Please suggest how to dissolve the naphthol in buffer for making standard curve.
Hi Kona,
The only way to discriminate between the two naphthols is HPLC.
What we do in my lab is the following.
HPLC separation of the two naphthols and fluorescence detection online.
HPLC consists of a 19-min run on C18 reverse phase column (usually 10-cm long cartridge) with a gradient between water and acetonitrile. The gradient conditions are: 15 min of linear gardient between 100% water and 60% acetonitirle followed by a 4-min plateau at 100% acetonitrile. Elution at 1 ml/min. Detection : fluoresence with excitation set at 288 nm and emmission set at 360 nm.
In these conditions, naphtol-1 elutes at 13.4 min, naphthol-2 elutes at 13.8 min and naphthalene elutes at 17 min.
Both authentic 1-naphthol and 2-naphtol are found at SIGMA. 1-Naphthol is product N2780 (82 euros for 10 grams) and 2-naphthol is product Aldrich 185507 (14 euros for 5 grams).
Good luck for your experiments!
Philippe Urban
Hello Philippe,
Thank you for your answer.
Actually I am estimating esterase enzyme activity in mosquitoes by 1/2-naphthyl acetate assay. The product 1/2-naphthol will be visible with fast blue staining and estimate by spectrometer at 570nm. Here I want to estimate enzyme activity by correlating the 1/2-naphthol standard curve (like protein estimation using BSA standard cure). When I tried to dilute the naphthol acetone solution in sodium phosphate buffer/water for making serial dilutions, naphthol was getting precipitate. So I would like to know how to dissolve naphthol in which buffer at what pH and for making standard curve. If you having information please help me...
Thank you...
Kona
Hi again Kona,
Oh I understand better.
Well, coudl you precsie me what are 1/2-naphthol concentrations you use (stock solution? final concentrations in the cuvettes for calibration?). Many thanks.
Usually, we dissolve naphtahlene and napthols in methanol or ethanol. Our stock solution is at 50mM naphthalene in MeOH. Naphthols being more soluble, we make stock solutions up to 100 mM in methanol. Then, we dilute this solution in buffer to be in the range 500 - 10 micromolar for calibrations. No precipitation occurs.
Best regards,
Philippe
Hi Philippe,
OK, I will also try to dissolve stock solution in methanol then dilute in buffer. May I know which buffer concentration and pH you are using for dilution?
When I contacted with some researcher, they suggested, dissolve 0.5mg/ml of 1/2-naphthol in 10% acetone buffer (first in acetone then KPO4 buffer for making final concentration). I tried with 0.02M KPO4 buffer pH: 7.2 once for dissolving, it was working but I found some turbidity, I am thinking I should optimize the concentration of buffer and pH. I will try methanol also.
Thank you for your suggestions for my research,
Kona
Hi again Kona,
The buffers we use are either Na/K phosphate buffers 50 or 100 mM pH7.0 to pH7.4 (we use several different phosphate buffers for different purposes). Another buffer I use frequently for enzymatic measurements is Tris-HCl 50 mM pH7.4. All our buffers contain EDTA 1 mM.
We use Na/K instead of all potassium because, Na/K phosphate buffer does NOT precipitate at cold (this is important for protein purification for instance) whereas K/K buffer precipitates at cold !!! Maybe the precipitate you see is not naphthol molecules but buffer molecules ?
Concerning naphthols. We make typically a 50 mM solution of naphthol in pure MeOH (a methanolic stock solution). Then we directly dilute it in Na/K phosphate buffer. This way to get a solution 100 micromolar in naphthols in buffer will needs to add 2 microliters of 50mM methanolic stock solution to 1 ml of buffer.
Best regards,
Philippe
Hi Philippe,
Thank you, This information will be useful.
Kona
hi
could you please tell me how could I prepare stuck solution of Na/K phosphate buffer? my final concentration that I need is 50 mM pH: 7 ....
I know it is easy but I would be glad if some one tell me how?
Hello Rosa,
Quite easy indeed.
Prepare two stock solutions of phosphate salts such as: 1 liter of 1M Na2HPO4 and 1 liter of 1M KH2PO4. Then use these two stock solutions to prepare 1 liter of each at 50 mM, you'll get 1L of 50 mM Na2HPO4 and 1 liter of 50 mM KH2PO4.
Transfer the 1 liter of 50 mM Na2HPO4 (basic salt of phosphate) to a 2-liter baker, put a pH electrode in that solution and stir with a magnetic bar. Then progressively add the 50 mM KH2PO4 (acidic salt of phosphate) solution to the other one until the pH reach the exact value you wish, in your case: 7.00 !! It should be about 600 ml.
You will get about 1.6 liters of a 50 mM Na/K phosphate buffer at pH 7.0.
Good luck and best regards,
Philippe Urban
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