Peak tailing and column radial heterogeneity in linear chromatography

The correlation between the radial heterogeneity of a column and the tailing of the elution profiles of chromatographic peaks was studied using a numerical method. A parabolic distribution of the linear flow velocity of the mobile phase and of the column efficiency in the radial direction were assumed. Moment analysis showed that peak tailing takes place under such experimental conditions and that it increases with increasing range of radial variations of the flow velocity and the column efficiency. It was also found that the higher the column efficiency, the larger the effect of a given degree of radial heterogeneity on the extent of peak tailing. Peak tailing behavior of columns having different efficiencies could be correlated with each other by an equation. Some characteristic features of tailing peaks were analyzed in connection with the column radial heterogeneity.     

Optimal peak area determination in the presence of noise

A method is presented for determining optimal peak integration intervals on the basis of known peak shapes and noise characteristics. General theoretical considerations lead to conditions yielding optimal integration intervals. Examples of frequently occurring peak shapes and noise types are given, such as gaussian or skewed peak shapes, with band-limited first-order noise or flicker noise superimposed. The optimal integration intervals are approximately independent of the signal-to-noise ratio, and they are considerably smaller than the integration intervals normally used. The resulting expected peak area estimation errors are compared with the estimation error resulting from peak maximum amplitude measurement. On the basis of this comparison, rules of thumb are proposed to determine whether piak maximum measurement or peak integration yields the best results. Simulation of different peak shapes with noise superimposed confirms the results obtained. A flexible method is presented for the optimal measurement of the area of peaks with an unknown shape. This on-line method is simple, and could be used as a simple peak-finding procedure. The method requires almost no computer memory, and can be implemented on a microcomputer. A simulated example of this procedure is given.     

Digital method of net peak area determination in activation analysis

A study of the precision by three methods of gamma-ray photopeak integration has been carried out. COVELL's, total peak area method and the proposed method have been considered. The latter was considered to be the most advantageous because of its relatively high precision and simplicity.     

X-ray chemical shift determination by energy dispersive detection

X-ray energy shifts due to differences in the chemical states of the emitting elements were investigated. The centroids of the Kα and Kβ characteristic lines produced from chromium and manganese compounds were measured and compared with the centroids of X-ray lines produced from the corresponding metals. Excitation was carried out using a ¹⁰⁹Cd radioactive source, and the X-rays were detected with use of a Si(Li) energy dispersive semiconductor detector. solver, a program included in Microsoft Excel for Windows, was used to approximate the form of the X-ray lines with a gaussian function. The results showed that energy dispersive X-ray detection can be used for chemical shift determinations.     

X-ray energy dependence of the properties of the focused beams produced by polycapillary X-ray lens

We investigated X-ray energy distribution in an X-ray microbeam produced by a polycapillary X-ray lens in combination with a sealed-type X-ray tube. This polycapillary X-ray lens has an output focal distance (OFD) of approximately 15 mm. The size of the X-ray microbeam and its OFD were estimated by using a wire scanning method. In our case, the sizes of the X-ray microbeams at the output focal distance were 49 microm for Mo L(alpha), 36 microm for W L(alpha), and 28 microm for Mo K(alpha). The spot sizes depend on the energy of the X-ray fluorescence. The reason for the energy dependence is that X-ray capillary optics is based on the principle of propagation through glass capillaries by means of X-ray total external reflection. The evaluated OFD values of Mo L(alpha) and Mo K(alpha) were slightly changed in 17 microm. However, a deviation of 100 microm from the OFD caused only a 3% increase of the focal spot size. Therefore, we concluded that the OFD showed no significant dependence on X-ray energy.     

Essential peak area normalisation for quantitative impurity content determination by capillary electrophoresis

Summary A number of papers have been published [1–5] which mention that normalisation of CE peak areas (ie division of peak areas by migration time) is necessary to ensure correct quantitation. However, there is a general unawareness of the impact of not performing this simple data manipulation upon impurity results when expressed as % area/area. An exercise has been conducted to exemplify the need for this normalisation using the separation of selected pharmaceuticals as illustrative examples. The impact of normalisation on % area/area results was demonstrated using the pharmaceutical ranitidine, and a synthetic precusor, as test solutes. The UV absorbance of each compound was determined and found to be equivalent. A solution of ranitidine hydrochloride was the spiked with a weighed amount of precursor. The % area/area CE results, when normalised, mateched the known weighed radio. Use of uncorrected peak area data in this instance would have resulted in a severe underestimation of impurity levels. A quantitative analysis of drug related impurities was conducted at three levels of operating voltage whilst keeping all other operating parameters constant. This produced electropherograms having identical peak profiles but each peak having a different retention time and peak area. However, when normalised, the results were identical at each level of operating voltage confirming the validity and necessity of normalisation. A chiral separation of a racemic pharmaceutical was conducted. The uncorrected peak area data indicated the test sample was not racemic whilst the corrected data correctly confirmed that the compound was racemic. The significance and impact upon reported purity data of not normalising peak area data has been clearly demonstrated in this paper.     

An estimation of the column efficiency made by analyzing tailing peak profiles

It has been shown previously that most columns are not radially homogeneous but exhibit radial distributions of the mobile phase flow velocity and the local efficiency. Both distributions are best approximated by fourth-order polynomial, with the velocity in the column center being maximum for most packed columns and minimum for monolithic columns. These distributions may be an important source of tailing of elution peaks. The numerical calculation of elution peaks shows how peak tailing is related to the characteristics of these two distributions. An approach is proposed that permits estimations of the true efficiency and of the degree of column radial heterogeneity by inversing this calculation and using the tailing profiles of the elution peaks that are experimentally measured. This method was applied in two concrete cases of tailing peak profiles that had been previously reported and were analyzed by applying this new inverse approach. The results obtained prove its validity and demonstrate that this numerical method is effective for deriving the true column efficiency from experimental tailing profiles.     

Evaluation of the temporal effect to the peak tailing in flow injection analysis

The deformation of a flow injection analysis peak from a Gaussian shape has two components: spatial and temporal. The former is mainly attributed to the Poisseulie effect in the tubular flow, whereas the latter is related to the observing position (of a fixed detector) at which signal is measured. The combination of the two makes a skewed peak track on the recorder chart. Therefore, an observed peak may imply a substantial fraction of a “false” tail due to the effect of non-simultaneous detection. An expanding-Gaussian model is proposed to simulate the purely temporal effect, and the asymmetric factors were compared with that of the experimental peak shapes. In most cases the peak deformation occurring in flow injection analysis should be regarded as “temporal”. The contribution of the spatial effects (Poisseulie profile and others) might not be as significant as it was thought previously.     

Titanium dioxide determination in sunscreen by energy dispersive X-ray fluorescence methodology

Nowadays there are many sun-protection cosmetics incorporating chemical and/or physical UV filters as active ingredients and there are no official methods to determine these kinds of compounds in sunscreen cosmetics. The objective of this work is to estimate TiO₂ concentration, without sample preparation, employing a portable energy dispersive X-ray fluorescence (EDXRF), aiming to estimate the sun protection factor (SPF) due to the physical barrier in sunscreen composition, and also identify the metals present in the samples. A portable EDXRF system was used for the analysis of fifteen commercial samples. It was also prepared three formulations estimated in FPS-30 using TiO₂ at 5%. Quantification was performed using calibration curves with standards from 1 to 30%. The physical barrier contribution in the SPF, associated to Ti concentration, was determined for all samples. The presence of some elements, like K, Zn, Br and Sr was detected in the sunscreen, identifying chemical elements that were not cited in the formulations. Three commercial samples were analyzed for total SPF determination and the result shows that the measured value is 10% lower than the nominal one.     

Zirconium and hafnium determination by energy dispersive X-ray fluorescence with solid phase preconcentration

Two preconcentration methods has been developed for simultaneous determination of zirconium and hafnium by energy dispersive X-ray fluorescence (EDXRF). The first method is a liquid-solid extraction procedure with the use of an anionic exchange resin modified with xylenol orange. The second is a precipitation procedure carried out in the presence of lanthanum. Both methods permit significant enhancement of sensitivity in comparison with direct measurement in the aqueous phase. The applicability of both procedures for the preconcentration of Zr and Hf prior to their determination by EDXRF was demonstrated by analyzing synthetic mixtures and a sample of zirconium ore. The results obtained with the use of the modified resin show relative standard deviation of about 4% and good agreement with those obtained by spectrographic analysis.     

An approach for measuring peak area of an overlapped peak pair

Summary Based on the exponentially modified Gaussian (EMG) function, a new approach is presented for the quantitation of a partially overlapped peak pair. This approach can be employed in a wide range of peak area ratio and asymmetry, provided that the relative valley between the two peaks is not greater than 55%. The first peak area is calculated by the use of the ratio of the front half-width at height fraction 0.1 to that at heigt fraction 0.5. The second peak area is calculated by subtracing the first peak area from the total area which is provided by a microprocessor-based integrator. The relative error for the first peak never exceeds ±5%, but this is not always the case for the second peak. The relative error for the second peak depends not only on the accuracy of the total area and that of the first peak area but on area ratio as well.     

Physical origin of peak tailing on C18-bonded silica in reversed-phase liquid chromatography

Single component isotherm data of caffeine and phenol were acquired on two different stationary phases for RPLC, using a methanol/water solution (25%, v/v, methanol) as the mobile phase. The columns were the non-endcapped Waters Resolve-C18, and the Waters XTerra MS C18. Both columns exhibit similar C18 -chain densities (2.45 and 2.50 micromol/m2) and differ essentially by the nature of the underivatized solid support (a conventional, highly polar silica made from water glass, hence containing metal impurities, versus a silica-methylsilane hybrid surface with a lower density of less acidic free silanols). Thirty-two adsorption data points were acquired by FA, for caffeine, between 10(-3) and 24 g/l, a dynamic range of 24,000. Twenty-eigth adsorption data points were acquired for phenol, from 0.025 to 75 g/l, a dynamic range of 3000. The expectation-maximization procedure was used to derive the affinity energy distribution (AED) from the raw FA data points, assuming a local Langmuir isotherm. For caffeine, the AEDs converge to a bimodal and a quadrimodal distribution on XTerra MS-C18 and Resolve-C18, respectively. The values of the saturation capacity (q(s,1) approximately equal to 0.80 mol/l and q(s,2) approximately equal to 0.10 mol/l) and the adsorption constant (b1 approximately equal to 3.11/mol and b2 approximately equal to 29.1 l/mol) measured on the two columns for the lowest two energy modes 1 and 2, are comparable. These data are consistent with those previously measured on an endcapped Kromasil-C18 in a 30/70 (v/v), methanol/water solution (q(s,1) = 0.9 mol/l and q(s,2) = 0.10 mol/l, b1 = 2.4 l/mol and b2 = 16.1 l/mol). The presence of two higher energy modes on the Waters Resolve-C18 column (q(s,3) approximately equal to 0.013 mol/l and q(s,4) approximately equal to 2.6 10(-4) mol/l, b3 approximately equal to 252 l/mol and b4 = 13,200 l/mol) and the strong peak tailing of caffeine are explained by the existence of adsorption sites buried inside the C18-bonded layer. It is demonstrated that strong interactions between caffeine and the water protected bare silica surface cannot explain these high-energy sites because the retention of caffeine on an underivatized Resolve silica column is almost zero. Possible hydrogen-bond interactions between caffeine and the non-protected isolated silanol groups remaining after synthesis amidst the C18-chain network cannot explain these high energy interactions because, then, the smaller phenol molecule should exhibit similarly strong interactions with these isolated silanols on the same Resolve-C18 column and, yet, the consequences of such interactions are not observed. These sites are more consistent with the heterogeneity of the local structure of the C18-bonded layer. Regarding the adsorption of phenol, no matter whether the column is endcapped or not, its molecular interactions with the bare silica were negligible. For both columns, the best adsorption isotherm was the Bilangmuir model (with q(s,1) approximately equal to 2 and q(s,2) approximately equal to 0.67 mol/l, b1 0.61 and b2 approximately equal to 10.3 l/mol). These parameters are consistent with those measured previously on an endcapped Kromasil-C18 column under the same conditions (q(s,1) = 1.5 and q(s,2) = 0.71 mol/l, b1 = 1.4 l/mol and b2 = 11.3 l/mol). As for caffeine, the high-energy sites are definitely located within the C18-bonded layer, not on the bare surface of the adsorbent.     

Assessment of coal and ash environmental impact with the use of gamma- and X-ray spectrometry

Gamma-ray spectrometry (GS), energy dispersive X-ray fluorescence (EDXRF) analysis methods and wavelength dispersive X-ray fluorescence (WDXRF) were applied for the studies of some coal components, e.g., sulphur, light and heavy metal element concentrations and naturally occurring radioactive isotope contents. Hundred fifty coal samples originating mostly from eight different coal mines from Upper Silesian Coal Basin and 150 samples of ash obtained from these coal samples in laboratory by total combustion at final temperature of 820°C, were analyzed. Such comparitive analyses can be helpful in selection of most suitable kind of coal for burning in electrical power and heat plants to minimize the environmental pollution.     

Interconversion of cyclosporin molecular form inducing peak broadening,tailing and splitting during reversed-phase liquid chromatography

Summary Reversed-phase liquid chromatography of cyclosporin A, a peptide immunosuppressant, at various temperatures produced remarkably different chromatograms. At 60°C one sharp peak was obtained, at 23°C this became a single broad peak and between 15° and 0°C this became one high sharp peak followed by a tailing or low plateau. Remarkably different chromatograms were produced also by varying the mobile phase flow-rate. The effects of both temperature and flow-rate on the chromatogram could be accounted for by interconversion between two forms of the cyclosporin molecule. Kinetic analysis showed that one form was converted by first-order kinetics with a half-life of 2.0 min at 20°C and that the apparent activation energy for the conversion was about 18 kcal/mol. The other two immunosuppressants, cyclosporin C and D, were also shown to undergo interconversion. This kinetic analysis of the interconversion should be helpful in clarifying the relationship between molecular structure and activity of the immunosuppressants.     

SAXS instrumental broadening and the order dependence of peak width

The width at half-height of SAXS discrete diffraction peaks has been used to compute the number of polyethylene lamellae in a stack. The dependence of this width on diffraction order reflects the nature and magnitude of lattice fluctuations. Within the literature there have been conflicting reports on this order dependence and hence on the nature of the lattice fluctuations. Previous studies have neglected the effects of instrumental broadening. These present studies show that instrumental effects can account for some 40% of the observed first-order peak width and drastically change the ratio of peak widths as a function of order. These studies, carried out on isothermally grown polyethylene single-crystal mats, also demonstrate the importance of the functions chosen to represent the various broadening factors. A mat was made up consisting of randomly stacked lamella with two distinct fold periods. The scattering from this mixed mat could not be described by either of the prevailing theories.     

Peak area measurement of severely overlapped pairs

Summary Based on the ratio of two apparent heights and an empirical correction factor, a method is presented for quantitation of peak areas of severely overlapped pairs. This method can be applied to a wide range of area ratios and peak asymmetries, provided there exists a clear and precise valley-except for shoulder peaks. The relative errors of the first peak are usually within ±3% and theoretical relative error limits are −7.0% to +5.5%. Peak asymmetry of a severely overlapped pair can be determined using the ratio of the front half-width to the rear half-width at 10% peak height of an overlapping profile. The asymmetry so determined is an apparent one and the relative errors are −4% to +5.3% for peaks with 90% relative valley, depending on area ratio, degree of overlap and asymmetry. An empirical area equation for the first peak involving area ratio, asymmetry, resolution and the area measured by a perpendicular drop algorithm is also developed.     

TLC technique for the detection and determination of gamma- BHC in biological material

Summary Five isomers present in technical benzene hexachloride were successfully separated by TLC on silica-gel G impregnated with silver nitrate solution. The -isomer was identified by running a control sample of -BHC. Measurement of the spot area was found suitable for the estimation of 5–20g of-BHC, and was applied to analysis of autopsy tissues. The limit of identification is 0.1g.

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Zusammenfassung Auf mit Silbernitratlösung imprägniertem Kieselgel G konnten dünn-schichtchromatographisch fünf Isomere aus technischem Hexachlorbenzol erfolgreich getrennt werden. Das-Isomere wurde mit Hilfe einer Vergleichsprobe identifiziert. Die Planimetrie der Flecken eignet sich zur Schätzung von 5–20 g-Hexachlorbenzol. Die Methode wurde zur Untersuchung von Autopsieproben angewendet. Die Nachweisgrenze beträgt 0,1g.