PubMed · 14970547
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Shawn Doonan. 2004. Making and changing buffers.. https://doi.org/10.1385/1-59259-655-x%3A91
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In this paper, we report the dependence of buffer pH and coating surfactant chain-length on electro-osmotic (EO) mobility in co-polyester microchannels. Thermoplastics co-polyester hydrolyzes to anionic functionality to create electrical double layer on the micro-channel walls. These negatively charged sites are partially or completely screened when long-chain surfactants are added into the buffer. This ancillary technique to modify surface charge polarity to avoid analyte adsorption is known as dynamic coating. We develop a theory to predict the EO mobility tendency on buffer acidity considering the combination of pH-dependent surfactant aggregation and surface dissociation. Our findings of pH-dependent EO mobility in coated channels, using three types of quaternary ammonium surfactants, lauryltrimethyammonium bromide (LTAB), trimethyl (tetradecyl) ammonium bromide (TTAB), and cetyltrimethyammonium bromide (CTAB), agree with our theoretical prediction. We also explain the chain-length dependence of mobility with a collaborative adsorption mechanism of surfactant aggregates.
A condition for multiplex polymerase chain reactions (PCRs) of which outcomes sensitively indicate the actual annealing temperature of thermal cycling is reported. The multiplex reaction was designed to produce four different amplicons of 200, 300, 400, and 480 bp. However, the degree of amplification of each amplicon sensitively responds to a small change in the annealing temperature, by which one can predict the actual annealing temperature of thermal cycling. Deviations between the actual and the designated annealing temperatures as small as 0.5 degrees C were manifested by the banding patterns of the multiplex PCRs in simple agarose gel electrophoresis. For prediction of temperatures in a more objective manner, capillary electrophoresis was also applied to obtain numerical expressions of the relative intensities of the amplicons. By optimizing the multiplex PCR conditions, where concentrations of buffer, dNTPs, and primer pairs were major factors, satisfactory sensitivity and reproducibility of the band patterning were achieved. Blind tests demonstrated the accuracy of the prediction of actual annealing temperatures within +/-0.5 degrees C. The multiplex PCR approach will be further refined and tested for realization of an easily accessible alternative to a physical temperature measurement device in testing the performance of thermal cyclers for PCR.
BACKGROUND: Although "buffering" as a homeostatic mechanism is a universal phenomenon, the quantitation of buffering action remains controversial and problematic. Major shortcomings are: lack of a buffering strength unit for some buffering phenomena, multiple and mutually incommensurable units for others, and lack of a genuine ratio scale for buffering strength. Here, I present a concept of buffering that overcomes these shortcomings. THEORY: Briefly, when, for instance, some "free" H+ ions are added to a solution (e.g. in the form of strong acid), buffering is said to be present when not all H+ ions remain "free" (i.e., bound to H2O), but some become "bound" (i.e., bound to molecules other than H2O). The greater the number of H+ ions that become "bound" in this process, the greater the buffering action. This number can be expressed in two ways: 1) With respect to the number of total free ions added as "buffering coefficient b", defined in differential form as b = d(bound)/d(total). This measure expresses buffering action from nil to complete by a dimensionless number between 0 and 1, analogous to probabilites. 2) With respect to the complementary number of added ions that remain free as "buffering ratio B", defined as the differential B = d(bound)/d(free). The buffering ratio B provides an absolute ratio scale, where buffering action from nil to perfect corresponds to dimensionless numbers between 0 and infinity, and where equal differences of buffering action result in equal intervals on the scale. Formulated in purely mathematical, axiomatic form, the concept reveals striking overlap with the mathematical concept of probability. However, the concept also allows one to devise simple physical models capable of visualizing buffered systems and their behavior in an exact yet intuitive way. CONCLUSION: These two measures of buffering action can be generalized easily to any arbitrary quantity that partitions into two compartments or states, and are thus suited to serve as standard units for buffering action. Some exemplary treatments of classical and non-classical buffering phenomena are presented in the accompanying paper.