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Author: Subject: Soluble/Insoluble oxalate in spinach
mcr90
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[*] posted on 6-6-2014 at 12:03
Soluble/Insoluble oxalate in spinach


I am a graduate student in nutrition. I have no lab experience but have had the opportunity to begin working in a Urology lab for my thesis research dealing with the bioavailability of oxalate in spinach. I have been assigned the task to isolate oxalate crystals in spinach and view/quantify them microscopically. Any ideas for a protocol?
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blogfast25
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[*] posted on 6-6-2014 at 13:00


Treat filtered (perhaps centrifuged) spinach juice with calcium chloride (or any other water soluble calcium salt), this will precipitate the acid as calcium oxalate (CaOx). Filter and wash.

Although poorly soluble it has enough solubility to be able to be displaced with sodium carbonate:

CaOx(s) + Na<sub>2</sub>CO</sub>3</sub>(aq) === > CaCO<sub>3</sub>(s) + Na<sub>2</sub>Ox(aq)

... because CaCO3 is much more insoluble than CaOx.

After filtration, this leaves you with water soluble sodium oxalate in solution. This can be quantified e.g. by titration with potassium permanganate in acid conditions.

Acc. the wiki entry on oxalic acid, the content in spinach is around 1 g / 100 g, not really microscopic.


[Edited on 6-6-2014 by blogfast25]




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thesmug
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[*] posted on 6-6-2014 at 13:08


As for getting the crystals you could either dry them in a dessicator or simply let them stand. Boiling the water might also work.



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blogfast25
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[*] posted on 6-6-2014 at 13:09


Quote: Originally posted by thesmug  
As for getting the crystals you could either dry them in a dessicator or simply let them stand. Boiling the water might also work.


There has to be a lot of other 'crap' in spinach juice. I think the OA needs to be isolated first.




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[*] posted on 6-6-2014 at 13:11


Yeah I didn't really think about that. Do you have any ideas on isolating the oxalate?



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[*] posted on 6-6-2014 at 13:20


You mean the acid? There's various ways to isolate it but I don't see the point for his purpose. You can see oxalic crystals under the microscope all day long and twice on Sunday: it doesn't teach you anything you can't learn from a jar of 'deck cleaner'.

It should be sufficient to isolate it in a quantifiable way, for instance as an oxalate.

[Edited on 6-6-2014 by blogfast25]




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[*] posted on 6-6-2014 at 14:07


I think that the OP requires examining the crystals as part of their project, but I don't see why. To OP: quantifying the crystals seems unnecessary since it is much easier to quantify oxalate in solution. Would you mind giving us more information?

[Edited on 6/6/14 by thesmug]




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[*] posted on 6-6-2014 at 14:55


I'd guess he is interested in isolating the crystals as they occur in the leaves, rather than making a solution of oxalate and precipitating the calcium salt from that.

I'm pretty sure you could have googled this yourself, as it appears in the first 3 results with trivial keywords but for the purpose of this thread:

Liovando Marciano da Costa et al Rev. Bras. Ciênc. Solo (2009) 33:3

"Extraction and concentration of biogenic calcium oxalate from plant leaves"

Quote:
The leaves were washed with distilled water to remove impurities and at last rinsed in deionized water. The leaves were cut in smaller pieces, filled in a heavy-duty blender, and covered with deionized water to 5 cm above the plant material. The device must work until obtaining a greenish juice of the leaves. The liquefied material was sifted through a 0.20 mm sieve. A sample of 20 crystals extracted from croton leaves was measured and the values varied from 0.004 to 0.018 mm. The 0.20 mm sieve was adequate to separate the fibrous leaf material from the plant juice and the CaOx crystals. The plant material retained in the sieve was ground again in the blender. The procedure must be repeated until the plant juice becomes totally clear.

The volume of extracted plant juice was placed in 1000 mL test tubes for sedimentation of the calcium oxalate crystals and plant material. The sedimentation process must be repeated until the supernatant is light-colored and free of chlorophyll, pigments as well as other organic water-soluble compounds. The solid material containing the calcium oxalate crystals and other not water-soluble impurities were at the bottom of the test tube. The supernatant was removed by draining off.

In the end of the extraction, the formed sediment can be transferred to smaller test tubes (100 mL) to concentrate the extracted material. After concentrating the suspension, the solid product was dried at 60 ºC.

Hydrogen peroxide (30 %) was added to the dry material and the suspension formed was left to rest for 24 h to remove the organic impurities. Hydrogen peroxide was added until it covering the solid material by 2 cm. After 24 h reacting with the organic substances the CaOx crystals were free of impurities. The optical microscope was used to observe the purity of the calcium oxalate crystals. The procedure with hydrogen peroxide can be repeated in case of any remaining impurities. The resulting solid material was washed with deionized water to remove impurities and dried at 60 ºC.




[Edited on 6-6-2014 by phlogiston]




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