5.3. The Introduction of Samples from HPLC
At this point it is noteworthy to recall the differences between GC and LC. Chapter 2 defined GC as a technique applicable to relatively volatile, thermally stable compounds. These restrictions greatly limited the types and number of compounds that could be analyzed by GC, and GC-MS. LC, discussed in Chapter 3, uses a mobile phase in the analysis of many of the compounds analyzed by GC, and also can be used to analyze the plethora of biomolecules that are non-volatile and thermally unstable at even slightly elevated temperatures. While the conditions used in LC greatly extends the applications of chromatography, it has historically suffered difficulties with mass spectrometry interfaces. Most of the various forms of LC, especially HPLC types discussed in Chapter 3, can be interfaced with MS today.
The largest difficulties in interfacing LC with MS is the removal of the mobile phase solvent prior to introduction to the MS mass analyzer and the transfer and ionization of nonvolatile analyte molecules into the gas phase. The first attempt at an LC-MS interface was to place the effluent droplets from the LC onto a supposed chemical resistant conveyer belt that transported the liquid into the MS ionization chamber. The conveyer belt was then cleaned and returned to the HPLC effluent for more sample. However, these early attempts resulted in inefficient removal of the analytes from the conveyer belt and analyte residue being left on and released from the belt during subsequent MS runs. This problem was significantly compounded with 4.5 mm diameter HPLC columns with flow rates in the range of 1 mL/min. The later use of 300 to 75 mm long capillary columns improved flow rate problems. The invention of Electro Spray Ionization (ESI) solved all of the major problems associated with sample introduction to MS. ESI was first conceived in the 1960s by Malcolm Dole at Northwestern University, but it was not put into practice until the early 1980s by John B. Fenn of Yale University (and resulted in his Noble prize in 2002). Its common use today has been one of the most important advances in HPLC and today allows routine identification of biological macromolecules.
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