Measurement of uncertainty in peptide molecular weight determination using size-exclusion chromatography with multi-angle laser light-scattering detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of biopolymers such as peptides or proteins is vital information for understanding their physical/chemical properties. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and size-exclusion chromotography-multi-angle laser light scattering (SEC-MALLS) techniques each with its advantages and limitations were used for molecular weight determination of the SPf66 peptide. The precision of each method was studied using a two-factor fully-nested design with all the analyses performed by the same operator on a single instrument. The overall precision corresponded to the time-different intermediate precision (i.e. time and repeatability conditions). In the case of SEC-MALLS, all potential uncertainty components were carefully evaluated in an Ishikawa diagram, then included and mathematically combined with the uncertainty arising from the accuracy assessment to provide the overall uncertainty. In this case, the refractive-index increment with solute concentration value (dn/dc) provided the most significant contribution to the combined uncertainty. A method for its quantitation is proposed. The accuracy of method B (SEC-MALLS) against reference method A (MALDI-TOF-MS) was assessed using the interval testing hypothesis to limit the risk of unacceptable bias. The results indicate that the bias of B is higher than the limit established at 5%, and is therefore not traceable to A under the studied conditions.     

Light-scattering and turbidimetric detection of silica colloids in size-exclusion chromatography

Silica colloids were separated by size-exclusion chromatography and monitored by fluorimetric and UV detection. In the former means of detection, silica colloids were visualized by light-scattering. The signal intensity based on the light scattering increased with increasing size of the silica colloids. The maximum intensity was observed at excitation wavelengths around 270–290nm. In UV detection, silica colloids were visualized based on turbidimetry, and the signal intensity also increased with increasing size of the silica colloids and with decreasing detection wavelength. The signal intensities for both light-scattering and turbidimetric detection were a linear function of the concentration of the silica colloids. The detection limit at S/N = 3 for 78-nm colloids was 0.06 ppm for light-scattering detection whereas the LOD was 2.3 ppm for UV detection. Effects of mobile phase conditions and flow rate on resolution and peak shape were examined. Use of phosphate buffer allowed the separation of silica colloids of different sizes in size-exclusion chromatography.     

Molecular mass distribution analysis of ethyl(hydroxyethyl)cellulose by size-exclusion chromatography with dual light-scattering and refractometric detection

Dual low-angle light scattering and refractometric detection coupled to size-exclusion chromatography provided proof for the presence of a low amount of stable aggregates/particles in ethyl(hydroxyethyl)cellulose. Unlike the correct size-exclusion chromatographic behavior of the parent polysaccharide itself, the aggregates exhibit variable size-dependent weak retention as a function of flow-rate and of ionic strength of the aqueous mobile phase. Therefore, determination of the molecular mass of non-aggregated polymer is possible in aqueous mobile phase containing 0.1 M NaCl under conditions at which aggregates are completely adsorbed on the column packing irrespective of the flow-rate used. Flow-rate and ionic strength-dependent variations of aggregate behavior as well as model size-exclusion experiments with latex particles indicate that they partly carry a minute charge and have a compact structure. Their weak retention under the separation conditions used suggests a difference in their surface chemistry when compared with the dissolved polymer coils which exhibit a correct size-exclusion behavior.     

Interlaboratory study of a liquid chromatography method for erythromycin: determination of uncertainty

Erythromycin is a mixture of macrolide antibiotics produced by Saccharopolyspora erythreas during fermentation. A new method for the analysis of erythromycin by liquid chromatography has previously been developed. It makes use of an Astec C18 polymeric column. After validation in one laboratory, the method was now validated in an interlaboratory study. Validation studies are commonly used to test the fitness of the analytical method prior to its use for routine quality testing. The data derived in the interlaboratory study can be used to make an uncertainty statement as well. The relationship between validation and uncertainty statement is not clear for many analysts and there is a need to show how the existing data, derived during validation, can be used in practice. Eight laboratories participated in this interlaboratory study. The set-up allowed the determination of the repeatability variance, s(2)r and the between-laboratory variance, s(2)L. Combination of s(2)r and s(2)L results in the reproducibility variance s(2)R. It has been shown how these data can be used in future by a single laboratory that wants to make an uncertainty statement concerning the same analysis.     

Packed column supercritical fluid chromatography with light-scattering detection. I. Optimization of parameters with a carbon dioxide/methanol mobile phase

Summary Evaporative light scattering detectors have, in recent years, gained acceptance in chromatography with dense mobile phases i.e. liquid and supercritical fluid chromatography. In the present work an instrument of this type has been used in packed column supercritical fluid chromatography with carbon dioxide/methanol mixtures. Detector response and signal-to-noise ratios have been determined using squalane as test compound. Nebulizer gas flow, evaporator temperature, photomultiplier sensitivity, and mobile phase composition were found to have an influence on instrument performance. With this type of detector the field of packed column SFC applications can be extended to include non-UV-absorbing substances even when mixed mobile phases or composition gradients are necessary for the separation.     

Estimation of uncertainty for the determination of mercury in food by CVAAS

This paper is aimed at reporting a full validation of a Cold Vapour Atomic Absorption Spectrometry (CVAAS) method for mercury determination in fishery products. Method precision, trueness, limit of detection and limit of quantification are evaluated. The uncertainty of measurement is estimated following the bottom-up approach in conjunction with the validation data as suggested for complex analysis. The precision is evaluated using a pooled relative standard deviation and the recovery is added to the measurement uncertainty budget. Uncertainties of weight of sample, dilution factor, calibration, work solution and analyte amount are also assessed. The most relevant uncertainty sources result those associated to amount of mercury determined in the final sample solution, to method precision and to recovery. The relative contributions have different weights according to the analyst decision regarding recovery correction of results. In conclusion, the adopted CVAAS method fully complies with EU requirements. Presented at AOAC Europe/Eurachem Symposium March 2005, Brussels, Belgium     

Packed column supercritical fluid chromatography with light-scattering detection. II. Retention behaviour of squalane and glucose with mixed mobile phases

Summary Evaporative light scattering detectors can be used to detect organic substances without chromophoric groups in packed column supercritical fluid chromatography (SFC). A detector of this type has been used to detect squalane and glucose after SFC with various packed columns and binary mobile phases. In this study, the amount of organic modifier in carbon dioxide/modifier mixtures was varied. The results give further insight into the mechanisms that influence retention behaviour in packed column separations with super- and subcritical mobile phases. Squalane is an ideal non-polar test solute which shows long retention times on non-polar columns while its elution can be accelerated by non-polar modifiers in carbon dioxide. Glucose is an extremely polar solute containing hydroxyl groups. Elution of this sugar can be improved with polar modifiers. Column packings with polar end groups lead to high capacity ratios and long retention times for glucose. Most columns used in this study contained silica-based packing materials. For purposes of comparison, a polymeric packing (HEMA RP-18) was also employed.     

HPLC assay of underivatized free amino acids with column switching and evaporative light-scattering detection

In this communication the authors report the HPLC SEPARATION OF 20 amino acids without derivatization. Separation was achieved by column swithing using a C-8 reversed-phase column and a cationic ion-exchange column. Detection of the underivatized amino acids was accomplished by means of an evaporative laser light-scattering (ELS) detector. The influence of the temperature of the diffusion tube and nebuolzation gas pressure on detector response is reported. The relationship of peak areas vs. mass show a good log-log linearity within a 1:10 range. Detechtion limit is in the order of 200 pmol for most amino acids under the conditions used.     

Simultaneous determination of eleven quinolones in animal feed by liquid chromatography with fluorescence and ultraviolet absorbance detection

A rapid and simple multi-residue procedure is described for assaying eleven quinolones (cinoxacin, ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid and sarafloxacin) in feeds at sub-additive levels (1–5 mg kg⁻¹). Five grams of sample were extracted by a metaphosphoric acid/acetonitrile mixture (70/30, v/v) and purified onto OASIS HLB cartridges. The determination was achieved by liquid chromatography (LC) using a GEMINI C18 analytical column both with fluorescence detection (FD) and photodiode-array (DAD). Limits of detection for each drug were in the range 0.04–0.8 mg kg⁻¹. Above the limit of quantification (LOQ), in poultry feed the recoveries were from 69 to 98% with relative standard deviations less than or equal 10%. Finally the measurement uncertainty was estimated using the bottom-up approach.     

Distribution analysis of ultra-high molecular mass poly(ethylene oxide) containing silica particles by size-exclusion chromatography with dual light-scattering and refractometric detection

Two different size-exclusion chromatography (SEC) systems, connected in-line either to a low-angle light scattering (LALS) or to a multiangle light scattering (MALS) detector, are employed for determination of molecular mass distributions (MMD) of poly(ethylene oxide) (PEO) samples having a weight average molecular mass up to eight millions. The detrimental effect of the presence of strongly scattering silica particles in the samples on the light scattering signal can be eliminated using a suitable sample dissolution procedure utilizing silica solubility in aqueous mobile phase. The selection of flow-rate and sample concentration have a large impact on the obtained results. Hydrodynamic retardation phenomena and nonlinearity effects are shown to introduce severe errors in the molecular mass distributions unless flow-rate and sample concentration are kept at sufficiently low levels. Self-compensating ability of the dual detection in flow-rate effects is shown to be the main advantage here. A good agreement between the results obtained using LALS and MALS detection is found provided that a carefully selected angular extrapolation procedure is used in the case of MALS data. Thus, using carefully selected experimental conditions, SEC with light-scattering (LS) and refractometric detection proved to be an efficient technique for MMD characterisation also of ultra-high molecular mass (UHM) PEO polymers.     

Quantitative analysis of hydrophobic pulmonary surfactant proteins by high-performance liquid chromatography with light-scattering detection

A new method for the separation and quantification of two hydrophobic lung surfactant proteins (SPs) is described. It is based on size-exclusion chromatography using the apolar stationary phase butyl silicagel with a pore size of 30 nm and isocratic elution with chloroform, methanol and trifluoroacetic acid. The samples were prepared from sheep lung lavage fluid by centrifugation and fractional extraction with butanol and chloroform–methanol. The chromatograms show three peaks in the elution order SP-B, SP-C and lipids. A small peak ahead of SP-B, which disappeared after reduction with 2-mercaptoethanol, was oligomeric SP-B. The response of the evaporative light-scattering detector was non-linear. For preparative high-performance liquid chromatography ultraviolet detection at 279 nm is recommended.     

Fractionation and characterization of phenolic resins by high-performance liquid chromatography and gel-permeation chromatography combined with ultraviolet, refractive index, mass spectrometry and light-scattering detection

HPLC and gel permeation chromatographic (GPC) characterization of complex phenol-formaldehyde resins is described. Reversed-phase HPLC fingerprints the phenolic monomers, dimers and some oligomers. The molecular masses of these phenolic compounds were determined using an ion trap mass spectrometer. GPC analyzes tetrahydrofuran-soluble phenolic polymers beyond HPLC capability. The molecular mass distribution and structural information of the phenolics was determined by both conventional and laser light-scattering calibration methods. GPC with both UV and refractive index detection provides weight concentration of phenolic resin and the molar concentration of the phenol unit in the oligomers or polymers.     

Characterization of dextrans by size-exclusion chromatography on unmodified silica gel columns, with light-scattering detection, and capillary electrophoresis with laser-induced fluorescence detection

Unmodified silica gel size-exclusion columns were used in an on-line combination with light-scattering detection for a size characterization of dextrans. The influence of electrostatic interactions on analyte retention was briefly investigated. Size-exclusion chromatography was also used for evaluation of the fluorescence labeling procedure for dextrans with 8-aminonaphthalene-1,3,6-trisulfonic acid. The derivatives obtained through this procedure were used for electrophoretic measurements with laser-induced fluorescence detection. A comparison between the size-exclusion data and capillary electrophoresis indicates that the effectiveness of fluorescent labeling decreases with molecular mass of the dextran analytes.     

Estimation of width of narrow molecular-weight distributions by size-exclusion chromatography with concentration and light scattering detection

A new method for the estimation of the weight-to-number-average molecular-weight ratio, Mw/Mn of polymers with a narrow molecular-weight distribution, approximated by log-normal distribution, is proposed using size-exclusion chromatography (SEC) with concentration and light-scattering detectors. From experimental data, the Mw/Mn ratios are calculated by two procedures: one using the concentration and light-scattering elution curve for the polymer measured, and the other based on the concentration elution curve and calibration line for a wide range of molecular masses. An iteration method has been developed making the two Mw/Mn ratios converge. The method was applied to a series of narrow molecular-weight distribution polystyrene standards.     

Estimation of measurement uncertainty for the determination of nonylphenol in water using solid-phase extraction and solid-phase microextraction procedures

We describe an estimation of measurement uncertainty calculated by the “bottom-up” approach for the determination of the oestrogenic compound nonylphenol in treated water samples by solid-phase extraction (SPE) and solid-phase microextraction (SPME) procedures and GC/MS detection. The results were compared and the different contributions to the uncertainty were evaluated. A study of the linear range was established and validation was performed for both methods using statistical analysis of several indicative parameters. In terms of validation data, precision (R.S.D. values <20%) and trueness (relative error <11%) were obtained for both methods under day-to-day conditions. The results of the estimation of measurement uncertainty obtained for both methods for concentrations higher than 1 μg/l have demonstrated that the time-consuming SPE method has a lower relative uncertainty (32%) than the SPME method (42.8%). The chromatographic uncertainty value was the main factor in the SPME method whereas the recovery factor (used to calculate the concentration) was the main contribution to uncertainty in the SPE method.     

Size exclusion chromatography with dual-beam refractive index gradient detection of polystyrene samples

Non-aqueous size exclusion chromatography (SEC) of polystyrenes (as model analytes) is examined using the microscale molar mass sensor (μ-MMS) for detection. The μ-MMS is combined with SEC to demonstrate this simultaneously universal and molar mass selective detection method for polymer characterization. The μ-MMS is based on measuring the refractive index gradient (RIG) at two positions (upstream and downstream) within a T-shaped microfluidic channel. The RIG is produced from a sample stream (eluting analytes in the mobile phase) merging with a mobile phase stream (mobile phase only). The magnitude of the RIG is measured as a probe beam deflection angle and is related to analyte diffusion coefficient, the time allowed for analyte diffusion from the sample stream toward the mobile phase stream, and the bulk phase analyte refractive index difference relative to the mobile phase. Thus, two deflection angles are measured simultaneously, the upstream angle and the downstream angle. An angle ratio is calculated by dividing the downstream angle by the upstream angle. The μ-MMS was found to extend the useful molar mass calibration range of the SEC system (nominally limited by the total exclusion and total permeation regions from ∼100,000 g/mol to ∼800 g/mol), to a range of 3,114,000-162 g/mol. The injected concentration LOD (based on 3 s statistics) was 2 ppm for the upstream detection position. The point-by-point time-dependent ratio, termed a ‘ratiogram’, is demonstrated for resolved and overlapped peaks. Within detector band broadening produces some anomalies in the ratiogram shapes, but with highly overlapped distributions of peaks this problem is diminished. Ratiogram plots are converted to molar mass as a function of time, demonstrating the utility of SEC/μ-MMS to examine a complex polymer mixture.     

Determination of inulin in meat products by high-performance liquid chromatography with refractive index detection

Inulin is a naturally occurring carbohydrate with beneficial nutritional and technological properties. A high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of these beta-fructans in meat products, containing this type of additive. The method includes extraction of inulin with hot water, followed by hydrolysis with inulinase enzyme, and determination of the released fructose by HPLC with refractive index detection. An internal standard of rhamnose was used to quantify fructose. The method incorporates a sample blank (without inulinase hydrolysis) for each specimen to subtract contributions of free fructose and fructose from sucrose. The results showed good precision with average RSDs of 2.4% for repeatability and 5.2% for reproducibility. Analytical recovery ranged from 102 to 106%. Satisfactory linearity (r=0.999) was obtained.     

Quantification of copper and zinc species fractions in legume seeds extracts by SEC/ICP-MS: validation and uncertainty estimation

Fractions of Cu and Zn species in legume samples (common white bean, pea, chick pea and lentil seeds and defatted soybean flour) were analysed by on-line hyphenation of size exclusion chromatography and inductively coupled plasma-mass spectrometry. Samples were extracted by 0.02 mol l⁻¹ Tris–HCl buffer solution, pH 7.5. The extraction efficiency lay in the region 60–90 and 60–80% for Cu and Zn, respectively. Quantification of elements in the individual chromatographic fractions was carried out by isotope dilution (ID) and external calibration (EC) techniques. For ID analysis the chromatographic effluent was mixed with the flow of ⁶⁵Cu and ⁶⁸Zn isotope enriched solution and the isotope ratio values ⁶³Cu/⁶⁵Cu and (⁶⁴Zn+⁶⁶Zn)/⁶⁸Zn were measured. In the case of EC technique calibration solutions of elements were injected to the flow of mobile phase by the second injector. Prior entering detector the effluent was mixed with the flow of internal standard solution (In, 50 μg l⁻¹). Both methods have similar precision, however the behaviour of both studied elements was not the same. The chromatographic analysis itself was the main source of variability in the case of Cu. For Zn species analysis, the extraction process and the manipulation with the extract, played the significant role too. It was probably caused by lower stability of the present zinc chelates. The total amounts of Zn found in all chromatographic fractions represented 85–95% of Zn in sampled extract whereas those of Cu approached 100%. In case of small peaks the results of ID and EC were not the same. The EC results were lower then ID results. The great deal of results uncertainty accounts for the precision.     

Simultaneous analysis of esters and acylglycerols in biodiesel by high-performance liquid chromatography with refractive index detection

Currently, gas chromatography (GC) is the most widely used analytical technique to verify the quality of biodiesel in relation to its glyceride and fatty acid methyl ester (FAME) contents, even though its use has some disadvantages, such as damage to the injector and column caused by the presence of trace levels of triacylglycerols in biodiesel, which means the column has to be replaced every 3 months; the need for the sample to be derivatized, which, while improving chromatographic separation, also increases analysis time; and the use of several imported standard solutions. The main aim of this work was to use high-performance liquid chromatography with refractive index detection (HPLC-RI) to simultaneously quantify the glyceride (mono, di, and triacylglycerol) and FAME contents of biodiesel. The proposed method showed satisfactory results when compared with those obtained by the reference method (GC), particularly when these results were within the working ranges of the reference method. The proposed method using HPLC-RI is therefore promising and could potentially be used instead of the reference method, since the results it yielded were statistically equivalent, with 95% confidence, to the results obtained by the reference method (GC) for the nine samples of commercial biodiesel analyzed in this study.     

Analysis of mono- and disaccharides in milk-based formulae by high-performance liquid chromatography with refractive index detection

A simple and reproducible method for the qualitative and quantitative analysis of free mono- and disaccharides (fructose, glucose, galactose, sucrose, lactulose and lactose) in milk-based formulae by high-performance liquid chromatography (HPLC) with refractive index (RI) detection was developed and validated. The method showed good linearity with determination coefficients exceeding 0.99. The limits of detection (DL) in these sugars were 0.17, 0.13, 0.06, 0.16, 0.05 and 0.25 mg/ml, respectively; and the limits of quantification (QL), 0.27, 0.24, 0.20, 0.26, 0.22 and 0.38 mg/ml. The relative standard deviations (R.S.D.s) for repeatability in fructose, sucrose, lactulose and lactose were 0.78, 0.99, 2.91 and 0.46 and the R.S.D.s for reproducibility were 4.8, 6.15, 7.04 and 2.49, respectively. Recoveries in all sugars were between 93 and 113%.