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Posted by Megan on September 5th, 2017 in Megan Hardie  ⟩  0 comments

Buffers are a class of solution-stabilizing molecules which existed long before contemporary lab technology. Natural buffer substances like bicarbonate and carbonic acid are manufactured by organisms and molecular interactions, functioning to maintain pH equilibrium.

After natural buffer systems were discovered, their balancing effects became indispensable in scientific exploration. Synthetic buffers were developed over decades to produce reliable reactions in experimental models, enhancing biochemical reactions and medicinal products.

New buffers are introduced every year, built from the fundamentals developed over a century ago. This article explores buffers beginning with the foundation which made them inseparable from biochemistry. We’ll then follow the construction and replacement of buffering systems among individual studies as procedures are continually refined.



First developments: Quantifications and medicine

Quantifying chemical behavior is essential to experimentation, so calculations paved the buffer system’s entry into biochemistry. In 1908, Lawrence Joseph Henderson formulated an equation which described carbonic acid as a natural buffer. Karl Albert Hasselbalch later reformulated Henderson’s work logarithmically to create the Henderson–Hasselbalch equation, a formula that measures pH derivation in terms of acidity. It became a valuable tool for estimating the equilibrium pH in acid-base reactions and what pH a buffer would constitute.

Buffers applied to other mediums had their own mathematics developed soon after. Published by M. Koppel and K. Spiro in 1914, “On the action of moderators (buffers)” studied the effects of these substances “in the shift of the acid-base equilibrium of biological fluids.” The paper introduced our basic concept of buffer value (P, relating to strong acids) and gave revolutionary calculations to buffering activity among different substance types: weak and dibasic acids, bases, ampholytes and buffer mixtures.

Another contribution was made eight years later when Van Slyke published a simplified calculation for buffer values in a solution. His quantification using β, the amount of strong base, was similar to Koppel and Spiro’s and had identical assumptions. Van Slyke’s focus tended towards the physiological aspects of buffer solutions whereas the previous authors intended their calculations for physicochemical mathematics. This algorithm for acid-base variation has since been useful for blood-based chemistry and determining the relationship between buffer concentration and capacity.

Early buffers focused on medicinal purposes like stabilizing biofluid pH. The 1928 invention of Tums maintained pH ranges in the digestive system via neutralization of stomach acid. Alka-Seltzer, made available in 1931, is still used as a lab demonstration in university lectures. 



Foundational experiments: 1960-1990s

Basic reagents are used in combination to produce the most potent buffer solutions. Once buffers transitioned into biochemistry, researchers began to establish what chemical mixtures were most productive for equalizing the pH of certain reactions.

Between the 1960s and 80s, a project for determining the best buffers resulted in a list that remains crucial in modern laboratories. “Good’s buffers” were produced or collected by Norman Good and his colleagues, and selected on a number of criteria that qualified application to research in the biological field. Some of the requirements were pKa between 6 and 8, high water solubility, stability and a lack of exchange with membranes or biochemical reactions. Good also prioritized substances that could be prepared easily and safely.

One of the lab world’s most valuable buffer agents, Tris – was first recognized by Good in the early 1960s. Known in therapeutics as THAM, Tris quickly adopted scientific roles. Tris and other reagents identified by Good continue to act as the equalizing agents within buffer mixtures by adjusting pH to a specified range. 



Recent developments: 2000s

After Good’s buffers became common knowledge, researches took advantage of flexible buffer technology. The ease of inventing compounds gives each laboratory license to pioneer new buffer mechanisms; furthermore, new technology and experimentation methods expand research opportunities. Recent decades have demonstrated the creativity of researchers who seek better devices for validating experiments. Their initiative to revise past substances advances biochemical techniques. We can visualize buffer advancement through a few novel systems, invented to improve research activity.

Retrieving results is the first priority of scientific research. Two studies in 2007 used new buffers to optimize data recovery from biochemical tests involving DNA. In an article on real-time polymerase chain reaction (PCR) analysis, researchers intended to improve the quantification and yield of replicated DNA using a combination of buffers with Tris. By using HEPES, MOPS or TAE alone or together in addition to Tris, the reaction’s efficiency was improved in terms of detection and measurement. Another project identified a pair of new buffers for use in plant DNA flow cytometry. Termed “general purpose buffer” (GPB) and “woody plant buffer” (WPD), these buffers produced quality results from samples. GPB was more applicable to softer tissue plants and WPD worked better with recalcitrant tissue. These original substances had higher productivity than buffers previously used for this technique, indicating a possible replacement for other solutions.

Inventing buffers relevant to multiple techniques can result in overall enhancements to biochemical analysis. All chemical substances have qualities that prevent them from being functional in all test circumstances. For buffers, limits are posed by cooling processes that reduce their efficacy. Cold temperatures can degrade buffer-maintained pH and damage frozen or refrigerated samples, but a 2008 research project designed a buffer which could combat the constraint. Buffers become either more acidic or more alkaline when cooled, so researchers combined solutions until they found the correct proportions to have a minimal pH shift of 0.2 (reduced from the 2 pH change seen in other buffers). Biochemical tests need maximally stable pH from which results can be confidently validated, so having this buffer mix at a lab’s disposal could optimize their experimentation.



(Click the image for an expanded version of the timeline)



Today’s researchers use decades of assembled knowledge to develop new, better compounds for stabilizing biochemical reactions. With such a reliable foundation for implementation, it’s obvious why buffers have proliferated throughout the field. Old buffer systems can be applied to emerging techniques, but more often new solutions are created to replace outdated ones. As Good’s team encouraged safe, inexpensive buffers, science encourages the discovery of productive buffers for conducting each test. We have high expectations for this category of substances – with the direction of visionary scientists, our methods can only improve.

Buffers purchasable from GoldBio



References

Ahmad, A., & Ghasemi, J. (2007). New buffers to improve the quantitative real-time polymerase chain reaction. Bioscience, Biotechnology, and Biochemistry, 71(8), 1970-1978. doi:10.1271/bbb.70164.

Ambler, J., & Rodgers, M. (1980). Two new non-barbiturate buffers for electrophoresis of serum proteins on cellulose acetate membranes. Clinical Chemistry, 26(8), 1221-1223. Retrieved August 1, 2017, from http://clinchem.aaccjnls.org.

Loureiro, J., Rodriguez, E., Dolezel, J., & Santos, C. (2007). Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Annals of Botany, 100(4), 875-888. doi:10.1093/aob/mcm152.

Roos, A., & Boron, W. F. (1980). The buffer value of weak acids and bases: origin of the concept, and first mathematical derivation and application to physico-chemical systems the work of M. Koppel and K. Spiro (1914). Respiration Physiology, 40(1), 1-32. doi:10.1016/0034-5687(80)90002-X.

Sun, H., Lau, K. M., & Fung, Y. S. (2010). A new capillary electrophoresis buffer for determining organic and inorganic anions in electroplating bath with surfactant additives. Journal of Chromatography A, 1217(19), 3244-3250. doi:10.1016/j.chroma.2010.01.011.

Thomas, J. M., & Hodes, M. E. (1981). A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH. Analytical Biochemistry, 118(1), 194-196. doi:10.1016/0003-2697(81)90178-0.

Williamson, J. D., & Cox, P. (1968). Use of a New Buffer in the Culture of Animal Cells. Journal of General Virology, 2, 309-312. doi:10.1099/0022-1317-2-2-309.



                Megan Hardie
           GoldBio Staff Writer

Megan Hardie is an undergraduate student at The Ohio State
University’s Honors Arts and Sciences program. Her eclectic
interests have led to a rather unwieldly degree title: BS in
Anthropological Sciences and BA English Creative Writing,
Forensics Minor. She aspires to a PhD in Forensic Anthropology
and MA in English. In her career, she endeavors to apply the
qualities of literature to the scientific mode and vice versa,
integrating analysis with artistic expression.

Category Code: 79101, 79102, 79105, 88221, 88241

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