Injection volume effect

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). 

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 5.16 LC-MS-MS signal responses" obtained from wheat forage matrix samples using various mobile-phase additives (injection volumes of 50 p,l). From Choi, B. K., Hercules, D. M. and Gusev, A. I., LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices , Fresenius J. Anal. Chem., 369, 370-377, Table 2, 2001. Springer-Verlag GmbH Co. KG. Reproduced with permission...

L. M. Surguchev, A. Koundin, and D. Yannimaras. Air injection— cost effective lOR method to improve oil recovery from depleted and waterflooded fields. In Proceedings Volume. SPE Asia Pacific Impr Oil Recovery Conf (APIORC 99) (Kuala Lumpur, Malaysia, 10/25-10/26), 1999, SPE Number 57296. 

For isocratic LC, the solute does not need to fully elute from the second-dimension column prior to the next sampling period. This is illustrated in Fig. 6.4, where it is shown that more than one sample can be resident in the column at one time. Using the chromatograms shown in Fig. 6.5, which show the effect of various injection volumes that will be discussed later, it is not necessary to wait for the full 2 min of sampling time. This significantly helps to speed up the sampling process and is most useful for SEC, where short elution time ranges are typically found and short times between the injection and nonretained peaks are typical of column operation. 

Parameters that should be tested in HPLC method development are flow rate, column temperature, batch and supplier of the column, injection volume, mobile phase composition and buffer pH, and detection wavelength [2], For GC/GLC methods, one should investigate the effects of column temperature, mobile phase flow rate, and column lots or suppliers [38], For capillary electrophoresis, changes in temperature, buffer pH, ionic strength, buffer concentrations, detector wavelength, rinse times, and capillaries lots and supplier should be studied [35, 36], Typical variation such as extraction time, and stability of the analytical solution should be also evaluated [37],... 

The molecular-weight distributions were measured using a Waters GPC in the dual-detector mode (DRI and UV). The UV detector was operated at 254 nm. The samples were prepared by dissolving 2 mg of polymer in 10 ml of THE The injection volume was 200 pi. Separations were effected using two Polymer Labs 10-gm PL mixed-B columns. THF was used as the mobile phase. The molecular-weight distributions were calculated relative to narrow polystyrene standards ranging from 102 to 4 x 106 M ,... 

Shintani et al. (1967) developed and proposed the methodology that currently seems to be more utilized. It uses the lateral vastus muscle and includes a methodology for evaluation, scoring, and grading of irritation. Additionally, Shintani et al. investigated the effects of several factors such as pH of the solution, drug concentration, volume of injection, the effect of repeated injections, and the time to maximum tissue response. This method also constitutes the USP method (USP, 1985). 

This work [34, 35] reinforces the view that chromatographic separation itself is not always sufficient to effect adequate clean-up to remove all matrix effects but that these will be reduced if the minimum possible injection volume is used. 

Fig. 5. Effect of the flow rate on the separation efficiency. Separation of a protein mixture at six different flow rates (40,80,120,160,200 and 240 ml/min) normalized to the elution volume. Conditions Column 80 ml CIM DEAE Tube Monolithic Column Mobile phase buffer A 20 mM Tris-HCl buffer, pH 7.4 buffer B 20 mM Tris-HCl buffer + 1 M NaCl, pH 7.4 Gradient 0-100% buffer B in 200 ml Sample 2 mg/ml of myoglobin (peak 1), 6 mg/ml of conalbumin (peak 2) and 8 mg/ml of soybean trypsin inhibitor (peak 3) dissolved in buffer A Injection volume 1 ml Detection UV at 280 nm. (Reprinted with permission from Podgornik A, Barut M, Strancar A, Josic D, Koloini T (2000) Anal Chem 72 5693)...

Fig. 14 a, b. Effect of gradient steepness on the very fast separation of polystyrene standards in a molded monolithic poly(styrene-co-divinylbenzene) column (Reprinted with permission from [121]. Copyright 1996 Elsevier). Conditions column, 50 mm x8 mm i.d., mobile phase, linear gradient from 100% methanol to 100% tetrahydrofuran within a 1 min b 12 s, flow rate, 20 ml/min, peaks represent polystyrene standards with molecular weights of 9200,34,000 and 980,000 (order of elution), 3 mg/ml of each standard in tetrahydrofuran, injection volume 20 pi, UV detection, 254 nm... 

The concept of a theoretical plate is based on the number of equilibria that may have taken place during the separation process and this number is related to the number of times the effective volume of a column is greater than the peak volume. The variance (a2) for the peak is a measure of the broadening of the injection volume while the square of the retention time (fR2) is a measure of the effective column volume for that compound. Hence the number of theoretical plates (AO may be calculated from the following equation ... 

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. 

It is critical when performing quantitative GC/MS procedures that appropriate internal standards are employed to account for variations in extraction efficiency, derivatization, injection volume, and matrix effects. For isotope dilution (ID) GC/MS analyses, it is crucial to select an appropriate internal standard. Ideally, the internal standard should have the same physical and chemical properties as the analyte of interest, but will be separated by mass. The best internal standards are nonradioactive stable isotopic analogs of the compounds of interest, differing by at least 3, and preferably by 4 or 5, atomic mass units. The only property that distinguishes the analyte from the internal standard in ID is a very small difference in mass, which is readily discerned by the mass spectrometer. Isotopic dilution procedures are among the most accurate and precise quantitative methods available to analytical chemists. It cannot be emphasized too strongly that internal standards of the same basic structure compensate for matrix effects in MS. Therefore, in the ID method, there is an absolute reference (i.e., the response factors of the analyte and the internal standard are considered to be identical Pickup and McPherson, 1976). 

FIGURE 10 Chromatograms showing the effect of sample solvent and injection volume on peak shape.The peak at 5 min is an impurity in a NCE. Column Supelco Discovery RP amide C16 mobile phase, 2.5% acetonitile in water detection, UV 190 nm injection 10, 20,30,50 and 100 pL sample solvent (A) 2.5% (B) 10% (acetonitrile in water). 

FIGURE 6 Effect of p-cyanophenol on the separation of perchlorate. Column 4x250mm lonPac ASII. Flow rate I.OmLmin. Injection volume 25pL. Detection suppressed conductivity utilizing the Anion Self Regenerating Suppressor (4mm), recycle mode. Ion I—perchlorate (20mgL" ). (a) Eluent lOOmM NaOH. (b) Eluent 50 mM NaOH and 5mM p-cyanophenol. 

FIGURE 4 Peak area precision study to evaluate the effect of injection volumes. The resolution of the sampling syringe was about 0.01 jlL as determined by the digital resolution of the stepper motor and the size of the sampling syringe. 

DLs for impurities should correspond to less than 0.1% of the main compound " compounds that take effect in lower amounts may require lower DLs. For example, imipramine N-oxide hydrochloride impurities and salicylamide impurities have been determined at the 0.01% level. DLs and QLs in CE are generally to some extent higher (in concentration) than in HPLC because of the small optical path (50—100 pm) used for ultraviolet (UV) detection and small injected volume (2—20nl) compared with HPLC 10 mm optical length and 10—200 pi injected volume. ... 

The reproducibilities of both the peak heights and peak areas are shown for replicate injections of a 100 ng mC solution in Fig. 5.9. The results reveal that the injection volume has no significant effect on precision for the standard solution, providing the volume does not exceed 200 pi. Peak area generally offers greater precision (relative standard deviation 2%) at all injection volumes. As the injection volume is decreased, so the width at halfheight decreases. 

Effect of Injection Volume. Table II shows the effect of injection volume on peak broadening and measured column efficiency. The bandwidths listed in Table II are due to injection volume alone, and were measured using an injector connected directly into the flowcell of a low-bandwidth detector. The plate reductions were then calculated for a 24,000 plate column, such as that represented by the bottom line of Table I, assuming 5 and 10 ml, respectively, for exclusion and total permeation volumes. Efficiencies of 23,000 plates at exclusion and 25,000 plates at permeation were actually measured for the column indicated in Table II. The effect of large injection volumes is thus to lose 25 to 50% of the potential column efficiency. 

Tatle II. Effect of injection volume on bandwidth ind realized efficiency of a Perkin-Elmer/PL Gel 5- m 100 Angstrom column eluted with THE at 1.0 ml/min... 

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