Mar 19, 2015
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So today in lab I was making a buffer. The recipe made a total volume of 1L, however, initially you add 900mL then add a few solid ingredients. After the solid ingredients are added you top off the volume to 1L and make sure the pH is 8.5.

Our lab manager claimed you should pH the solution before you top the final volume off to 1L (so after you add the solid ingredients). I didn't argue with her or anything, but that doesn't make sense to me. Adding extra water will change the pH and bring it closer to 7,no?

I'm only asking because this is bothering me, not because I'm mad about a small detail or the interaction. Anyone care to explain who was correct/why? Thanks for the replies
 
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Mar 19, 2015
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You are correct. That person probably just wants to double check you added everything before filling it up to the point you can't add anymore stuff to fix it.
That makes sense. I had a grad student tell me the same thing as the lab manager a few weeks ago so I thought I was misunderstanding a concept from chemistry. Thanka for the clarification, it is much appreciated.

Edit: I added water to the solution while it was still in the pH meter. The pH increased by .05. Why is this?
 
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breezy16

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It's in the buffer range; adding 100mL of water will not alter the pH dramatically, if at all. The reason you pH before you QS is so that if you need to add ~mLs of HCl or NaOH, you're not messing with molarity by having a final volume of 1010mL or something.
 
OP
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Mar 19, 2015
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It's in the buffer range; adding 100mL of water will not alter the pH dramatically, if at all. The reason you pH before you QS is so that if you need to add ~mLs of HCl or NaOH, you're not messing with molarity by having a final volume of 1010mL or something.
Ahhh. I completely overlooked the buffer range. Thanks for clarifying/disproving me
 
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If you are using miliQ or a properly distilled source, you should be modifying pH before you reach final volume. Adding more pure water will make absolutely no difference in your pH. Conversely, diluting your buffer with acids/bases to ameliorate pH issues will cause concentration deviations.

Your superior is correct.

EDIT:

Sidenote!-- If you are using TRIS buffer it is temperature sensitive so be sure to pH your buffer at the temperature you intend to use it at!!!!
 
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Mar 19, 2015
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If you are using miliQ or a properly distilled source, you should be modifying pH before you reach final volume. Adding more pure water will make absolutely no difference in your pH. Conversely, diluting your buffer with acids/bases to ameliorate pH issues will cause concentration deviations.

Your superior is correct.

EDIT:

Sidenote!-- If you are using TRIS buffer it is temperature sensitive so be sure to pH your buffer at the temperature you intend to use it at!!!!
So does water not alter pH because the HH equation? pH= pka + [A]/[HA]....so when we add water we are adding an equal concentration of hydronium ion and hydroxide ion...thus[A]/[HA]=1 and pH and therefore not affected?
 

MaxPlancker

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Yes the superior is correct although the difference is negligible most of the time...

Not because an equal amount of H3O+ or OH- is being added, but because the ratio of A- to HA is constant as you add water (i.e. the change in factors to A- is the same for HA).

IMO, in most buffers for molecular biology/biochemistry, its fine to just take 1L water and add the solids...lol perhaps the lazy way but really a lot of biology is not that specific except for certain techniques such as ITC.
 
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Yes the superior is correct although the difference is negligible most of the time...

Not because an equal amount of H3O+ or OH- is being added, but because the ratio of A- to HA is constant as you add water (i.e. the change in factors to A- is the same for HA).

IMO, in most buffers for molecular biology/biochemistry, its fine to just take 1L water and add the solids...lol perhaps the lazy way but really a lot of biology is not that specific except for certain techniques such as ITC.
Do not listen to this poster. There is no excuse for poor laboratory practice. As the lab manager for several years I would ask this undergraduate to leave and never return. With this mindset, I can't be sure when they would choose to follow correct procedure or when they would disregard it -- a pervasive danger to everyone's experiments.

To answer your question, there is nothing wrong with thinking of pure water as equally concentrated with H3O+ and HO- (that is exactly what it is). I'm not sure why maxplancker insists you are incorrect in making this conclusion. It is, by definition, the only substance with this unique type of equilibrium. (Kw)
 

MaxPlancker

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Do not listen to this poster. There is no excuse for poor laboratory practice. As the lab manager for several years I would ask this undergraduate to leave and never return. With this mindset, I can't be sure when they would choose to follow correct procedure or when they would disregard it -- a pervasive danger to everyone's experiments.

To answer your question, there is nothing wrong with thinking of pure water as equally concentrated with H3O+ and HO- (that is exactly what it is). I'm not sure why maxplancker insists you are incorrect in making this conclusion. It is, by definition, the only substance with this unique type of equilibrium. (Kw)
Not poor laboratory practice. Calm your panties. As I said above, certain experiments require more exactness than others. In 3 labs I've been in, none have taken the longer, more technically correct route albeit for a few sensitive experiments (and these are big shot labs). It becomes a matter of practicality, and time is priceless.
 
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Careful @MaxPlancker , your disgraceful machismo is showing. Notwithstanding your cleaver method for making buffers more quickly! What a guy.

I'm going to have to disengage my conversation with you. Making your buffer in the wrong order doesn't save time and I don't have the patience to continue the back and forth.
 
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MaxPlancker

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Careful @MaxPlancker , your disgraceful machismo is showing. Notwithstanding your cleaver method for making buffers more quickly! What a guy.

I'm going to have to disengage my conversation with you. Making your buffer in the wrong order doesn't save time and I don't have the patience to continue the back and forth.
It's not the wrong order. I would hate to have a lab manager as close minded as you. It is possible (perhaps not for someone of your intelligence) to calculate how much solid can be added to a liter to produce, theoretically and for all practical intent, the same buffer as you describe. How you would do that, I will let you try to puzzle it out. You call it machismo...I call it laughing at you.
 

Romz

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Do not listen to this poster. There is no excuse for poor laboratory practice. As the lab manager for several years I would ask this undergraduate to leave and never return. With this mindset, I can't be sure when they would choose to follow correct procedure or when they would disregard it -- a pervasive danger to everyone's experiments.

To answer your question, there is nothing wrong with thinking of pure water as equally concentrated with H3O+ and HO- (that is exactly what it is). I'm not sure why maxplancker insists you are incorrect in making this conclusion. It is, by definition, the only substance with this unique type of equilibrium. (Kw)
A little harsh though, lol.