We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome C and myoglobin generated Mascot MOWSE scores of 107 for cytochrome C and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively.

Diagonal capillary electrophoresis is a form of two-dimensional capillary electrophoresis that employs identical separation modes in each dimension. The distal end of the first capillary incorporates an enzyme-based microreactor. Analytes that are not modified by the reactor will have identical migration times in the two capillaries and will generate spots that fall on the diagonal in a reconstructed two-dimensional electropherogram. Analytes that undergo enzymatic modification in the reactor will have a different migration time in the second capillary and will generate spots that fall off the diagonal in the electropherogram. We demonstrate the system with immobilized alkaline phosphatase to monitor the phosphorylation status of a mixture of peptides. This enzyme-based diagonal capillary electrophoresis assay appears to be generalizable; any post-translational modification can be detected as long as an immobilized enzyme is available that reacts with the modification under electrophoretic conditions.

An ink jet printer valve and nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by separation of a set of standard peptides. That separation generated up to 40,000 theoretical plates in less than three minutes. Detection limits were 500 amol using an ABI TOF-TOF instrument and 2 fmol for an ABI Q-TOF instrument. Over 70% coverage was obtained for the tryptic digest of α-lactalbumin in less than 2.5 minutes.

Metal oxalate synthesis and pyrolysis provides an opportunity for students to (i) learn stoichiometry, (ii) experience the consequences of proper stoichiometric calculations and experimental techniques, and (iii) be introduced to the relevance of chemistry by highlighting oxalates in context, for example, usages and health effects. At our institution, general chemistry students synthesized the hydrated form of either iron(II), nickel(II), or manganese(II) oxalate. Synthesis was followed by oxalate pyrolysis and subsequent determination of the pyrolysis product’s identity, of three possible, using stoichiometric calculations and comparisons of theoretical and actual yields. Striking color changes that accompany these pyrolysis reactions were well received by students and served to highlight the chemical changes involved. Student actual yield values for both the iron and manganese pyrolysis products compared favorably with theoretical yield values and, in addition, 78% and 76% of students would have chosen the correct iron or manganese pyrolysis product, assuming correct stoichiometric and experimental calculations. Flaws in students’ experimental techniques were especially noticeable during pyrolysis of the nickel oxalate. Specifically, students did not heat to constant weight, did not heat to high enough temperature to achieve complete pyrolysis, and ended the pyrolysis prematurely, that is, at an intermediate yellow-colored product.

A novel calorimetry experiment that (i) provides “real-world” connections, (ii) assists students in learning thermochemistry, and (iii) uses equipment commonly found in an undergraduate general chemistry laboratory was implemented at our institution. Ultimately, students design a custom heat pack to treat the frostbitten hand of an Alaskan pipeline maintenance worker. First, students measure and use the specific heat of Vienna sausage to approximate the hand’s specific heat. Students obtained a mean specific heat of 3.1 ± 0.5 J g−1 °C−1 for Vienna sausage, which compares favorably with generally accepted values for the human body’s specific heat. Second, students qualitatively determine heats of salt dissolution and identify a salt for their heat pack. Next, students quantitatively determine their chosen salt’s heat of dissolution. Finally, using the Vienna sausage’s specific heat, students estimate the heat required to raise the temperature of the hand from 15 °C (frostbite risk) to 37 °C, calculate the amount of salt and water needed for their heat pack, and fashion and test their heat pack. To gauge student perceptions toward this experiment, a survey was administered. Results indicate that the majority of students enjoyed this experiment (69.3%) and felt that the experiment had “real-world” connections (59.4%).