The Effect of Sample Volume

The dispersion effect of the sample volume was discussed in Chapter 4 and little more needs to be said about it. It will be seen later that the sample volume controls both the concentration and the mass sensitivity of the chromatographic system and thus, should be made as large as possible. This means that all other sources of band dispersion must be kept to an absolute minimum to permit the maximum possible sample volume to be used. A better understanding of the causes of band dispersion has resulted in 

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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. 

It has also been shown that the effect of sample volume on peak width will be most significant for the early peaks (the most narrow peaks). In addition, the degrading effect of sample volume will progressively decrease as the capacity ratio of the peak becomes larger. However, the resolution of both late and early peaks are equally important and, consequently, the limiting sample volume will be that which restrains the dispersion of the first peak to 5% or less. 

The effect of sample volume on peak width has been considered and treated theoretically in Chapter 6 however, it is of interest to determine the maximum sample volume that can be tolerated with modern columns packed with small particles. The maximum sample volume is defined by the following equation,... 

To minimize the effect of sample volume on dispersion, and ensure that there was minimum dispersion from the valve and valve connections, a 0.2 pi Valeo internal... 

Calculate the ratios of the counting efficiencies for the various energies at each sample volume relative to those for the 480-mL volume in the cylindrical container to observe the effect of sample volume on efficiency. 

In industrial-scale chromatography, one very desirable aspect is the ability to handle large amounts of samples, without any increase in Hs. For sample volumes of up to a few percent of the total column volume, Hs remains constant, but it then increases with the sample volume. It has been reported that the effect of sample volume on Hs should rather be treated as the effect of the sample injection time to (s) [7]-... 

Figure 20.7 The effect of sample volume on the linear range of a thermometric glucose sensor with a 0.6 mm x 10 mm CPG column with glucose oxidase/catalase. The flow rate was 50 /xl min . ... 

The pH of the reagent carrier was however adjusted to 4.5 to obtain maximum peak height and minimum iron (III) interference in the analysis. In order to proceed with the final system design, the effect of sample volume, mixing coil length and flow-rate were studied using Morin at fixed concentration of 2.5xlCH M and pH 4.5. 

To minimize the effect of sample volume on dispersion, and ensure that there was minimum dispersion from the valve and valve connections, a 0.2 jxl Valeo internal loop valve was employed to place the sample on the column. In addition, the sample valve was used with an intra-column injection system [2] that ensured the sample was placed in the center of the column about 5 mm below the packing surface. TMs device also determined that, even if the packing settled, there would be no void above the point of injection and the effective length of the column was not changed. All connecting tubes were 0.007 in. I.D. and their lengths were kept to a minimum (Le., <5 cm). The detector employed was the Perkin Elmer LC-85B fitted with a cell 1.4 pi volume and was used in conjunction with an electronic amplifier that had an effective time constant of 24 millisec. The columns employed had internal diameters of 8-9 mm to ensure the peak volumes were very large compared to any dispersion introduced by extra-column dispersion. As a result, the extra-column dispersion was maintained at a level of less than 2% of the peak volume for all measurements and, in most instances, was maintained at a level of less than 1%. 

What is the minimum activity the system can sense And discriminate What is the effect of sample volume on the measurement Is the reactive surface of the microelectrode totally immersed in the substance being studied What substances are present that may interfere with the true reading In what way and to what extent do they alter the true reading Also, can the calibration fluid be made more representative of the experimental fluid and thereby negate the need for or reduce the magnitude of the corrective factor (k Int) . n/z... 

The effect of sample volume on quantitative analysis by SPME. 

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