Column, capillary dead volume

It is clear that the separation ratio is simply the ratio of the distribution coefficients of the two solutes, which only depend on the operating temperature and the nature of the two phases. More importantly, they are independent of the mobile phase flow rate and the phase ratio of the column. This means, for example, that the same separation ratios will be obtained for two solutes chromatographed on either a packed column or a capillary column, providing the temperature is the same and the same phase system is employed. This does, however, assume that there are no exclusion effects from the support or stationary phase. If the support or stationary phase is porous, as, for example, silica gel or silica gel based materials, and a pair of solutes differ in size, then the stationary phase available to one solute may not be available to the other. In which case, unless both stationary phases have exactly the same pore distribution, if separated on another column, the separation ratios may not be the same, even if the same phase system and temperature are employed. This will become more evident when the measurement of dead volume is discussed and the importance of pore distribution is considered. 

The idea of the effective plate number was introduced and employed by Purnell [4], Desty [5] and others in the late 1950s. Its conception was evoked as a direct result of the introduction of the capillary column or open tubular column. Even in 1960, the open tubular column could be constructed to produce efficiencies of up to a million theoretical plates [6]. However, it became immediately apparent that these high efficiencies were only obtained for solutes eluted at very low (k ) values and, consequently, very close to the column dead volume. More importantly, on the basis of the performance realized from packed columns, the high efficiencies did not... 

The contribution of the equipment between injection unit and detector cell should be negligable in relation to the column for a sufficient column characterization short connections with narrow capillaries and zero dead volume unions are the precondition for reliable plate numbers. Every end fitting of a column causes additional band broadening. In the past a column type was offered that could be directly combined without any capillary links unfortunately, it has disappeared from the market. 

The problems associated with coupling packed columns to a mass spectrometer are s re severe than those encountered with capillary columns. Conventional pacdced columns are operated at much higher flow rates, 20 to 60 al/ain, and although this diminishes the influence of dead volumes in the interface on sample resolution, it poses a problem due to the pressure and volume flow rate restrictions of the mass spectrometer. The interface must provide a pressure drop between column and mass spectrometer source on the order of 10 to 10, it must reduce the volumetric flow of gas into the mass spectrometer without diminishing the mass flow of sample by the same amount, and it must retain the integrity of the sample eluting from the column in terms of the separation obtained and its chemical constitution [3,25,26]. To meet the above requirements the interface must function as a molecular separator. 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Carrier-gas is transferred from the column through a heated metal capillary, which minimizes the dead volume at the end of the column and prevents condensation of the column effluent prior to its entry into the scrubbing unit. The tube carrying the liquid stream is joined to the gas stream tube, at a T-junction that is joined to the mixing coil by a glass-to-metal seal. Furfural is transferred from the gas stream into the liquid stream and the colour develops the two phases are then separated by the debubbling unit and the liquid stream is re-sampled through the flow cell of the colorimeter. A Technicon peristaltic... 

The concept of the effective plate number was introduced and employed in the late nineteen fifties by Purnell (7), Desty (8) and others. Its introduction arose directly as a result of the development of the capillary column, which, even in 1960, could be made to produce efficiencies of up to a million theoretical plates (9). It was noted, however, that these high efficiencies were were only realized for solutes eluted close to the column dead volume, that is, at very low k values. Furthermore, they in no way reflected the increase in resolving power that would be expected from such high efficiencies on the basis of the performance of packed columns. This poor performance, relative to the high efficiencies produced, can be shown theoretically ( and Indeed will be, later in this book) to result from the high phase ratio of capillary columns made at that time. That is the ratio of the mobile phase to the stationary phase in the column. The high phase ratio was... 

Sample analysis was performed by using an Applied Biosystems (Foster City, CA) API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source and an Agilent 1100 capillary HPLC system (Palo Alto, CA). The capillary HPLC system included a binary capillary pump with an active micro flow rate control system, an online degasser, and a microplate autosampler. The analytical column was a 300 pm I.D.x 150 mm Zorbax C18 Stablebond capillary column (pore size 100 A and particle size 3.5 pm). The injection volume was 5 pL, and a needle ejection rate of 40 pL/min was used. The pLC flow rate was 6 pL/min. In order to minimize dead volume before the column, the autosampler was programmed to bypass the 8 pL sample loop 1.5 min after injection. The mobile phase consisted of (A) 2 mM ammonium acetate (adjusted to pH 3.2 with formic acid) in 10 90 acetonitrile-water, and (B) 2 mM ammonium acetate in 90 10 acetonitrile-water. The percentage of mobile phase B was held at 32 % for the first minute, increased to 80 % over 8 min, and then increased tol00% over the following 1 min. 

Some argue that miniaturized tools for both chemical synthesis and analysis need to be integrated onto a single chip to gain the true benefits of miniaturization [57], not least because of the problems associated with subsystem interconnectivity, dead volumes and chip-to-world interfaces. Demonstrations toward such a goal include, for example, a hyphenated mixing reaction channel coupled to a capillary electrophoresis column [58]. 

The choice of ICP-MS is mainly due to the low detection limits and high elemental specificity. Because the sample must be physically transported from the separation column or capillary into the plasma, the interface is important. High analyte transport efficiency is desirable to obtain low detection limits, but care must also be taken in designing the interface so that the separation is not degraded. Dead volume and induced laminar flow due to the interface [398] must be considered. 

Once suitable ionization conditions have been established, LC separation can be optimized. As with any LC system, attention needs to be paid to column choice, correct tubing diameters, zero dead volume connections, use of guard columns, and mobile phase filtration and de-gassing. Modem LC pumps can deliver reliable gradients at low flow rates but for capillary LC, precolumn flow splitting or specialized pumps may be necessary. Mobile phase composition and pH should be chosen to... 

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