll end up with a soup containing a small amount of ethanol and a bunch of other complex junk. Teasing it out, chemically, without an LC/GC is practically impossible.

O3


The term ferment-able sugar was used by a vet, my interest in actually trying to reduce the amount of sugar in the spring in grass.

I would like to see if its possible to apply ferts and that limit the amount the amount of sugar produced in the grass./.

Unlikely i know, but experimentation is fun even if it dosnt work out!
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[*] posted on 21-6-2016 at 17:25


I do make my own Ethanol but normally i just use yeast and cane sugar. I wouldnt mind trying out some of the bacteria that make Ethanol. I am fascinated by micro organisms and how they metabolize things, i wont be doing biology in the new term though :(.

I would like to try out some wild yeasts and glucose and see what they produce, but like you say without a GC there isnt much i can tell.
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[*] posted on 21-6-2016 at 17:27


The urine sugar assay seems interesting, so looks like its time to buy some aspirin!! Finally got a good use for the nitric acid :D
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[*] posted on 24-6-2016 at 12:43


Ok i have the wrong Acid!

Is there another acid i can use instead? or a synth to make it? The new burete is in Cm3 and looks like never used despite being pretty old (very old box with it), should do the trick though :D.

Made a real mess of the copper stuff i was doing! so sorting that out at the weekend and hopefully can do a titration if i can get another Acid.
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