Injection, reproducible

Hot needle or solvent flush method. Rapid injection. Reproduce injection time as close as possible. 

Cold split Column independent 100-fold increase in sensitivity with respect to conventional GC injection Reproducible and accurate sampling Controlled evaporation No discrimination on basis of b.p. Rapid transfer of sample to column Low volume of CIS liners (2-3 xL)... 

Sternson et al. [58] used a high performance liquid chromatographic method for the analysis of miconazole in plasma. Miconazole was extracted from alkalinized plasma with n-heptane-isamyl alcohol (98.5 1.5) and separated by high performance performance liquid chromatography on p-Bondapak Ci8 with ultraviolet detection at 254 nm. The mobile phase was methanol-tetrahydrofuran-acetate buffer (pH 5) (62.5 5 32.5) containing 5 mmol octanesulfonate per liter. The flow rate was 2 mL/min. Recovery was 100%. The relative standard deviation for injection-to-injection reproducibility was 0.4% and that for sample-to-sample variation was 5% at high miconazole concentrations (30 pg/mL) and 1% at low (1 pg/mL) concentrations. The limit of detection was 250 ng/mL. 

The flow injection technique is based on three main principles sample injection, reproducible timing, and controlled dispersion [128]. The dispersion can be described as limited, medium, or large in a colorimetric system based on a reaction between the sample and a suitable reagent, a medium dispersion is preferred. Thus in the flow injection determination of nitrate, the reductor column should not excessively increase the dispersion. In a copperised cadmium reductor, more than 90% of the total nitrate is reduced within 1 - 2 s with minimum risk of further reduction of nitrite [167]. Consequently, the reductor can be made very small, which results in a minimal increase of dispersion. 

The maximum injection volume depends on the volume of the sample loop in the injection valve. The reproducibility of manual injection depends on the skill of the operator. The use of a small sample loop and an overflow injection of the sample solution so that the loop is fully flushed with sample are basic requirements for quantitative analysis. The highest injection reproducibility can be obtained by an auto-injector with a fixed sample loop. The smallest reasonable injection volume is 1 (A. A nl-scale injection valve can be constructed however, the memory effect at the surface of contact parts affects quantitative analysis compared with the use of a /d-scale injection valve. For a semi-micro system, a low hold-up volume injection valve is desired. The minimum injection volume is 80 nl. For a preparative-scale injection, the sample loop can be easily replaced with a larger-volume loop, such as a 200 jA, instead of the standard 20 /A loop. 

FIGURE 1.25 HPLC determination of impurities in a levothyroxin (L-T4) formulation. Experimental conditions Column, Chiralpak QN-AX (150 rum x 4 rum ID) mobile phase, acetonitrile-50 mM ammonium acetate (60 40, v/v) (pHa 4.5) flow rate, 0.7 mLmiu UV detection, 240 nm temperature, 25 C. Sample, T4-200 tablets (Uni-Pharma, Greece) containing 0.2 mg L-T4 sodium per tablet the tablet was pulverized, suspended in methanol-10 mM sodium hydroxide (1 1 v/v) and after ultrasonication for 5 min the residues were removed by filtration. An aliquot of 10 xL of the filtrate was directly injected. (Reproduced from H. Gika et al., J. Chromatogr. B, 800 193 (2004). With permission.)... 

This test could conceivably be performed holistically, as part of the injection reproducibility test (defined later). Using retention time as an indicator, such an approach would work provided the retention time for a test component was very stable from week to week. However, this is often not that easy to control consistently, since one often encounters column/mobile phase variations and other variables. Therefore, flow accuracy is typically not tested in this manner. 

The Groton Biosystems protein analyzer tests injection reproducibility at three pressures (e.g., 25mbar at 0.2min, 50mbar at 0.1 min, and lOOmbar at 0.05min). Each injection pressure is injected five times and the complete injection linearity relative standard deviation (RSD) needs to be less than 5%. In addition, the carryover from successive injections is investigated. 

Apart from the qualification dossiers provided by vendors there seems, at present, to be very little information published on the performance of an operational qualification for capillary electrophoresis (CE) instruments other than a chapter in Analytical Method Validation and Instrument Performance. The chapter, written by Nichole E. Baryla of Eli Lilly Canada, Inc, discusses the various functions (injection, separation, and detection) within the instrument and provides guidance on the type of tests, including suggested acceptance criteria, that may be performed to ensure the correct working of the instrument. These include injection reproducibility and linearity, temperature and voltage stability, detector accuracy, linearity, and noise. 

Figure 12.3. Typical electropherogram from an injection reproducibility test. CE conditions capillary, 50 qm ID x 50 cm (40 cm to detector) temperature, 20°C detection, 325 nm with 10-nm bandwidth injection, 3 s (5 kPa) 1 mM 4-hydroxyacetophenone applied voltage, +30 kV separation buffer, 20 mM borate at pH 9.2 conditioning, 2-min high-pressure rinse (100 kPa) with 20 mM borate buffer pH 9.2 between runs.

Injection parameters. If the injection reproducibility or linearity results are problematic, ensure that the sample vial cap is put on correctly. Sometimes, if the cap is put on incorrectly, the vial cannot be pressurized and injection either fails or is irreproducible. Also, check to make sure that no air bubbles are present in the sample vial. If air is injected into the capillary, poor results will be obtained. 

Acceptance criteria for tailing factor, percent injection reproducibility, relative standard deviation of calibration and/or control standard... 

Calculation of peak area versus weighing for injection reproducibility... 

Calculation of injection reproducibility for calibration and control standard and assessment against predefined acceptance criteria... 

Figure 2.4—Direct vaporisation injector used for packed columns. Typical septum injector with a microseal option (the Merlin microseal) that can be used for thousands of injections (reproduced by permission of Hewlett-Packard).

Consider a sudden loss of injection-to-injection reproducibility. With only a little experience, the analyst without an integrator will look to injection technique, a leak almost anywhere (syringe, septum, column fitting, external gas connection, etc.) or perhaps a contaminated column. Where one looks first depends on experience with the instrument and the analysis and... 

All system suitability criteria for injection reproducibility, standard confirmation, resolution, sensitivity determination, and peak tailing must be met on each day. 

Sample is introduced into the sample loop in a variety of ways the vial may be pressurized to force the sample out, or a syringe may be used to draw the sample out. The syringe is usually controlled by a stepping motor so that different sample volumes can be injected reproducibly by partially filling the sample loop. Once the sample loop has been loaded, the valve is electrically actuated. Some autosamplers also include other features such as sample heating or cooling, and the ability to perform standard additions, thereby improving the precision of the analysis. 

With hydrostatic injection mechanisms, injection reproducibility can be better than 1-2% RSD. The volume of sample loaded is a function of the capillary dimensions, the viscosity of the buffer, the applied pressure, and the time, and it can be calculated using... 

Fig. 5.32. Alkylation and GC of aqueous inorganic halides. A, authentic salts B, solution prepared on a cation-exchange column. Peaks 1 = methylfluoride 2 = water 3 = methyl chloride 4 = methyl bromide 5 = trimethylamine 6 = methyl iodide. Conditions stainless-steel column, 10 ft. x 1/4 in. O.D., Chromosorb 101 (80-100 mesh) helium flow-rate, 75 ml/min column temperature, 125°C injection port temperature, 360°C 6 til of an aqueous solution of tetramethylammonium halides (0.25 M each) injected. (Reproduced from Anal. Chem., 42 (1970) 1672, by courtesy of J. MacGee and the American Chemical Society.)...

Above, saline injected control animals below, adipokinetic hormone (200 pmoles/animal) injected. Reproduced with permission from Ref. 23. Copyright 1983 Academic Press. 

Figure 7. Amperometric response of the AuNP/AChE electrode (a) after injection of 10 pL of a 20 mM ATC1 solution, and (b) after injection of 20 pL of 1 mM carbofuran. The final concentrations of ATC1 and carbofuran were 66.4 and 6.6 pM, respectively. The arrows denote the time of injection. (Reproduced with permission from Du et al. A. Shulga et al. Electrochem. Commun. 2007, 9, 935-940).

Figure 10.7 Comparison of the results of numerical calculations of the relaxation and of the parabolic models with experimental results. Solid lines experimental results by Seidel-Morgenstern [41]. Dot-dashed line profiles calculated with the relaxation model. Dotted lines profiles calculated with the parabolic approximation. The figure on each subfigure indicates the duration of the rectangular injection. Reproduced from F. James, M. Pastel, M. Sepulveda, Physica D, 138 (2000) 316.(Fig. 8).

Check the autosampler, check if the correct injection volume is requested by the control unit, or check the manual injection. See also the sample volume check at Poor Injection Reproducibility above. 

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