Dual-column method

Grant et al. (2002) designed a parallel system employing two HTLC columns (Cyclone, 50 x 1 mm, Cohesive Technologies) connected to one analytical column (Zorbax SB-C18, 50 x 2 mm, Hewlett Packard) on a 2300 HTLC. A polyarylethyl ketone (PAEK) six-port Valeo (Valeo Instruments, Texas) was used to increase switching speed and reduce carry-over. Peak focusing was used when the analyte was flushed from the TFC column into the analytical column by aqueous dilution. Compared to the dual column method, the overall time reduction was 1.5 to 4 min per sample with comparable data quality at the linear range of 0.1 to 100 ng/mL. 

Samples are injected onto the turbulent-flow column similar to single-column methods. The analytes of interest are retained in the turbulent-flow column while the large macromolecules are eluted to waste. Once the analytes are separated from the matrix, the samples are then eluted into the analytical column. The characteristics of the analytical column determine the peak shape and separation seen at the MS detector. Flow rates which are compatible with the mass spectrometer can then be used and the chromatograms are based on conventional HPLC parameters. The key to dual-column methods is that the retentive properties of the analytical column must be sufficiently stronger than that of the turbulent-flow column the dual-column approach is performed in such a manner so that the mobile-phase composition needed to elute the analyte from the turbulent-flow column does not elute the analyte from the analytical column. The sample is then focused at the head of the analytical column until the mobile-phase conditions are changed to elute the analyte. The choice of columns is critical to the success of dual-column methods. Table 10.2 lists some of the applications of dual-column methods found in the literature. 

Figure 10.5. Dual-column method configuration (a) sample load and cleanup (b) sample elution.

Fig. 2. Chromatogram from dual column method Column 1, 6-ft molecular sieve 5A, 98°F Column 2, 10-ft molecular sieve 5A, — 110°F.

Broske [69] investigated the correlation between the method of pretreatment of the fused-silica surface and the retention characteristics of components with different polarity. Broske used in his work the so-called dual column method (see, for instance, ref. [70]). This was accomplished by connecting a section of fused-silica tubing downstream to a standard 25 mx 0.32 mm capillary column. A direct correlation was found between the retention characteristics on uncoated tubing and the inertness measured on the chromatography column obtained. 

Gas Chromatographic Determination of Alcohol in Blood by the Dual Column Method... 

While capillary columns have improved the resolution of pyrolyzate compounds, the type of stationary phase is still important in the discriminatory power of PGC. A dual-column method has been reported in an effort to further improve the discrimination of PGC of paint samples (248). This method uses a polar and a nonpolar capillary column connected to the same injection port of a gas chromatograph. The pyrolyzate vapors are split between the two columns, and a separate, different pyrogram is generated simultaneously for the same sample. 

Many IC techniques are now available using single column or dual-column systems with various detection modes. Detection methods in IC are subdivided as follows [838] (i) electrochemical (conductometry, amper-ometry or potentiometry) (ii) spectroscopic (tJV/VIS, RI, AAS, AES, ICP) (iii) mass spectrometric and (iv) postcolumn reaction detection (AFS, CL). The mainstay of routine IC is still the nonspecific conductometric detector. A significant disadvantage of suppressed conductivity detection is the fact that weak to very weak acid anions (e.g. silicate, cyanide) yield poor sensitivity. IC combined with potentiometric detection techniques using ISEs allows quantification of selected analytes even in complex matrices. The main drawback... 

In general, a comprehensive separation strategy implies the desire to resolve/analyze all components within a sample. In the specific context of a multidimensional chromatographic method, the term is more narrowly applied to indicate that all analytes introduced to the first-dimension separation are also subjected to a second-dimension separation. There are two basic configurations used by our laboratory to carry out comprehensive multidimensional (IEX/RP) protein separations—IEX— Dual Column RP system and IEX—Dual Trap RP system (Figs. 13.1 and 13.2), respectively. 

For separation, high resolution capillary columns are used, and in most circumstances each extract is examined by a dual column technique. This is to add a degree of confirmation into the method. 

As pointed out above, IC is a well-established method for the analysis of inorganic anions and has become the method of choice in many application areas. Many techniques are available using singlecolumn [46] or dual-column systems with various detection modes. IC can be used both for analytical and preparative purposes. Large sample volumes, up to 1300 pul, can be injected to determine trace anions and cations and to attain detection limits of 10-400 ng/1. For determinations at a pig/1 to mg/1 level, a sample size of 10-50 xl is sufficient. Preconcentration is necessary for lower concentrations (an additional column, a sample pump, an extra valve and an extra time are the disadvantages of this approach [47]). With an IEC column and isocratic... 

Preparation of Resins. Maltenes (1 g) obtained after removal of asphaltenes (obtained either by C5 or C7) and solvent were dissolved in cyclohexane and the solution was run on a dual column (Davison Silicagel grade 62 and Alcoa F 20 alumina, according to USBM-API method)(25). After eluting the saturate and aromatic fractions ( deasphalted oil ) with appropriate solvent, resins on the column were extracted once with a 50/50 (v/v) mixture of ether and methanol, then with a 75/25 (v/v) mixture of chloroform and methanol, last with a 75/25 (v/v) mixture of carbon tetrachloride and methanol (26, 27). 

The dual-phase nature of these RAM materials allows the direct injection of the biological sample matrix onto the column without pretreatment. Some disadvantages with the use of RAM columns are that retention times can be long (> 10 minutes) the column must be washed between injections and the mobile phases are not always compatible with some ionization techniques used in LC-MS/MS. Dual-column RAM techniques are also used. These methods use an analytical separation column placed in series downstream from the RAM column. A general overview of the use of RAMs in LC has been published in two parts [117,118]. 

Figure 12.18 shows a typical chromatogram obtained from an on-line product gas analysis for the oxidation of CH4. The chromatograms obtained from the TCD were typically characterized by steady baselines with well-resolved peaks. The product spectrum indicates that both partial and total combustion reactions of CH4 are present. Both CO2 and H2O are baseline-resolved using a Hayesep R column while the O2, N2, CH4, and CO are baseline-resolved on a molecular sieve column. The overall analysis time requires about 9 min. Some gaps exist in the retention times for the various components, but no attempt was made to reduce these by additional optimization of the temperature program. The overall analysis time could have been reduced if the dual-oven method had been used as described elsewhere (Delaney and Mills, 1999 Nicole et al., 2000). 

A method verified by the Intersociety Committee in a manual on Methods of Air Sampling and Analysis has been used to separate and determine O2, N2, CO, CO2, and CH4 in gas samples by GC. A dual column/dual thermal conductivity detector system is employed. The first column contains a very polar stationary liquid phase and retains CO2 only, while the second column is packed with molecular sieve 13X and separates the rest of the components. A tube filled with 10/20 mesh Indicating Drierite is installed between the sample introduction system and the first column to retain water in the sample. The analysis is operated slightly above ambient temperature to obtain the best precision results. The detection limits for CO2 and O2 are 250 and 300 ppm, respectively. The separation can be completed within 8.5 min. 

A method to select the appropriate stationary phase for analysis of a sample mixture is to consider the polar characteristics of the analytes and select a stationary phase of similar polarity. An analyte with similar polar character to the stationary phase will be well retained, the principle of like attracts like applies, and useful retention is then likely to occur leading to adequate selectivity and separation of the analytes primarily on the basis of volatility. Conversely, if the solute is immiscible with the stationary phase then little or no retention difference will be obtained. Further useful indication of retention characteristics may be obtained by analysing a sample on a non-polar and polar column, for example, a dual column GC fitted with Apiezon/OVlOl and Carbowax 20M columns temperature programmed 50-220°C at 10°Cmin with a final hold of 10 min. The chromatogram will indicate the polarity of stationary phase required for the components and the analysis can be repeated with columns of differing polarity, e.g. OV17, OV1701. Tables of RIs for various classes of compounds have been published, mainly for squalane, the reference non-polar stationary phase and Apiezon L, with RI values for Carbowax 20M as a reference polar stationary phase [10]. 

In this chapter, we described an easily implemented, effective, and highly reproducible dual-column HPLC prefractionation method that we have developed for improving routine proteomic analyses. The approach results in multifold increases in the numbers of proteins that can be confidently identified by LC-MS of whole cell lysates without fractionation. We outline the key steps in the overall procedure, from sample preparation through to MS/MS and attendant data analysis, using yeast soluble protein extract as a test mixture. 

A much more efficient, but less sensitive method for the determination of cobalt in sea water, tap water and waste waters by flame AAS with on-line column preconcentration was proposed by Fang et al.[14]. The column was packed with quinolin-8-ol immobilized on controlled pore glass. With dual columns, 48-fold sensitivity enhancement was achieved at a sampling frequency of 60 h , achieving a detection limit of 0.2 //g 1 ... 

The dual-column and single-column ion-exchange precoilcentration flame AAS systems as well as the DDC-Cis sorbent extraction flame AAS and ETAAS systems used for the determination of cadmium were also used for the determination of copper in water samples. The detection limit for the dual-column system was 0.07-0.09 /ig l at 60 samples h [7] for the sorbent extraction preconcentration flame AAS system, 0.2 /xg r at 120 samples h [9] for the sorbent extraction ETAAS system, 0.02 /xg 1 at approximately 20 samples h [10]. The sensitivity of sorption column preconcentration methods with flame AAS detection should be sufficient for the determination of copper in most natural water samples. 

Conventional reversed phase HPLC is unable to separate ara-cyti-dine, ara-uridine, ribo-cytidine and ribo-uridine whereas a dual column procedure utilising an Ultrasphere ODS column in series with a Partisil PXS 10/25 SCX column gave excellent separation of these compounds (Sinkule and Evans, 1983). The accuracy of the method allowed comparative pharmacokinetic and pharmacodynamic studies to be carried out. 

It is possible to use the liquid nature of the stationary phase to gain advantages that have no equivalent in classical LC. For example, the roles of the phases can be switched during a run the mobile phase becomes the stationary phase, and vice versa. This way of working with a CCC column is called the dual mode method (see Octanol-Water Distribution Constants Measured by CCC, p.l616). It is also possible to flush the content of a CCC column, ensuring a complete recovery of all parts of the injected sample. 

If the retention volumes of some constituents of the sample are too large, different methods using the liquid stationary phase can be employed to speed up the separation. For example, the dual mode method can be used. The retained constiments are eluted in the reversed mode. It is absolutely certain that no part of the sample can stay trapped inside the CCC column. 

Arsenobetaine is considered relatively nontoxic but is a common form of arsenic in some foods such as seafood. Slingsby and coworkers developed a dual column selectivity method and electrospray mass spectrometry detection to determine several common arsenic species at high sensitivity [17]. Both anionic and cationic species of arsenic were determined. 

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