Baseline noise and measurement uncertainty in liquid chromatography.

The stochastic properties of baseline noise in HPLC systems with a UV photo-diode array, photo-multiplier and gamma-ray detector were examined by dividing the noise into auto-correlated random process (Markov process) and an independent process (white noise). The present work focused on the effect of the stochastic noise properties on a theoretical estimation of the standard deviation (SD) of area measurements in instrumental analyses. An estimation theory, called FUMI theory (Function of Mutual Information), was taken as an example. A computer simulation of noise was also used. It was shown that the reliability (confidence intervals) of theoretical SD estimates mainly depends on the following factors: the ratio of the white noise and Markov process occurring in the baselines; the number of data points used for the estimation; the width of a target peak for which the SD is estimated.     

Investigations of artifact peaks in sensitive high-performance liquid chromatography methods

Chromatograms of sensitive HPLC methods often show disturbing peaks, which mostly can be traced back easily. But sometimes such peaks are difficult to reproduce and therefore hard to trace. This paper describes two cases where artifact peaks arose due to contamination by the septum and by the sampling equipment, respectively, leading to misinterpretation of impurities and erroneous quantification. Furthermore, troubleshooting and ways to overcome these deficiencies are outlined.     

Urinary testosterone measurement by gas chromatography after solid-phase extraction and high-performance liquid chromatography.

A method was developed for the rapid determination of testosterone in urine. The procedure consists of solid-phase extraction (SPE) followed by high-performance liquid chromatographic (HPLC) clean-up before gas chromatographic determination. Recovery was evaluated by adding [3H]testosterone (10(4) cpm) to urine samples; the mean recovery of radioactivity after SPE and HPLC was 82%. Precision was estimated by repeated measurement of testosterone in four different urine samples; the coefficient of variation was 7.9% (95% confidence limits 6.1-11.4%). Accuracy was evaluated by standard addition and dilution assays; a linear relationship was found between the expected and observed values (r2 = 0.982). The method is rapid, effective and suitable for routine analysis.     

Indirect detection in high-performance liquid chromatography

Indirect detection is a technically simple method to follow and quantify compounds without inherent detector response in high-performance liquid chromatography. The underlying principle can be expressed in simple equations. These equations indicate clearly how the detection sensitivity can be optimized and how disturbance effects can be avoided when the mobile phase gives detector response.     

Monolithic columns in high-performance liquid chromatography

Monolithic media have been used for various niche applications in gas or liquid chromatography for a long time. Only recently did they acquire a major importance in high-performance column liquid chromatography (HPLC). The advent of monolithic silica standard- and narrow-bore columns and of several families of polymer-based monolithic columns has considerably changed the HPLC field, particularly in the area of narrow-bore columns. The origin of the concept, the differences between their characteristics and those of traditional packed columns, their advantages and drawbacks, the methods of preparation of monoliths of different forms, and the current status of the field are reviewed. The actual and potential performance of monolithic columns are compared with those of packed columns. Monolithic columns have considerable advantages, which makes them most useful in many applications of liquid chromatography. They are extremely permeable and offer a high efficiency that decreases slowly with increasing flow velocity.     

Low-energy interactions in high-performance liquid chromatography

Liquid chromatographic systems with very weak excessive analyte-adsorbent interactions have been studied. These systems consisted of a homologous series of n-alkanes as both analytes and mobile phases with a C18 reversed-phase adsorbent. A linear decrease of the analyte retention volume with an increase of the number of analyte carbon atoms was found. Corresponding increases of analyte retention with an increase in the number of eluent carbon atoms was also discovered. An explanation of these two effects on the basis of adsorption theory is proposed. A good correlation of column hold-up volume calculated by interpolation of the retention dependencies for above mentioned systems with that measured by the minor disturbance method has been shown. A study of the temperature dependencies of these alkane systems has shown entropy-governed retention dependencies.     

Stability-indicating high-performance column liquid chromatography and high-performance thin-layer chromatography methods for the determination of olopatadine hydrochloride in tablet dosage form

This paper describes two simple, specific, accurate, and precise methods for estimation of olopatadine hydrochloride (OLO) in tablet dosage form. The first method is a stability-indicating isocratic RP-HPLC method. The analysis is performed on an RP-18 column using 0.1% orthophosphoric acid (adjusted to pH 4.5 with triethylamine)-acetonitrile (75 + 25, v/v) mobile phase at a flow rate of 1 mL/min. Paracetamol (PAR) was selected as the internal standard. Retention times of OLO and PAR were 11.30 +/- 0.02 and 4.70 +/- 0.03 min, respectively. For the HPTLC method, precoated silica gel 60 F254 aluminum sheets were used as the stationary phase; the mobile phase was methanol-chloroform-ammonia (8 + 2 + 0.1, v/v/v). The detection of the analyte band was carried out at 301 nm, and its Rf value was 0.46 +/- 0.03. The analytical methods were validated according to International Conference on Harmonization guidelines. Linear regression analysis data for the calibration plots showed a good linear relationship between response and concentration in the range of 0.1-1 microg/mL and 0.1-0.9 microg/band for HPLC and HPTLC, respectively.     

Application of high-performance liquid chromatography to the measurement of cortisol secretion rate

After administration of [3H] cortisol, the specific radioactivity of the urinary cortisol metabolites is determined by chemical conversion of the 17-hydroxycorticosteroid conjugates to 17-ketosteroids, extraction with Sep-Pak C18 cartridges, derivatisation with 2,4-dinitrophenylhydrazine and isocratic reversed-phase high-performance liquid chromatography. This allows on-line measurement of ultra-violet absorbance; fractions are collected and 3H is measured by scintillation counting. Alternatively extraction and chromatography are done after enzymic hydrolysis of the steroid conjugates, and the major metabolites are determined colorimetrically in the fractions. The results from the two methods were consistent with each other and with published data.     

Comparison of different methods to estimate the uncertainty in composition measurement by chromatography

Natural gas is a mixture that contains hydrocarbons and other compounds, such as CO₂ and N₂. Natural gas composition is commonly measured by gas chromatography, and this measurement is important for the calculation of some thermodynamic properties that determine its commercial value. The estimation of uncertainty in chromatographic measurement is essential for an adequate presentation of the results and a necessary tool for supporting decision making. Various approaches have been proposed for the uncertainty estimation in chromatographic measurement. The present work is an evaluation of three approaches of uncertainty estimation, where two of them (guide to the expression of uncertainty in measurement method and prediction method) were compared with the Monte Carlo method, which has a wider scope of application. The aforementioned methods for uncertainty estimation were applied to gas chromatography assays of three different samples of natural gas. The results indicated that the prediction method and the guide to the expression of uncertainty in measurement method (in the simple version used) are not adequate to calculate the uncertainty in chromatography measurement, because uncertainty estimations obtained by those approaches are in general lower than those given by the Monte Carlo method.     

Limited sample recovery in coupled methods of high-performance liquid chromatography of synthetic polymers

Complex polymer systems, which exhibit multiple distributions in their molecular parameters can be characterized by coupled liquid chromatographic methods. The latter combine entropic (exclusion) and enthalpic (interaction) retention mechanisms. However, recent experimental results suggest that some coupled liquid chromatographic methods may suffer from incomplete sample recovery. This refers, for example, to liquid chromatography under critical conditions of enthalpic interactions and to eluent gradient liquid chromatography. Sample recovery in both latter methods was investigated for selected model systems applying adsorption retention mechanism. Reduced sample recovery was confirmed for both methods. It was revealed that even very high final strength of mobile phase may be insufficient for complete elution of polymer samples in eluent gradient polymer liquid chromatography.     

Estimating the measurement uncertainty in forensic breath-alcohol analysis

The evidentiary weight attributed to forensic breath alcohol results in drunk-driving prosecutions requires that measurement uncertainty be established and shown to be fit-for-purpose. The principal components contributing to breath alcohol measurement uncertainty include: (1) biological/sampling, (2) instrumental, (3) traceability and (4) the water/air partition coefficient for control standards. Employing duplicate breath results from over 92,000 subjects to estimate the biological/sampling component and assuming reasonable forensic values for the other components, the combined and expanded uncertainty is determined for a practical example. The combined uncertainty for an unbiased single determination breath alcohol measurement was: . Employing the expanded uncertainty (k = 2.58), the 99% confidence interval for a mean breath alcohol concentration of 0.0935 g/210 L was 0.0866 to 0.1004 g/210 L. The proportion of combined uncertainty associated with each component was determined to be: biological/sampling 73%, analytical 10%, traceability 13% and water/air partition coefficient 4%. These are forensically acceptable estimates and demonstrate fitness-for-purpose of breath alcohol measurement when employing appropriate elements of quality control.     

Determination of diastereomerization barrier of some flavanones by high-performance liquid chromatography methods

The rate constants and activation energy barriers DeltaG# of diastereomerization reaction of flavanones: naringin, narirutin, hesperidin and neohesperidin were determined. The stopped-flow HPLC (SFM-HPLC), dynamic HPLC (D-HPLC) and enantioselective HPLC combined with the classical kinetic method were applied for determination of these parameters. It was found that the rate constants of diastereomerization were about eight times higher for naringin and narirutin (1.9 x 10(-5) s(-1)) than for hesperidin and neohesperidin (2.4 x 10(-6) s(-1)). No significant differences in the rate of diastereomerization were found between neohesperidosides and corresponding rutinosides.