Injection volume, tolerable

A quality control laboratory had a certain model of HPLC in operation. One of the products that was routinely run on the instrument contained two compounds, A and B, that were quantitated in one run at the same detector wavelength setting. At an injection volume of 20 /tL, both compounds showed linear response. The relatively low absorption for compound B resulted in an uncertainty that was just tolerable, but an improvement was sought. 

The injection volume chosen for analysis must represent a compromise between the amount of sample needed to properly detect the eluting material, and the amount of extra-column dispersion the analyst is willing to tolerate. It is also important that the same injection volume be used for both samples and standards, and that sample injection be properly synchronized with the start of data acquisition. 

The maximum injected volume was 50 mL kg" [25]. LD50 was thus limited by the iodine concentration of the solution in the case of P743 and P840 and the NS-CA iobitridol. Thus, the intravenous acute systemic tolerance by the route intended for use in clinical practice was found to be satisfactory (Table 5). 

The low flow rate in the microbore column ensures sample volumes compatible with the secondary conventional column and permits the injection of a small volume onto the secondary column, making the transfer of incompatible solvents possible without peak shape deterioration or resolution losses [63], The possible disadvantage could be the lower sample capacity of microbore LC columns. However, in LCxLC, a sensitivity enhancement can be obtained if the formation of compressed solute bands at the head of the secondary column is achieved during the transfer from the first to the second dimension. Moreover, a larger volume can be injected into the first-dimension microcolumn, used as a highly efficient pre-separation step, and a limited decrease in efficiency due to a large injection volume can be tolerated. 

For determining the robustness of a method a number of parameters, such as extraction time, mobile-phase pH, mobile-phase composition, injection volume, source of column lots and/or suppliers, temperature, detection wavelength, and the flow rate, are varied within a realistic range and tlie quantitative influence of the variables is determined. If the influence of a parameter is within a previously specified tolerance, this parameter is said to be witliin the robustness range of the method. These method parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment. 

If the sample is dissolved in a solvent that is weaker than the mobile phase, then the sample can be enriched on the head of the column without penetrating into the column bed. This compression effect is particularly important for capillary LC applications, since it permits significantly larger injection volumes. A substantial increase in sensitivity results, and conventional autosamplers with 20-jnl loops can be used.16 However, sample solubility and recovery, miscibility of the sample with the mobile phase, and the maximum tolerable loss in column efficiency and resolution must all be assessed experimentally for optimum on-column focusing.16... 

The drag is administered into the subcutis of test animals (mouse, rat, rabbit, or dog) at the latero-dorsal area of the thoracic wall. The injection volume has to be adjusted to the size of the animal. The overall evaluation of findings is done according to the scoring described for intramuscular tolerance testing (Stotzer 1989). 

Indocyanine green was introduced by J. Caesar et al. in 1961 as a liver function test. Anionic tricarbocyanine dye is referred to as an ideal test substance (1.) it is tolerated very well there have been no reports of any incidents so far, and even paravenous injection is tolerated (2.) it is excreted unchanged by hepatocytes in the bile as there is no bio transformation - this is why ICG clearance is valid as a measure of hepatocellular uptake and transport processes (3.) there is no interference with drugs (except rifamycin), haemolysis, bilirubin (up to approx. 4 mg/dl) or hyperlipidaemia (4.) the substance is not subject to the enterohepatic circulation (J.) the rapid elimination, which depends on hepatic perfusion ( flow-limited ), allows the calculation of the hepatic flow volume as a whole based on ICG clearance (6.) the method is simple to perform. 

Maximal tolerated dose for STPP liposomes is established by injecting Balb/C mice with 0.45, 1.5, 4.5, and 15 mg/kg of STPP in injection volume. 

When an initially painful intravenous or intramuscular injection must be administered repetitively, patient reluctance develops. Injection pains are usually accompanied by hemorrhage, edema, inflammation, and tissue necrosis." Among the factors responsible for painful injections, the most important are the drug solubility in aqueous medium, the viscosity, the pH and the hypo- or hyperosmotic character of the injected drug solution, the amount of the injected volume, the site of injection, the pain tolerance of the patient, and the technique of administration. Other factors include precipitation of the drug at the injection site, and localized cell lysis. ... 

You can calculate the maximal tolerable injection volume from the variance contributions of all effects to band broadening. This is possible because HPLC consists of stochastic independent processes. However, who has got the time and leisure to do this Therefore, please find in the following paragraph some numbers and recommendations. Without being given additional explanations just believe it ... 

With Eq. 2.26 both the theoretical peak volumes for representative conventional HPLC and UHPLC column formats as well as at a given value of 0 (Table 2.6) can be calculated. The resulting injection volumes may appear relatively low, but they should be considered for making best use of the respective column efficiency. If a loss in N of 10% can be tolerated, one can inject twice this volume. 

Flow injection analysis is an analytical technique based on injecting a known volume of sample into carrier or reagent streams that are transported in small-diameter conduits (typically 0.5-0.8 mm internal diameter) under laminar flow conditions (Ruzicka and Hansen, 1988 Taljaard and van Staden, 1998 McKelvie, 2008 van Staden and van Staden, 2012 Worsfold et ah, 2013). In this moving stream, the sample does not decompose it is physically and chemically converted into a detectable species that causes a detector response downstream of the injection point (Ruzicka and Hansen, 1988 Taljaard and van Staden, 1998 Saurina, 2008 Saurina, 2010 van Staden and van Staden, 2012). The results will be reproducible on condition that all critical parameters (reproducible injection, controlled reaction time, and controlled dispersion) are held within certain tolerance levels (Ruzicka and Hansen, 1988 Taljaard and van Staden, 1998 van Staden and van Staden, 2012). The basic FIA instrument is composed of a multichannel pump, an injection valve, a flow-through detector, and a signal output device (originally a recorder, lately a computer) (van Staden and van Staden, 2012). Before FIA, relevant variables such as flow rates, reactor dimensions, injection volume(s) and chemical (reaction) conditions... 

Similar to HPLCs, UHPLC injection loops vary in size and type and can be readily switched out. As with the mixer, choice of sample loop size depends on balancing the injection volume required and the amount of extra system volume that can be tolerated. The smaller the sample loop, the less loss of sample and the smaller the total flow pafh. Some instruments include the needle in the flow pafh, and some instruments... 

Because UHPLC injections tend to be smaller (more in the range of 0.1-10 piL), the tolerances for calibration on the expelled injection volumes have to be much tighter. A tolerance of 0.3 piL would have very little effect on 25 or 10 p,L injections, but would be useless at 0.2 and 0.5 p.L injection volumes, which are not uncommon for UHPLC. 

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 is seen that columns having diameters less than 2 mm will only tolerate a maximum sample volume of a fraction of a microliter. Although larger volume valves can be used to inject sample volumes of this size, the dispersion from the valve is still likely... 

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