Mini-column Chromatography Research Articles

Fully automated minicolumn chromatography

Miniaturized chromatography columns (minicolumns) operated by automated liquid handlers are an integral part of bioprocess purification development. However, these systems can be limited in both their efficiency and accessibility. Because the minicolumn chromatography operation itself is higher throughput, the lower throughput pre- and post-operation activities become the bottleneck of the workflow. Additionally, method writing and operation of the systems while varying multiple parameters, using a design of experiments approach for example, can be error-prone and resource intensive. Here, we have developed a fully automated minicolumn chromatography system to both address these bottlenecks and improve the accessibility of these systems by allowing users to enter chromatography-relevant information through a simplified user interface. Methods have been developed to automate buffer preparation and protein solution titration leveraging modeling and integrated pH probes with feedback control. Chromatogram generation and fraction pooling has additionally been automated to improve the efficiency of post-chromatography operations. We have also demonstrated the flexibility of the system through an example run where both bind-and-elute chromatography and flowthrough chromatography experiments were performed in parallel. Additionally, all methodology and parameters to operate the system have been shared. We hope this will help interested parties improve the efficiency and accessibility of their minicolumn chromatography systems.

Evaluation of alternative methods for radiochemical purity testing of indium-111 capromab pendetide

ObjectiveTo evaluate alternative methods for radiochemical purity testing of indium (In)-111 capromab pendetide after the discontinuation of instant thin-layer chromatography silica gel (ITLC-SG) strips. DesignDescriptive experimental study. SettingUnited States in 2009. ParticipantsNot applicable. InterventionPaper chromatography using Whatman 3MM strips, paper chromatography using Whatman 31ET strips, and mini-column chromatography using silica Sep-Pak cartridges were evaluated in the radiochemical purity testing of 13 consecutive vials of In-111 capromab pendetide prepared for clinical patient imaging procedures. These methods also were evaluated by testing seven aliquots of In-111 capromab pendetide that had been spiked with 5% to 15% In-111 pentetate (diethylene triamine pentaacetic acid). Main outcome measuresCorrelation coefficients, radiochemical purity values for each method compared with ITLC-SG, and procedure times. ResultsCorrelation coefficients of 0.988, 0.996, and 0.979 were found for ITLC-SG compared with Whatman 3MM, Whatman 31ET, and silica Sep-Pak, respectively. Compared with ITLC-SG, mean (±SD) radiochemical purity values differed by −0.8± (range −2.5 to 0.4) for Whatman 3MM, −0.8± (–2.3 to 0) for Whatman 31ET, and −0.5± (–3.6 to 0.7) for silica Sep-Pak. The approximate time required to develop the chromatography strip or elute the mini-column was 3, 14, 5, and 6 minutes for ITLC-SG, Whatman 3MM, Whatman 31ET, and silica Sep-Pak, respectively. ConclusionAll three methods evaluated were acceptable alternatives to ITLC-SG for radiochemical purity testing of In-111 capromab pendetide. Based on its slightly higher correlation to ITLC-SG, slightly tighter SD and range, and slightly shorter development time, Whatman 31ET is preferred in our facility.

Polyphenolic Composition of Marion and Evergreen Blackberries

: The polyphenolics in aqueous extracts of Marion (Rubus sp. hyb) and Evergreen (Rubus laciniatus) blackberries and blackberry seeds were isolated by solid-phase extraction using C18 cartridges and polyamide mini-column chromatography, and characterized using reversed-phase high-performance liquid chromatogra-phy/diode array spectral techniques. Flavonols (primarily quercetin glycosides) were the major phenolics in berries, whereas procyanidins (catechin and epicatechin) and ellagic acid derivatives predominated in seeds. No flavonols were found in seeds. Evergreen berries contained higher level of epicatechin, ellagic acid derivatives, and flavonols than Marion berries, whereas Marion berries and seeds were higher in anthocyanins, total phenolics, and antioxidant properties as measured by oxygen radical absorbance capacity and ferric reducing antioxidant power.

GC/MS analysis of polybrominated diphenyl ethers in fish collected from the Inland Sea of Seto, Japan

Development of an analytical method for polybrominated diphenyl ethers (PBDEs) in fish and their concentration in Japanese marine fish were investigated. Fish homogenate was extracted with diethyl ether/hexane (1+3). The extract was cleaned up by automated gel permeation chromatography (GPC) and then by mini-column chromatography, which consisted of three layers of silica gel and sulfuric acid-impregnated silica gel. The PBDE fraction was concentrated and injected into a GC/MS with negative chemical ionization (NCI). Recoveries of the 15 individual PBDEs (BDE-15, 28, 37, 47, 66, 71, 75, 77, 85, 99, 100, 119, 153, 154, and 209) each at a fortification level of 4 ng/g lipid were in the range of 88–128% and the relative standard deviations (RSD) were 0.43–7.6% ( n= 4). Seven species of marine fish (conger eel, flounder, gray mullet, horse mackerel, red sea bream, sea bass, and yellowtail) were collected from the Inland Sea of Seto, and were analyzed with the developed method. Seven PBDEs (BDE-28, 47, 66, 99, 100, 153, and 154) were detected in all the samples. The most abundant PBDE congener was BDE-47 found in all the samples. Relatively high levels of PBDEs were found in the gray mullets and yellowtails.

Studies on neurosteroids X. Determination of pregnenolone and dehydroepiandrosterone in rat brains using gas chromatography-mass spectrometry-mass spectrometry.

An assay method for pregnenolone and dehydroepiandrosterone in rat brains is developed using gas chromatography (GC)-electron ionization-mass spectrometry (MS)-MS. The extract of the rat brain homogenate containing deuterated internal standard with organic solvent is purified by silica gel minicolumn chromatography. The obtained fraction is derivatized into methyloxime, treated with dimethylisopropylsilylimidazole, and then subjected to GC-MS-MS. The method is applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which are divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increases more than three times after stress, dehydroepiandrosterone in both regions is not so clearly influenced by stress.

Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry.

The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.

Studies on neurosteroids: VII. Determination of pregnenolone and its 3-stearate in rat brains using high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

An assay method for pregnenolone and its 3-stearate in rat brains has been developed using LC–atmospheric pressure chemical ionization (APCI) isotope dilution MS. The extraction of the rat brain homogenate containing deuterated internal standards (ISs) with organic solvent followed by silica gel mini-column chromatography gave two fractions containing pregnenolone and its 3-stearate together with the respective IS. Each fraction was derivatized into 3-acetate-20-methyloxime and 20-methyloxime, respectively, followed by silica gel mini-column chromatography to remove the excess reagents, and then each derivatized fraction was applied onto reversed-phase LC–APCI-MS. The method was applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which were divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increased more than five times after stress, its 3-stearate decreased after stress.

Gel Permeation and Florisil Chromatographic Cleanup and Gas Chromatographic Determination of Organochlorine Pesticides in Eggs

Cleanup of residual organochlorine pesticides (OCPs; alpha-, beta-, gamma-, and delta-BHC; aldrin; dieldrin; p, p'-DDE; o,p'-DDT; p,p'-DDD; and p,p'-DDT) in eggs by gel permeation chromatography (GPC) and Florisil minicolumn chromatography is described. Ten OCPs in purified extract are determined by gas chromatography with electron capture detection. The lipids extracted from whole egg are cleaned up first by GPC with an Envirogel column and an ethyl acetate-cychlohexane (1 + 1, v/v) mobile phase and then by Florisil minicolumn chromatography with 15% (v/v) diethyl ether-hexane eluant. Cleanup is highly efficient. Average recoveries of 10 spiked OCPs (0.0025-0.0125 ppm) ranged from 81 to 101%, with coefficients of variation between 1 and 14%. The detection limit was 0.001 ppm for the 10 OCPs.

Single-step separation of organochlorine pesticide residues from fatty materials by combined use of solid-matrix partition and C 18 cartridges

A rapid single-step partition step between n-hexane and acetonitrile on a two-cartridge system composed of Extrelut-3 with a C 18 cartridge connected downstream has been developed for the separation of organochlorine (OC) pesticide residues from oils and fats. The extract is cleaned up by Florisil minicolumn chromatography before gas chromatography with electron-capture detection. Carry over of fatty material through the partition step is in the order of 4–40 mg for ca. 1.0 g of different oil or fats loaded on the two-cartridge system. Recoveries of 15 OC pesticides from ca. 1 g of soya oil spiked were between 70 and 103% (except HCB=ca. 60%) at spiking levels in the range 0.02–0.1 mg/kg.

Rapid method for the determination of multiple pyrethroid residues in fruits and vegetables by capillary column gas chromatography

A rapid and economical simplified multi-residue method is described for the determination of multiple pyrethroid insecticides in fruits and vegetables. The residues are extracted from crops with methanol and the crop co-extractives are removed by toluene partitioning and Florisil—charcoal minicolumn chromatography. The final extract is analysed by capillary column gas chromatography with electron-capture detection. The recoveries were determined by fortifying six different crops (apples, oranges, cabbages, pears, peppers and tomatoes) with eleven pyrethroids (Py-115, allethrin, biphenthrin, fenpropathrin, cyhalothrin, permethrin, cyfluthrin, flucythrinate, fluvalinate, fenvalerate and deltamethrin) at three levels, 0.01–0.07, 0.10–0.70 and 1.0–7.0 mg/kg. Three determinations were made at each level for each crop. Recoveries of the eleven pyrethroids ranged from 70.4 to 110.0% at the three different levels. The practical determination limit of the method was in the range 3.0–30.0 μg/kg for all the pyrethroid insecticides. The proposed method had major advantages that simplified steps were achieved for the extraction and the clean-up, the solvent consumption was reduced and the analysis time was shortened.

Quantitative analysis of platelet-activating factor in human plasma: Application to patients with liver cirrhosis and disseminated intravascular coagulation

A simple and reliable analytical procedure was developed for determination of platelet-activating factor (PAF) in human plasma using radioimmunoassay (RIA). The assay system consisted of lipid extraction with 2-propanol, lipid separation by Amprep octadecyl minicolumn chromatography and thin-layer chromatography and RIA (charcoal method), and was suitable for quantitation of 30–1000 pg of PAF. The sensitivity of RIA for PAF was notably higher than that for sn-2-short-chain PAF-like phosphatidylcholines. This assay system was then applied for measurement of PAF in human plasma. The normal level of plasma PAF was 54 ± 40 pg/ml ( n = 35), whereas plasma PAF levels in patients with liver cirrhosis (LC) and disseminated intravascular coagulation (DIC) were significantly elevated to 238 ± 314 pg/ml ( n = 14) and 591 ± 328 ml ( n = 14), respectively. The values obtained using this assay system were comparable to those obtained by gas chromatography/mass spectrometry analysis and bioassay. These results indicate that our new assay system is useful for determining changes in the level of plasma PAF associated with diseases such as LC and DIC.

Purified haemoglobin preparations in the evaluation of HbA1c determination by ion exchange chromatography.

The use of ion exchange resins for the estimation of HbA1c in clinical samples rests on the assumption that HbA1c is effectively and efficiently separated from other N-terminally modified haemoglobins and from HbA0. To test this assumption, we applied highly purified preparations of HbA(1a+1b, HbA1c and HbA0 to ion exchange minicolumns, using conditions of application simulating actual blood samples and the first and second elution buffers provided by the manufacturer. The authenticity and purity of the applied haemoglobin preparations were documented by high performance liquid chromatography, gel electrophoresis and carbohydrate content. About 40% of the applied HbA(1a+1b) eluted in the first fraction; 45% eluted in the second fraction, and 10% to 15% required 1 mol/L NaCl to elute from the column. Of the applied HbA1c, 65-80% eluted where expected in the second fraction, about 20% required 1 mol/L NaCl to elute from the column, and the remainder eluted with HbA(1a+1b). Some 3-6% of pure HbA0 applied to minicolumns emerged in the second fraction, with the remainder eluting as expected after making the buffer 1 mol/L in NaCl. The results indicate that the fraction eluting from ion exchange minicolumn chromatography that is designated 'HbA1c' contains HbA(1a+1b), and that a substantial portion of the HbA1c in an applied sample does not elute in this fraction.

Comparative Evaluation of Commercially Available Aflatoxin Test Methods

Five qualitative methods and 1 quantitative aflatoxin analytical method were compared with the Holaday-Velasco (HV) minicolumn and thin-layer chromatography (TLC) methods for corn in an evaluation involving 4 U.S. Department of Agriculture Federal Grain Inspection Service (USDA-FGIS) laboratories, 1 laboratory at the University of Georgia, and 1 laboratory at the University of Arizona. Samples analyzed included 1 set of artificially contaminated corn containing both aflatoxin B1 and B2 (ratio of B1:B2 of 92:8), 1 set of artificially contaminated corn containing only aflatoxin B1, and 1 set of naturally contaminated corn. Levels of total aflatoxin tested were 0, 10, 15, 20, 25, 30, and 40 ppb. Results of analysis of these samples with each method evaluated are reported. Chi-square analyses indicated that performance of the Afla-20-Cup, Aflatest, EZ-Screen, OXOID, and SAM-A methods was not statistically different from that of the HV minicolumn. Agri-Screen results were not statistically different from those obtained with TLC.

Minicolumn chromatography for the detection of sterigmatocystin in agricultural commodities

A simple method for detecting sterigmatocystin in agricultural commodities by minicolumn chromatography was developed. Clean-up as well as semi-quantitative estimation could be done on the same column. The method is suitable for the analysis of samples of rice, wheat, maize and sorghum contaminated with sterigmatocystin and could be used as an effective preliminary screening technique in public health laboratories.

Biliary alkaline phosphatase measured by mini-column chromatography on DEAE-cellulose: application to detection of hepatobiliary diseases.

Using chromatography on diethylaminoethyl (DEAE) cellulose, we measured biliary alkaline phosphatase (BALP; EC 3.1.3.1) activities in sera from 182 patients, most with hepatobiliary disorders but some with non-hepatobiliary diseases. Relative BALP activities were extremely low in otherwise healthy carriers of hepatitis B virus (mean: 5.4 U/L) and in patients with non-hepatobiliary diseases (mean: 5.3 U/L). Although BALP activities were detectable in some cases of liver cirrhosis and chronic hepatitis, these values were generally low (respective means: 12.6 and 12.0 U/L). High BALP activities were detected in patients with primary hepatocellular carcinoma, secondary metastatic liver tumors, and obstructive jaundice: mean values were 27.2, 37.2, and 73.6 U/L, respectively. There was no correlation between BALP activity and bilirubin concentration in patients with obstructive jaundice, nor between BALP activities in obstructive jaundice caused by stones and in those caused by extrahepatic tumor. Some patients with primary hepatocellular carcinoma had high BALP but low alpha-fetoprotein (AFP) values, some others the reverse. Based on AFP alone, the sensitivity for detecting hepatocellular carcinoma was 79%; adding BALP in parallel improved the sensitivity to 97%. We found minicolumn chromatography on DEAE-cellulose useful for determining BALP activity in hepatobiliary diseases.

Characteristics of Visual, Minicolumn and TLC Methods in Detecting Aflatoxin Contaminated Loads of Farmers Stock Peanuts

Abstract Results from two 1980 experiments were used to compare the performance of the visual, minicolumn and thin-layer chromatography (TLC) methods in detecting loads of farmers stock peanuts with aflatoxin. The first experiment was conducted to establish variability of the methods under ideal conditions. The second experiment was conducted to evaluate the performance of the three methods under commercial conditions. In this paper, data and empirical models were used to explore potential improvements in detecting low-level contaminated lots. The minicolumn and TLC methods were very sensitive and provided consistent measurements. Operation characteristic (OC) curves developed by using empirical models (Logistic and Gompertz) were compared to statistical distribution functions used by Whitaker and coworkers. Assuming a desired probability of acceptance of 15%, both the minicolumn and TLC methods would accept lots with 60 ppb aflatoxin, while the visual methods would accept lots with 150 ppb aflatoxin. In crop years similar to 1980, the currently used method would not segregate peanuts to meet the 1–5 ppb (total) tolerance levels and would provide excessive sheller risk. Because of their objectivity, precision and higher sensitivity, the minicolumn and TLC have potential for improving the detection of contaminated loads of farmers stock peanuts.

Pesticide Residues in Imported Australian Meats

A gas chromatographic method for quantitative analysis of organochlorine pesticides and chlorpyrifos in meats has been established. The extracted fats was subjected to silica gel dry column chromatography and further cleaned up by Florisil minicolumn chromatography. As a result of investigation of imported Australian meats, aldrin, heptachlor and chlorpyrifos were not detected in any meat, and dieldrin was found over the tentative maximum residue limit (0.2 ppm) in only one case of cattle. Among the 4 kinds of meats investigated, cattle and sheep meat showed similar low residue levels but the residue levels of p, p′-DDT, p, p′-DDE, dieldrin and heptachlor epoxide in horse meat were relatively high compared to the other meats.

Labelling of monoclonal antibodies 10B, 8C, and M2A with Indium-111

Monoclonal antibodies 10B, 8C and M2A were conjugated with cyclic DTPA anhydride to a substitution level of 1–2 mol DTPA/mol antibody. The DTPA-conjugated antibodies were purified by size-exclusion mini-column chromatography and labelled with [ 111In]In acetate to a specific activity of 37–74 kBq/μg (1–2 μCi/μg). The radiochemical purity of the antibody preparation was 76–81%. The immunoreactive fraction of the radiolabelled antibodies ranged from 0.17 to 0.31, significantly lower than that of their radioiodinated analogs.

Rapid determination of organochlorine pesticide residues in feeds by capillary gas chromatography

AbstractA simple micro‐method is described for determination of 15 organochlorine pesticide residues in mixed feeds. Clean‐up of the petroleum ether extract is by basic alumina minicolumn chromatography and quantitative identification is by capillary gas chromatography with electron capture detection. Cyanopropyl‐polysiloxane chemically bonded fused silica capillary columns have been used to perform high resolution analyses in short time as well as to overcome possible interferences by PCBs. Recovery, repeatability, and detection limits of the proposed procedure are checked and results are also reported for non random samples of 180 feeds collected in Italy.

Improved separation of fetal hemoglobin by mini-column chromatography.

We present chromatographic conditions for separating and measuring fetal hemoglobin on a 5-cm column of Sephadex CM-50. A distinct fetal band is evident, even when present at less than 1% of total hemoglobin. Precision (CV) of better than 5% is achievable.