Peak area precision injection volume

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... 

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. 

Autosampler precision can be checked by replicate injections of a control sample, with wash injection intervals between every two sample injections. The repeatability of peak areas, mathematically expressed as RSD, is used as a criterion for autosampler precision. For example, when 10 consecutive injections of 10 pL of a solution are performed, the expected RSD for peak area precision ranges from 0.5% to 1.0%. Single injections of different volumes, such as 5, 10, 50, or 80 pL, can also be used, simultaneously checking the linearity of the injector, the detector, and the data system. Another approach to qualify the autosampler involves the gravimetric determination of the average volume of water per injection withdrawn from a tared vial after six 50-pL injections. The procedure... 

Figure 5.6. Diagram illustrating typical autosampler precision vs. injection volumes. CCA, 4 -chloro-3 -sulfamoyl-2-benzophenone carboxylic acid, is a hydrolysis product tor chlorthalidone. Note that peak area precision increases (lower RSD) with larger injection volumes. This was caused by the finite sampling volume precision of the autosampler sampling syringe and stepper motor, which was about 0.01 pL for this particular autosampler. Reprinted with permission from reference 12. See the same reference for additional experimental details.

The inherently lower injection volumes associated with UHPLC can be prone to injection-to-injection peak area imprecision. Injection precision of <0.5% RSD (typically required of pharmaceutical analyses) are readily achievable for injection volumes of >5 p,L. This poses a challenge for method translation from HPLC. Taking into account the scaling factor for conversion of a method from a 4.6 to 2.1 mm diameter coluum, any method with an injection volume of <24 p,L will scale to one where Vinj < 5 p,L. 

Experiments 10-27 are designed to check the autosampler injection precision, pump repeatability and detector/system linearity. One programs the system to automatically inject multiple replicate volumes of a certified test standard. One typically injects 6-10 replicates per volume. The standard component s peak areas are used for calculated injection precision (reproducibility) and system linearity whereas, the retention times are used to calculate pump repeatability. 

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. 

Autosampler The autosampler should have an injector capable of injecting sample volumes from 1 to 100 pL. The injector should have a precision of <1.5% RSD. The injector carryover should be <0.5% of peak area. The autosampler should pick the correct vial. The autosampler should use relays and/or contacts to communicate with the laboratory CDS. Temperature-controlled sample racks should be capable of maintaining samples in the temperature range of 4-15 °C (+3°C). 

Precision. The ability of the injector to draw the same amount of sample in replicate injections is crucial to the precision and accuracy for peak-area or peak-height comparison for external standard quantitation [10,11]. If the variability of the sample and standard being injected into the column is not controlled tightly, the basic principle of external standard quantitation is seriously compromised. No meaningful comparison between the responses of the sample and the standard can be made. The absolute accuracy of the injection volume is not critical as long as the same amount of standard and sample is injected. 

Precision. The precision of multiple injections was evaluated using triplicate injections (at each nominal volume) of the same hGH/desamido hGH mixture that was used for the linearity study (Table I). The percent of desamido hGH in the mixture was calculated as the ratio of its peak area to the total peak area raw peak areas must be corrected for differences in migration velocity as previously described (15). 

There are several conclusions that can be drawn from these results. First, the peak areas have a pooled RSD of about 5%, a value which is much better than that expected for densitometric scanning of electrophoresis gels. Second, note that the standard deviation of the peak area measurements was essentially independent of the volume injected. Thus, the relative standard deviation of the peak area dramatically decreases as the injection volume increases. An injection volume of at least 10 nL is required to obtain good precision (2-3%, excluding a single poor replicate... 

The observed precision is comparable to the values we previously reported for biosynthetic human insulin (16). It also is similar to independent results obtained using a totally automated system (2.9% RSD) and much better than that reported for manual injection (11.8% RSD), both using a hydrodynamic injection technique (21). Finally, the observed precision for the percent desamido, which is really an area ratio similar to what would be obtained by comparison to an internal standard, is excellent for the 10-nL or larger injections. Although the data are insufficient to make a definitive conclusion, it suggests that the observed error is comparable to that obtained from many chromatographic techniques. It also suggests that one of the predominant sources of error is imprecision in the injection volume. The error in injection volume was recently characterized (19). They also reported approximately 1-3% RSD in peak areas for vacuum injection of various compounds. 

An example of the use of an internal standard is the precision enhancement for univariate quantitative chromatography based on peak area. With manual sample injection, the reproducibility of volume size is not consistent. For this approach to be successful, the internal standard peak must be well separated from the peaks of any other sample constituent. Use of an internal standard can be avoided with an autosampler. 

In contrast to the IS method, external standardization may be used in which several standard solutions of varying concentrations of the sample are prepared. Following constant volume injection of each standard solution, a plot of peak area (or height) versus concentration is made, and unknown sample concentrations are obtained from interpolation of the calibration curve. The success of this technique, however, is dependent upon the precision of injection volume, readily accomplished with automatic injection but less so when manual microliter syringes are used. 

Because of the small injection volumes used in CE, peak area reproducibility is poorer than HPLC. The use of high sample concentrations and internal standards makes CE precision values more comparable to HPLC. The precision of solute migration times can also suffer because of variations within the capillary between injections, affecting the EOF. This can be alleviated by careful preparation of buffers and samples and the use of dynamic capillary coating systems. 

Principles and Characteristics Chester et al. [89] have identified some eleven essential considerations for accurate and precise trace analysis by means of capillary SFC, matching HPLC precision. The key to trace analysis below 1 ppm with an FID is providing an injection volume of sufficient size (with complete avoidance of splitting). By injecting volumes up to 0.5 /u.L relative standard deviations of less than 0.3% for the injected volume are achieved with little or no sacrifice of chromatographic performance RSDs for solute areas of 2% are quoted. FID detection permits quantitation of well-shaped peaks as low as approximately 100 pg in mass, thus providing quantitation of sub-ppm solutes in the injection solvent. Packed column SFC, which uses standard size HPLC columns and hence standard HPLC injection systems, yields more reproducible quantitative results than cSFC. Cfr. also ref. [3a]. 

A repeatable volume of the specimen to be analyzed is precisely injected into a gas chromatograph equipped with a flame ionization detector (FID). The peak area of each impurity is measured. Concentration of each impurity is determined from the linear calibration curve of peak area versus concentration. Purity by gas chromatography (GC) is calculated by subtracting Ae sum of the impurities found from 100.00. Results are reported in weight percent... 

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