Courtney P. Mudd

A computer-controlled all-tantalum stopped-flow microcalorimeter with microjoule resolution

A new all-tantalum differential stopped-flow heat-conduction microcalorimeter with microjoule resolution has been developed. The instrument consists of two matched channels, each of which has two reagent inlet lines. A computer is used to process the data and control the syringe drive system which runs the samples through the calorimeter. The reagents are mixed in 0.6 s in ratios of 1:1, 1:2, 1:2.5, 1:4, 1:5, or 1:10. The priming volume from the loading port to the mixer is 1 ml and the reaction volume of the detection tube is 160 microliters. The instrument has a sensitivity of 1.60 J/V.s and a differential baseline stability of 100 nJ/s (p-p) over a 4 h period. The sample size can be reduced to 27 microliters with only a 12% loss in sensitivity. With an electrical step power input, the 10-90% response is 40 s. By using a data decomposition scheme, the response time can be improved to 1 s which allows the direct measurement of moderately fast reaction kinetics. With water/water mixes, differential heats of mixing are typically (+/-) 2 microJ with a standard deviation of (+/-) 2.5 microJ. Reaction heats in the 20-50 microJ range can be measured with a standard deviation of (+/-) 3 microJ. A fast reaction, e.g. HCl dilution, can be completed in 150 s. When loading and priming times are included, 25 reactions can be completed in 120 min. A chilled water jacket is used to allow operation over a temperature range of 4 degrees C to 50 degrees C.

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements

Heat conduction calorimeters are widely used in the biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015 degrees C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003 degrees C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box. An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell and cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution in the cell can be the dominant factor in determining the rise-time of the instrument. The heat conduction calorimeter described here has improved characteristics: a baseline stability of 200 nJ x s-1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ x s-1; a sensitivity of 6.504 +/- 0.045 J x V-1 x s-1 referred to the sensor output; and a rise-time of 122 s for the 10-90% response.

Temperature‐controlled vacuum chamber for x‐ray diffraction studies

In order to apply the osmotic stress method for direct measurement of forces between membranes or between macromolecules, we have designed and built an x‐ray camera which can control the sample temperature from 0 °C to 70 °C while confining the path of diffracted x rays to an evacuated space between the sample and film. The system uses a linear feedback sensor which provides ±0.1 °C accuracy and a base‐line stability of 0.02 °C over the entire operating range. The controller uses solid‐state thermoelectric modules to regulate the temperature of the sample and is capable of automatically shifting from the heating to cooling mode of operation to regulate at temperatures near room temperature. The sample solutions are mounted between two Mylar windows in a removable cell which can be cleaned and loaded outside the instrument. The film plate is mounted on a slide which can be positioned between 4 and 22 in. from the sample. A beam stop is also mounted on the film plate holder and can be adjusted 1 in. both ve...

A stopped-flow mixer device for a batch microcalorimeter application to NAD-NADase reaction

A new molded polypropylene, diamond-like carbon (DLC)-coated mixing cell has been developed for use in the batch microcalorimeter. Reagent volume can be varied from 25 microliters to 100 microliters. A 10 microcalorie reaction heat can be measured to 5%. Repeat reactions can be done as often as every 10 min for a fast reaction. Reactions can be started within 1 h or less after loading. A pre-equilibrator and a temperature-controlled syringe drive unit permit solutions to be stored at 4 degrees C while being run at any temperature from -20 degrees C to 40 degrees C. The kinetics and enthalpy of reaction of NAD-NADase have been measured. delta H is about 21 kcal/mol endothermic.

An electronic interface for microcomputer-based flow cytometry: design, operation and performance characteristics

Abstract An inexpensive electronic interface for the acquisition of asynchronous flow cytometry data using a commercially available microcomputer and A/D converter is described. In the design of this interface, the major objectives were to minimize the amount of custom hardware necessary between the computer and flow cytometer and to maximize usable sensitivity and resolution by implementing a gated integrator and trigger hold-off approach to acquire the asynchronous data pulses from the cytometer. The major error sources which can occur during data acquisition are identified and their effects on system performance are shown. A simple protocol is described to minimize the effects of these errors on the acquired data. The influence of computer-processor clock speed on data acquisition rates is also documented.

An iterative method for the deconvolution of microcalorimeter thermograms

An iterative method for the deconvolution of microcalorimetry thermograms suitable for small digital computers is presented. The method employs a measured impulse response function directly as the deconvolution kernel, thus explicit system simulation is not required. Data are presented showing the performance of the method and the exchange of signal-to-noise ratio for time resolution that is made when deconvolution techniques are employed. An improvement in the system time resolution of fifty times is demonstrated with measured data.