Sample on-column

Due its robustness, particularly toward uncleaned samples, on-column, splitless injection is the most widely applied technique for sample introduction. 

Tp Direct injection of plasma samples on column TSK LS-410, 20-32 urn, treated with human plasma 60x4 ACN-phosphate bufferfpH 7.4) 181... 

On-column injection can also be applied for large volume sample introduction. However, especially when the sample volume is large, special attention has to be paid on the cleanup of the sample. On-column injection has also been used as a transfer technique in an online combination of extraction, liquid chromatographic cleanup and gas chromatography. ... 

Column switching is an elaborate form of sample preparation in which a sample is loaded onto column 1 with a mobile phase generated by pump 1. Once the unwanted contaminants, etc., are flushed through column 1 to waste, the flow path is switched so that pump 2 now elutes sample on column 1 in the opposite direction and onto column 2, where further separation occurs. Often columns 1 and 2 and mobile phases from pumps 1 and 2 are very different in order to trap the analytes of interest on column 1 and then separate them on column 2 with a very different mobile phase. 

For samples that decompose easily, an on-column injection may be necessary. In this method the sample is injected on the column without heating. The column temperature is then increased, volatilizing the sample with as low a temperature as is practical. 

Ohm s law the statement that the current moving through a circuit is proportional to the applied potential and inversely proportional to the circuit s resistance (E = iR). (p. 463) on-column injection the direct injection of thermally unstable samples onto a capillary column, (p. 568) one-taUed significance test significance test in which the null hypothesis is rejected for values at only one end of the normal distribution, (p. 84)... 

Cool on-column injection is used for trace analysis. Ah. of the sample is introduced without vaporization by inserting the needle of the syringe at a place where the column has been previously stripped of hquid phase. The injection temperature must be at or below the boiling point of the solvent carrying the sample. Injection must be rapid and no more than a very few, usuahy no more than two, microliters may be injected. Cool on-column injection is the most accurate and reproducible injection technique for capihary chromatography, but it is the most difficult to automate. 

The maximum allowable dispersion will include contributions from all the different dispersion sources. Furthermore, the analyst may frequently be required to place a large volume of sample on the column to accommodate the specific nature of the sample. The peak spreading resulting from the use of the maximum possible sample volume is likely to reach the permissible dispersion limit. It follows that the dispersion that takes place in the connecting tubes, sensor volume and other parts of the detector must be reduced to the absolute minimum and, if possible, kept to less than 10% of that permissible (i.c.,1 % of the column variance) to allow large sample volumes to be used when necessary. 

The sample concentration also should be kept as low as possible, particularly in analyses of polymers on columns packed with small particle size resins. The maximum sample concentration to achieve maximum resolution decreases as the sample molecular weight becomes higher and the resin particle size becomes smaller. It is usually in the range of 0.05-5 mg/ml, depending on the sample molecular weight and resin particle size. 

FIGURE 4.47 Dependence of HETP on sample volume. Column Toyopearl HW-55F, 22 mm X 30 cm. Sample 0.1% myoglobin. Elution 14 mM Tris-HCI, pH 7.9, in 0.3 M NaCI. Flow rate 52 cm/ hr. Detection UV at 220 nm. Legend to. sample injection time Z, column length u. linear velocity. 

The use of totally permeated flow markers in aqueous GPC offers similar advantages along with many of the same shortcomings that one finds in nonaqueous GPC. One problem commonly found in aqueous GPC is that salt peaks due to the on-column ion exchange of counter ions of a polyelectrolyte with dissimilar ions in the GPC mobile phase will occur at or near the total permeation volume of the column. These salt peaks will often obscure the flow marker used in the analysis. Short of preconditioning the sample to exchange... 

C. A. Monnig and J. W. Jorgenson, On-column sample gating for high-speed capillary zoneelectiophoresis . Awn/. Chem. 63 802-807 (1991). 

Figure 11.4 Chromatograms of plasma samples on a silica-chiralcel OJ coupled column system (a) plasma spiked with oxprenolol (internal standard) (b) plasma spiked with 040 p-g/ml metyrapone and 0.39 p-g/ml metyrapol (racemate) (c) plasma sample obtained after oral administration of 750 mg metaiypone. Peaks are as follows 1, metyrapone 2, metyrapol enantiomers 3, oxprenolol. Reprinted from Journal of Chromatography, 665, J. A. Chiarotto and I. W. Wainer, Determination of metyrapone and the enantiomers of its chfral metabolite metyrapol in human plasma and urine using coupled achfral-chfral liquid cltro-matography, pp. 147-154, copyright 1995, with permission from Elsevier Science.

Traditionally, LC and GC are used as separate steps in the sample analysis sequence, with collection in between, and then followed by transfer. A major limitation of off-line LC-GC is that only a small aliquot of the LC fraction is injected into the GC p. (e.g. 1 - 2 p.1 from 1 ml). Therefore, increasing attention is now given to the on-line combination of LC and GC. This involves the transfer of large volumes of eluent into capillary GC. In order to achieve this, the so-called on-column interface (retention gap) or a programmed temperature vaporizor (PTV) in front of the GC column are used. Nearly all on-line LC-GC applications involve normal-phase (NP) LC, because the introduction of relatively large volumes of apolar, relatively volatile mobile phases into the GC unit is easier than for aqueous solvents. On-line LC-GC does not only increase the sensitivity but also saves time and improves precision. 

J. Slobodnik, O. Ostezkizan, H. Lingeman and U. A. Th Brinkman, Solid-phase extraction of polar pesticides from environmental water samples on grapliitised carbon and Empore-activated carbon disks and on-line coupling to octadecyl-bonded silica analytical columns , J. Chromatogr. 750 227-238 (1996). 

Figure 14.15 Typical SFC chromatogram of total olefins in gasoline 1, saturates 2, aromatics 3, olefins t(L), time of loading sample on to columns and eluting saturates t(AR), time of eluting aromatics t(C), time of eluting remaining saturates from olefin trap t(BF), time of eluting olefins by back-flush.

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