Sample preparation was performed according to our standard operating procedure, previously described by Rouvroye et al. [29 (link)]. In short, a calibrated scale (Mettler Toledo, AT 261 Delta Range, Columbus, OH, USA) was used to weigh approximately 500 mg of fecal and frozen urine sample. Subsequently, the sample was transferred to a glass vial (20 mL, Frickenhausen, Germany), which was restored in a −20 °C freezer [22 (link)]. The fecal and urine samples were sent to the BioMedical Sensors Lab, School of Engineering, University of Warwick (Coventry, UK) on dry ice (−80 °C) for VOC analyses.
At261 deltarange
Manufactured by Mettler Toledo
Sourced in United States
The AT261 DeltaRange is an analytical balance from Mettler Toledo. It is designed to provide precise weighing measurements with a maximum capacity of 210 grams and a readability of 0.1 milligrams.
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9 protocols using at261 deltarange
1
Standardized Sample Preparation for VOC Analysis
2
Multimatrix Trace Element Analysis
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Invertebrate, sediment and fish tissue samples were transferred into pre-weighed PP tubes and the total weights were assessed (Mettler Toledo AT261 Delta Range). Procedure blanks and standard reference material including mussel tissue (NIST-2976; National Institute of Standards and Technology, USA)) and estuarine sediment (BCR-277R) were included. The samples were freeze-dried for four days in a Thermo Scientific, Heto PowerDry LL3000 freeze dryer, which was connected to a Thermo Savant VL P80 Valu Vacuum Pump. The samples were removed and stored until digestion. They were grouped into two categories according to dry weight and appropriate acid volumes were added.
Samples were transferred to 10 ml glass tubes, and a mixture of HNO3 (69%) and HCl (37%) (1:3; Aqua Regia) was added to keep mercury in a stable solution, hereafter they were left at room temperature for 24h. Subsequently, 200 μl of H2O2 was added to digest the fat tissue. The samples were digested in two steps at 120°c and 160°c respectively in a pressurized microwave system (CEM Discover SP-D; CEM Corporation, Matthews, NC 28106, USA). After digestion, the samples were diluted with Milli-Q water. For water samples, a mixture of HNO3 and HCl (1:3) was added and stored at -20 °C until analysis (Mataba et al., 2016 (link)).
Samples were transferred to 10 ml glass tubes, and a mixture of HNO3 (69%) and HCl (37%) (1:3; Aqua Regia) was added to keep mercury in a stable solution, hereafter they were left at room temperature for 24h. Subsequently, 200 μl of H2O2 was added to digest the fat tissue. The samples were digested in two steps at 120°c and 160°c respectively in a pressurized microwave system (CEM Discover SP-D; CEM Corporation, Matthews, NC 28106, USA). After digestion, the samples were diluted with Milli-Q water. For water samples, a mixture of HNO3 and HCl (1:3) was added and stored at -20 °C until analysis (Mataba et al., 2016 (link)).
3
Optimizing Fecal VOC Profiling Conditions
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The influence of
fecal sample mass, number of thaw–freeze cycles, and duration
of storage at room temperature were assessed by comparing the VOC
profiles derived from the subsamples taken from the original fecal
sample of each HC and IBD subject. The subsamples were weighed on
a calibrated scale (Mettler Toledo, AT 261 Delta Range, Ohio, United
States), labeled, and returned to a −24 °C freezer until
further handling. We compared the variables of interest with our standard
operating procedure (reference standard), defined as a mixture of
500 mg of feces diluted with 10 mL of tap water and kept at room temperature
for 10 min prior to analysis. These reference standard settings were
chosen since they were used in several previous studies on fecal VOC
profiling in a range of gastroenterology diseases and have provided
us with positive results.14 (link),22 (link)
fecal sample mass, number of thaw–freeze cycles, and duration
of storage at room temperature were assessed by comparing the VOC
profiles derived from the subsamples taken from the original fecal
sample of each HC and IBD subject. The subsamples were weighed on
a calibrated scale (Mettler Toledo, AT 261 Delta Range, Ohio, United
States), labeled, and returned to a −24 °C freezer until
further handling. We compared the variables of interest with our standard
operating procedure (reference standard), defined as a mixture of
500 mg of feces diluted with 10 mL of tap water and kept at room temperature
for 10 min prior to analysis. These reference standard settings were
chosen since they were used in several previous studies on fecal VOC
profiling in a range of gastroenterology diseases and have provided
us with positive results.14 (link),22 (link)
4
Standardized Fecal Sampling for VOC Analysis
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From each original collected fecal sample, 500 mg (with a deviation of 5%) was weighted on a calibrated scale (Mettler Toledo, AT 261 Delta Range, Columbus, OH, USA). This sample amount was chosen based on results of a previous sampling method study for VOC-pattern recognition using field asymmetric ion mobility spectrometry (FAIMS), showing that samples of 500 mg provide an optimum ratio of VOCs to the headspace to distinguish the scent profiles of IBD patients [41 (link)]. The subsample was then transferred into a labelled 3 mL sealed vacutainer (BD vacutainer, Franklin Lakes, NJ, USA) and stored once again in a −24 °C freezer. Fecal samples were thawed to room temperature (18 °C) 30 min prior to analysis to allow VOCs to fill the headspace.
5
Measuring Fluid Capacity of Fiber Pads
6
Rearing Black Soldier Fly Larvae
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BSF larvae were provided by Bio.S Biogas (Grimma, Germany). The larvae were fed ad libitum with PKM and maintained in 19.5 × 16.5 × 9.5 cm (l × w × h) polypropylene containers with a density of 150 mg eggs per container at 27 ± 1 °C and 60 ± 10% relative humidity (RH) in darkness [3 (link),27 (link),28 (link),29 (link)]. The PKM was provided by PT Alternative Protein Indonesia (Tebet, Indonesia) and stored in a dry and dark place until it was fed. The developmental stage was determined by the weight (AT261 DeltaRange, Mettler, Giessen, Germany), length, and head capsule width (Keyence VHX-2000 digital microscope, Keyence, Osaka, Japan) as described elsewhere [30 (link)].
7
Residual Water Determination in Freeze-Dried Products
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Karl Fischer coulometric titration was used to determine residual water content. Two vials for each LC experiment and six vials for each LS experiment (three edge vials and three center vials, respectively) were used for analysis. Only non-TC vials were used for the residual moisture analysis. Between 40 and 80 mg of sample aliquots were filled into glass vials under dry atmosphere (glove box, 20–25°C, relative humidity < 1%) and crimped. The exact sample weight was recorded by weighing the empty and filled vials using a Mettler Toledo (AT261 DeltaRange®) analytical balance. Sample vials were placed in an 874 Karl Fischer Oven Sample Processor (Metrohm, Filderstadt, Germany) at a temperature of 80°C. The oven temperature was evaluated to be sufficient for complete water extraction of the processed freeze dried products in this study. Higher oven temperatures did not result in an increase in the extracted water content during the analysis. The water was transferred into a Karl Fischer Moisture Analyzer 831 KF (Metrohm, Filderstadt, Germany) by purging the vial headspace with dry nitrogen at 60 mL/min, and moisture content was recorded in percent. The precision of the measurement was verified using a crystalline water standard with 1% water content (Hydranal™ Water Standard KF Oven).
8
Trace Metal Analysis in Fish Tissues
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For the acute exposure experiment, whole bodies were acid digested, whereas for the subacute exposure experiment the gill arches were collected for metal analysis. The samples were dried at 60°C for 48 hours. They were set to cool down in a desiccator before the dry weight was taken on a precision balance (Mettler AT261 DeltaRange). Next, trace-metal-grade HNO3 (69%, Seastar Chemicals) was added to the samples, blanks and standard reference material (SRM-2976, Mussel tissue, National Institute of Standards and Technology), and left to settle after which H2O2 (29%, Seastar Chemicals) was added. Volumes for the digestions were adjusted according to tissue weight, for the carcasses (5ml HNO3 followed by 0.5ml of H2O2) and the gills (1.5ml of HNO3 followed 0.05 ml of H2O2). The digestion process was initiated at room temperature for 12 hours, followed by a 30 minute-incubation in a hot block (Environmental Express) at 115°C. The total metal concentration and major ion levels (Na, K, Mg, and Ca) in the digested tissues and water samples were determined with a quadrupole inductively coupled plasma mass spectrometer (ICP-MS; iCAP 6000 series, Thermo Scientific). The calculated recoveries of the reference material were 102.67, 94.62 and 100.07% for cadmium, copper and zinc respectively. The recoveries for the major ions K, Mg, Na and Ca were 98.06, 89.65, 92.89 and 94.41 respectively.
9
Thermoanalytical Evaluation of Polymer Films
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TGA Method. TGA was carried out using a Q5000 TGA instrument (TA instruments, USA). The accurately weighed polymer gel (25 ± 0.01 mg; as prepared in the BPreparation of Thin Polymer Films^section), which had approximately the similar thickness as the samples used in oven drying, was placed in the center of the platinum sample pan and heated isothermally at 40, 60, and 80°C for different durations in a nitrogen atmosphere (50 ml/min); weight loss was then determined over a period of time. The TGA apparatus was also used to dry films isothermally at different temperatures before DSC and microscopic analysis, as described in the following section. The TGA apparatus was previously calibrated for weight and temperatures following the manufacturer's guidelines. The results were analyzed using TA universal analysis software (version 4.5A).
Laboratory Convection Oven. A laboratory convection drying oven (UFE 500, Memmert, Germany) was used to study the drying kinetics of the polymer films. Rectangular HPMC and PVA multicomponent films (4 × 10 cm 2 ) were placed in the oven immediately after the casting process and heated isothermally at 40 ± 0.5, 60 ± 0.5, and 80 ± 0.5°C with an air flow rate of 0.5 ± 0.02 m/s. The humidity was <5%. The weight loss at various time points was measured using a sensitive balance (AT261 Delta range, Mettler, Switzerland).
Laboratory Convection Oven. A laboratory convection drying oven (UFE 500, Memmert, Germany) was used to study the drying kinetics of the polymer films. Rectangular HPMC and PVA multicomponent films (4 × 10 cm 2 ) were placed in the oven immediately after the casting process and heated isothermally at 40 ± 0.5, 60 ± 0.5, and 80 ± 0.5°C with an air flow rate of 0.5 ± 0.02 m/s. The humidity was <5%. The weight loss at various time points was measured using a sensitive balance (AT261 Delta range, Mettler, Switzerland).
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