A chemically stable piperazinic buffering agent that is used as the best buffer in cell culture media and cryopreservation media. It is utilized in biological researches related to the fields of biochemistry and molecular biology .
HEPES buffer (C8H18N2O4S) basically represent a chemical system that properly maintains the considerable number of specific ions present in the specific medium. It is stated to be a pH buffer when it adjusts the number of hydrogen ions present in the solution .
The considerable amount of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid in the preferred medium typically varies from 10mM to 25mM. The frequently used quantity is about 25Mm.
It is obtainable under the descriptive names of different labels of 4-(2-Hydroxyethyl) piperazine-1-ethane sulfonic acid, HEPES Free Acid, HEPES Monosodium Salt, HEPES, 99%, for biochemistry, and MFCD00006158 in the local market .
HEPES buffer (C8H18N2O4S) is available in the sustainable packaging of 25g, 50g, 100g, 250g, 500g, and 1kg in poly bottles. It is also available in the market in bulk quantities of 5kg, 25kg, and 50kg which are sold in the packaging of poly drums to save it from the external environmental conditions .
The structure of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid is shown below.
Figure 1: Structure of HEPES 
The procedure of making 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES buffer) in the laboratory is discussed below in detail  .
Equipment: The equipment used in the formation of HEPES buffer are given.
Stir bar (used to agitate liquid)
Sterile bottle (100mL)
Sterile Filter paper (2μm)
Reagents: The reagents required for the synthesis of HEPES buffer solution are described.
238.30 grams of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES buffer) in powdery form.
Distilled water (purified water with no minerals and contaminations in it).
Preparation Method: Following steps are necessary for the preparation of one liter of HEPES buffer solution in the laboratory.
Separate 850mL of distilled water from the prepared stock in a suitable container.
Now mix 238.3 grams of HEPES buffer (C8H18N2O4S) powder in the solution and stir it well by using a stirring bar.
Add 10N sodium hydroxide in the solution to maintain the desired pH of a solution.
Filter the solution by using Sterile Filter paper of 2μm in a sterile bottle.
Add distilled water until the final volume of the solution becomes 1 liter. The final HEPES buffer solution should be clear and transparent.
Store the solution at 4 degrees Celsius for future use .
The practical applications of HEPES buffer (C8H18N2O4S) in various fields are eloquently described.
Buffering capacity: when added to the cell culture media, HEPES buffer (C8H18N2O4S) undoubtedly enhance the buffering capacity of the media which, outside the incubator, requires elongated periods of manipulation .
Non-coordinating buffer with non-metal ions: HEPES buffer (C8H18N2O4S) do not coordinate with non-metal ions present in the solution to form complex compounds. That’s why it is declared as a non-coordinating buffer with non-metal ions .
Ampholytic separator: 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid is equally used as an Ampholytic separator in the cell culture media to adequately maintain a pH gradient during electro-focusing.
Stabilize pH: HEPES buffer (C8H18N2O4S) stabilizes the physiological pH of the solution or cell culture media.
Radical formation: HEPES buffer has the exceptional ability to instantly form radicals in a sustainable solution. Due to this ability, it is not utilized in oxidation-reduction reactions .
Rat insulinoma cells: HEPES buffer (C8H18N2O4S) is also used in culturing rat insulinoma cells along with sodium pyruvate and RPMI-1640, etc. .
Chromatography and Electrophoresis: HEPES buffer (C8H18N2O4S) is additionally used in the effective techniques of chromatography and electrophoresis .
Following are the specifications of 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid that are discussed below in the table .
Hazard and safety measures of HEPES buffer are described below .
Personal Protective Equipment:
Effective protection of Eyes: Properly use protective face shields or standard glasses while typically dealing with HEPES in the laboratory.
Necessary protection of Skin: To typically prevent direct contact with exposed skin, wear proper clothing that provides protection against exposure like lab coats, impervious gloves, and boots.
Respiratory Protection: Perform biochemical researches that typically involve the practical use of HEPES buffer (C8H18N2O4S) in a well-ventilated laboratory.
Proper storage conditions:
Cool temperature: carefully store the remaining solution at a well-ventilated place with a cool temperature.
Tightly Sealed: Use tightly sealed containers to avoid contamination that may be present in the atmosphere or in the surrounding places.
First Aid Measures: The first aid measure in possible cases of direct contact are described below.
Eye Contact: If the odorless liquid or powder form of HEPES buffer (C8H18N2O4S) accidentally contacted with sensitive eyes. Immediately wash the eyes of the identified victim with excess water. Carefully keep washing your face and eyes for 15-20 minutes. In specific cases of stinging, redness, or petty irritation, seek medical attention.
Inhalation: In the case of inhalation of the powdery form of HEPES buffer (C8H18N2O4S), mild irritation in the mucus membrane of the respiratory system may occur. Inhale fresh air. If the unfortunate victim is not breathing, instantly give him artificial breath. Promptly seek medical advice on excessive exposure to HEPES buffer (C8H18N2O4S).
Skin Contact: In case of accidental contact with skin, gently wash the contaminated body parts immediately. Shed the contaminated clothes. Seek medical attention according to the typical symptoms.
Ingestion: In case of ingesting it unintentionally, do not induce vomiting. Drink plenty of water. Seek medical advice immediately .
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