I had the opportunity to use the Beckman System Gold® HPLC with a UV/Vis detector and a C18 column for the analysis and characterization of recombinant and synthetic proteins with sequences identical to portions of HIV proteins. I studied these recombinant and synthetic proteins in order to characterize them chromatographically, immunologically, and spectrally.
The HIV recombinant and synthetic proteins were stored in acetic acid after elution from affinity columns were then diluted to the dilution which was expected to fall within the limits of the detectors for sequencing. I found the hardware of the Gold® HPLC similar in many respects to the reverse phase HPLC Millipore system that I used while working at the Mayo Clinic some 5-6 years earlier. The Gold® HPLC is nicely designed with respect to the reservoirs for the mobile phase integration as well as other features concerning the layout and integration of the components. The reservoirs are conveniently placed; there are plexiglass sealed areas of the unit which help to lower noise; the software caters to every whim of the chromatographer (from the FTIR scanning of elution time and fixed wavelength measurement at every elution time, to the ability to change axis’s absorption levels during data manipulation). I also found the system to be especially well-designed with respect to the Fourier transform approximation electronics or the ability to detect with a single wavelength at any desired retention time. Unfortunately, the software/hardware did not have the ability to do both a single frequency measurement and scan a spectrum of the sample simultaneously for one injection. If these could be performed simultaneously at all elution minutes, then a second injection or multiple injections would not be necessary (this is important to synthetic peptides). The additional injections took a significant amount of time.
The software was designed well enough to have a flow steadied rate with two 180 degrees out-of-phase pumps. I found the design logic of the software to be nicely organized as well. However, I would have liked to have had the software more flexible with respect to moving around in the system. The layout of the software was like a decision tree, with only one way in and out. Because of the complexity and thoroughness of the software, one could be several steps into the system and have to back out to the main shell in order to move onto another task.
The unit I was using did not have an auto-sampler or fraction collector. However, for my purposes, it performed well and allowed me to gather the spectrum concerning the extinction coefficient of the protein, spectra absorbance fingerprint, and elution characteristics. Afterwards, I compared extinction coefficients and the theoretical coefficient was close to the actual.
The system did an excellent job of allowing the researcher the flexibility to manipulate the data in every way imaginable. A drawback of the computer system was that it was slow in accessing tasks. Also, the system did not have the ability to do both a UV/Vis single frequency measurement and scan a spectrum of the sample simultaneously. It would have been nice to have the flexibility to have other detectors easily added for chromatographically labeled analytes and other purposes.
System Gold® Microbore HPLC From Beckman Coulter
My favorite feature was that the system truly had a way to fingerprint an elution time from the samples. With the proper control, a wavelength scan could positively identify your sample without any other analysis (e.g. for a sample such as a specific synthetic peptide).
I wished for numerous ways to move around in the software as well as wanted to move around faster. I also wished that there was page within the index which had a box and bubble chart showing the layout of the software.
The Bottom Line
I found the system very powerful with respect to it ability to differentiate between small increments in elution times, Fourier Transform spectra and the ability to view the spectra of the samples which I ran. I highly recommend the system from a number of different aspects: Detector, integration of software, and overall design of the system.