Buffer Preparation Guide

Tips and Hints

A Guide to Biological Buffer Preparation

For Weighing, pH Measurement and Pipetting
Introduction

Researchers in Life Sciences carry out work on biological molecules,

such as antibodies or proteins, in order to modify them for their pur-
poses, often in the search for disease cures and prevention.

Almost all biological processes are pH dependent. The purpose of a

buffer in a biological system is to maintain intracellular and extracel-
lular pH within a very narrow range and resist changes in pH in the
presence of internal and external influences.

The rather complex laboratory buffer preparation process involves

a number of different steps, including amount calculation, substance
weighing, pH solution value control and liquid pipetting. Over the years,
METTLER TOLEDO has gained a deep understanding of weighing, pH
determination and liquid pipetting and we have developed this buffer
preparation guide, containing useful tips and hints for the use of bal-
ances, pipettes and pH Meters, in order to pass on some of this knowl-
edge and support you on optimizing your buffer preparation workflow.
Content
Introduction 3
1. The Buffer Concept 4
2. Optimizing Buffer Preparation Process
and METTLER TOLEDO Solutions 6
3. pH Measurement Tips and Hints 10
4. Weighing Tips and Hints 13
5. Pipetting Tips and Hints 16

3
Buffer Concept 1. The Buffer Concept

1.1. Buffer Definition

Buffers are aqueous systems that resist changes in pH when small

amounts of acid or base are added and are composed of a weak acid
and its conjugate base. A buffer keeps the pH of a solution constant
by asorbing protons that are released during reactions or by releasing
protons when they are consumed by reactions. The discovery that
partially neutralized solutions of weak acids or bases are resistant
to changes in pH, when small amounts of strong acids or bases are
added, led to the concept of the “buffer“.

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

1.2. Biological Buffers

Different inorganic substances were originally used as buffers

(e.g. phosphate, cacodylate, borate and bicarbonate) and later, weak
organic acids were also used. Many of these buffer substances, how-
ever, have the disadvantage of not being inert and have lasting effects
on the system under investigation (e.g. inhibition of enzymes or inter-
actions with enzyme substrates etc.). Biochemical reactions are espe-
cially sensitive to pH. In all multi-cellular organisms, the fluid within
the cell and the fluids surrounding the cells have a characteristic and
nearly constant pH. The purpose of a biological buffer in a biological
system, therefore, is to maintain the intracellular and extracellular pH
within a very narrow range and resist changes in pH in the presence
of internal and external influences.

4
 1.3. General Requirements of Biological Buffers

Biological buffers used in cell cultures, isolation of cells, enzyme

assays and other biological applications must possess the following
distinctive characteristics.

1. Solubility
The buffer should be freely soluble in water and poorly soluble in other
solvents.

2. Permeability through biological membranes

The buffer should not be able to permeate biological membranes to
prevent concentration in the cell or organelles.

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).

5
Laboratory Solutions 2. Optimizing Buffer Preparation Process
and METTLER TOLEDO Solutions

The buffer preparation procedure consists of around 7 process steps,

including calibration and performance check of instrument, weighing,
pipetting and pH determination processes. METTLER TOLEDO solutions
make this procedure easy and reliable with the help of:
• XPR or XSR Precision Balances
• LabX laboratory software
• SevenExcellence™ pH Meters
• Rainin Pipet-Lite or Rainin AutoRep™ pipettes
• Label printers

Step 1: Calibration and performance check

Calibrate instruments easily

METTLER TOLEDO offers simple and reliable routine testing solutions
for balances, pH meters and pipettes.

• CarePacs® test weights for smooth routine

balance testing.

• Evaporation traps for occasional pipette

performance tests.

• Buffers of known pH value for exact meter pH

calibration.

www.mt.com/BuffersAndMore

6
Step 2: Recipe selection and calculation

Calculate the amount of compounds to weigh in

Most labs use more than 20 buffers. Use the correct
recipe for the planned buffer preparation. Buffer
solution recipes can be saved directly on the bal-
ance as weighing methods or via LabX software.
LabX software provides full step-by-step SOP user
guidance on the balance screen. Depending on
the required volume of buffer, the weights for the
compounds is automatically recalculated ensuring
reliable results.

www.mt.com/LabX

Step 3: Weighing-in

Weigh in compounds accurately with XPR or XSR

Precision Balances
Buffer solution recipes can be saved directly on the
balance as weighing methods or via LabX software.
At the start of each method, the required volume of
the buffer solution is entered and the component
quantities are calculated automatically. Accurate
and easy to clean SmartPan™ of XPR or XSR bal-
ance enable precision weighing. There is no need
for a draft shield, even when weighing in a fume
hood, making weighing easier and more ergonomic.

www.mt.com/xpr-precision

7
Laboratory Solutions

Step 4: Addition of solvents

Add solvent quickly with Rainin AutoRep Pipette

The AutoRep pipette saves time and speeds up long
pipetting series whilst remaining fatigue-free and
precise. The flexible volumes and wide selection of
easy-to-exchange tips ensure optimal performance
on any given volume from 1 μl – 50 mL.

www.mt.com/autorep

Step 5: Dissolving and pH adjustment

Check and adjust pH value correctly

The SevenExcellence™ pH Meter, with its large
clear color display and the state-of-the-art touch
screen operation, makes pH measurement simple
even for untrained users. With the addition of the
InLab® Routine Pro pH Electrode, with its integrated
temperature probe for correct automatic temperature
compensation, checking and adjusting pH becomes
a quick and accurate task.

www.mt.com/SevenExcellence
www.mt.com/electrodes

8
Step 6: Buffer volume adjustment

Adjust buffer to final volume with precise

pipetting
The Rainin Pipet-Lite is the most ergonomic, precise
and durable manual pipette on the market for rou-
tine pipetting avoiding tired hands and repetitive
strain injuries.

www.mt.com/pipettes

Step 7: Label and report generation

Generate labels and save results automatically

LabX Software automatically stores buffer results
and prints labels for buffer solutions. This ensures
safe data transfer and clearly readable labels.

www.mt.com/LabX

9
pH Measurement 3. pH Measurement Tips and Hints

3.1. Tips and Hints for Buffer Preparation

Exact and reliable pH Measurement is important for the correct prepa-

ration of buffers. Here are some useful tips and hints for pH determina-
tion when preparing buffers.

1. Select the correct electrode for your sample

pH electrodes play a very impor­tant role in performing correct pH value
determinations, since they are responsible for the actual pH measure-
ment. Based on your ap­plication, select the most suitable electrode.

2. Use a pH electrode with integrated temperature probe

Combined electrodes (figure 2) with an inner pH sensor and an outer
reference element are very commonly used. They house a temperature
sensor in the same body as the pH and reference elements in order to
further simplify pH measurements. This allows temperature compen-
sated measurements to be made.

Fig. 2: Combined electrode with integrated temperature probe

3. Immerse electrode no longer than necessary

It is important that the pH electrode is not left immersed in solution
any longer than necessary especially when using a solution that con-
tains proteins. After several pH measurements of solutions containing
proteins, rinse the electrode in a mild alkali solution and then wash
several times with de-ionized water.

10
 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.

4. Prevent any bacterial or fungal growth

Autoclave or sterile-filter the buffer solution in order to prevent any
bacterial or fungal growth. This is important when large quantities of
buffers are prepared and stored over a long period of time.

3.2. Tips and Hints for pH Determination

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

[H+] (M) 1 10−2 10−4 10−6 10−8 10−10 10−12 10−14

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“.

2. Clean electrode before beginning

Prior to beginning; make sure the electrode is well rinsed with deion-
ized water. To clean the electrode, rinse it with de-ionized water after
each measurement but never wipe it clean with a tissue.

11
pH Measurement

3. Calibrate with at least two standard buffer solutions

The buffers used for calibration must be selected according to the
sample’s pH. For instance, if a sample is expected to have a pH
of 7.45, the calibration must include pH buffers 7.00 and 9.21
(or similar).

4. Get rid of bubbles inside the electrode

Bubbles on the inside of the electrode will make measurements unsta-
ble. To get rid of any bubbles, gently shake the electrode with a vertical
motion, such as with a fever thermometer.

5. Maintain the identical stirring conditions when calibrating and

measuring
Use the same stirring conditions when calibrating and measuring,
i.e., if no stirring takes place during calibration, do not stir during
measurements.

6. Maintain pH electrode regularly to prolonging its lifetime

Electrodes should always be stored in aqueous and ion-rich solutions.
The electrode should never be stored dry or in distilled water as this
will affect the pH-sensitive glass membrane and thus shorten the life-
time of the electrode. If not sure which storage solution to use, check
the electrode’s manual.

7. Use the TroubleShooter if performance problems occur

A correctly selected pH electrode that has been working properly may,
nevertheless, suddenly start performing badly. The pH TroubleShooter
gives a detailed electrode diagnosis, as well as tips and hints on how
to resolve problems.

pH troubleshooter: www.electrodes.net

12
 4. Weighing Tips and Hints

4.1. Tips and Hints for Buffer Preparation

When weighing in all the different buffer solution components, care

must be taken to use the right amount of the right component. The
actual weight of each component must be logged, and, if recording
results by hand or entering the values into a computer, care must be
taken to avoid transcription errors. Here are some useful tips and hints.

1. Choose the right balance

Depending on how much of each of the buffer solution components is
required and the minimum weight of the balance, it may be necessary
to use different balances. The right balance can be recommended by
GWP® Recommendation.

2. Using weighing methods

The buffer solution recipes can be saved on the balance as weighing
methods. By saving the most commonly used buffer solution recipes
on the balance, it is quick to retrieve the method needed and start
working. The balance provides step-by-step SOP, so operators can
easily follow the process of buffer preparation.

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.

13
Weighing

4.2. Tips and Hints for accurate weighing

Balances are sensitive to external influences, which can significantly

affect weighing accuracy. There are several factors which can influence
the weighing, such as air currents, electrostatic charges and magnetic
stirrers. Here are some useful tips and hints.

1. Choose the optimum location

The place of installation and the weighing bench must be stable. These
are usually the vibration-free areas of a building.

Temperature: Keep the temperature of the room as constant as pos-

sible. Weighing results are strongly influenced by rapid changes of
temperature. Do not weigh near radiators or windows.
Atmospheric humidity: Ideally the relative humidity (% RH) should be
between 45% and 60%. Balances should never be operated above or
below the measuring range of 20% to 80% RH.
Light: If possible, place the balance near a window-free wall. Direct
sunlight (radiant heat) will influence the weighing result. Place the
balance a significant distance from lighting fixtures to avoid heat
radiation. This especially applies to light bulbs. Use fluorescent tubes
(if possible).

+30 °C max.

Optimal

−5 °C min.

al-Bereich 20 –8
xim 0%
Ma

14
2. Proper operation of the balance

Standby / Switch on: Do not disconnect the balance

from the power supply and always leave it in standby
mode. This allows the balance to maintain thermal.
Levelling: To ensure and document that the bal-
ance is correctly levelled every time we recom-
mend the XPR and XSR balances with the built-in
“LevelControl” automatic warning function.

3. User Ergonomics

When working at a balance for a long period of

time, ergonomics plays an important role. Users
should ensure that they adopt the correct sitting or
standing position. To ensure that measurements can
be read easily by all operators, the balance should
incorporate a large-digit display, and the brightness,
contrast and display angle should be adjustable.

Learn more: Weighing the right way

15
Pipetting 5. Pipetting Tips and Hints

5.1. General Tips for pH Titration

The following tips help to ensure that buffer pH is properly adjusted.

1. Use Precision Pipettes instead of glass transfer Pasteur pipettes

The addition of concentrated acids and bases to a buffer in order to
achieve the correct pH is often associated with some guesswork,
especially for first time buffer preparers. Traditionally, glass transfer
Pasteur pipettes have been popular for titration but these do not offer
precise knowledge of the volume added. Therefore, we recommend the
use of precision pipettes to aid the development of reproducible titra-
tion protocols.

2. Use electronic pipettes for reproducible titration protocols

Aspiration and dispensing are initiated on electronic pipettes by
pressing a trigger rather than using a plunger. By using an electronic
pipette, users can improve sample pick-up and dispensing consis-
tency, which, in turn, eliminates most user-to-user technique variability
and improves repeatability. Electronic pipettes, such as the Rainin
E4 XLS+ (figure 4), often have a “Titrate“ mode, which shows the user
precisely how much acid / base was added to a buffer during titration.
This speeds up development of reproducible titration protocols.

Fig. 4: E4 XLS+
16
 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.

4. Make notes for future buffers

Make a note of the volume of acid or base being added during each
buffer titration in order to perform the same operation much faster
in the future. Sometimes, the correct pH is reached with acid / base
remaining in the pipette tip. In this case, it can be difficult to estimate
how much acid / base was added to the buffer and we recommend
using the titration function on an electronic pipette which keeps track
of the dispatched volume displaying very precise information concern-
ing how much reagent was used during titration.

5.2. Tips and Hints for Precise Pipetting

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.

1. Choose the right pipette

For greatest accuracy, work within a pipette range which is 35% of the
nominal volume or higher.

2. Keep immersion angle vertical

Keep the immersion angle as close as possible to the vertical other-
wise the vertical liquid column becomes smaller and too much sample
will be aspirated.

17
Pipetting

3. Use the right immersion depth

If the tip is immersed too far, more liquid will be aspirated due to
increased pressure. Liquid retained on the tip surface can also distort
results. If the tip is not immersed far enough, air can be drawn in
resulting in air bubbles and inaccurate volume.

4. Use the correct dispensing technique

For most applications, it is recommended to dispense with the end of
the tip resting against the vessel wall. This reduces or prevents sample
remaining in the tip after dispensing. Remove the pipette by sliding the
tip end up the side-wall in order to release any remaining droplet
inside the tip.

5. Use the reverse pipetting mode for viscous liquids

For dense and viscous liquids, such as glycerol or Tween 20 use the
reverse pipetting mode or a positive-displacement pipette. Before sam-
ple pick-up, the plunger is pressed completely down to the blowout
position. The selected volume of a liquid plus an excess is aspirated
into the pipette tip. To dispense, the plunger is pressed only down to
the zero position.

18
Enhance Daily Research with Useful
Tips and Tricks

Discover more lab solutions for life science research from METTLER TOLEDO
on our Academia Internet page. Download useful information, such as
technical reports, practical examples, application brochures and guides
providing active support for daily research work allowing beginners and
professionals alike to benefit from our life science solutions and know-how.

www.mt.com/academia

19
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