Column microbore

Having established that a finite volume of sample causes peak dispersion and that it is highly desirable to limit that dispersion to a level that does not impair the performance of the column, the maximum sample volume that can be tolerated can be evaluated by employing the principle of the summation of variances. Let a volume (Vi) be injected onto a column. This sample volume (Vi) will be dispersed on the front of the column in the form of a rectangular distribution. The eluted peak will have an overall variance that consists of that produced by the column and other parts of the mobile phase conduit system plus that due to the dispersion from the finite sample volume. For convenience, the dispersion contributed by parts of the mobile phase system, other than the column (except for that from the finite sample volume), will be considered negligible. In most well-designed chromatographic systems, this will be true, particularly for well-packed GC and LC columns. However, for open tubular columns in GC, and possibly microbore columns in LC, where peak volumes can be extremely small, this may not necessarily be true, and other extra-column dispersion sources may need to be taken into account. It is now possible to apply the principle of the summation of variances to the effect of sample volume. 

Steven Carr (SmithKline Beecham) has used microbore columns to desalt proteins prior to ES-MS (32). The pore diameter of PolyHEA used (usually 200 A) was selected so that all proteins of interest would elute at Vo with 50 mM formic acid. Only the Vo peak was allowed to flow into the ES-MS nebularizer the rest of the SEC effluent (including the salts) was diverted to waste by opening a microdumper valve between the column and the nebularizer. The properties of the mobile phase were quite compatible with ES-MS analysis. 

Chromatograms Demonstrating the Concentration Effect of the Microbore Column Sampling Apparatus... 

HPLC columns with reduced diameters (microbore columns) are now available. The flow rate from such columns required to give a desired flow rate at the same linear solvent velocity (and thus retention time) as a 4.6 mm i.d. column operating at 1 mlmin is given by the following equation ... 

The use of microbore columns is not yet routine as much more rigorous control of parameters, such as flow rate and instrument dead volume, is required to ensure that degradation of chromatographic performance does not occur. It is therefore experimentally more difficult. 

Mobile phase flow rates from nlmin to in excess of 1 mlmin can be used with appropriate hardware, thus allowing conventional and microbore columns to be employed. 

Capillary column This term refers to a chromatographic column of small diameter and is used in both gas and high performance liquid chromatography. In HPLC, the term is usually apphed to columns with internal diameters of between 0.1 and 2 mm. The term microbore column is often used synonymously to describe these columns but is more correctly applied to columns with internal diameters of 1 or 2 mm. 

Microbore Column Chromatography A Unified Approach to Chromatography, edited by Frank J. Yang... 

There are at least three approaches to fast GC/MS (1) use of microbore columns with time-of-flight mass spectrometry (TOFMS) (2) use of low-pressure (LP)-GC/MS to aid separations at increased flow rate " and (3) use of supersonic molecular beam mass spectrometry (SMBMS) (also known as supersonic GC/MS), which can accept increased flow rates and short analytical columns."... 

An advantage of the microbore gas chromatrography/time-of-flight mass spectrometry (GC/TOFMS) method over the other two approaches is that separation efficiency need not be compromised for speed of analysis. The rapid deconvolution of spectra ( scan rate ) with TOFMS makes it the only MS approach to achieve several data points across a narrow peak in full-scan operation. However, the injection of complex extracts deteriorates performance of microbore columns quickly, and an increased LOD and decreased ruggedness result. Microbore columns may be used in water analysis if the LOD is sufficiently low, but they can rarely be used in real-life applications to complicated extracts. 

F. J. Yang (Ed.), "Microbore Column Chromatography. A Unified Approach to Chromatography", Delcker, New York, NY, 1989. 

Schwartz, H. E., Karger, B. L., and Kucera, F, Gradient elution chromatography with microbore columns, Anal. Chem., 55, 1752, 1983. 

Microbore and packed capillary HPLC column technology has not yet met the requirements for breakthrough of new technologies [554]. On commercial instruments in general efficiency and detectability with microbore columns are lower than with normal-bore columns. Microbore and capillary HPLC suffer from... 

LC-PB-MS is especially suited to NPLC systems. RPLC-PB-MS is limited to low-MW (<500 Da) additives. For higher masses, LC-API-MS (combined with tandem MS and the development of a specific mass library) is necessary. Coupling of LC via the particle-beam interface to QMS, QITMS and magnetic-sector instruments has been reported. In spite of the compatibility of PB-MS with conventional-size LC, microbore column (i.d. 1-2 mm) LC-PB-MS has also been developed. A well-optimised PB interface can provide a detection limit in the ng range for a full scan mode, and may be improved to pg for SIM analyses. 

LC-NMR hyphenation consists of a liquid chromatograph (autosampler, pump, column and oven) and a classical HPLC detector. The flow of the detector is brought via an interface to the flow-cell NMR probe. Using commercial NMR flow-cells with volumes between 40 and 180 p,L, in connection with microbore columns or packed capillaries, complete spectra have been provided from 1 nmol of sample. These micro-cells allow expensive deuterated solvents to be used, and thus eliminate solvent interference without excessive cost. The HPLC eluent can be split in order to allow simultaneous MS detection. 

HPLC coupled to MS was used for the determination of dimethyl xanthine metabolites in plasma.82 There have also been a number of methods published on the use of HPLC with a PDA detector. In 1996, Mei published a method for the determination of adenosine, inosine, hypoxanthine, xanthine, and uric acid in microdialysis samples using microbore column HPLC with a PDA detector.63 In this method, samples were directly injected onto the HPLC without the need for any additional sample treatment. 

One advantage of small bore columns is that they are operated at much lower mobile phase flow rates than the 4.6 mm columns, so there is a large reduction in solvent consumption and hence operating costs of the chromatograph. The efficiency of hplc columns does not depend on their diameter but it does depend on the velocity of the mobile phase in the column, so microbore columns are operated at velocities corresponding to the flow rates used with larger columns. If / (cm3 min-1) is the flow rate in a column with diameter d cm, and the mobile phase velocity is v cm min-1, / and v are related by ... 

You may have noticed that some of these are the same sort of advantages that are claimed for microbore columns. 

Because of their small internal volume, they require equipment with low extra-column dispersion. This is not, however as serious a limitation as it is with microbore columns, and 3x3 columns can often be used satisfactorily in conventional instruments. 

Utilizing the difference in selectivity between a monolithic silica-C18 column (2nd-D) and another particle-packed column of C18 phase (lst-D), 2D HPLC separation was shown mainly for basic compounds and other species (Venkatramani and Zelechonok, 2003). The authors also reported other examples of reversed-phase 2D HPLC, using amino- and cyano-derivatized particle-packed columns for 2nd-D separation. The combination of normal-phase separation for the 1 st-D and reversed-phase separation on monolithic Ci g column for the 2nd-D was reported (Dugo et al., 2004). The use of a microbore column and weak mobile phase for the lst-D and a monolithic column for the 2nd-D was essential for successful operation. Improvement in the 2D separation of complex mixtures of Chinese medicines was also reported (Hu et al., 2005). Following are practical examples of comprehensive 2D HPLC using monolithic silica columns that have been reported. 

Kohne, A.P., Welsch, T. (1999). Coupling of a microbore column with a column packed with non-porous particles for fast comprehensive two-dimensional high-performance liquid chromatography. J. Chromatogr. A 845, 463-469. 

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