A Guide To Biological Buffer Preparation: For Weighing, PH Measurement and Pipetting
A Guide To Biological Buffer Preparation: For Weighing, PH Measurement and Pipetting
A Guide To Biological Buffer Preparation: For Weighing, PH Measurement and Pipetting
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Buffer Concept 1. The Buffer Concept
O O
CH3 C–OH CH3 C–O – +H +
=
=
weak acid
Predominates Negligible
Fig. 1: Most buffers are weak acids and their salts dissociate in a solution as shown
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1.3. General Requirements of Biological Buffers
1. Solubility
The buffer should be freely soluble in water and poorly soluble in other
solvents.
3. Ionic strength
The buffer should not alter the ionic strength of the system.
4. Dependence of pK a value
The pK a value of a buffer should be influenced as little as possible by
the buffer concentration, the temperature and the ion composition of
the medium.
5. Inert substances
The buffer should not be subject to either enzymatic or non-enzymatic
changes, i.e. it should not be an enzyme substrate or enzyme inhibitor
and should not react with metabolites or other components. The buffer
should, therefore, be inert.
6. UV absorption
Buffers should not absorb any light at wave-lengths longer than
230 nm, since many spectrophotometric investigations are performed
in this range (determination of DNA, RNA and protein concentrations).
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Laboratory Solutions 2. Optimizing Buffer Preparation Process
and METTLER TOLEDO Solutions
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Step 2: Recipe selection and calculation
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Step 3: Weighing-in
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Laboratory Solutions
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Step 6: Buffer volume adjustment
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pH Measurement 3. pH Measurement Tips and Hints
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3. Clean protein contaminated electrode with a pepsin / HCI solution
Junctions contaminated with proteins can often be cleaned by immers-
ing the electrode into a pepsin / HCI (5% pepsin in 0.1 mol/L HCl) solu-
tion for several hours.
Ensure that you always measure the correct pH value by following the
tips and hints below.
pH −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Acidic Basic
Neutral
Fig. 3: pH scale
1. Warm-up pH Meter
A pH meter may require a warm up time of several minutes. When a
pH meter is routinely used in the laboratory, it is better to leave it “ON“
with the function switch set at “standby“.
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pH Measurement
pH troubleshooter: www.electrodes.net
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4. Weighing Tips and Hints
3. Weighing-in guide
As each ingredient is weighed in, the SmartTrac weighing guide of
the XPR or XSR balance displays the actual weight against the target
weight graphically. The colored bar turns green as soon as the weight
of the added component is within the predefined tolerance range. This
enables analysts to weigh in more quickly and with greater certainty.
4. Documentation
The final buffer solution must be carefully labelled with all information
to avoid any mix-ups. The expiry date is important to ensure the buffer
solution is still effective when it is used. All data relating to the buffer
solution preparation must be logged and stored securely for future ref-
erence and traceability.
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Weighing
+30 °C max.
Optimal
−5 °C min.
al-Bereich 20 –8
xim 0%
Ma
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2. Proper operation of the balance
3. User Ergonomics
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Pipetting 5. Pipetting Tips and Hints
Fig. 4: E4 XLS+
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3. Follow “general guideline” for pH adjustments
As a general guideline, it is safest to start titration very slowly, by add-
ing a volume of acid/base <0.01% of your total buffer volume. Note
the rate of pH change and adjust your titration accordingly. Another
option is to use a repeater pipette, which combines the advantages of
positive displacement, speed and precision. Consider using a large
volume pipette or serological pipette if larger volumes need to be
added (>1 mL) to get any change in pH. When very close to target pH,
slow down the addition of reagent.
Pipettes are used in buffer preparation when adding water and addi-
tional liquid chemicals to the solution. The tips and hints below explain
proper pipetting techniques when preparing buffers.
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Pipetting
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