Anal. Chern. 1BB1, 63,1467-1473
1467
Nanoscale Packed-Capillary Liquid Chromatography Coupled with Mass Spectrometry Using a Coaxial Continuous-Flow Fast Atom Bombardment Interface M. Arthur Moseley,’ Leesa J. Deterding, and Kenneth B. Tomer* Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O.Box 12233, Research Triangle Park, North Carolina 27709
James W. Jorgenson* Department of Chemistry, University of North Carolina, C.B. 3290,Chapel Hill, North Carolina 27514
Nanoscale packed-caplllary llquld chromatography (LC) columns have been coupled wlth mass spectrometry (MS) udng a coaxial continuoubflow fast atom bombardment Interface. The combined system has been appiled to the analysis of mixtures of peptides, lncludlng synthetic mixtures of bbactlve peptldes and tryptk digests of proteins. Name cab packeccCapllbty cdunrrr,Mer two prhklpai advantages for LC/MS analysis-high chromatographic separatlon effE clencles and low mobiie-phaso flow rates. The high separatkm etfklencles faditate the separation of complex mtxtwes, and the low mobliaphaso How rates reduce problems with coupllng the LC effluent with the high-vacuum, high-voltage envlronment of sector MS ion sources. The columns used in tMs work were 50- or 75-pn I.d., 1-2 m long, packed with 10-pm C18 particles, rwlng mobleghaw flow rates of 50-350 nL/mln.
Separation science methodologies have greatly benefitted from the trend toward miniaturization. The best example of this is the well-known advantages of capillary gas chromatography over conventional packed-column gas chromatography (GC). Smaller inner diameter gas chromatography columns yield significantly better separation efficiencies,yield shorter analysis times, and greatly facilitate the coupling of GC with mass spectrometry. Similar advantages are found in liquid chromatography when miniaturization of the columns occurs. As with GC,the highest separation efficiencies are obtained with open tubular liquid chromatography (OTLC) columns, which have the stationary phase coated onto the wall of the column. Due to the much higher viscosity of liquids as compared to gases and the resultant increase in resistance to mass transfer in liquid mobile phases compared with gas mobile phases, the optimum OTLC column inner diameter is significantly smaller than the optimum GC column inner diameter. Theory predicts (1)that the optimum OTLC column inner diameter, over a range of analysis times and pressures, will be 1.5-3 pm. This very small optimum OTLC column inner diameter results in a small column wall surface area and, therefore, a small amount of stationary phase. This results in a low capacity factor, which reduces the dynamic range of the amount of analytes that can be injected onto the column before overloading occurs. Nanoscale packed-capillary LC columns offer a solution to this capacity problem due to the high surface area to volume ratio of columns packed with porous silica particles. This improves the analysis of polar analytes due to an increased *Authors to whom correspondence should be sent. A h at the Department of Chemistry, University of North Carolina. 0003-2700/91/0363-1467$02.50/0
capacity factor and also permits the injection of large volumes of dilute aqueous sample onto reversed-phase columns, with focusing of the anal* onto the front of the stationary-phase packing. Also, the columns can be fabricated from commerically available packing materials, offering a wide selection of stationary-phase chemistry. A number of research groups have successfully produced packed capillary columns with inner diameters between 150 and 350 pm (2-8).While such columns will typically have minimum reduced plate heights equivalent to conventional LC columns, flow dispersion (eddy diffusion) and/or resistance to mass transfer tends to be more significant than is found with conventional LC columns (9). This has been attributed to the increased contribution of the “wall effect”, where the column wall influences the structure of the packing bed, decreasing the particle density and, thus, reducing mobile-phase flow resistance. In LC columns, there are two discreet regions of packing bed structure, a less densely packed region at the wall of the column, extending approximately 5 particle diameters from the column wall, and a more densely packed region in the remainder of the column bed in the center of the column. Eddy dispersion will increase as the wall effect region increases in its percentage contribution to the total bed volume (10).In capillary LC columns with 350-150-pm i.d. packed with 5-pm particles, a high percentage (=5-25%) of the packing bed structure will be influenced by the wall effect. In 1988, Karlsson and Novotny reported an evaluation of packed-capillary columns with inner diameters as low as 44 pm (11) where it was found that such columns showed an improvement in separation efficiency over the 150-350-pm4.d. columns. The packing bed structure in columns with this inner diameter would be expected to be homogeneous, with the entire column bed being influenced by the wall effect. Kennedy and Jorgenson (12)performed a comprehensive study on the effect of column diameter on column performance and found that packed capillary LC columns with a column inner diameter to particle size ratio of
