pH

[sudhis]

generally we adjust ph of buffer at 25°c with ±0.05 range . but we operate hplc sometimes above 50°c .so please justify the same since there will be change in pH as temperature increases.

[amaryl]

generally we adjust ph of buffer at 25°c with ±0.05 range . but we operate hplc sometimes above 50°c .so please justify the same since there will be change in pH as temperature increases.

If buffer pH changes then why will it be a buffer than?


Temperature as a variable in LC
Ø Decreases viscosity of the mobile phase and hence lowered backpressure over the column, allowing higher speed or longer columns with higher plate numbers.
Ø Increase analyte diffusivity due to increase mass transfer, resulting in improved column efficiency and reduces analysis time.
Ø There is a risk of increased column bleeding at high temperatures, increasing the background noise.

Regards,

Amaryl.

[jitender]

Thats true pH of buffer will change with change in temperature. However, I wont be bothered for a method in which both the temperature at which buffer was prepared (pH was measured) and temperature at which HPLC separation is achieved are defined. In that case its just a change of numeric value of pH with change in temperature.

[Bill Tindall]

Knowing the value of pH in a separation is unimportant. All that is important is that each time the separation is done, the pH must be the same.

Hence, if you reproducibly prepare a buffer under some condition and then operate under some different condition that will change the value of the pH under the new condition, this change is unimportant if the change is the same each time.

Changes in pH are an every day occurance in LC. Buffers are prepared in water at room temperature at some pH and then the buffer is mixed with an organic solvent and perhaps the temperature is changed. The operating pH may be several pH units different from what was measured when the buffer was prepared. But it does not matter if the chage is reproducible.

The most precise way to prepare your buffer is to not use the pH meter. Determine how many grams or mL of buffer components are required to get the proper buffer for the separation. Simply prepare the buffer by adding these specified weights of components each time. This method of buffer preparation is orders of magnitude more precise than what you are doing.

[amaryl]

Knowing the value of pH in a separation is unimportant. All that is important is that each time the separation is done, the pH must be the same.

Hence, if you reproducibly prepare a buffer under some condition and then operate under some different condition that will change the value of the pH under the new condition, this change is unimportant if the change is the same each time.

Changes in pH are an every day occurance in LC. Buffers are prepared in water at room temperature at some pH and then the buffer is mixed with an organic solvent and perhaps the temperature is changed. The operating pH may be several pH units different from what was measured when the buffer was prepared. But it does not matter if the chage is reproducible.

The most precise way to prepare your buffer is to not use the pH meter. Determine how many grams or mL of buffer components are required to get the proper buffer for the separation. Simply prepare the buffer by adding these specified weights of components each time. This method of buffer preparation is orders of magnitude more precise than what you are doing.

Sir, could you please elaborate this method of preparation of buffer with an example.

If buffer pH changes with temperature. Whats the relation, inverse or direct?

Regards,

Amaryl.

[Victor]

Bill, I agree with what you are saying about buffer preparation.

At times I have need to know accurately the pH of a solution I have prepared. I have used a number of calculation systems which tell me what the pH of the solution I have prepared (by weighing) should be. However, on measuring the pH I have noted there may be a difference in pH of +/- 0.1 units beween the measured and predicted values. I feel I can measure pH to within +/- 0.04 or thereabouts using a thermostat water bath and calibrant buffers. I suspect therefore that the problem is approximations in the calculation programs-is this misplaced arrogance? What is your opinion of this?

Note I am talking about the pH of buffers used in HPLC like TFA, not highly studied calibrant buffer solutions like potassium hydrogen phthallate. THese remarks also refer to purely aqueous solutions (apart from the small amount of organic additive (e.g TFA).

These remarks have no relevance to most people, who like you say, mostly need reproducible buffers, not accurate buffers.

[Bill Tindall]

Victor, I am about to leave on a trip. I will make a detailed reply to your question by Wed evening. It is not a simple question to answer.

[adam]

Bill

I know that in many previous threads, you have advocated weighing the buffer components as being more accurate than adjusting pH with a meter. At the risk of exasperating you, I think there is one potential problem with that approach: which is that many buffers are hygroscopic, and pick up moisture. So when the analyst weighs them they may actually have less buffer than they think, because some of the weight comes from water.

[Mark Tracy]

Moisture control is what dessiccators, drying ovens and moisture balances are all about. If precision is important, use them.

[tom jupille]

I think there is one potential problem with that approach: which is that many buffers are hygroscopic, and pick up moisture. So when the analyst weighs them they may actually have less buffer than they think, because some of the weight comes from water.

The first time I met Bill face-to-face, I asked him the same question. As I recall, the subsequent conversation went something like this:

Bill: "Tom, what kind of a function is pH?".

Me: "Uhhh, logarithmic?"

Bill: "Right, which means that a 1 pH unit change is what percentage change in hydronium?"

Me: "Ummm, ten times, which I guess means 1000%?"

Bill: "Right again. Now, do you know how big a weighing error you'd have to make to be off by a tenth of a pH unit?"

I'll leave the calculation as an exercise, but the answer is here:
http://www.lcresources.com/wiki/index.p ... Q:PHAdjust

[JA]

Some of the phosphates can form huge hydrates at over twice the mol weight. What is ideal then, purchase only the anhydrous materials, store in an evacuated desiccator and dry before use?

[jitender]

Weighing high grade chemicals should give the more accurate pH values. pH meters are calibrated using the buffers (some uses commercially available tablets) and some make their own calibration solutions from dessicated/dried and accurately weighed chemicals.
Commercial Tablet is compacted mass of buffer components weighed accurately.

[HW Mueller]

There are ~99.9% purity hydrated phosphates, etc., available. They should do, they are easy to weigh.
In my experience, pH meters have been the most difficult of all instruments to handle. For instance, I havn´t completely figured out, in years of using the same pH meter, why sometimes it has an unacceptable drift, other times not. I said this before: Bill convinced me on the weighing matter. Still I generally run the solutions on the pH meter, just to see and to keep the pH meter and me in "training". Also, if one has to run a series on different pH it is sometimes faster to just mix two solutions (acidic and basic version at same concentration) and checking it with the pH meter. It appears that if one is "trained" on a pH meter in the mentioned way the discrepancies between weighing and the pH meter are not such that they bother the LC too much.
Still: Since Bill, and also due to my (practical) experience with pH meters I would rather trust the weighing than a pH meter.

[Rafael Chust]

Quite curious... although I never did any special thought about this, I always prepared buffers by weighing and just "confirm" with the pH meter.

Concerning the change of temperature, it is a direct correlation - there are even tables about it published around...

And, just some food for thought: a variable that changes in a predictable manner is a constant, right?

[JS]

Hello, first post.

Is it correct then, that commercially available high purity hydrated phosphates will be less hygroscopic or will have this property reduced to the point that the weighing error involved has negligible effect on the resulting concentration?