TWO-DIMENSIONAL HPLC SYSTEMS 197 with large through pores completely filling the tube The monolith column formed is then treated with a silylation reagent to form a bonded phase within the silica pores Commercial columns, such as the Chromolith SpeedRodTM, run with efficiency of a 2–3-mm particulate column, but can be run at flow rates of 6–8 mL/min without exceeding approximately 2,500 psi back-pressure, greatly decreasing run times These columns offer the potential for creating a hybrid-silica monolith, which can be run on existing HPLC systems at high flow rates, that are temperature and pH resistant By their very nature, these columns would be void free and the only column killers that they would suffer from would be particulates and bound organics They probably could be reverse flushed for particulate wash out and bound materials could be washed off with strong solvents 16.4 MICRO-PARALLEL HPLC SYSTEMS A variation of the microfluidics HPLC-on-a chip mentioned in Chapter 15 is the polymeric BrioTM cartridge system A replaceable 8- or 24-channel BrioTM cartridge contains side-by-side 30-cm × 0.5-mm i.d column channels packed with standard HPLC stationary phase The cartridge is run loaded into an autosampler-equipped VeloceTM gradient chromatography system A BrioTM cartridge inserted into the VeloceTM system makes the same HPLC separations in all channels with simultaneous UV and/or FL detection As a quality control or cost-per-test instrument, the 100-run cartridge allows accelerated assessment of compound purity, stability, and other physiochemical properties Running this many 15 sec/sample separations on a single instrument can rapidly reduce a laboratory’s sample load 16.5 TWO-DIMENSIONAL HPLC SYSTEMS Much of the pressure to develop automated sequential HPLC separations has come from the necessity to separate complex biological mixtures, especially protein mixtures Traditionally, complex mixtures of proteins have been separated using two-dimensional gel electrophoresis (2D GEP) The first dimension gel separation is carried out with electrophoresis buffers, the gel plate is rotated 90° and the second SDS-PAGE separation is carried out under denaturing conditions, using sodium dilauryl sulfate The separated spots are then visualized, scraped off the plate, and then extracted for further analysis Protein analysis by MALDI time-of-flight mass spectrometry starts with this time- and labor-intensive 2D GEP separation mode In theory, combining two HPLC modes sequentially would provide an online LC/LC/MS/MS and speed the analytical procedure Bands from the first separations could be detected and collected with an automated loop-and-valve injector, and then individual bands could be passed to the second LC for 198 NEW DIRECTIONS IN HPLC separation using a different separation mode A model first mode for LC1 would be an affinity separation of antibodies followed by a partition separation in LC2 of the purified effluent cuts passed from the affinity column, with the peaks from the bonded-phase column being analyzed by the MS unit This sequence would benefit from removal of salt or small molecules used to displace the antibody proteins from the first column in the break through of the reverse phase column The main problem that has been encountered so far in the development of a LC/LC system is the large differences seen in the resolving power of the various HPLC modes Partition separations are dramatically more efficient than either ion exchange or size separations, the other modes normally used for separating proteins Attempts to two-dimensional sequential partition separation using different types of bonded-phase columns have not provided significant improvements in separating power to justify the technique Only affinity separations seem to provide a specific enough first separation to provide a useful feedstock for the partition dimension purification 16.6 THE PORTABLE LC/MS Advances in the art of chromatography and micro-miniaturization of hardware components and electronics are rapidly approaching the point where an HPLC in a suitcase is a real possibility Components such as high-charge density batteries, nano-flow syringe pumps, the chip HPLC, monolith cartridge HPLC columns, and tiny portable computers leave only a compact RI or UV detector as the missing element Add to this a chip LC interface and a miniaturized quadrupole or ion trap MS detector and the picture is complete Already, high vacuum fist-size turbo pumps and 5-in long quadrupole analyzers are available The system would be put together in a vented case with the fluidics on the bottom A syringe pump, pre-loaded with solvent before leaving the laboratory, the battery, and a waste vessel would be in the first layer Stacked above it would be a nebulizer helium lecture bottle, chip ISI interface, the internal loop injector, the column, and the mass spectrometer The MS unit would also be evacuated with a roughing pump before leaving the lab and the turbo-pump spun up and left running on the internal battery The final layer would be the touch-pad flat screen portable to control the MS unit and display MW annotated TIC chromatograms A portable thermal dye sublimation printer would be sold as an option, but chromatography reports could be printed on return to a central laboratory.The whole unit could be powered by its internal battery, but most likely would draw most of its power from an automobile hot point charger The demand for such an LC/MS luggable would come from the field environmental chemist, from the arson investigator, and obviously from your local forensic CSI and drug enforcement teams It would avoid the problem of THE PORTABLE LC/MS 199 sample aging and delays in compound analysis by providing an immediate answer on-the-spot, based on tables of retention times and molecular weights for suspect compounds I hasten to add that this system does not exist at the moment You probably not want to include it in your equipment proposal for this year But, it is rapidly becoming a viable option for development And, if successful, would the portable linear ion trap (LIT) based LC/MS/MS, for definitive compound identification by searching a MS database, be far behind? APPENDICES I have enclosed seven items as parts of the Appendix The first, a separations guide, points out starting points for chromatographic separations and also suggests trends in usage of columns, mobile phase, and detectors The second item is a list of frequently asked questions (FAQs) that I have encountered while serving as an in-house trouble-shooting resource for customers and as an instructor for extension courses I taught 30 two-day HPLC course in seven states before I began teaching at the University of Missouri–St Louis Many of these answers arose as responses to problems raised by students in my classes In the third appendix, I have added tables of solvents and volatile buffers important for use in LC/MS Nonvolatile buffers cause problems when you remove solvent and ionize effluent for injection into the mass spectrometer and need to be avoided when this detector has been selected The fourth item is a glossary of HPLC terms I have tried to include much of the terminology and buzzwords used in the field The fifth part is a trouble-shooting quick reference It is not intended to replace the systematic trouble-shooting discussion in Chapter 10 When things go wrong, however, you may find it helpful I have arranged it in the way things flow through the system: from pumps to the integrator or data acquisition computer The sixth item is a series of three HPLC laboratory experiments The first familiarizes the student with getting a system up and running and calibrating a column with standards The second experiment shows how to clean a column and pacify a system The last is a first, quick look at methods development HPLC: A Practical User’s Guide, Second Edition, by Marvin C McMaster Copyright © 2007 by John Wiley & Sons, Inc 201 202 APPENDICES These are three areas where I feel new users tend to get up or ignore when first approaching an HPLC system The last appendix is a selected reference list It is not intended to be exhaustive, but simply to give you a point to enter the literature in the field To stay current you probably want to subscribe to American Laboratory, LC/GC Magazine, the Journal of Liquid Chromatography, and Chemical Abstracts— HPLC Selects Chemical Abstracts are also on-line as part of Dialog’s computer database I have found Google to be a very usefully search engine when I am trying to learn about a new instrument or obtain background information about a new technique HPLC literature is extensive, published in many and surprising places, and of variable quality and reproducibility APPENDIX A PERSONAL SEPARATIONS GUIDE Application Vitamins (water soluble) Vitamins (fat soluble) Steroids Triglycerides Phospholipids Column Detector C18 C18 C18 C8 Si UV (254 nm) UV (280 nm) UV (230 nm) UV (220 nm) UV (206 nm) Prostaglandins C18 UV (192 nm) Bromphenacyl acids Krebs cycle acids C18 RNH2 Monosaccharides CX-Ca 10 Polysaccharides TSKpw 11 Nucleic acids 12 Nucleosides CX-Na C18 UV (254 nm) UV (210), RI, CAD UV (195 nm), RI, CAD UV (195 nm), RI, CAD UV (254 nm) UV (254 nm) 13 Nucleotides C18 UV (254 nm) 14 PTH amino acids C18 UV (254 nm) 15 OPA amino acids C18 Fl (230/418) Conditions a,b 8% AN/H2O, C7SO3 80% AN/H2O 60% MeOH/H2O 60% AN/H2O 130/5/1.5AN/MeOH/ 85% H3PO4 35% AN/H2O, PO4 pH 2.5 15–80% AN/H2O 25–250 mM PO4, pH 2.5 H2O (80°C) H2O (