Sample volume, significance

GF separates proteins with differences in molecular size. The technique is ideal for the final polishing steps in a purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient Figure 41). Buffer conditions are varied to suit the sample type or the requirements for further purification, analysis or storage step, since buffer composition does not directly affect resolution. Proteins are collected in purified form in the chosen buffer. 

A correction for dilution must be made if the amount of standard added changes the total sample volume significantly. Additions of analyte ranging from twice to one-half the amount of analyte present in the original sample are optimum conditions. 

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

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. 

FIGURE 7.4 Separation of a standard protein mixture on a Fractogel EMD BioSEC-column (600-16 mm) after incubation with 30% acetonitrile. The sample contained BSA ( ), ovalbumin ( ), and cytochrome c (A) (sample volume 500 ftl flow rate 1.0 ml/min). No significant shifts of the retention times and no loss of the resolution were observed even after 900 hr of exposure. 

Ideally, the sample should be injected onto the column as an infinitely thin disc, which covers the total cross section of the column. Because this is impossible, PSS has injected finite volumes onto the columns. In theory, these injection volumes should be as low as possible. In order to be able to detect the sample with significance, a certain (high) concentration of the sample has to be injected. This concept works well for low molar mass compounds, which do not generate much sample viscosity. However, when working with samples... 

Solids content is a general test. All solid materials in the oil are measured as a percentage of the sample volume or weight. The presence of solids in a lubricating system can significantly increase the wear on lubricated parts. Any unexpected rise in reported solids is cause for concern. 

To determine the band dispersion that results from a significant, but moderate, sample volume overload the summation of variances can be used. However, when the sample volume becomes excessive, the band dispersion that results becomes equivalent to the sample volume itself. In figure 10, two solutes are depicted that are eluted from a column under conditions of no overload. If the dispersion from the excessive sample volume just allows the peaks to touch at the base, the peak separation in milliliters of mobile phase passed through the column will be equivalent to the sample volume (Vi) plus half the base width of both peaks. It is assumed in figure 10 that the efficiency of each peak is the same and in most cases this will be true. If there is some significant difference, an average value of the efficiencies of the two peaks can be taken. 

Blood collection from the tail vein is a simple and rapid, nonsurgical method which does not require anesthesia. A relatively large number of serial samples can be obtained within a short period of time. However, this method is limited to relatively small sample volumes (<250 pi per sample). Although larger volumes can be obtained by placing the rat in a wanning chamber, this procedure could significantly influence the disposition of the test compound and therefore is not recommended for routine studies. Blood collected from the cut tail has been shown to provide valid concentration data for numerous compounds. 

At now, the samples volumes necessary for certain analysis can be significant, which can preclude the use of integrated sequence on microscale chromatography. 

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