Injection volumes

Because of the strong interactions, lEC can focus big injection volumes at the column head very well. It is also possible to highly load ion exchangers. Loading depends on many parameters (all the parameters described above), but it is particularly dependent on the size of the sample molecules and the static and dynamic 

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The column is swept continuously by a carrier gas such as helium, hydrogen, nitrogen or argon. The sample is injected into the head of the column where it is vaporized and picked up by the carrier gas. In packed columns, the injected volume is on the order of a microliter, whereas in a capillary column a flow divider (split) is installed at the head of the column and only a tiny fraction of the volume injected, about one per cent, is carried into the column. The different components migrate through the length of the column by a continuous succession of equilibria between the stationary and mobile phases. The components are held up by their attraction for the stationary phase and their vaporization temperatures. 

Once injection water treatment requirements have been established, process equipment must be sized to deal with the anticipated throughput. In a situation where water injection is the primary source of reservoir energy it is common to apply a voidage replacement policy, i.e. produced volumes are replaced by Injected volumes. An allowance above this capacity would be specified to cover equipment downtime. 

Calibration curves are usually constructed by analyzing a series of external standards and plotting the detector s signal as a function of their known concentrations. As long as the injection volume is identical for every standard and sample, calibration curves prepared in this fashion give both accurate and precise results. Unfortunately, even under the best of conditions, replicate injections may have volumes that differ by as much as 5% and often may be substantially worse. For this... 

Precision The precision of a gas chromatographic analysis includes contributions from sampling, sample preparation, and the instrument. The relative standard deviation due to the gas chromatographic portion of the analysis is typically 1-5%, although it can be significantly higher. The principal limitations to precision are detector noise and the reproducibility of injection volumes. In quantitative work, the use of an internal standard compensates for any variability in injection volumes. 

Injector The sample, typically 5-200 )J,L, is placed in the carrier stream by injection. Although syringe injections through a rubber septum are used, a more common means of injection is the rotary, or loop, injector used in ITPLC and shown in Figure 12.28 of Chapter 12. This type of injector provides reproducible injection volumes and is easily adaptable to automation, a feature that is particularly important when high sampling rates are desired. 

The majority of FI A applications are modifications of conventional titrimetric, spectrophotometric, and electrochemical methods of analysis. For this reason it is appropriate to evaluate FIA in relation to these conventional methods. The scale of operations for FIA allows for the routine analysis of minor and trace analytes and for macro-, meso-, and microsamples. The ability to work with microliter injection volumes is useful when the sample is scarce. Conventional methods of analysis, however, may allow the determination of smaller concentrations of analyte. 

The curves in Figure 17 show that as the injection volume is increased, so the retention volume of the peak also increases. The retention volume of the small negative peak produced by the smallest charge will be the same as that for a sample... 

Figure 17. Vacancy Elution Curves from Different Injection Volumes on a Column of 500 Theoretical Plates...

For best results, sample injection volume should not exceed... 

For best results, use the flow rate, injection volume, and column sample capacity guidelines in Table 3.11. Conditions outside these guidelines may be used, but poor resolution between proteins may result from extensive deviations from these guidelines. 

Small particle size resins provide higher resolution, as demonstrated in Fig. 4.41. Low molecular weight polystyrene standards are better separated on a GIOOOHxl column packed with 5 /u,m resin than a GlOOOHg column packed with 10 /Ltm resin when compared in the same analysis time. Therefore, smaller particle size resins generally attain a better required resolution in a shorter time. In this context, SuperH columns are best, and Hhr and Hxl columns are second best. Most analyses have been carried out on these three series of H type columns. However, the performance of columns packed with smaller particle size resins is susceptible to some experimental conditions such as the sample concentration of solution, injection volume, and detector cell volume. They must be kept as low as possible to obtain the maximum resolution. Chain scissions of polymer molecules are also easier to occur in columns packed with smaller particle size resins. The flow rate should be kept low in order to prevent this problem, particularly in the analyses of high molecular weight polymers. 

The injection volume should be kept as small as possible to attain maximum resolution in analyses. This is particularly important in analyses on columns packed with small particle size resins such as SuperH. Injection volumes of 0.1 % or less of the total column volume are recommended on SuperH columns. A few times larger injection volumes may be applied to other series of H type columns. 

Larger injection volumes, e.g., 2% of the total column volume, are sometimes advantageous in the preparative fractionation of polymers (33). More samples can be injected using larger injection volumes with a slight decrease in resolution. When the same amount of sample is injected with a smaller injection volume and a higher sample concentration, the resolution decreases more significantly. 

In order to achieve the best efficiency the SEC column should be operated at optimized operating parameters. The most important ones are flow rate [cf. van Deemter equation for band-broadening effects (21)], sample viscosity (depends on molar mass and concentration of the sample), and injection volume (7). 

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... 

Molar mass (example) Flow rate (example) Injection volume (example) Concentration (example)... 

Using other methods for the calculation of plate count can result in different numbers, depending on peak shape. It should also be kept in mind that many other operational parameters, such as eluent viscosity, column temperature, flow rate, and injection volume, will influence the results of the plate count determination. 

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. 

The peak width increases with injection volume. Therefore this parameter has to be fixed for comparative measurements. It has become the custom to inject low molecular weight test samples in very small volumes at very high concentrations, occasionally even as pure compounds. This extreme is not recommended as it is more important to inject a constant sample amount, reproducibly, in a precisely kept volume. Typical GPC injections are between 50 and 200 /a1. It is better to inject a larger volume of a lower concentration polymer solution. GPC units are often not designed for injection volumes lower... 

SEC measurements were made using a Waters Alliance 2690 separation module with a 410 differential refractometer. Typical chromatographic conditions were 30°C, a 0.5-ml/min flow rate, and a detector sensitivity at 4 with a sample injection volume of 80 fil, respectively, for a sample concentration of 0.075%. All or a combination of PEO standards at 0.05% concentration each were used to generate a linear first-order polynomial fit for each run throughout this work. Polymer Laboratories Caliber GPC/SEC software version 6.0 was used for all SEC collection, analysis, and molecular weight distribution overlays. 

In HOPC there are several parameters users can adjust for their own needs after a column is selected concentration of the polymer, injection volume, and... 

Column internal diameter Volumetric flow rate Injection volume UV-deteaor cell volume Sensitivity improvement... 

Figure 12.11 Coupled SEC-RPLC separation of compound Chemigum mbber stock (a) SEC ti ace (b) RPLC trace of fraction 1, dibutylphthalate (c) RPLC trace of fraction 2, elemental sulfur. Coupled SEC conditions MicroPak TSK 3000H (50 cm) X 2000H (50 cm) X 1000 H (80 cm) columns (8 mm i.d.) eluent, THE at a flow rate of 1 mL/min UV detection at 215 nm (1.0 a.u.f.s.) injection volume, 200 p-L. RPLC conditions MicroPak MCH (25 cm X 2.2 mm i.d.) column flow rate, 0.5 mL/min injection volume, lOpL gradient, acetonitrile-water (20 80 v/v) to 100% acetonitrile at 3% acetonitrile/min UV detection at 254 nm (0.05 a.u.f.s.). Reprinted from Journal of Chromatography, 149, E. L. Jolmson et al., Coupled column cliromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

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