For the range of pH 4-6, you could use citric acid-sodium phosphate buffer or citric acid-sodium citrate buffer. Tables for their preparation are here:

http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html

I recommend you the use of "Buffer Maker Software". It a special tool for wide range to buffer preparation. Regards

Here is an additional piece of useful information for convenient buffer preparation:-

After you have made the buffer you  can dilute it with water to whatever MES concentration you require.  Within limits the pH will not alter significantly. A 10-fold dilution should give a buffer with virtually the same pH as the original solutioneven though the MES concentration is one-tenth. You can test this easily, before use, for any particular buffer that you are using and should be done especially if the buffers are very dilute.

many thanks for your useful information

Please see the following ring:

https://www.goldbio.com/documents/3550/MES+Buffer+0.5M+Stock+Solution+with+table.pdf

Best regards

Vit

Peter Jackson, adjusting the pH of MES is done with NaOH not 2-(N-morpholino)ethanesulfonic sodium salt.

If you start with a solution of the sodium salt of MES then adding NaOH will raise the pH to a value at which the MES has little or no buffering capacity. If you start with a solution of the acid form of MES, then, as you indicated, you can adjust the pH of the solution with NaOH to a pH at which the MES will act as buffer. However it is more convenient to make two individual solution of MES one of the acid form and the other of the sodium salt . You can simply mix them together to obtain a solution of MES at the buffer pH required, The concentration of MES in both of the starting solutions ( the acid from and the sodium salt form) should be the same. There is no need to measure the volumes of the two solutions when mixing them. The final solution will be an MES buffer at the chosen MES concentration and at the pH chosen. In addition there will be less temperature change in the solution using this method compared with adjusting the pH with NaOH solution.

Hi Dr. Jackson,

How can I prepare 0.1M MES buffer with pH 4.5 from the another MES buffer which has a pH 6.1 ?

Thank you

You have to add acid to lower the pH. Unfortunately, since base was already added to raise the pH, you are going to end up with a solution containing both MES and some salt. For example, if you used NaOH to adjust the pH to 6.1 and then use HCl to bring it down to 4.5, you will have NaCl in the MES buffer.

Since pH 4.5 is lower than the useful range of MES (5.5 to 6.7), however, there is really no point in making this adjustment. You should use a more suitable buffer, such as acetate or citrate.

Make a 0.1M solution of the acid form of MES. Measure the pH. If the pH is much lower than pH4.5 then you can add the solution to your buffer which is at pH6.1. If the pH is close to pH 4.5 ( and lower than pH4.5) then you will need to add a large volume of the 0.1m MES acid form solution to lower the pH to 4.5. Try on a small scale first. If the pH is greater than 4.5 then you will not be able to lower the pH to 4.5.using only MES solution.You will need to add acid as indicated in the previous answer but then you will have a buffer containing a salt as well as your MES.

Make a 0.1M solution of the acid form of MES. Measure the pH. If the pH is much lower than pH4.5 then you can add the solution to your buffer which is at pH6.1. If the pH is close to pH 4.5, and lower than pH4.5, then you will need to add a large volume of the 0.1m MES acid form solution to lower the pH to 4.5. Try on a small scale first. If the pH is greater than 4.5 then you will not be able to lower the pH to 4.5.using only MES solution.You will need to add acid as indicated in the previous answer but then you will have a buffer containing a salt as well as your MES.

Thank you very much Dr. Shapiro and Dr. Jackson for your valuable suggestions.

Yes, if by MES you mean 2-(N-Morpholino)ethanesulfonic acid, then the concentration of MES is important. The greater the concentration of MES then the more buffering capacity there is in the buffer solution. That is, at greater MES concentrations then the pH of the buffer will change less when a specific quantity of acid or base is added than for lesser MES concentrations.

More Information:

The MES is the buffer ion and this molecule determines the pH range over which the buffer solution is an effective buffer; that is, the range where the MES molecule has significant buffering capacity. An MES buffer will have its highest buffering capacity at pH6.1, which is its pK. The pK is the pH at which, on average, 50% of the MES molecules in the buffer solution are protonated. Any individual MES molecule can be protonated at any time. At the pK any individual molecule will be protonated 50% of the time. The pK is determined by the chemical structure of the buffer molecule.

(All buffers have their highest buffering capacity at the pK of each specific buffer reagent).

The recommended range for MES is pH5.5 to 6.7 (see suppliers catalogues (et al.) for data on the recommended buffering ranges for buffers). This range is 0.6 pH units higher and lower than the pK of MES, which is 6.1.

For information on the maths of buffers refer to the Henderson-Hasselbach equation.

The following information may be of use to you for the making of buffers.

The method described here is a user friendly alternative to using strong acid or alkali solutions to adjust the buffer pH .

When making MES buffers it is best to use the MES monohydrate as the water content is defined.

As an example:

To make a solution in which the final concentration of MES is 0.1M, and the required buffer pH is 5.9 and the counter ion is sodium.

1, Make a 0.1M solution of MES monohydrate acid form: this is solution A

2. Make a separate solution of 0.1M MES sodium salt: this is solution B

3. Take a small volume of solution A and stir the solution whilst measuring its pH using an accurate pH-meter that has been standardized with standard buffer solutions ( available commercially).

4. Add solution B carefully while stirring until the pH reaches pH5.9 and is stable.

You will then have the buffer required. This buffer is correctly described as: “0.1M 2-(N-Morpholino)ethanesulfonic acid buffer, sodium salt, pH 5.9”.

It is not necessary to measure the volumes of either solution A or B. Neither is it necessary to adjust the volume of the final buffer solution with water. The concentration of MES is 0.1M since both solutions mixed have that concentration of MES

To obtain any other pH, just mix solutions A and B until that pH is reached.

There may be some warming during the mixing. A pH meter fitted with a temperature compensation probe will compensate for the effect on the pH electrode of the warming by the buffer solution. It will NOT compensate for the warming effect on the pH of the actual buffer. The buffer temperature coefficient can be used to calculate the actual buffer pH at a specific temperature if that is different from the temperature at which the buffer is being measured.

The best way of making MES buffer buffer is to start with a solution of MES free acid at required molarity and adjust the pH with NaOH. The sodium salt is made internally and the molarity will remain unchanged.. By definition buffer molarity is the additive of the molarities of acid and its conjugate base.

A note of caution, although buffering capacity is higher at higher concentrations, in principle, the buffer concentration must be kept at the bare minimum. Buffer components should remain as silent spectators without interfering with the components of the assay mixture.

Peter Jackson I was also following a protocol which uses MES buffer at pH 4.8 and on checking online NaOH was suggested for adjusting the pH. I was using the MES hydrate buffer (acid-hydrate) at 100mM concentration which has pH around 3 and used NaOH to raise the pH up to 4.8. Do you think this would be useful as now I'm reading that the useful pH range for MES buffer is 5.5 to 6.7? Thank you!

4-Morpholineethansulfonic acid ( MES) has a pKa of 6.1 ( at 25oC), which means that it is most effective as a buffer ( has its highest buffering capacity) at pH 6.1. Consequently it's useful buffer range is from pH 5.5 to pH 6.7. At pHs higher or lower than this range MES will not be an effective buffer.

If you need to buffer a solution at pH 4.8 then you need to use a different buffer reagent. Commercial suppliers of buffer reagents will usually have information on buffer reagents and the useful pH ranges at which they buffer effectively.

Peter Jackson I want to use MES buffer at pH 5. I understand it's useful range is 5.5 to 6.7, and there are other buffers like acetate and citrate buffers are better options for pH 5. But my problem is I want to use very low conductivity buffers but the acetate and citrate buffers are very conductive even at concentration 0.1mM, which raised the current values than the limit of my power supply and stop the process (electrophoresis). I want to know would MES not work at all at pH 5.

At pH 5 MES buffer will not be effective as a buffer. Citrate has 3 possible ionisable carboxyl groups and , depending on the the pH can carry more current than acetate that has only one ionisable group. . For a specific concentration of acetate buffer , the lower the pH the less conductive the buffer. The optimum buffer pH for an acetate buffer is at its pK which is approximately pH 4.7

Peter Jackson :Can you suggest if 1:10 dilution of the MES buffer (0.5 Molar; to 0.05 Molar), with pure water (MQ, pH around 5.6), would have impact on the pH of the 1:10 diluted MES buffer? Would this change the original pH of the buffer, which is pH 5?

I suggest you do the dilution and measure the change in pH if there is any.

The useful pH range for MES buffer is from pH5.5 to 6.7. At pHs higher or lower than this range MES will not be an effective buffer.

Dr. Peter Jackson

I have MES Buffered Saline Pack. Their characteristic is:

0.1M MES [2-(N-morpholino) ethanesulfonic acid], 0.9% sodium chloride, pH=4.7, when pouch contents dissolved in a final volume of 500 ml de-ionized water.

Now, I want to make 0.5M MES Buffer in pH=6.1 from this buffer. I have some questions:

1- if I pouch contents in 100 ml distilled water(D.W) ( instead of 500 ml), do I made a solution 0.5 M??

2- How can I adjust pH=6.1 from this solution?

3- Do you think, Can one package dissolve in 100 ml D.W instead of 500 ml? If it did not dissolved, Can I use sonicator for dissolving it?

4- If I made Buffer solution, How long is stable and i can use? Dose it unstable and I should prepare it when I need?

Thank you so much in advance. Peter Jackson

When you dissolve the contents of the pouch in water so that the final volume is 500ml the concentration of the MES is 0.1M. If you dissolve the contents of the pouch in so that the final volume of the solution is 100ml then the concentration of MES will be 0.5M However the pH may not be 4.7. Also the concentration of sodium chloride will be 4.5%. (w/v) .

If the only counter ion the MES ( acid form) is sodium then you could add aqueous sodium hydroxide solution:

Dissolve the contents of the pouch in approximatley 90 ml of water and then add some NaOH solution to adjust the pH to 6.1 then add more water to bring the final volume to 100mL. I do not know if the pouch contents will be soluble in 100mL

Thank you so much Dr. Peter Jackson.

when I want to add NaOH solution to buffer solution for adjusting pH at 6.1, Should I make a NaOH solution with pH6.1 then add to buffer solution?

Peter Jackson

If you dissolve the contents of the pack in 100ml water the MES concentration will be 0.5M . However the concentration of sodium chloride will be 4.5%. .

If the NaCl concentration of 9% is acceptable then to raise the pH from 4.7 to 6.1 you can add sodium hydroxide solution providing that the only counter ion to the MES is the sodium cation. However if you add NaOH solution you will reduce the concentration of MES .

To ensure that the final MES concentration is 0.5M you need to dissolve the solid in less that 100ml water ( try 90ml) and then add NaOH solution until the pH is 6.1. Then add water to bring the solution to final volume of 100mL Adding the water will not change the pH significantly. The NaCl concentration will be 4.5%

I do not know if MES is soluble in water at 0.5M Try it and see . The final temperature of the buffer must be that at which you will use it . Altering the temperature of the buffer will alter its pH

The following are the ways you can prepare MES buffer pH 6.0 and pH 5.0. Since you have not mentioned the molarity so I kept it at 100mM. In the reaction tube the final concentration is generally kept at much lower than 100mM concentration. Dilution of the buffer in this range will not change the pH significantly. Using stock buffers and adding a small volume of the stock buffer to the reaction mixture is a general practice in molecular biology.

MES is not a good buffer for pH 4 because the pKa of MES is 6.1. Buffers work better within 1 unit of the pKa.

2/22/2021 8:55:39 AM

Compound name: MES buffer

pKa: 6.1

pH: 6 Molarity: 0.1M Volume: 0.1 Moles salt: 0.0044268837 Moles Acid: 0.0055731163

Henderson Hasselbalch calculations:

Totalmoles = Total volume(L) * Molarity = 0.01

pH = pKa + Log [Salt]/[Acid]

6 = 6.1 + Log [Salt]/[Acid]

10 ^ (pH - pKa) = 10 ^ -0.0999999999999996 = [Salt] / [Acid] = 0.79433 = ratio

Moles Salt = Total moles * (ratio/(ratio + 1)) = 0.00442688366237708

Moles Acid = Total moles * (1/ratio + 1) = 0.00557311633762293

You can prepare this buffer by the following methods:

1. Dissolve 1.952 grams MES free acid, molwt.: 195.24)in 60 ml water.

Adjust the pH to 6 with 10N NaOH.

It will consume approximately 0.44 ml 10N NaOH.

Adjust the volume to 100 ml with water.

2. Dossolve 1.088grams MES (free acid molwt: 195.24) and 0.962

grams Sodium salt of MES (Sodium salt molwt: 217.25) in 80 ml water.

Verify pH and adjust the pH if required with 1N HCl or 1N NaoH over a small range.

2/22/2021 9:04:08 AM

Compound name: MES buffer

pKa: 6.1

pH: 5 Molarity: 0.1M Volume: 0.1 Moles salt: 0.0007358756 Moles Acid: 0.0092641244

Henderson Hasselbalch calculations:

Totalmoles = Total volume(L) * Molarity = 0.01

pH = pKa + Log [Salt]/[Acid]

5 = 6.1 + Log [Salt]/[Acid]

10 ^ (pH - pKa) = 10 ^ -1.1 = [Salt] / [Acid] = 0.07943 = ratio

Moles Salt = Total moles * (ratio/(ratio + 1)) = 0.000735875561175736

Moles Acid = Total moles * (1/ratio + 1) = 0.00926412443882427

You can prepare this buffer by the following methods:

1. Dissolve 1.952 grams MES free acid, molwt.: 195.24)in 60 ml water.

Adjust the pH to 5 with 10N NaOH.

It will consume approximately 0.07 ml 10N NaOH.

Adjust the volume to 100 ml with water.

2. Dissolve 1.809grams MES (free acid molwt: 195.24) and 0.160

grams Sodium salt of MES (Sodium salt molwt: 217.25) in 80 ml water.

Verify pH and adjust the pH if required with 1N HCl or 1N NaOH over a small range.

Thank you so much Dr. Peter Jackson Peter Jackson

Your guidance helps me a lot.

Yes, I can dissolve the content pouch in 90 ml easily and also measured the pH. The pH was 4.9.

Now I want to change pH from 4.9 to 6.1.

Thank you so much Dr. Babu Venkanna Bassa Babu Venkanna Bassa

Your answer help me to understand the relations in best form.

Thank you so much.

Dear Babu Vennkana Babu Venkanna Bassa

I want to make 0.5 M MES buffer. The pH is 4.73 and the initial volume MES buffer is 40 ml. The final volume is 50 mL. I want to change pH and arrive pH=6.1 at final volume.

So, I should add 10 mL NaOH solution to arrive pH=6.1.

I do the below calculation.

Compound name: MES [2-(N-morpholino) ethanesulfonic acid], 0.9% sodium chloride

pKa: 6.1

pH: 6.1

Molarity: 0.5M

Volume: 0.05 litre

Total moles = Total volume(L) * Molarity = 0.05*0.5=0.025

pH = pKa + Log [Salt]/[Acid]

6.1 = 6.1 + Log [Salt]/[Acid]

[Salt] / [Acid] = 1 = ratio

Moles Salt = Total moles * (ratio/(ratio + 1)) = 0.0125

Moles Acid = Total moles * (1/ratio + 1) = 0.0125

Now, I have a solution MES buffer 40 ml with pH = 4.73.

Final volume is 50 mL.

I do not know How much (10M,0.1M) NaOH solution should I add?

Thank you so much in advance.

Sara

The NaOH solution of 0.1N is too low especially for MES buffer. Use the NaOH at about 2.5N concentration and always use the pH meter for adjusting the pH.

4/6/2021 7:51:19 AM

Compound name: MES buffer

pKa: 6.1

pH: 6.1

Molarity: 0.5M

Volume: 0.05 L

Moles salt: 0.0125000000

Moles Acid: 0.0125000000

MES sodium salt stock concentration:

MES free acid stock concentration:

Henderson Hasselbalch calculations:

Total moles = Total volume(L) * Molarity = 0.025

pH = pKa + Log [Salt]/[Acid]

6.1 = 6.1 + Log [Salt]/[Acid]

10 ^ (pH - pKa) = 10 ^ 0 = [Salt] / [Acid] = 1.00000 = ratio

MolesSalt = Total moles * (ratio/(ratio + 1)) = 0.0125

MolesAcid = Total moles * (1/ratio + 1) = 0.0125

You can prepare this buffer by the following methods:

1. Dissolve 4.881 grams MES free acid, molwt.: 195.24)in 30 ml water. Adjust the pH to 6.1 with 2.5N NaOH. It will consume approximately 5.00ml of 2.5N NaOH. Adjust the volume to 50ml with water. Mix well. Please use more concentrated NaOH solution for higher volumes and higher concentrations of the buffer.

Or

2. Dissolve 2.441grams MES (free acid molwt: 195.24) and 2.715grams Sodium salt of MES (Sodium salt molwt: 217.25) in 40 ml water. Verify pH and adjust the pH if required with 1N HCl or 1N NaOH over a small range. Adjust the volume to 50ml. Mix well.

Please do not add the entire volume of NaOH at one time. Please use a pH meter and a stirrer and adjust the pH gradually. The volume of the NaOH solution given here is only a guide.

Dear Babu Vennkana Babu Venkanna Bassa

Thank you so much.