Elution of Sample Components

The potential that exists across this interfacial region is called the zeta potential, , and may be described by Eq. 12.4  

Equation 12.5 indicates that the electroosmotic flow rate is large when is large and when the interfacial region is small. 

Taking electroosmosis into account, the velocity and residence time of the analyte can be recalculated as 

By using Eqs. 12.6 and 12.7, the separation efficiency, or number of theoretical plates, N, becomes 

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Unfortunately, this technique is not selective and all components are affected in the same way so that if exact co-elution of sample components occurs, this may not be detected. Using a single quadrupole instrument limits the user to using in-source fragmentation, but in many cases this can provide enough information to identify unknowns. 

From Eq. 12.2, we can see that the fast elution of sample components requires high voltages and/or short capillaries. 

FIGURE 6.14 (a) Principle of SBCD, elution with five interstitial volumes on 4-cm distance (5x4 cm) is faster than single development on 20-cm distance (thick line), (b) Rp values of sample components plotted as a function of modifier concentration. Optimal concentration (Y) for SBCD (5x4 cm) is lower than for development on the full distance of 20 cm (X). (Modified from Soczewinski, E., Chromatographic Methods Planar Chromatography, Vol. 1, Ed., Kaiser R.E., Dr. Alfred Huetig Verlag, Heidelberg, Basel, New York, 1986, pp. 79-117.)... 

As plant extracts mainly comprise large amonnts of ballast substances (e.g., lipids and chlorophylls), their purification is often a priority in the analysis. Such purification can be expensive in terms of both time and solvent consumed and can lead to losses of sample components. Online purification and separation of extracts contaminated with plant oil, can be readily performed by TLC in equilibrium chambers [1] that enable the use of continuous elution. 

Other systems with similar components can also be used, provided they can be operated at flow rates that will be compatible with the column-operating pressures. For some systems, additional column fittings may be required to facilitate connection of the Superose 6 column. If the purpose of the gel-filtration step is to exchange buffers, then the column should be equilibrated and eluted with the buffer that the sample is to be exchanged into. Optimal separation of sample components can be achieved with a sample volume of 200 pL. For desalting or buffer exchange, a sample volume of up to 2 mL can be used. 

Appropriate SPE sorbent selection is critical to obtaining efficient SPE recovery of semivolatile organics from liquids. Henry [58] notes that an SPE sorbent must be able to sorb rapidly and reproducibly, defined quantities of sample components of interest. Fritz [73] states that successful SPE has two major requirements (1) a high, reproducible percentage of the analytical solutes must be taken up by the solid extractant and (2) the solutes must then be easily and completely eluted from the solid particles. The sorption process must be reversible. In addition to reversible sorption, SPE sorbents should be porous with large surface areas, be free of leachable impurities, exhibit stability toward the sample matrix and the elution solvents, and have good surface contact with the sample solution [68,73],... 

Mixtures of benzodiazepines and thiazide diuretic drugs were separated by gradient elution CEC and identified using ESI-MS by Taylor and Teale [38], They used 330-500 x 50-75 pm i.d. colums packed with Hypersil ODS and Apex ODS and gradients of 50-80% acetonitrile in 5 mmol/1 aqueous ammonium acetate to elute the sample components. Benzodiazepines were detected in the positive ion mode using 1% acetic acid as the sheath liquid, whereas the thiazide diuretics were detected in the negative ion mode with 80% isopropanol in water as sheath liquid. 

The approach of Jandera and Chura ek allows the optimization of the resolution of one given (arbitrary) pair of sample components and the minimization of the retention volume of another (arbitrary) solute. It requires knowledge of the isocratic retention vs. composition relationships of these three solutes. The information needed may be acquired from gradient elution experiments performed as part of the optimization procedure, or from separate isocratic experiments. The selection of the three arbitrary solutes considered during the optimization process appears to have a large effect on the result and the resolution cannot be optimized throughout the chromatogram. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

Mobile phase volume. The volume of solvent in a packed column, given by the amount of mobile phase required to elute a sample component which does not interact with the packing material (VM also known as the void volume, T0)-... 

As was discussed in Chapter 3, the smaller the molecule, the greater the number of pores it can enter in the packing material. Larger molecules are excluded from some or all of the pores. The more pores a sample component enters, the longer it takes to elute from the column. This selective permeation into the pores is based on molecular size and results in the separation of the various sizes of sample components in a highly predictable manner. 

This predictability provides a major advantage over other LC modes because the maximum amount of solvent required to elute all sample components is equivalent to one column volume, and the elution volume of any molecule can be predicted for a given column if a calibration curve is available. Clearly, the most crucial ingredient for success in using SMGPC is selecting the proper type of column with the appropriate calibration curve. 

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