Biological Industries Israel
Beit Haemek Ltd.
Version 1.0
Page 1 of 2 Pages
Revision Date: 09/2009
Product Profile
Product Name:
Hepes Buffer Solution(1M)
Product Catalog Number:
03-025-1
Unit Size Availability:
(B)100g;(C) 20ml
Formulation:
Liquid Formulation
Defined Storage Conditions:
15-30°C (Room Temperature)
Stability: (Under Ideal Handling & Storage)
Please Refer To Product Label
Important Note! Please read the MSDS and Product Profile carefully in their entirety before using this material for possible safety
precautions and potential hazards.
Product Description:
When considering biological pH, the Hepes molecule is zwitterionic, a buffer with the capability of carrying a positive charge at one end of the
molecule and a negative charge at the other. It has been described as one of the best all-round, multi-purpose buffers available for biological
research. It is a commonly used buffer ideal for most cell culture work as it helps to maintain pH levels, especially in basal media in culture.
This is mainly due to its ability to maintain physiological pH despite the changes in CO2 concentration which occurs normally in cell culture due
to cellular respiration. When compared to bicarbonate buffers also commonly used in cell culture, It is claimed that Hepes may not only be a
more effective buffering agent for maintaining enzyme structure and function at lower temperatures but also is reportedly superior to NaHCO3
in controlling pH in tissue or organ cultures. pH buffering is deemed necessary not only due to the fact that the growth of many cells is
restricted to within narrow pH limits, but also because cellular metabolism frequently alters pH. However, the choice of buffers is dependent
upon many factors including optimal milieu conditions, nutrient niche requirements, specific cell line, general circumstances and most of all, the
researcher's experience. Buffer strength for cell culture applications is usually within the range of from 10-25 mM. Diligence and care must be
to the utmost in maintaining appropriate osmolality and toxicity in media with respect to the specific cell line. Hepes provides a buffer within the
pH range of 6.15-8.35 showing its wide applicability and its most efficacious nature (i.e., pK + 1, as a general rule). Although the most
commonly used buffering system is bicarbonate due to its nutritional benefits in spite of its reduced buffering capacity at physiological pH, the
supplementation of Hepes provides an additional advantage to cell culture medium within a pH range that is commensurate with the
concentration utilized. Although Hepes offers no nutritional benefit to cells, it is supplemented solely for its extra buffering capacity when the
cell culture may require manipulation for extended periods outside the CO2 incubator. Biological buffers are not only utilized in in vitro cell
culture, but also in enzyme assays and some electrophoretic applications at physiological pH. Universally applicable buffers for Biochemical
applications must have some of the following characteristics:
Must be H2O-Soluble
Show Non-Interference with Biological Processes or Biological Membranes in terms of(e.g. Surface Adsorption, Solubilization,
Penetration)
Have Known Complex-Forming Tendency with Metal Ions
Are Non-Toxic
Have a Low U.V. Absorption Wavelength
Buffers play a vital role in the control of the pH of not only intracellular and extracellular fluids of the body but also in almost all biological
processes. The human body has been described as essentially an acid factory producing lactic acid in our muscles, pyruvic acid as
carbohydrates are metabolized, to the hydrochloric acid of our stomachs and the carbonic acid in the blood acquired in the CO2 from the air we
breathe. All these biological processes are pH sensitive and therefore dependent on a very narrow pH range for optimal physiological
metabolic equilibrium and stability by getting rid of or neutralizing these acids effectively. Even the minutest change in pH may result in
metabolic acidosis (pH <7.35) or alkalosis (pH>7.45). A buffer solution is one in which the pH remains relatively constant even when there is
an increase or decrease in the presence of an acid or base. The purpose of a buffer in any biological system is to help maintain a normal
equilibrium of the extracellular and intracellular pH within the narrowest of ranges that resist superficial changes in the pH level in the presence
of external and internal influences. As cells undergo metabolic processes especially that of glucose, acids and CO2 (e.g. lactic, pyruvic) are
produced with a resultant pH decrease. For example, the pH indicator, Phenol Red, assists in rapidly identifying even superficial changes from
neutral to acidic pH values by measuring gradual nutrient depletion. Cultured cells require not only a sterile environment but also unique niche
nutrient requirements for growth. In addition, the cell culture environment must maintain its stability to optimally grow and this is where
temperature and pH come to the fore. Most cells require pH conditions within a 7.2-7.4 range and controlling pH is a key, essential factor for
creating the most optimum cell culture milieu. There are major variations to this optimum, as the ideal pH at culture initiation should be nearer
to 7.4, but should not fall below 7.0 during the culture. A pH below 6.8 usually inhibits cell growth. Continuous transformed cell lines (e.g.
Hybridoma Cell Lines) prefer more of an acidophilic milieu (7.0-7.4). Thus, the regulation of pH is critical immediately following cell seeding
when the new culture is acclimatizing itself to its new environment. This is usually achieved by one of two buffering systems:
•
The normal buffer system from the cell culture media constituents analogous to the blood in which the gaseous CO2 stabilizes with
the CO3 /HCO3 content
•
Improved buffering and pH stability using a zwitterion buffer such as Hepes either in addition to or instead of bicarbonate
Biological Industries, Kibbutz Beit Haemek 25115 Israel
Telephone: 972-4-9960-595 Fax: 972-4-9968-896
Web Site: www.bioind.com
1
E-Mail: [email protected]
Biological Industries(BI)
Page 2 of 2 Pages
In basal media for instance, the salts are primarily responsible for providing physiological pH among other factors such as osmotic pressure
and membrane potential. Generally, culture media are buffered to compensate for the cellular production of CO2 and lactic acid as by-products
of cellular metabolism. The traditional role of buffering culture media is with bicarbonate (HCO3). As cells grow and generate CO2 naturally, an
accumulation of CO2 in the headspace will prevent CO2 diffusing out of the medium with a resultant increase in weakly-dissociated NaHCO3
producing an excess of H+ ions and a concomitant decrease in pH. When this CO2 is dissolved, it forms a buffering system with the
bicarbonate. If a situation exists where cell density is low or the cells have entered a lag phase, they may not produce enough CO2 to maintain
optimal pH levels. In order to counter such problems, bicarbonate-buffered media require the use of incubators with a 5-10% CO2 atmosphere.
Some media with lower bicarbonate levels (e.g. MEM) or with higher levels (DMEM) require ~5% CO2 and ~10% CO2 respectively to maintain
pH. The most important factor being the correct percent CO2 based upon the particular medium's bicarbonate level, irrespective of cell type.
Cell culture media turns acidic as it becomes exhausted as the acid end products from carbohydrate fermentation lower the pH causing the
Phenol Red to turn from a reddish-purple hue (alkaline) to a yellowish hue (acid). The pH status of the medium is continually monitored as
indicated by the color. The breakdown of carbohydrates during fermentation may occur especially by contamination by microorganisms such
as bacteria and yeasts yielding incompletely oxidized products. Some forms of fermentation may occur in the absence of oxygen in which case
ATP is generated in reaction pathways when organic compounds act as both acceptors and donors of electrons. Factors affecting pH stability
of the cell culture medium include:
Glucose Concentration
Headspace Above the Medium to Prevent CO2 Loss and an Increase in Hydroxyl Ions
Buffer Capacity & Type
In essence, we see that the buffer system is what prevents such a large change in pH. There are many important buffer solutions and most
biochemical reactions, whether they occur in the laboratory or from within the human body, are carried out in buffered solutions. Hepes, in
comparison to the sodium bicarbonate buffer system is more suitable as a buffer when a physiological pH range of 7.2-7.6 is desired and may
be used with or without a CO2 atmosphere, whereas the bicarbonate buffering system requires the use of a CO2 incubator.
Some of the Predominant Characteristics of Hepes Solution 1 M include:
•
•
•
•
•
Zwitterionic Buffer
pKa range between 6.0 and 8.13
Liquid Formulation with high solubility
Easy-To-Use
For Biochemistry, Cell-Culture and Molecular Biology Applications
Instructions/Procedure:
1)
Take a bottle from the defined storage conditions at 15-30°C and read the label.
2)
Wipe the outside of the bottle with a disinfectant solution such as 70% ethanol.
3)
Using aseptic/sterile technique under a laminar-flow culture hood and work according to established protocols.
Storage & Stability:
This product should be stored under specified conditions (15-30°C) and used within the time indicated on the label. Do not use after the
expiration date as specified on the label. Deterioration of liquid media may be recognized by any of the following characteristics, among
others including: (a). color change, (b). granulation/ clumping, (c). insolubility,(d). And/or decrease in expected performance parameters.
Any material described above should not be used and therefore discarded. Hepes is light-sensitive. Please store in the dark for optimal
performance.
It is recommended to review the extensive literature concerning cell-culture media and its supplementation and the physiological parameters
required for each specific cell-line as per their essential requirements.
Quality Control:
Test
Specification*
Appearance:
Clear Solution
pH:(@ 37°C)
7.29-7.31
Sterility:
Test & Record
Please note that all batches/lots will differ to an extent.
Auxiliary Products
Product Name
Catalog Number
Storage Temperature
Dulbecco's Phosphate Buffered Saline(DPBS) without Calcium and
02-023-1
Room Temperature
Magnesium
(15-30°)
Amphotericin B 250 micrograms/ml
03-028-1
-20°C
Penicillin-Streptomycin Solution
03-031-1
-20°C
Penicillin-Streptomycin Nystatin Solution
03-032-1
-20°C
Phenol Red Solution
03-100-1
Room Temperature(15-30°)
Penicillin G Sodium Salt Cell Culture-Tested Biochemical
41-5012-8°C
Note: For a list of Serum, other Antibiotics, or Biological Industries'
Products Reagents and Supplements, please refer to our Product
Catalog/Product Profiles/ Product Guides and Internet Site.
References:
1)
14th Edition Of Merck Index,pps.1224,1514
2)
Current Editions USP/E Ph
3)
Biological Industries(BI )Specifications
4)
Biological Industries, Kibbutz Beit Haemek 25115 Israel
Telephone: 972-4-9960-595 Fax: 972-4-9968-896
Web Site: www.bioind.com
2
E-Mail: [email protected]