34 May / June 2016


excellent radial and structural homogeneity of the phase [9].


Figure 8. Scanning electron micrograph of a hybrid organic silica monolith.


Chromolith Performance RP-18e (4.6 mm i.d., 10 cm), had the effi ciency as a column packed with 3.5-4 µm particles but with a pressure drop (or permeability) equivalent to a column packed with 7-8 µm particles. They are prepared by hydrolytic polymerisation of e.g. tetramethoxysilane in aqueous acetic acid in the presence of polyethylene glycol (PEG). Mesopores in the structure are formed by treatment with aqueous ammonia. First generation monolithic silica columns (rod and capillary) had ca. 2-8 µm through-pores and 1-2 µm skeletons, which were too large to generate high effi ciency and the external porosity was too high. Through pores even of 2 µm resulted in a large mobile-phase mass transfer contribution to band broadening. A dilemma however exists if improvement in effi ciency is desired-reduction in the size of the through pores and skeleton will decrease the permeability, this being one of the original advantages of the monolith structure. In so- called ‘second generation monoliths’ the size of the through pores (1.1-1.2 µm) was decreased by ~40% in Chromolith HR compared with the fi rst generation ‘Chromolith Performance’. The additional improved radial homogeneity in the structure from the centre to the outer portion has resulted in a claimed increase in column effi ciency of up to 50% compared with the fi rst generation columns, with H values as low as 5 µm. This value would suggest that up to 20,000 theoretical plates could be expected in a 10 cm column. However, about 2.5 times the pressure is required to achieve the same fl ow velocity compared with fi rst generation monoliths. An advantage of monolith columns is the absence of retaining frits that are necessary in particle packed columns. This lack of frits could partially explain the claimed resistance of monoliths to samples with a high concentration of matrix compounds. The structure of a hybrid capillary silica monolith is shown in Figure 8; note the apparently


Monoliths have suffered from competition with very small particle packed and especially shell columns; their performance particularly for fast analysis is inferior. A problem with the rod form of monoliths of i.d. comparable to typical packed HPLC columns (i.d. 2-5 mm) is the necessity of enclosing or cladding the structure in a suitable material such as polyether ether ketone (PEEK) without leaving void spaces that could disrupt the radial fl ow profi le of the column and thus cause deterioration in effi ciency. The stability of typical cladding procedures to high pressure is considerably less than that of stainless steel particulate columns, being typically only 200-300 bar. Further efforts are necessary to improve the effi ciency of monoliths without increasing the back pressure, for instance by further enhancements in the homogeneity of the structure of the material. Nevertheless, there may be a demand for the use of long capillary monoliths for the separation of complex biological samples e.g. in peptide analysis, where columns of length several meters can generate over 1 million theoretical plates at acceptable back pressures.


Polymer monolith columns complement silica-based monoliths in that they have been applied to the separation of high molecular weight solutes and biologically active compounds. The interested reader is directed to an excellent review of these materials recently published by Svec and Lv [10].


Conclusions.


Small particle columns operated at high(er) pressures can generate the same effi ciency as conventional HPLC columns with considerably reduced analysis time and solvent consumption. However, these columns require sophisticated instrumentation with low extra-column bandspreading as well as higher pressure capability. Their use is not without practical diffi culties, which include frictional heating and change in separation selectivity with increased pressure. Nevertheless, pressure can also be considered as an additional tool to manipulate selectivity.


Shell particles give further performance enhancements allowing use of somewhat larger ~2.5 µm particles at lower pressures instead of sub-2 µm particles to give similar high effi ciency. Alternatively, sub-2 µm shell particles can generate superior effi ciency to totally porous particles of the same size at similar pressures. Shell particles suffer less from detrimental frictional heating effects than totally porous particles, due to the enhanced heat dissipation that results from


the higher thermal conductivity of their solid cores.


Shell particles seem to have few disadvantages even with regard to overloading, which was the major limitation of the original pellicular particles. These contained a much smaller proportion of porous material. They appear to be currently the best choice for fast effi cient separations.


The original fi rst generation monolithic silica columns, which became available at the turn of the century, initially offered great promise due to their improved effi ciency and lower back pressure compared with the 5 µm particle size materials most popular at the time. However, these columns have suffered competition in terms of speed of analysis and effi ciency especially compared with that of shell columns. More recent second generation silica monoliths give improved effi ciency over fi rst generation materials due to smaller through pores/skeletons and enhanced structural homogeneity. However, this improved effi ciency is at the expense of higher back pressure.


Acknowledgements


The author thanks Dr Bill Barber (Agilent Technologies) for provision of Figure 1, the authors of reference 3 for Figure 2, and the authors of reference 9 for Figure 8.


References 1. N. Tanaka, D.V. McCalley, Anal. Chem. 88 (2016) 279-298.


2. W. Chen, K. Jiang, A. Mack, B. Sachok, X. Zhu, W.E. Barber, X. Wang, J. Chromatogr. A 1414 (2015) 147-157.


3. R. Hayes, P. Myers, T. Edge, H.F. Zhang, Analyst 139 (2014) 5674-5677.


4. M.M. Fallas, S.M. C. Buckenmaier, D.V. McCalley, J. Chromatogr. A 1235 (2012) 49-59.


5. K. Vanderlinden, K. Broekhoven, Y. Vanderheyden, G. Desmet, J. Chromatogr. A 1442 (2016) 73-82.


6. S. Fekete, J.L. Veuthey, D.V. McCalley, D. Guillarme, J. Chromatogr. A 1270 (2012)127- 138.


7. M.M. Fallas, N. Tanaka, S.M.C. Buckenmaeir, D. V. McCalley, J. Chromatogr. A 1297 (2013) 37-45.


8. J.C. Heaton, X. Wang, W.E. Barber, S.M.C. Buckenmaier, D.V. McCalley, J. Chromatogr. A 1328 (2014) 7-15.


9. H. Lin, L. Chen, J. Ou, Z. Liu, H.W. Wang, J. Dong, H. Zou, J. Chromatogr. A 1416 (2015) 74-82.


10. F. Svec, Y. Lv, Anal. Chem. 87 (2015) 250-273.


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