Peak shape reproducibility

Figure 4.8 Influence of sample solvent on peak shape [reproduced with permission from N. E. Hoffmann, S. L. Pan and A. M. Rustum, J. Chromatogr., 465,189 (1989)]. Conditions sample, phenylalanine mobile phase, buffer pH 3.5-acetonitrile (92 8) stationary phase, reversed phase UV detector, 210 nm. The sample is dissolved in buffer with (a) 0%, (b) 30%, (c) 50%, (d) 70% acetonitrile.

If the mobile phase is buffered at pH 3, then both of the acids will be neutral species and hence more hydrophobic. This would result in adequate peak shape, reproducible retention times, and longer retention times. Because both compounds are neutral and more hydrophobic, the affinity for the stationary phase will be much greater. An increase in the organic modifier may help to resolve this issue if the retention time is excessive, but may result in a loss of resolution. 

Flgvire 4.5 The influence of endcapping on peak shape and retention of soee PTH-anino acids using a reversed-phase separation system. Peak identification 1 PTH-histidine, 2 PTH-arginine and 3 PTH-valine. (Reproduced with permission from ref. 71. Copyright Preston Publications, Inc.)... 

The pH used in the first C18 separation dimension was rather high (pH 10), however, no peptide loses or carryover, due to on-column precipitation, were observed. Peak shape was comparable to peptide analysis at low pH. Modern stationary phases, based on hybrid silica and stable alkyl bonding chemistry, are well suited for chromatography at extreme pH without compromising column lifetime or analysis-to-analysis reproducibility (Wyndham et al., 2003). 

At times it is necessary to add reagents such as buffers, ion-pairing reagents, or other modifiers such as triethylamine to the mobile phase to improve reproducibility, selectivity, or peak shape. 

Fig. 11.20. Theoretical peak shape for a hypothetical singly charged protein ion of Mr = 15,300 at different settings of resolution. Reproduced from Ref. [98] by permission. John Wiley Sons, 1992.

Reproducible slope ( = sensitivity, response factor) linearity Wavelength accuracy Efficiencies, peak shapes... 

A major problem with viscous fingering is that reproducible peak shapes, albeit distorted, may be observed this may be highly misleading in interpreting SEC results. To obtain reliable results, molecular weight distributions of samples should be obtained as a function of polymer concentrations to arrive at a value in which peak distortion is not present or peak shape does... 

When a particular component eluting at a certain retention volume is to be estimated, this approach can be outlined as follows. Since SEC is extremely reproducible, the peak shape, peak width and peak height are dependent on the amount of the species in the sample volume injected, sample volume and retention time. From these factors the SEC peaks can be simulated or elution pattern of any species within the separation range can be plotted as a function of mass vs. retention volume. The analysis data supplies the concentration of this particular species over two or more 0.5 ml intervals. A match-up computer program has to be developed so that it can pick up the peak shape and concentration based on 3 or 4 data points at known Intervals. 

The success of this modeling can be ascertained by the ability to replicate the observed peak shapes using Gaussian peaks centered at peak positions suggested by the modeling (Fig. 3). For the case where n = 3/2, five peaks were needed 1 for the monomeric A1 and 2 peaks each for each of the two dinuclear A1 species. These peaks were combined to successfully replicate the observed NMR peaks recorded for this sample. When n = 2, the data were reproduced using only 4 peaks, two each for each of the two dinuclear A1 species. Our earlier predictions (11) showed that HCl could combine with either of the dinuclear Al-species in three different positions, which showed different acid strengths. 

Column equilibration ( 10 column vol. recommended) ensures baseline stability, good peak shape, and reproducible retention times. For the specific column chosen in this unit for isoflavone analysis, at least 18 ml total is needed to equilibrate the system with mobile phase. The common practice is to purge the pump system and connect the inlet end of the column to the injector outlet. The initial pump flow should be set at 0.1 ml/min and increased to 0.6 ml/min in 0.1 ml/min increments. Once a steady backpressure and baseline have been achieved, the column is ready to use. Before injecting samples, it is suggested to run a blank gradient first to clean the column and help check for the possibility of impurity peaks. 

Day-to-day reproducibility depends on the lifetime of the different parts of the pumping unit. Corrosion with some additives, such as cetrimide, is well known. Metal particles from frits or leaking from the pump hardware may dramatically affect peak shape or baseline in ion chromatography. 

The separation of safflower oil (SFO)-linseed oil (LSO) methyl esters is shown in Fig. 16. Free fatty acid methyl ester elution reproducibility, resolution, and baseline stability were maintained at sample sizes of 17-170 /zg, although capacity factors (k) decreased approximately 25% between the 17- and 170-/zg sample sizes. The trend of longer retention times with smaller sample sizes was consistent throughout their studies. Peak distortion, such as observed when gas chromatographic columns are overloaded, was not observed in their system. Perhaps larger FAME samples compete for silver ion sites the same way the ACN cosolvent competes for those sites. Excellent peak shapes were obtained, even with sample elution times of 1.5-2.0 h. 

Responses in standard solutions were tested for lead, cadmium, and zinc (see Fig. 7.5). The results obtained show well-defined and single peaks for all of the metals. Sharper peaks were obtained for lead and cadmium compared to zinc. Detection limits of 23.1, 2.2, and 600 pgL-1 were estimated for lead, cadmium, and zinc, respectively, based on the signal-to-noise characteristics of these data (S/N = 3). The reproducibility of the Bi-GECE was also tested and found to be 2.99%, 1.56%, and 2.19% for lead, cadmium, and zinc, respectively. The difference in peak shapes (sharper for lead and cadmium) and in detection limits of these heavy metals can be explained by the binary and multi-component fusing alloys formation of lead and cadmium with bismuth [40]. According to these results, it was deduced that zinc competes with bismuth for the surface site rather than involving an alloy formation with this metal. 

Isocratic focusing techniques, in conjunction with turbulent-flow chromatography, are also explored in this chapter. Isocratic focusing is used to improve chromatographic peak shape, enhance sensitivity, and optimize reproducibility. Isocratic focusing has also led to the development of generic methodologies that eliminate the need... 

Sample introduction is a major hardware problem for SFC. The sample solvent composition and the injection pressure and temperature can all affect sample introduction. The high solute diffusion and lower viscosity which favor supercritical fluids over liquid mobile phases can cause problems in injection. Back-diffusion can occur, causing broad solvent peaks and poor solute peak shape. There can also be a complex phase behavior as well as a solubility phenomenon taking place due to the fact that one may have combinations of supercritical fluid (neat or mixed with sample solvent), a subcritical liquified gas, sample solvents, and solute present simultaneously in the injector and column head [2]. All of these can contribute individually to reproducibility problems in SFC. Both dynamic and timed split modes are used for sample introduction in capillary SFC. Dynamic split injectors have a microvalve and splitter assembly. The amount of injection is based on the size of a fused silica restrictor. In the timed split mode, the SFC column is directly connected to the injection valve. Highspeed pneumatics and electronics are used along with a standard injection valve and actuator. Rapid actuation of the valve from the load to the inject position and back occurs in milliseconds. In this mode, one can program the time of injection on a computer and thus control the amount of injection. In packed-column SFC, an injector similar to HPLC is used and whole loop is injected on the column. The valve is switched either manually or automatically through a remote injector port. The injection is done under pressure. 

Despite the several detailed procedures reported for the fabrication of packed columns for CEC [14,17,20,27,30,47-50], column fabrication may still be regarded as an art. A reliable and reproducible performance of a column depends on the column fabrication. Poorly packed columns can lead to low efficiency, poor resolution, and asymmetric peak shapes. The capillary tubes typically used to fabricate CEC columns are fused silica tubes with inner diameters of 100 pm or less, with 50 and 75 pm I.D. being the most popular. The small inner diameter allows for heat dissipation, which is generated by the applied electric field. Packing such columns is an elaborated process and a skill that requires experience. 

The reduction in energy spread means that double focusing is unnecessary. Micromass achieves excellent peak shape and reproducibility with single (directional) focusing. So the lens system is virtually identical to that used in TIMS, accelerating the ions through a stack that starts with an extraction plate at —900 V and ends with a source slit at —8 kV. 

Quantification of aroma compounds using GC and internal reference has long been a problematic issue.81 Detection response factor, peak shape, discrimination phenomenon at the injector port, and, of course, disproportion during sample preparation were more or less unavoidable. Stable isotopes used as internal standards combined with an MS detector have realized reproducible and far more accurate quantification. The major drawback of this method is the tedious process of preparing the isotope-labeled standards. 

As early as 1997, Taylor et al. [57] demonstrated the gradient separation of corticosteroids in extracts of equine urine and plasma (Figure 10). The sample were purified using solid-phase extraction and automated dialysis, respectively. A reproducibility study revealed that peak broadening occurred only after the analysis of 200 urine extracts. Later, Stead et al. observed that on-line sample concentration could be easily achieved, as longer injection times had minimal influence on peak shape [58]. They demonstrated that the CEC separation of steroids in plasma was superior to HPLC. Several other groups also reported successful CEC separations of drugs and major metabolites in extracts of urine and plasma [59-62],... 

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