{"id":650,"date":"2018-10-06T16:09:16","date_gmt":"2018-10-06T20:09:16","guid":{"rendered":"https:\/\/www2.whoi.edu\/site\/repetagroup\/?page_id=650"},"modified":"2022-12-23T13:06:30","modified_gmt":"2022-12-23T17:06:30","slug":"elemental-isotopic-spectral-and-chromatographic-data-for-hmwdom","status":"publish","type":"page","link":"https:\/\/www2.whoi.edu\/site\/repetagroup\/projects\/dom-and-microbial-ecology\/elemental-isotopic-spectral-and-chromatographic-data-for-hmwdom\/","title":{"rendered":"Elemental, isotopic, spectral and chromatographic data for HMWDOM"},"content":{"rendered":"\n\n\t

Elemental, isotopic, spectral and chromatographic data for HMWDOM<\/h1>\n

On this page:\u00a0Sampling protocols<\/a>\u00a0|\u00a0Elemental and isotopic analysis<\/a>\u00a0|\u00a0Nuclear magnetic resonance spectroscopy<\/a>\u00a0|\u00a0Proton NMR<\/a>\u00a0|\u00a0Carbon-13 NMR<\/a>\u00a0|\u00a0Phosphorous-31 NMR<\/a>\u00a0|\u00a0Heteronuclear Single Quantum Coherence (HSQC) Spectroscopy<\/a>\u00a0|\u00a0Size Exclusion Chromatography<\/a>\u00a0|<\/p>\n\t

Sampling protocols
\n<\/b><\/h3>\n\n

Large-volume seawater samples were drawn from the 20m intake pipe at the Natural Energy Laboratory in Kona, Hawaii (19.727o<\/sup>W, 156.063o<\/sup>N) in February 2013.\u00a0 The samples were filtered to remove bacteria and small particles using a cleaned (10% HCl) Suporflow dual-stage (0.8 and 0.2 microns) Gelman polyether sulfone (PES) cartridge filter (Chisolm-Pall) fitted to an Advanta stainless-steel housing.\u00a0 The high molecular weight dissolved organic matter fraction (HMWDOM) was collected using two spiral wound ultrafiltration systems consisting of a stainless-steel centripetal pump and membrane housings and a fluorinated high-density polyethylene reservoir. Each system was plumbed with Teflon tubing and PVDF valves, and fitted with two ultrafiltration membranes (Osmonics GE-H4040C; Separation Engineering) that nominally retain organic matter with a relative molecular weight >1 kDa (><\/strong>\u00a099% rejection of vitamin B12<\/sub>\u00a0). Membranes were cleaned using isopropanol, detergent (0.01% micro), HCl (0.01 M), and NaOH (0.01 M); stored in sodium azide (0.55 mM); and rinsed with water immediately before use. Approximately 32,241 liters of seawater were concentrated over a period of 14 days.\u00a0 Each day, samples (~1150 L per system) were concentrated to 20 L, filtered through a 0.2 micron PES cartridge filter (Propor demi-cap) into a fluorinated HDPE carboy and stored in a -20o<\/sup>C freezer.\u00a0 The sample was cooled, but did not freeze during storage.\u00a0 The following day, this concentrate was combined with the new day’s concentrate, reduced in volume to 20L, and again stored at -20o<\/sup>C.\u00a0 This process was repeated daily.\u00a0 At the end of the sampling period, the pooled samples were frozen, and returned to Woods Hole for further processing. \u00a0\u00a0Dissolved organic carbon measurements showed 22% of TOC was recovered in the concentrate.\u00a0 In Woods Hole, samples were thawed, filtered through a 0.2 micron PES filter, then filtered again through a stirred cell system fitted with a 30 kD cellulose fiber (ultracell) filter to remove viruses. Samples were desalted by a smaller ultrafiltration system fitted with Osmonics GE-E4010 filters.\u00a0 Twenty liters of sample was concentrated to 2L, to which 2L of ultra-pure water was added.\u00a0 The diluted sample was again reduced in volume to 2L, and the process repeated a total of 12 times, until the no visible precipitate was observed with the addition of sample to 10 mg\/mL silver nitrate. Desalted samples were lyophilized to a fluffy white powder.<\/p>\n

 <\/p>\nRelated Files\u00a0<\/strong>
\n\u00bb Sampling_protocol_180324.docx<\/a>\n\t

Elemental and isotopic analysis<\/h3>\n\n

Samples (typically 4-8 mg) were weighed into tin capsules (9 x 10 mm; Costech), which were carefully folded and submitted for analysis to the University of California, Davis, Stable Isotope Facility (http:\/\/stableisotopefacility.ucdavis.edu\/<\/a>).\u00a0 A description of the instruments and handling protocols can be found at: (http:\/\/stableisotopefacility.ucdavis.edu\/13cand15n.html<\/a>\u00a0).\u00a0 Each sample was run in triplicate with numerous sample blanks (folded tin capsules with no sample inside) interspersed throughout the analysis.\u00a0 In all cases, carbon and nitrogen in blanks were below the limit of detection.\u00a0 The freeze-dried samples were 30% by weight carbon with C\/N of 13.1, d13<\/sup>C = -21.68\u2030, and d15<\/sup>N = 6.88\u2030.<\/p>\nRelated Files\u00a0<\/strong>
\n\u00bb Elemental_and_isotopic_characterization_180365.docx<\/a>\n\t

Nuclear magnetic resonance spectroscopy<\/h3>\nRelated Files\u00a0<\/strong>
\n\u00bb Nuclear_magnetic_Resonance_Spectroscopy_180345.docx<\/a>\n\t\t\t\t\n\t\t\t\t\"Proton\n\t\t\t\t<\/a>\n\t

Proton NMR<\/h3>\n

Proton<\/em>\u00a0nuclear magnetic resonance spectroscopy:\u00a01<\/sup>H NMR was performed on a Bruker AVANCE instrument at 400.13 MHz in D2<\/sub>O solvent with solvent suppression (zgpr).\u00a0 Chemical shifts were referenced to water at 4.8 ppm.\u00a0 Spectra (Figure 1) are similar to prior spectra collected on HMWDOM with major peaks at 5.21 ppm (O-C-O), 3.75 ppm (68%; H-C-OH), 2.77 ppm (2%; C-N-CH3<\/sub>), 2.04 ppm (11%; OOC-CH3<\/sub>), and 1.29 ppm (20%; O-C-CH3<\/sub>).<\/p>\n\tRelated Files\u00a0<\/strong>
\n\u00bb Figure_1_1HNMR_180264.jpg<\/a>\n\t\t\t\t\n\t\t\t\t\"Carbon-13\n\t\t\t\t<\/a>\n\t

Carbon-13 NMR<\/h3>\n

Carbon-13<\/em>\u00a0NMR spectra were collected with broad-band decoupling at 100.61 MHz in D2<\/sub>O and a 200 Hz line broadening.\u00a0 Spectra (Figure 2) were similar to prior solid state spectra collected on HMWDOM with major peaks at 174.8 ppm (8%; O=C-N), 101.6 ppm (8%; O-C-O), 70.8 and 61.5 ppm (71%; H-C-OH; H-C-NH; C-O-CH3<\/sub>), 22.7 ppm (NOCCH3<\/sub>), and 16.7 ppm (O-C-CH3<\/sub>).\u00a0 Peaks at 22.7 ppm and 16.7 ppm contributed 12% of total carbon.<\/p>\n\t\n\t\t\t\t\n\t\t\t\t\"Phosphorous-31\n\t\t\t\t<\/a>\n\t

Phosphorous-31 NMR<\/h3>\n

Phosphorus-31<\/em>\u00a0NMR spectra were collected with broad-band decoupling at 161.98 MHz in D2<\/sub>O.\u00a0 Spectra (Figure 3) were similar to prior solid-state spectra collected on HMWDOM referenced to phosphate at 0 ppm with major peaks at 27.8 and 23.7 ppm (23% total area; C-P (phosphonate), 0 ppm (64%; C-O-P (phosphate and phosphate esters), and -10.2 ppm (14%; O-P-O-P (pyrophosphate))<\/p>\n\tRelated Files\u00a0<\/strong>
\n\u00bb Figure_3_phosphorus_labeled_180284.jpg<\/a>\n\t\t\t\t\n\t\t\t\t\"Heteronuclear\n\t\t\t\t<\/a>\n\t

Heteronuclear Single Quantum Coherence (HSQC) Spectroscopy<\/h3>\n

Heteronuclear Single Quantum Coherence (HSQC) spectroscopy<\/em>.\u00a0 13C\/1H heteronuclear correlation spectra (Figure 4 and 5) show strong cross peaks for fucose (15. 96 x 1.98 ppm) and rhamnose (17.23 x 1.25 ppm), N-acetyl amino sugars (H-C-N(O)C-C<\/strong>H3<\/sub>; 22.76 x 2.04 ppm and , H-C<\/strong>-N(O)C-CH3<\/sub>; 56.0 x 3.40 ppm and 58.4 x 3.36 ppm), and O-methyl sugars (C-O-C<\/strong>H3<\/sub>; 60-63 x3.47- 3.74 ppm).<\/p>\n\tRelated Files\u00a0<\/strong>
\n\u00bb Figure_4&5_HSQC_180265.jpg<\/a>\n\t

Size Exclusion Chromatography<\/h3>\nRelated Files\u00a0<\/strong>
\n\u00bb Chromatographic_Characterization_180364.docx<\/a>\n\t\t\t\t\n\t\t\t\t\"Molecular\n\t\t\t\t<\/a>\n\t

The molecular weight distribution of HMWDOM was determined by size exclusion chromatography (SEC) using two 8 mm x 300 mm NOVEMA Max 100 \u00c5 10 micron columns (polymer standards services, Amherst, MA, USA) connected in series with an 8×50 mm guard column.\u00a0 The SEC columns were calibrated with pullulan standards ranging in molecular weight from 342 Da (DP-2) to 708000 Da (DP-4370) eluted in ultra pure water and detected by refractive index (Figure 6, top panel).\u00a0\u00a0 To minimize interaction with the SEC column, HMWDOM was eluted in 0.1M NaCl in 0.1% trifluoroacetic acid (Figure 6, middle panel), and the results transformed into the molar mass distribution (Figure 6, bottom panel). Size exclusion chromatography shows a well-defined peak in molecular mass at ~2000 Da, and a broad distribution of molecular weights up to 1 x 105<\/sup>\u00a0Da, the upper weight range of the column.\u00a0 Different molecular weight fractions were collected and characterized by proton NMR.\u00a0 Each fraction showed the same NMR spectra, suggesting\u00a0 aggregation is significant.\u00a0 SEC calibration data supplied by Derek Lohman at PSS.<\/p>\n\tRelated Files\u00a0<\/strong>
\n\u00bb Figure_6_SEC_180304.jpg<\/a>\n\n","protected":false},"excerpt":{"rendered":"

Elemental, isotopic, spectral and chromatographic data for HMWDOM On this page:\u00a0Sampling protocols\u00a0|\u00a0Elemental and isotopic analysis\u00a0|\u00a0Nuclear magnetic resonance spectroscopy\u00a0|\u00a0Proton NMR\u00a0|\u00a0Carbon-13 NMR\u00a0|\u00a0Phosphorous-31 NMR\u00a0|\u00a0Heteronuclear Single Quantum Coherence (HSQC) Spectroscopy\u00a0|\u00a0Size Exclusion Chromatography\u00a0| Sampling protocols Large-volume seawater samples were drawn from the 20m intake pipe at the Natural Energy Laboratory in Kona, Hawaii (19.727oW, 156.063oN) in February 2013.\u00a0 The samples…<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":628,"menu_order":1,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/pages\/650"}],"collection":[{"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/comments?post=650"}],"version-history":[{"count":3,"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/pages\/650\/revisions"}],"predecessor-version":[{"id":1272,"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/pages\/650\/revisions\/1272"}],"up":[{"embeddable":true,"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/pages\/628"}],"wp:attachment":[{"href":"https:\/\/www2.whoi.edu\/site\/repetagroup\/wp-json\/wp\/v2\/media?parent=650"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}