Gel Permeation Chromatographic Separation Research Articles

Simultaneous determination of hexabromocyclododecanes, polybrominated diphenyl ethers, and dechlorane-related compounds in boxed sushi meals using a developed analytical method

We investigated the concentrations of halogenated flame retardants (HFRs), which include hexabromocyclododecanes (HBCDDs), polybrominated diphenyl ethers (PBDEs), and dechloranes and related compounds (DRCs), in 25 typical ready-made boxed sushi meals (each divided into seafood and non-seafood portions) using a developed simultaneous analytical method involving accelerated solvent extraction and gel permeation chromatographic separation. The developed method yielded good recoveries of surrogates (72–122 %). HBCDDs, PBDEs, and DRCs were detected in all seafood portions. While DRCs were also frequently detected in non-seafood portions, HBCDDs and PBDEs were hardly detected. The estimated dietary intakes of HBCDDs, PBDEs, and DRCs from boxed sushi meals were well below the corresponding health-based guideline values. In conclusion, our study suggests that the intake of HFRs from boxed sushi meals poses low concern for consumer health and that the developed simultaneous analytical method is highly useful for determining HFRs in seafood-based meals.

Probing Aggregation Tendencies in Asphaltenes by Gel Permeation Chromatography. Part 2: Online Detection by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Inductively Coupled Plasma Mass Spectrometry

This work is the second installment of a study that probes the aggregation behavior of asphaltenes by gel permeation chromatography (GPC). In part 1, analysis of GPC aggregate fractions collected from the 2017 PetroPhase asphaltene sample by direct infusion revealed an inverse correlation between aggregate size and aromaticity. However, characterization of the largest aggregate fractions by direct infusion was hampered by solvent contaminant peaks and dynamic range limitations due to the extremely low ionization efficiencies of the larger, more aliphatic species that comprise those fractions. Here, we couple the GPC separation with online detection by positive atmospheric pressure photoionization ((+)APPI) 21 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to overcome those problems and reveal that the most abundant species that comprise the largest aggregate segment are indeed the most aliphatic. The ability to characterize difficult-to-analyze samples, like asphaltenes, is the first major advantage of online coupling. Another benefit is the increased chromatographic resolution afforded by online coupling, which enables a finer examination in the most aggregated region and reveals a local trend opposed to the global trend. The very first species to elute in the largest aggregates were more condensed polyaromatic compounds, and the larger, more aliphatic species elute shortly thereafter in much greater relative abundance.

Silicon Photonic Microring Resonator Arrays for Mass Concentration Detection of Polymers in Isocratic Separations.

Molecular weight distribution (MWD) is often the most informative analytical parameter in polymer analysis, with gel permeation chromatography (GPC) being the most common approach for determining the MWD for polymer samples. Many industrially relevant polymers lack chromogenic or fluorogenic signatures, precluding use of spectroscopy-based detection. Universal detectors, such as evaporative light scattering and charged aerosol detectors, are nonlinear, limiting quantitative polymer analysis. Differential refractive index (dRI) detectors show linear mass concentration sensitivity but are limited for some analyses given that they are incompatible with gradient-based separations, have a limited dynamic range, and require extended thermal equilibration times. In this study, we investigated the utility of silicon photonic microring resonator arrays as a quantitative mass concentration detector for industrial polymer analysis. Microring resonators have optical properties that are sensitive to changes in refractive index, offer an extended dynamic range, have a broad solvent compatibility, and have a linear mass concentration detection for a range of molecular weights. Linear mass concentration detection for microrings was demonstrated through a series of isocratic GPC separations using narrow MWD polystyrene (PS) standards. This detection technology was then utilized in conjunction with conventional GPC detectors to analyze a series of broad MWD PS standards, with results in good agreement with dRI and UV/visible. These results demonstrate the potential of the microring resonator platform as a detector for industrial polymer analysis.

Selective determination of poly(styrene) and polyolefin microplastics in sandy beach sediments by gel permeation chromatography coupled with fluorescence detection

Microplastics generated by plastics waste degradation are ubiquitous in marine and freshwater basins, posing serious environmental concerns. Raman and FTIR spectroscopies, along with techniques such as pyrolysis-GC/MS, are typically used for their identification. We present a procedure based on gel permeation chromatography (GPC) coupled with fluorescence detection for semi-quantitative selective determination of the most common microplastics found in marine shoreline sediments: poly(styrene) (PS) and partially degraded polyolefins (LDPEox). By operating the detector at either 260/280 or 370/420 nm excitation/emission wavelengths PS can be distinguished from LDPEox upon GPC separation. Semi-quantitative determination of microplastics contents is also possible: dichloromethane extracts of PS and LDPEox yield linear plots of fluorescence peak area vs concentration (0–5.0 mg/mL range) and were used as reference materials for quantification of the microplastics content in sand samples collected in the winter berm and dune sectors of a Tuscany beach in Italy.

Characterization of aromatic hydrocarbons and sulfur heterocycles in Saudi Arabian heavy crude oil by gel permeation chromatography and ultrahigh resolution mass spectrometry

The characterization of petroleum crude oil samples remains an overwhelming task for any single analytical technique. Additionally, the compositional complexity of crude oil typically increases with increase in sulfur content and decrease in API gravity. In the present investigation we employed gel permeation chromatography (GPC) for separating Arabian Heavy crude oil into clearly defined fractions, and atmospheric pressure photo ionization Fourier transform-ion cyclotron resonance mass spectrometry (APPI FT-ICR MS) for elucidating the structural commonalities through in-depth characterization of the fractions. The work involved the development of a GPC method by the selection of suitable columns, mobile phase, and optimization of separation conditions. Toluene was found to be suitable as a mobile phase for the analysis of nonpolar aromatic compounds as well as compatible with the subsequent APPI FT-ICR MS measurement. The GPC fractionated aromatic compounds based on the number of carbon atoms in alkyl chains. Compounds with a higher number of carbon atoms in their alkyl chains elute earlier in the GPC separation. This observation was true for aromatic hydrocarbons (HC), mono-sulfur (S1) and di-sulfur (S2) containing compounds. Using respective planar aromatic cores as parent structures, we systematically calculated the average number of alkyl carbon atoms present in each GPC fraction. The average number of alkyl carbon atoms were found to be 36, 27, 17, 9 and 5 in successive GPC fractions (from early to late elution), with only two alkyl carbon atoms overlap between successive fractions.

(Invited) New Cyclic Porphyrin Arrays ~ Preparation and Specific Elution Behavior on Modified Silica-Gel Columns

Functionalized nanorings are expected to provide unique reaction fields in which specific and selective molecular transformation occurs. Here, we designed new cyclic porphyrin trimers (CP3s) in which three porphyrin moieties were connected through 2,2’-bipyridyl groups at their 3,3’-positions. CP3s were synthesized by nickel-mediated reductive coupling reaction of bis(2-chloro-4-methoxycarbonyl-6-pyridyl)porphyrinatozinc derivatives (1).(ref. 1) In a CP3, three bipyridyl parts are directed toward the center of the ring. Unlike metal-organized porphyrin cages formed from tetrapyridyl substituted porphyrins and metal ions, the three bisnitrogen pairs on the three bipyridyl moieties in CP3 are not used to construct the macrocyclic structures. Therefore, active metal species, such as coordinatively unsaturated complexes can be generated inside the rings, whereas generation of such active species in metal-oraganized nanocages is generally difficult because of destabilization of the skeletons. As well as the three bidentate 2,2’-bipyridyl ligands, three tetradentate porphyrinato parts gives metal ligands. The two kinds of metal ligands connected in a circular manner to provide a macrocyclic ligand for multicofacial and multinuclear complexes within a one-nanometer cavity. In fact, three π-allyl palladium complexes were incorporated into a zincporphyrin nanoring to give a hexametallic complex. During the synthesis of CP3s, an efficient separation method by using modified silica-gel columns was discovered. Reductive coupling of bis(chloropyridyl)porphyrin 1 gave mixtures of acyclic and cyclic porphyrin oligomers. They were eluted in the order of hydrodynamic volume of the oligomers on gel permeation chromatography (GPC), and repeated GPC processes were necessary to obtain pure material of CP3. To reduce the time- and solvent-consuming processes, several modified silica-gel columns, COSMOSIL series supplied from Nacalai Tesque, PYE (pyrene-modified), π-NAP (naphtalene-modified), PBB (pentabromobenzene-modified), NBE (nitrobenzene-modified), and CN-MS (cyanopropyl), were tested. When a mixture of pyridine and toluene was used as an eluent, extremely specific retention behavior of CP3 was observed on the modified silica-gel columns. Under the conditions, other acyclic oligomers were eluted without retention. Therefore, the target CP3s can be much easily isolated from reaction mixtures compared with GPC separation. The observed specificity is probably related to molecular recognition between CP3s and substituents on the used modified silica-gel columns. Reference 1) Ohkoda, Y., Asaishi, A., Namiki, T., Hashimoto, T., Yamada, M., Shirai, K., Katagami, Y., Sugaya, T., Tadokoro, M. and Satake, A., Chem. Eur. J., 2015, 21, 11745–11756. doi:10.1002/chem.201501854.

Silicon nanoparticles synthesised through reactive high-energy ball milling: enhancement of optical properties from the removal of iron impurities

Alkyl-terminated silicon nanoparticles (SiNPs) were prepared through reactive high-energy ball milling with contamination of iron from the steel milling materials. Iron impurities in the form of iron nanoparticles cause a decrease of photoluminescence intensity and an increase of the UV absorption. The iron impurities were removed either by gel permeation chromatography separation or by treatment with an aqueous HCl solution. The photoluminescence properties of the SiNPs were enhanced after the removal of iron. Transmission electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and proton nuclear magnetic resonance were used to determine morphology, elemental composition and surface passivation of SiNPs.

Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides

Rapeseed protein isolate (RPI) was hydrolyzed with Alcalase followed by reverse-phase high performance liquid chromatography (RP-HPLC) purification of bioactive peptides. The rapeseed protein hydrolysate (RPH) obtained after 4h digestion with Alcalase had a degree of hydrolysis (DH) of ∼11%. Gel permeation chromatography separation showed high contents of low molecular weight peptides in the RPH when compared to the RPI. After preparative and analytical RP-HPLC separations, three peptides (LY, TF and RALP) were purified and amino acid sequence determined by tandem mass spectrometry. LY (IC50, 0.11mM) was the most potent (p<0.05) against ACE activity when compared to TF (IC50, 0.81mM) and RALP (IC50, 0.65mM). However, RALP (IC50, 0.97mM) was the most potent (p<0.05) against renin activity when compared to LY (IC50, 1.87mM) and TF (IC50, 3.1mM). Single oral administration (30mg/kg body weight) to spontaneously hypertensive rats showed LY and RALP to be the more effective hypotensive agents with maximum blood pressure reduction of −26 and 16mmHg, respectively when compared to TF (−12mmHg). The results suggest that the higher number of hydrophobic amino acid residues LY and RALP contributed to their higher in vitro and in vivo activities when compared to TF.

Albumin-Mediated Selenium Transfer by a Selenotrisulfide Relay Mechanism

In this study, we demonstrated a human serum albumin (HSA)-mediated selenium transfer; the selenium exported from red blood cells (RBCs) was bound to HSA through the selenotrisulfide and then transferred into the hepatocyte. After the treatment of the RBCs with selenite, the selenium efflux from the RBCs occurred in an HSA concentration-dependent manner. Pretreatment of HSA with iodoacetamide almost completely inhibited the selenium efflux from the RBC to the HSA solution. The selenium efflux experiment was carried out in an HSA solution (45 mg/mL), and subsequently the HSA solution was subjected to gel permeation chromatographic separation. The peak fraction of the selenium content was consistent with that of the HSA. The selenium bound to HSA in this solution was completely eliminated by a treatment with penicillamine (Pen), which resulted in the generation of penicillamine selenotrisulfide, PenSSeSPen. The selenium efflux from the RBCs was also occurred in a Pen solution, and PenSSeSPen was observed in the resulting Pen solution. The selenium exported from the RBC was thought to bind to the HSA via a selenotrisulfide linkage with its single free thiol. A model of the selenium-bound HSA was prepared by the reaction of the HSA with PenSSeSPen. The selenium from PenSSeSPen can bind to HSA by a thiol exchange between Pen and the free thiol of HSA, which produces the selenotrisulfide-containing HSA (HSA-SSeSPen). When HSA-SSeSPen was incubated with isolated rat hepatocytes, the selenium content in the hepatocytes increased along with its decrease in the incubation medium. To verify the results from the model experiments using HSA-SSeSPen, we conducted the HSA-mediated selenium transfer experiment from RBC treated with selenite to the hepatocytes. The selenotrisulfide-containing HSA was able to transport the selenium into the hepatocyte. Overall, the selenium transfer from the RBC to the hepatocytes involves a relay mechanism of thiol exchange that occurs between the selenotrisulfide and thiol compounds (selenotrisulfide relay mechanism: R-SSeS-R + HSA-SH --> HSA-SSeS-R + R'-SH --> R-SSeS-R').

Improved separation of palladium species in biological matrices by using a combination of gel permeation chromatography and isotachophoresis.

The binding of palladium to high-molecular-mass compounds in palladium-treated lettuce is investigated as an example for a biological matrix. The total palladium concentration in lettuce leaves is 10.3 ng/g wet weight. After homogenization, high-molecular-mass compounds (> 10 kDa) are isolated by ultrafiltration. For separation of these palladium species a combination of preparative gel permeation chromatography (GPC) and preparative isotachophoresis (ITP) is used. Palladium is determined in separated fractions by using a highly sensitive total reflection X-ray fluorescence (TXRF) method after preconcentration. After GPC separation, four main fractions of palladium species are collected, each containing palladium in ng quantities (3-10 ng). Two of these fractions are further separated by ITP, yielding at least three main peaks per GPC fraction, each containing palladium in the range of 0.3-3 ng. These palladium containing peaks are characterized by high-performance size exclusion chromatography (HPSEC) and capillary isotachophoresis (cITP) in parallel. HPSEC enables the estimation of the molecular mass of six main palladium peaks, covering a molecular mass range of 69-200 kDa. It is also shown that the estimation of molecular mass after separation is more reliable than the respective estimation directly in the first GPC run. However, cITP reveals that each of the separated peaks is still a mixture of at least five different compounds.

Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides.

Hyaluronan was partially depolymerized on a large-scale quantity using bacterial hyaluronidase (E.C. 4.2.2.1) for preparation of chemically fully O-sulfated oligosaccharides. The hyaluro-oligosaccharide (HAoligo) mixture obtained by partial digestion was repeatedly applied to low pressure gel permeation chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer. The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance ((1)H NMR) spectroscopy, capillary electrophoresis (CE) on normal polarity mode, and a newly established separation method by normal phase chromatography with Amide-80 column. The purified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulfation. Characterization of chemically fully O-sulfated HAoligos was performed by both chemical compositional analyses after hydrolysis and (1)H NMR spectroscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAoligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase (bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ranged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfated hyaluronan (MW 100 kDa) through both competitive and noncompetitive effects.

GPC separation of polymer samples for MALDI analysis

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important technique to characterize the average molecular weights, oligomer repeat units, and end groups of polymer materials. Although MALDI methods have been developed for a broad variety of different synthetic polymers, MALDI continues to struggle with polymer samples having broad polydispersity (PD). We have combined MALDI and gel permeation chromatography (GPC) analyses for broad PD polymer samples with the use of a liquid chromatography (LC) interface. The LC interface uses heated sheath gas and a capillary nozzle to remove most of the mobile phase and deposit the GPC eluants on the precoated matrix on a moving MALDI plate. Our experiments demonstrate that the combination of GPC-LC interface-MALDI can aid in the characterization of broad PD samples, the verification of the presence of low-intensity, high-mass oligomers, and the detection of minor series in polymer samples.

Fractionation and characterization of mushroom dietary fiber (nonstarch polysaccharides) as potential nutraceuticals from sclerotia of Pleurotus tuber-regium (Fries) singer.

The nonstarch polysaccharides (NSPs) in the total dietary fiber (TDF) from the sclerotia of Pleurotus tuber-regium (tiger milk mushroom) were fractionated by the sequential use of chemical solvents. About half of the TDF was solubilized and two major alkali-soluble fractions (1 and 4 N sodium hydroxide) that contained 126 and 293 g/kg TDF were obtained. Sugar analysis and infrared spectroscopy indicated that the NSPs in these alkali-soluble fractions were mainly beta-glucans and chitin. These alkali-soluble NSPs were further purified by anion-exchange chromatography followed by gel permeation chromatographic separation. Methylation analysis revealed that these purified glucans were highly branched and contained a mixture of sugar linkages of beta-1,3, beta-1,6, and beta-1,4. The potential use of these sclerotial beta-glucans as nutraceuticals was discussed.

REE bound polysaccharides in leaves of Dicranopteris dichotoma by MAA

The binding of REEs with polysaccharides (PSs) in the leaves of fernDicranopteris dichotoma (DD) has been studied by molecular activation analysis (MAA). The PSs of different solubilities in the leaves of DD, which were named cold-water-soluble, hot-water-soluble, acidic-soluble and basic-soluble PSs, were obtained using biochemical separation techniques. After deproteinization and gel permeation chromatography separation, the absorption curves of the PSs were obtained by colorimetry. The eight REEs (La, Ce, Nd, Sm, Eu, Tb, Yb and Lu) were also detected in the PSs by neutron activation analysis (NAA). The results obtained showed that the REEs were bound firmly with the PSs in the leaves of DD. The molecular weight measurement on Sephadex G-200 demonstrated that those PSs bound with REEs were mostly small molecular weight polysaccharides (10 000 to 20 000 u).

On-line multicomponent determination of organic compounds in water following gel permeation chromatographic separation

Gel permeation chromatography with on-line multicomponent determination of analytes in the column effluent is described. The system includes simultaneous measurement of organic carbon, organic nitrogen and organic halogens based on segmented flow analysis and on-line UV oxidation. Quantitative recoveries of various organic model compounds were obtained. Additionally the diode-array spectrophotometric detection applied gives UV-visible absorption spectra providing fingerprints for the specific components present in water samples. The system was applied for characterization of wastewater from chemical industry.

Chromatographic separations of metal chelates present in commercial fertilizers: I. Development of a gel permeation chromatographic separation method for the identification of metal chelates in commercial fertilizers

The separation of iron(III) chelates of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxy-2-ethylenediaminetriacetic acid (HEEDTA), ethylenediaminedi( o-hydroxyphenyl acetic acid (EDDHA) and ethylenediaminedi( o-hydroxy- p-methylphenyl)acetic acid (EDDHMA) and the Cu(II), Zn(II) and Mn(II) chelates of EDTA by gel permeation chromatography on Bio-Gel P2 and Fractogel HW-40(S) was investigated. The chelates FeDTPA, FeHEEDTA, FeEDDHA and FeEDDHMA were separated on a 35 × 1 cm I.D. Fractogel column using 0.05 M NaCl-0.005 M KH 2PO 4 (pH 8.0) with a total analysis time of 3 h. FeEDTA was not completely resolved from FeDTPA, and FeHEEDTA, CuEDTA, ZnEDTA and MnEDTA were found to co-elute with FeDTPA. The method is applicable to the qualitative analysis of fertilizers containing a single trace element in combination with a single chelating agent. Positive identification of a chelate is achieved by comparison of retention time data and identification of the metal present by atomic absorption spectrometry or inductively coupled plasma mass spectrometry. Quantification can be achieved by comparison of peak height or area with that of standards.

Separation and Quantitation of Monoclonal Antibody Aggregates by Asymmetrical Flow Field-Flow Fractionation and Comparison to Gel Permeation Chromatography

During the downstream processing of monoclonal antibodies (Mab) the formation of dimers and/or oligomers may occur. Today, the most common technique for determination of aggregate content is gel permeation chromatography (GPC) but it has limited resolution and separation speed. Oligomers are particularly difficult to resolve. Asymmetrical flow field-flow fractionation (FFF) is a new analytical method for the separation of Mab aggregates. The monomer and dimer peaks were well resolved and three additional peaks, thought to be trimers, tetramers, and pentamers were partially resolved. The total time for a separation can be kept as short as 6 min. The GPC separation takes about 15 min with less resolution. A study of the reproducibility for the two techniques revealed that the precision was slightly better for GPC. Investigation of the dependence of sample concentration showed that the load limit for FFF was about 4 μg of Mab. The GPC technique requires prefiltered samples in order to avoid clogging of the column. For FFF it is possible to inject samples containing precipitated material without any pretreatment. The flow conditions can be adjusted so that the precipitated material elutes with the front, well separated from the monomer peak.

Evaluation of gel permeation chromatographic techniques and diode-array UV detection for the characterization of biotechnological fermentation substrates and broths

Gel permeation chromatography (GPC) was used for the analysis and characterization of fermentation substrates and broths. Chemical or enzymatic hydrolysis of wood and waste waters from the pulp industry were used as the substrate in ethanol fermentation. Sample pretreatment was carried out by liquid-liquid extraction with dichloromethane and water. The two phases were subjected to analysis by organic and aqueous GPC using a polymer-based GPC column, a silica support and a GPC column with dichloromethane, water and water-miscible tetrahydrofuran as the mobile phases. respectively. Diode-array UV spectrophotometric detection (DAD) of substrates and broths was utilized for characterization complementary to the GPC separations. The DAD method will give UV absorption spectra for different samples providing unique fingerprints for the specific compounds present.

Certain physical properties of water soluble non-starch polysaccharides from wheat, rye, triticale, barley and oats

The yield of water soluble non-starch polysaccharides (WSNSP) prepared from whole grains of wheat, rye, triticale, barley and oats was 0.6, 1.8, 0.7, 1.2 and 1.0% respectively of the dry matter. The WSNSP from wheat, rye and triticale contained mainly pentoses (74, 86 and 81% respectively of the total monosaccharides), while in the WSNSP of barley and oats glucose (74 and 69% respectively of the total monosaccharides) was the main monosaccharide. All the WSNSP samples contained some amount of protein (7% in rye-WSNSP to 18% in triticale-WSNSP) attached to their low molecular weight fractions as observed in the gel permeation chromatographic separation. The major amino acids in these samples were aspartic and glutamic acids. No hydroxyproline was found in these samples. Their water binding capacity determined as the amount of unfreezable water using differential scanning calorimetry showed that the WSNSP of wheat, rye, triticale, barley and oats was 0.41, 0.47, 0.42, 0.49 and 0.44 g/g dry matter respectively. The WSNSP of wheat and barley showed higher emulsifying capacities determined as the amount of oil emulsified by one gram of the sample in water. The intrinsic viscosity of the water solutions of these non-starch polysaccharides shows a direct relation to their molecular weight and their simha shape factors indicated that all the samples were of an extended shape. However, the intrinsic viscosity of the water solution of rye-WSNSP was significantly higher than that of wheat, triticale, barley and oats.

Preparation and Characterization of the High Molecular Weight [3H]Hyaluronic Acid

Two methods for the preparation of high molecular weight [3H]hyaluronic acid were investigated. In the first one, hydrogen atoms in the molecule were replaced by tritium. This isotopic substitution was performed in aqueous solution using Pd/CaCO3 as the catalyst. In the second method, the high molecular weight hyaluronic acid was alkylated with [3H]methyl bromide in liquid ammonia at a temperature of -33.5 °C. High-performance gel permeation chromatographic separation method was used for the isolation and characterization of the high molecular weight [3H]hyaluronic acid. Molecular weight parameters for the labelled biopolymers were Mw = 128 kDa, Mw/Mn = 1.88 (first method) and Mw = 268 kDa, Mw/Mn = 1.55 (second method). The high molecular weight [3H]hyaluronic acid having Mw = 268 kDa was degraded further by specific hyaluronidase. Products of the enzymatic depolymerization were observed to be identical for both, labelled and cold biopolymer. This finding indicates that the described labelling procedure using [3H]methyl bromide does not induce any major structural rearrangements in the molecule.