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[MUSIC PLAYING]

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PROFESSOR: Many chemistry
and biochemistry procedures

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will require you to perform
a reaction at a specific pH.

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To accomplish this
task, you must

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understand how to
both make a buffer

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and how to use a pH meter.

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This video will demonstrate two
methods of buffer preparation

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as well as proper
use of the pH meter.

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A buffer is an ionic compound
that resists changes to its pH

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when strong acids or
bases are added to it.

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The amount of strong
acid or strong base that

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can be neutralized by
a particular buffer

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is dictated by the
buffer's capacity, which

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is determined by the
concentration of weak acid

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and weak base in the buffer.

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A higher concentration
of buffering reagents

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translates into greater
buffering capacity.

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In the first part
of this video, we

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will walk through two methods
of buffer preparation.

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In the first example,
you will need

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to prepare 1 liter of
a 50 millimolar buffer

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for a protein that
is stable at pH 7.3.

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The first step is choosing
an appropriate buffer system.

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This buffer system should
produce a stable pH

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in the range needed and should
have a capacity large enough

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to accommodate your solutions.

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The weak acid used
for the system

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must have a pKa that is plus
or minus 1 unit of the desired

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pH--

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the closer the better.

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From this abbreviated
list of weak acids,

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the phosphate ion,
with a pKa of 6.82,

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is clearly the best
choice for a buffer

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with a desired pH of 7.3.

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Therefore, the weak acid used
for this system is H2PO4 minus,

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and the conjugate
base is HPO42 minus,

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which correspond to the salts
sodium phosphate monobasic

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and sodium phosphate dibasic.

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After determining an
appropriate buffer system,

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the Henderson-Hasselbalch
equation

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is used to calculate
the ratio of acid

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to conjugate base needed
at your desired pH.

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If your desired pH of 7.3 and
the pKa of the phosphate ion

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are substituted
into this equation,

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then the ratio of
conjugate base to acid

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is calculated to be 1.6.

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Therefore, your buffer must
contain 1.6 times the amount

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of sodium phosphate dibasic
to sodium phosphate monobasic.

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After determining
the appropriate ratio

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of conjugate base to acid, the
exact amount of each reagent

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is calculated in
the following way.

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Because you are planning to
make 1 liter of a 50 millimolar

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phosphate buffer at
pH 7.3, 50 millimolar

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is equal to the concentration
of acid plus the concentration

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of conjugate base.

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In addition, you already know
that the ratio of base to acid

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is 1.6, so the
concentration of base

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is equal to 1.6 times the
concentration of acid.

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By substituting Equation
2 into Equation 1,

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you can see that the
concentration of acid

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plus 1.6 times the
concentration of acid

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is equal to 50 millimolar.

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Through manipulation
of this equation,

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the concentration of acid needed
for this particular buffer

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is 19.2 millimolar.

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And the concentration of
base is 30.8 millimolar,

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which corresponds to 2.30
and 4.37 grams respectively

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for a 1-liter solution.

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To make the buffer, simply
dissolve 2.30 grams of sodium

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phosphate monobasic and 4.37
grams of sodium phosphate

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dibasic in 1 liter
of distilled water.

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An alternative method
of buffer preparation

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is simple, accurate, and
requires much less math.

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To demonstrate
this second method,

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we will prepare 1 liter of a
1.0 millimolar Tris buffer at pH

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7.60.

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The first step involves
the determination

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of the amount of weak acid,
or in this example, weak base,

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needed in the system.

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To make 1 liter of a 1.0
millimolar Tris buffer,

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1.0 millimoles or 121
milligrams of Tris are required.

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Add approximately 800
milliliters of distilled water

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as well as a stir
bar to the beaker.

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Then weigh 121
milligrams of Tris

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and transfer it to
a 1-liter beaker.

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Ensure that all of the
material has been transferred

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by rinsing the weigh
boat and transferring

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the rinses into the beaker.

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While stirring, monitor the
pH of this solution and add

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a sodium hydroxide solution
dropwise until the pH is

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exactly 7.60.

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The concentration of the
sodium hydroxide solution

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doesn't really matter.

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However, a solution
that is too dilute

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may require an excessive volume
to be added to the buffer,

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while a solution that
is too concentrated

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may cause you to
overshoot the desired pH.

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If you do happen to
exceed the desired pH,

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then add a hydrochloric
acid solution

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to bring the pH back down.

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Transfer this solution to
a 1-liter volumetric flask.

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Wash the beaker with
distilled water several times,

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and add these washings
to the volumetric flask.

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Dilute the buffer to the
mark on the volumetric flask

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and mix the solution
by inverting the flask

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several times.

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For the second method
of buffer preparation,

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you needed a pH meter.

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The final part of this video
will discuss the proper use

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of this equipment.

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A pH meter allows the
determination of hydrogen ion

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activity by a
potentiometric measurement,

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using a glass electrode
coaxially joined

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to a silver/silver-chloride
reference electrode.

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When the probe is
immersed in solution,

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the reference
electrode makes contact

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with the sample, which
completes the electrical contact

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between the reference electrode,
sample, and pH electrode.

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If the unknown solution
has different pH

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from the solution in the
probe, an electrical potential

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results, which is
registered on the meter.

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This video will demonstrate the
use of an Orion SA520 pH meter.

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The important
components of a pH meter

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include the display, the
probe, and the electrode tip.

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The electrode tip contains
a fragile glass bulb.

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Be extremely careful
not to hit the probe

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on the side of a beaker
and when using stir bars.

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To operate the pH
meter, first make

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sure that the electrode
is connected to the meter.

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Then calibrate the pH meter.

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The pH meter must
be recalibrated

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every time you turn it on
and after three to four hours

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of use.

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We will perform a
two-point calibration.

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This method involves
calibrating the pH meter

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with two different
buffers of known pH.

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Turn on the instrument
and select the pH mode

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as 0.1 or 0.01.

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Then press the ISO button and
verify that the pH reads 7.00.

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Rinse off the probe with
distilled water into a waste

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beaker and gently blot
it with a chem wipe.

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Place the probe into
the first buffer

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and press the CAL button.

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The number 1 should
appear on the display.

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The display will alternate
between 1 and the pH

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value of the solution.

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Wait until the display
stabilizes and press Enter.

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The number 2 should then
appear on the display.

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Rinse the probe with
distilled water,

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blot it with a chem
wipe, and place it

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into the second buffer.

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If your solution has an acidic
pH, then use the 4.01 buffer.

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If it has a basic pH,
use the 10.01 buffer.

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The display will alternate
between 2 and the pH

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value of the solution.

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Wait until the display
stabilizes and press Enter.

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The letters pH
will be displayed,

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indicating that the pH meter is
now ready to take measurements.

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Before taking a
measurement, make sure

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that your sample buffer is at
the same temperature you'll

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be using in your
experiments because pH

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is temperature dependent.

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Rinse and blot the probe and
place it into the buffer.

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Wait for the display to
stabilize and record the pH.

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Correct buffer preparation
and pH meter technique

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are essential to ensure
that your proteins

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and other sensitive chemicals
live long functional lives.

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