[RASMB] 99% versus >99% guanidine HCl

[Thomas Jowitt]

Hi

It is definitely necessary to find the best possible guanidine, but in my experience that is not good enough either. If you are using any fluorescence or absorbance optical techniques involving high concentrations of guanidine it is best to purify it with charcoal first. We do a double purification of 8M in activated charcoal and then re-assess the exact molarity by refractometry. I have added the protocol we use here. The end result is pristine pure guanidine which is far better when using absorbance optics. It can be stored for a long time or it can be scaled down but I would recommend doing a reasonable amount at once.

Guanidine purification

For 6.5litres of 8M GuHCl 
Empty  5Kg  GuHCl (Sigma G4505) into a bucket and add water to approx 6.5 litres.
Stir at room temp for 24hrs.
Add approx 30g activated charcoal powder (Sigma-Aldrich 24-227-6 G60-100mesh).
Stir overnight at 4ºC.
Filter using double filter paper in a large funnel.
Add 30g of charcoal.
Stir at room temp overnight.
Filter using a double filter paper in a large funnel
Filter through 0.2um nitrocellulose and 0.2um nylon filter membranes using a Buchner flask and vacuum.
N2 filter using a PM10 filter (optional)
Check molarity by refractive index  (water 1.3333, 8M GuHCl 1.465)
Use formula,  M = 60.4396n - 80.5495	n = refractometer reading at 23ºC

Thanks

Tom Jowitt

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of kaltofen at uni-potsdam.de
Sent: 18 May 2010 07:17
To: rasmb at server1.bbri.org
Subject: Re: [RASMB] 99% versus >99% guanidine HCl

Hi everybody,
depending on the methods you want to use, there are huge differences  
even between "ultrapure" GuHCl from different companies and even  
between different lot-numbers from the same supplier. We do lots of  
fluorescence spectroscopy and found it worth trying different  
suppliers (Thermo and Pierce are suitable). 99% is surely ok for  
protein purification but unfortunately the impurities even in  
ultrapure GuHCl absorb UV light and emitt in the same wavelength  
region as tryptophane. Same thing holds true for urea. That should be  
considered when planning fluorescence spectroscopy.

All the best,

Sabine


> Hi,
>> 99% is typically also considered to be spectroscopically pure and is
> recommended for spectroscopic measurements/assays such as protein
> unfolding/refolding. It is typically less than 0.1 Abs at a concentration
> 6M. If you need it for e.g. inclusion body resolubilization, etc. the less
> expensive one is just as good.
> Best
> Titus.
>
> -----Original Message-----
> From: rasmb-bounces at server1.bbri.org [mailto:rasmb-bounces at server1.bbri.org]
> On Behalf Of Avi Chakrabartty
> Sent: Monday, May 17, 2010 3:25 PM
> To: rasmb at server1.bbri.org
> Subject: [RASMB] 99% versus >99% guanidine HCl
>
> Dear RASMB community,
>
> Has anybody found a real difference between 99% guanidine HCl from Aldrich
> compared to >99% guanidine HCl from Sigma?
>
> -------------------------------------------
> Avi Chakrabartty, Ph.D., Professor
> Departments of Medical Biophysics and Biochemistry,
> University of Toronto, Ontario Cancer Institute,
> MaRS Centre, 101 College St., Toronto, Ontario, M5G 1L7, CANADA
> Phone: 416-581-7553 Lab: 416-581-7555 Fax: 416-581-7555
> email: chakrab at uhnres.utoronto.ca
> --------------------------------------------
>
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Sabine Kaltofen
PhD student

Universität Potsdam
Department of Physical Biochemistry
Institute of Biochemistry and Biology
Karl-Liebknecht-Str. 24-25, Haus 25, Raum B/0.05
D-14476 Potsdam-Golm
Telefon: +49-(331)-977-5245
Email: kaltofen at uni-potsdam.de



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