University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Classification: Agricultural -> grain -> barley

Citations 14

"Size Exclusion Chromatographic Determination Of β-glucan With Post-column Reaction Detection"
J. Chromatogr. A 1993 Volume 632, Issue 1-2 Pages 105-110
T. Suortti

Abstract: An extract of barley or malt was analyzed at 65°C on columns of µHydrogel 250 and 2000 in series, with aqueous 50 mM NaOH as mobile phase. A solution of calcofluor in 50 mM NaOH was mixed with the eluate, and the product of the specific reaction of β-glucan with the dye was measured fluorimetrically at 445 nm (excitation at 415 nm) or by UV absorbance. Results showed good correlation with those obtained by flow injection analysis with calcofluor as reagent. Using the specific interaction of β-glucan with calcofluor dye as a detection method for size-exclusion chromatographic analysis, very simple direct extractions can be used for sample preparation. Either fluorescence or UV detection is employed. The calibration graphs are linear between 200 and 20 mg/l, although much more sensitive analyzes are possible. The response remained independent of relative molecular mass in the range studied (1.7 x 10(6)-1.85 x 10(5)). Results for barley and malt show good correlation with those obtained by flow injection analysis using calcofluor.
Chromatography Spectrophotometry Post-column derivatization

"New Continuous-flow-monitoring System For Radioactive Amino-acids"
Agric. Biol. Chem. 1981 Volume 45, Issue 8 Pages 1881-1884
Satoshi MORI

Abstract: A continuous flow system consisting of a high-speed amino acid analyzer and a radioliq. analyzer is described for determining radiolabeled amino acids. The flow cell of the radio-liq. analyzer is packed with Li glass scintillator that fluoresces upon b-irradn. The amino acid analyzer was a column (4.5 x 300 mm) packed with Durrum DC 4A resin, with Li citrate buffer as eluant, and ninhydrin as detection reagent at 570 or 440 nm. The system was tested on the assimilation of proline-U-14C in barley roots. The system had a counting efficiency for 14C of ~45% and a detection limit for 14C of 0.25 nCi. In barley roots, the order of specific activity after 60 min was proline > glutamic acid > glutamine > GABA > aspartic acid > asparagine. After 120 and 180 min, the order was proline > aspartic acid > glutamic acid > glutamine > GABA > asparagine.
Amino Acids Glutamic acid Glutamine Aspartic acid l-asparagine 4-aminobutyric acid Radiochemical

"Physical And Structural Properties Of Barley (1 Fwdarw 3),(1 Fwdarw 4)-β-D-glucan. 1. Determination Of Molecular Weight And Macromolecular Radius By Light Scattering"
Carbohydr. Poly. 1997 Volume 32, Issue 1 Pages 7-15
C. Gómez, A. Navarro*, P. Manzanares, A. Horta and J. V. Carbonell

Abstract: (1->3),(1->4)-β-D-Glucan purified from the 65°C water extract of barley flour, commercial β-glucans and samples obtained by controlled depolymerization of the former, covering a range from 9 times 10^-3 to 6 times 10^-5 dalton, were characterized by size exclusion chromatography and light scattering. The online measurement of molecular weight with a multiangle laser-light scattering photometer gave reproducible and consistent values according to: (a) the process followed in sample preparation; (b) their chromatographic elution in terms of hydrodynamic volume; and (c) the kinetics of depolymerization. However, batch light scattering measurements of β-glucan molecular weights did not give concordant and reproducible results. These last data, together with the study of the very early stages of enzymatic degradation of high molecular weight β-glucans by barley endo-β-glucanases as monitored by the Calcofluor-FIA method, seem to suggest the formation of very labile molecular aggregates.
β-d-Glucan Fluorescence Method comparison

"Quantification Of High Molecular Weight (1->3)(1->4)-β-D-glucan Using Calcofluor Complex Formation And Flow Injection Analysis. II Determination Of Total β-glucan Content Of Barley And Malt"
Carlsberg Res. Commun. 1988 Volume 53, Issue 5 Pages 287-296
Kim G. Jørgensen, Sten Aastrup

Abstract: Complete solubilisation of barley or malt β-glucan was obtained by extraction with dilute acid. Investigation of the extraction procedure by high performance size exclusion chromatography indicates that originally water insoluble high molecular weight β-glucan is solubilized by degradation to smaller fragments. The fluorescence emitted by the specific complex formed between Calcofluor and β-glucan in solution was used to measure the total β-glucan content of barley and malt samples. Examination of the residues after the extraction with dilute acid by degradation with (1 fwdarw 3)(1 fwdarw 4)-β-D-glucanase and by fluorescence microscopy of Calcofluor stained residues confirmed the complete extraction of the β-glucan. Comparison between the Calcofluor-FIA method and the enzymatic method of McCleary and Glennie-Holmes (20) revealed a high correlation between the values obtained by the two methods. 39 barleys and 57 malts analyzed for β-glucan content with the two methods and linear correlation coefficients of r = 0.977 and r = 0.986, respectively, were found. The system enables a large number of barley or malt-β-glucan determinations to be performed within a short period of time with a minimum of manpower. The method is suitable for standard routine analysis in quality control laboratories.
β-d-Glucan Fluorescence Biotechnology

"Molecular-weight Distribution And (1->3)(1->4)-β-d-glucan Content Of Consecutive Extracts Of Various Oat And Barley Cultivars"
Cereal Chem. 1997 Volume 74, Issue 4 Pages 476-480
Michael U. Beer (), Peter J. Wood (,), and John Weisz ()

Abstract: The content and molecular weight (MW) of β-glucan in extracts from a selection of oat and barley cultivars were compared using flow injection analysis and high performance size-exclusion chromatography From 60 to 75% of the β-glucan was extracted from oat and waxy barley by hot water (90°C) containing heat-stable α-amylase, whereas just 50-55% was extracted from nonwaxy barley. Consecutive extractions with hot water and dimethylsulfoxide (DMSO) extracted 65% (nonwaxy barley) or 75-80% (oat and waxy barley) of the total β-glucan. An extraction with sodium hydroxide and sodium borohydride (NaOH/NaBH4) increased the percentage of β-glucan extracted to 86-100% but decreased the MW. The MW of β-glucan in the oat cultivars selected was significantly higher than those in the barley cultivars. The β-glucan extracted from the nonwaxy barley cultivars showed significantly higher peak MW than that from the waxy barley cultivars. 26 References
β-Glucan Sample preparation Extraction

"Application Of The Calcofluor Flow Injection Analysis Method For Determination Of β-glucan In Barley, Malt, Wort, And Beer"
J. Am. Soc. Brew. Chem. 1988 Volume 46, Issue 3 Pages 76-81
Sten Aastrup, and Kim G. Jørgensen

Abstract: Mixed linkage (1?3)(1?4)-?-d-glucan, the main constituent of barley endosperm cell walls, has been known for decades to be a troublesome compound in malting and brewing. Great efforts have therefore been made in order to assay ?-glucan. A new convenient and simple method of extracting the total ?-glucan in barley and malt is described in this paper. The ?-glucan in solution is subsequently assayed using flow injection analysis, by which ?-glucan and the fluorochrome, Calcofluor, are mixed. ?-Glucan in wort and beer can be assayed directly. The increase in fluorescence intensity is observed using a spectrofluorimeter. This method offers advantages over other methods with respect to the number of operational steps involved and the number of samples that can be analyzed per day. High correlation was found between the new method and enzymatic methods recently described.
β-Glucan Fluorescence Sample preparation Extraction

"Analysis Of β-glucans In Barley And Malt: A Comparison Of Four Methods"
J. Am. Soc. Brew. Chem. 1991 Volume 49, Issue 3 Pages 110-115
S. E. Ullrich and J. A. Clancy J. G. Cuti, Jr., and C. M. Tompkins

Abstract: Interest in analyzing β-glucans in barley and malt has been increasing. β-Glucan analyzes are time-consuming and expensive. However, newer, faster methods are being developed. The present study compared four methods of measuring β-glucans in barley and malt flour, including three enzymatic procedures developed at Washington State University (WSU), Cornell University (CU), and Biocon (Australia), and the flow injection analysis (FIA) system using the fluorochrome Calcofluor. Two- and six-row barleys were analyzed, providing a range of β-glucan levels of 3.5-10%. Fully malted to partially malted flours had a range of 0.2-2.5%. Accuracy and precision varied among methods. Absolute β-glucan percentage differed considerably among methods. The rank among methods was consistent for barley (CU gt FIA gt WSU gt Biocon) and different for malt (CU gt WSU gt Biocon = FIA). The methods were in generally good agreement for relative β-glucan values, (r gt 0.98 for comparison of any two methods over all barley and malt samples). However, r values ranged lower within barley (r = 0.89-0.95) and malt (r = 0.81-0.98) subsets. Choice of method will depend on the importance of cost, speed, accuracy, precision, and the type and intended use of the material being analyzed.
β-d-Glucan Method comparison

"Relationships Among The β-glucan Contents Of Barley, Malt, Malt Congress Extract, And Beer"
J. Am. Soc. Brew. Chem. 1998 Volume 56, Issue 4 Pages 164-168
M. J. Edney, D. E. LaBerge, and D. E. Langrell

Abstract: Malting barley breeders have directed a sustained effort toward reducing β-glucan problems through selection of lines exhibiting low barley β-glucan content. This study determined β-glucan contents in barley, malt, and Congress ext. in an effort to define this relationship and to determine which best predicts levels obtained in beer. The impact of environment and genotype on this relationship and prediction was also considered. In total, 60 samples of barley and the corresponding malts, Congress fine-ground extracts, and beers were analyzed for β-glucan content using the Calcofluor-flow injection method of anal. β-Glucan contents of malt and Congress ext. were highly correlated with levels detected in the beer produced from those malts. The correlation between the β-glucan content of barley and beer was significantly lower. This study also demonstrated the strong influence of environmental growing conditions of the β-glucan contents of barley, malt, Congress ext., and beer, although the genetic component was also significant for malt, Congress extract, and beer.
β-Glucan Fluorescence

"Determination Of Total (1->3),(1->4)-β-glucanase Activity By A Calcofluor Flow Injection Analysis Method-D-glucan In Barley And Malt Flour Samples"
J. Cereal Sci. 1996 Volume 23, Issue 3 Pages 293-296
Manzanares P. and Sendra J. M.

Abstract: Flour (0.2 g for barley, 1 g for malt) was mixed with 20 mL 0.05 M HNO3 and heated on a boiling water bath for 20 min. After cooling, the mixture was centrifuged, the supernatant was filtered and the residue was re-extracted with 5 mL 0.05 M HNO3. Total (1->3),(1->4)-β-D-glucan was determined by the fluorimetric Calcofluor-FIA method (J. Inst. Brewing, 1991, 97, 101). Results were compared to those obtained by the European Brewery Convention method (Jorgensen and Aastrup, Carlsberg Res. Communications, 1988, 53, 287) which involves extraction with hot water and H2SO4. Recoveries were 8-24% and 17-27% higher for barley and malt, respectively, using the HNO3 extraction. The RSD was 1.02%.
(1->3),(1->4)-β-d-Glucan Fluorescence Standard method Method comparison

"Modified Fluorimetric Flow Injection Analysis Method For The Determination Of (1->3)(1->4)-β-D-glucan"
J. Inst. Brew. 1987 Volume 93, Issue 5 Pages 396-398
Erika Mekis, György Pintér, György Béndek

Abstract: Aqueous solution of barley β-glucans are analyzed by a modification of the method of Joergensen et al. (Proc. 20th Congr. Eur. Brew. Conv., 1985, 403). The method involves use of the fluorescent dye Calcofluor as specific reagent for (1->3)(1->4)-β-D-linkages. The single-line system used involves only one pump, sample solution being directly injected into the reagent stream. The reaction gives increased intensity of fluorescence, which is detected at 420 nm (excitation at 366 nm) by a flow-through-cell fluorimeter. Analysis takes 20 s.
β-d-Glucan Fluorescence Optimization

"EBC Methods For Determination Of High-molecular-weight β-glucan In Barley, Malt, Wort And Beer"
Monatsschr. Brauwiss. 1989 Volume 42, Issue 4 Pages 162-166
Munck, L., Jorgensen, K.G., Ruud-Hansen, J., und Hansen, K.T.

Abstract: Two methods were evaluated in a collaborative test with 14 participating laboratories. The methods were; (i) that described by, e.g., Joergensen (Carlsberg Res. Commun., 1988, 53, 277), involving flow injection analysis with fluorimetric detection by using the fluorichrome Calcofluor, which binds specifically to β-glucan; and (ii) that described by, e.g., McCleary and Nurthen (J. Inst. Brew., 1986, 92, 168), involving enzymatic hydrolysis. Barley, malt and beer samples were analyzed for six concentration. of β-glucan. Acceptable levels of repeatability and reproducibility were achieved for both methods in the collaborative test and results for the two methods correlated well. Both methods are accepted by the Analytical Committee as Standard Methods.
β-Glucan Fluorescence Standard method Method comparison

"Evaluation Of The Dissolving Properties Of Malts Using The Friabilimeter, Calcofluoride And FIA Method"
Monatsschr. Brauwiss. 1996 Volume 49, Issue 7-8 Pages 220-225
Wackerbauer, K., Hardt, R., Hirse, U.

Abstract: This article describes the most common methods for the determination of the cytolytic dissolving of brewing malts in current usage. On the basis of malting tests and the study of trade malts, the authors assess the information provided from the friabilimeter and calcofluoride method and the determination of beta glucanes in the congress wort using flow injection. The benefits and drawbacks of the various methods are compared.

"Effects Of Ecological And Cropping Factors On β-glucan Content In Oats"
Rostl. Vyroba 1996 Volume 42, Issue 1 Pages 29-33
Hubik, K.; Tichy, F.

Abstract: Exact field trials were carried out mostly with registered oat genotypes at Bystrice nad Pernstejnem and Krukanice in 1993. Effects of the location, genotype and nitrogen fertilization on the content of water-soluble β-glucans in grain dry matter were investigated. It was determined on the basis of increasing calcofluor fluorescence emission (Sigma-Aldrich, USA) caused by binding this dye to β-glucans. A method of flow injection analysis as modified by Jorgensen (1988) using a device of his own construction was used. Barley β-glucans were used as a standard. Results were expressed in percentage. Experimental data were processed by analysis of variance using STATGRAPHICS program. Results of the trial are summarized in Tab. II. At significance level of P = 0.05, β-glucan content in oat grain was the most affected by the genotype and then by the location. Effects of fertilization were not significant. Furthermore, Tab. II suggests highly significant (P = 0.05) interactions, such as genotype x location and genotype x fertilization, where the genotype x location interaction shows a higher proportion as determined by F-ratio. Statistical results obtained by analysis of variance are presented in greater detail in Tabs. III to V. Tab. III evaluates effects of the genotype on β-glucan content in oat grain. On the average of two locations, the highest content of β-glucan was determined in the genotype Adam (3.0%) which significantly differed from the other genotypes studied. The genotype Zlatak and line KR 9506 showed tile lowest average β-glucan contents both being 1.9%. The other genotypes can be divided into two groups between which is a significant difference. The first group includes genotypes David, Auron and Ardo. The last one is a transition to the other group which consists of Abel and Flamingsnova. Effects of fertilization variants on β-glucan content is given in Tab. V. The difference between the average β-glucan content in oat grain in all of investigated genotypes in the variant with the highest effect, i.e. N-a and N-bl and the variant with the lowest effect, N-0, was 0.2% only. That corresponds with the results in Tab. II which showed insignificant effects of the fertilization variant on β-glucan content in oat grain. The other fertilization variants, N-c and N-b, were transitional ones and did not show significant influence. The last factor which can affect β-glucan content in oat grain was the location. Tab. VI shows highly significant difference in the average β-glucan content of all genotypes studied at two locations. The effect of the location on the average β-glucan content in grain of eight genotypes is illustrated in Fig.2. Six genotypes (Abel, Ardo, Auron, David, Zlatak and KR 9046) had average β-glucan contents higher at Bystrice nad Pernstejnem. The highest difference as compared to the location Krukanice was found in the genotypes Ardo and Auron. In contrast, Adam and Flamingsnova reached the highest β-glucan content in grain at the location Krukanice. Among all genotypes, the genotype Adam showed the highest difference (1%). With regard to highly significant differences in β-glucan content in grain of grown genotypes at both locations, it is necessary to study this character under different soil and climatic conditions, and then to elaborate variety zoning.
β-Glucan Fluorescence

"β-Glucan In Barley, Malt, And Congress Wort By Fluorescence"
J. Am. Soc. Brew. Chem. 1990 Volume 48, Issue 4 Pages 145-146
J. Cuti; J. Dufour; S. Home; M. Izawa; B. Jones; W. Ladish; S. Lie; S. Paeschke; and D. Thomas

Abstract: (1->3)(1->4)b-D-Glucan was determined in barley, malt, and congress wort using the fluorochrome Calcofluor and flow injection in collaborative anal. The repeatability standard deviation was acceptable but the reproducibility coefficient of variation was unacceptably high.
β-Glucan Fluorescence Method comparison