Injection volume for

Loading capacities in size exclusion chromatography are very low because all separation occurs within the liquid volume of the column. The small diffusion coefficients of macromolecules also contribute to bandspreading when loads are increased. The mass loading capacities for ovalbumin (MW 45,000) on various sizes of columns can be seen in Table 10.5. The maximum volume that can be injected in size exclusion chromatography before bandspreading occurs is about 2% of the liquid column volume. The maximum injection volumes for columns of different dimensions can also be seen in Table 10.5. 

Table 5.15 Relative signal responses from various injection volumes for the LC-MS-MS analysis of a wheat forage matrix sample. Reprinted from J. Chromatogr., A, 907, Choi, B. K., Hercules, D. M. and Gusev, A. L, Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression , 337-342, Copyright (2001), with permission from Elsevier Science...

TABLE 12.2 Typical Injection Volumes for Various Column Diameters... 

Figure 4 shows peak area precision vs. injection volume for a typical autosampler. Note that excellent peak area precision of 0.2% RSD was readily achievable for an injection volume >5 J,L. Precision levels are much poorer (0.5-1% RSD) for sampling volumes <5 J,L, attributable to the finite resolution of the sampling syringe and associated digital stepper motor. To obtain optimum peak area precision, the analyst must avoid potential problem situations such as an overly fast sample... 

Ideally, one should inject between 1% and 2% of the total volume of the capillary. For example, a 0.5 psi (34.5 mbar) injection for 8 s (Table 2) in a capillary with 75 pm internal diameter and 60 cm total length represents 2.02% of capillary volume to the window or 44.7 nL of injected volume. For a capillary with a diameter of 50 pm and under the same conditions, it represents 0.9% and 8.83 nL of injected volume, which is five times less. For a 75 pm capillary of 120 cm, the same conditions produce a sample plug of 0.39% and 0.63 nL. 

Table 3 Suggested Flow Rates, Loads, and Injection Volumes for a Given Column Size...

Various injector geometries (simple cross, double-T, triple-T) were investigated on a glass microchip. The triple-T injector allowed for a selection of different injection volumes. For instance, a triple-T injector allowed injection of three different volumes depending on whether a cross, double-T, or triple-T configuration was used (see Figure 4.6) [529]. Pinched injection has been used consecutively to inject two samples into the same separation channel (see Figure 4.7) [557]. 

The injection volume for these analyses is 1-50 yl. Under the conditions noted in the preceding table, benzidine elutes from the column in approximately 8.5 minutes. Following the elution of benzidine, a gradient elution program to 100% methanol in 5 minutes is effected to clean the column of later eluting compounds. This cleanup step is of critical importance in the analysis of bulk dye samples because of the presence of extensive contaminating material in the chloroform extract. For quantitation, the area of the sample peak is compared with that of appropriate standards. The limit of detection is 10 ng benzidine/injection. However, due to the relatively poor ex-... 

NOTE The injection volume for all sample extracts, blanks, quality control (QC) samples and calibration solutions shall he the same. 

Chang, H.-T. Maximization of injection volumes for DNA analysis in capillary electrophoresis. Electrophoresis 2001,... 

However, the reproducibility of the analyses is less certain than in HPLC because there are many subtle factors that can affect the precision of the injected volume. For hydrostatic introduction, relative standard deviation is of the order of 2 per cent, if an internal standard is used. When the efficiency N of the separations is sufficiently large, it becomes possible to separate isotopes of an element (Figure 8.11). 

The sample is loaded at atmospheric pressure into an external or internal loop, or groove in the valve core and introduced into the mobile phase stream by a short rotation of the valve. The volume of sample injected is normally varied by changing the volume of the sample loop or by partially filling a sample loop with a fraction of its nominal volume. External sample loops have volumes from about 5 p.1 up to about 5 ml, although typical injection volumes for conventional diameter columns are 10-50 xl. Injections from 1 p,l to about 40 nl require micro-injection valves equipped with replaceable internal loops [7,32-34]. Injection volumes less than about 40 nl are performed by positioning a split vent between the injector and the column. Typical injection volumes that preserve column efficiency for packed columns of different internal diameters are summarized in Table 5.1. For packed capillary columns with internal diameters < 0.2 mm direct injection will usually require the use of a split vent to minimize volume overload unless on-column focusing is possible. Injection volumes about 5 times larger than those indicated in Table 5.1 are sometimes used to increase sample detectability but with some decrease in the column separation power. 

Simple steps in sample preparation such as mixing, pretexing, filtration, dilution, making aliquots, etc. are already implemented in many instruments. Some manufacturers even allow extremely small injection volumes for p-bore techniques... 

In general, there is a compromise between sensitivity and injection volume for a given column. This stems from the fact that both column overloading and band-spreading effects associated with the injection volume may affect separation performance (as expressed by resolution between adjacent peaks or... 

Fignc 155 Plot of the logarithm of the plate count versus the logarithm of the reduced injection volume for columns of different plate counts. 

Table 12 Solution concentrations and injection volumes for SEC (data courtesy of Polymer Laboratories Ltd)...

Maximum Sample Injection. Volume for a Specific Column... 

The value of N should be determined under the same experimental conditions for SEC measurements. For example, sample concentration of 0.1Z and sample injection volume of 0.1 - 0.2 ml are for ordinary SEC conditions. However, because the value of N Increases much more with decreasing the injection volume down to 0.01 ml, the sample injection volume for the column performance specification by suppliers, is usually 0.01 ml. 

For sample introduction, six-way injection valves are commonly used. The reconstituted sample extract is loaded in an external or internal loop (e.g., 20 pL loop), and introduced into the mobile phase by rotation of the valve. The external loop (>5 pL) is interchangeable in order to adjust the injection volume. For injection volumes below 5 pL, internal loops should be used. The typical injection volume is 1-20 pL onto the column. To... 

Assuming standard column configuration and injection volume for a favoured analyte 2004 (US prices are usually significantly lower)... 

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