Dec 28, 2022
Version 1
Measurement of dissolved carbohydrate V.1
- Ying-Yu Hu1,
- Zoe V. Finkel1
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. Measurement of dissolved carbohydrate. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6168zvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2022
Last Modified: December 28, 2022
Protocol Integer ID: 66472
Keywords: Dissolved total carbohydrate, Dissolved polysaccharide, Dissolved monosaccharide, TPTZ method, Ferricyanide, hydrolysis
Funders Acknowledgement:
Simons Collaborative on Ocean Processes and Ecology
Grant ID: 723789
Simons Collaboration on Computational Biogeochemical Modeling of Marine Ecosystems
Grant ID: 549937
Abstract
Here we describe a protocol to measure the dissolved carbohydrate, including total dissolved monosaccharides and total dissolved polysaccharides.
For total dissolved carbohydrate measurement, freeze-dried dissolved carbohydrate samples are initially vortexed in 9 M H2SO4 for 15 s. The solution is diluted for a final H2SO4 molarity of 1.6 M and hydrolyzed for 3 hours at 90 °C. The hydrolysate is alkalinized by adding 12 M NaOH to the hydrolysate, the ratio of [H+] from hydrolysate to [OH-] from NaOH is 0.82. The alkalinized hydrolysate is oxidized by ferricyanide solution. The absorbance of TPTZ-Fe2+ complex is measured in microtiter plate at 595 nm.
For total dissolved monosaccharide measurement, freeze-dried dissolved carbohydrate samples are alkalinized by 12 M NaOH and then oxidized by ferricyanide solution. The absorbance of TPTZ-Fe2+ complex is measured in microtiter plate at 595 nm.
Our method has shown high reproducibility in aldohexoses, ketohexoses, deoxysugars, aldopentoses, uronic acid and amino sugars. The low limit of detection is 0.024 µg C/mL.
Protocol materials
K3[Fe(CN)6] Fisher ScientificCatalog #AC424120050
Step 22.1
TPTZMerck MilliporeSigma (Sigma-Aldrich)Catalog #T253-5G
Step 22.3
D-glucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270-100G
Step 5.1
NaOHFisher ScientificCatalog #BP359-500
Step 18
Na2CO3VWR InternationalCatalog #97061-972
Step 18
Sodium acetate anhydrous Fisher ScientificCatalog #BP333-500
Step 19.1
Citric acid Merck MilliporeSigma (Sigma-Aldrich)Catalog # 251275-500G
Step 19.1
Acetic acid Fisher ScientificCatalog #M1000632500
Step 19.1
Safety warnings
Ferric waste should be disposed into trace metal waste container.
Waste acid should be neutralized before disposed into sink.
Sample collection
Sample collection
12h
12h
GFF filter is combusted for 04:00:00 at 450 °C
Glass filter holder is combusted for 02:00:00 at 500 °C
Glass filter funnel, flask and 10 mL centrifuge tubes are combusted for 06:00:00 at 500 °C
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU
Tube caps are acid-washed.
Equipment
Polypropylene Screw Caps
NAME
Linerless, 15-415
TYPE
Kimble Chase
BRAND
73805-15415
SKU
12h
Filter microalgae sample and collect the filtrate, using gentle vacuum pressure (130 mm Hg).
Transfer 5 mL filtrate into centrifuge tube and flash freeze.
Note
Three tubes for total dissolved monosaccharide and three tubes for total dissolved carbohydrate measurement.
Freeze dry samples before measurement.
Glucose standards
Glucose standards
Primary standard solution
In a 2 mL microtube, weigh 1 ~ 2 mg D-glucoseD-glucoseVWR InternationalCatalog #G8270-100G
Add Milli-Q for a final concentration of 1 mg/mL (>600 µL).
Secondary standard for total dissolved carbohydrate
Add 45 µL primary solution into a 2 mL microtube
Add 955 µL Milli-Q and then vortex for a good mix
In 10 mL centrifuge tubes, prepare the following standard solutions:
| SD | Secondary solution (uL) | Milli-Q (uL) | |
| TCHO-SD1 | 0 | 100 | |
| TCHO-SD2 | 20 | 80 | |
| TCHO-SD3 | 40 | 60 | |
| TCHO-SD4 | 60 | 40 | |
| TCHO-SD5 | 80 | 20 | |
| TCHO-SD6 | 100 | 0 |
Secondary standard for total dissolved monosaccharide
Add 10 µL primary solution into a 2 mL microtube
Add 990 µL Milli-Q and then vortex for a good mix
In 12 mL amber vials, prepare the following standard solutions:
| SD | Secondary solution (uL) | Milli-Q (uL) | 12 M NaOH (uL) | |
| MCHO-SD1 | 0 | 984 | 16 | |
| MCHO-SD2 | 10 | 974 | 16 | |
| MCHO-SD3 | 20 | 964 | 16 | |
| MCHO-SD4 | 50 | 934 | 16 | |
| MCHO-SD5 | 100 | 884 | 16 | |
| MCHO-SD6 | 150 | 834 | 16 |
Equipment
Storage Vials and Closures
NAME
12 mL amber
TYPE
Thermo Scientific
BRAND
B7800-12A
SKU
VWR 66030-686
SPECIFICATIONS
Hydrolysis of total dissolved carbohydrate
Hydrolysis of total dissolved carbohydrate
20s
20s
Prepare water bath 95 °C
Add 100 µL Milli-Q to each tube with freeze-dried sample.
Use reverse pipetting technique, add 100 µL 18 M H2SO4 to standard solution/sample, immediately vortex for 00:00:15 (monitored by timer or stopwatch)
Note
Do not cap the centrifuge tube!
15s
Add 900 µL Milli-Q, tightly cap the centrifuge tube, and vortex for 00:00:05 .
5s
Place tube into water bath, log the time.
Note
Hydrolysis duration for each sample/blank/standard should be accurately monitored.
After all samples are in the water bath, reduce temperature to 90 °C .
Label amber vials for TPTZ measurement with white oil based sharpie.
# of vials = # of samples + # of blanks + # of standards
As soon as hydrolysis duration reaches 3 hours, remove the tube from water bath, let it sit in the tap water bath with ice to quickly stop hydrolysis.
Keep all hydrolysate in a dark cabinet at Room temperature .
Prepare TPTZ reagents
Prepare TPTZ reagents
12 M NaOH
Add 15 mL Milli-Q water into a 50 mL Falcon tube.
Add 12 g NaOH pellet into the water, swirl and have the pellets completely dissolved, let it cool down to Room temperature .
Transfer the solution into a 25 mL PP volumetric flask, rinse the tube three times by small amount of Milli-Q and combine the rinsed water into flask, top with Milli-Q water to 25 mL.
Alkaline solution for potassium ferricyanide
Dissolve 400 mg NaOH and 20 g Na2CO3 in volumetric flask and top to 1 L by Milli-Q. Store at room temperature.
NaOHVWR InternationalCatalog #BP359-500
Na2CO3VWR InternationalCatalog #97061-972
Sodium acetate solution
Dissolve 164 g sodium acetate, 42 g citric acid and 300 g acetic acid in a 1 L volumetric flask and top to1 L with Mill-Q water.
Note
In this solution, sodium acetate, citric acid and acetic acid is 2 M, 0.2 M and 5 M respectively.
Sodium acetate anhydrous VWR InternationalCatalog #BP333-500 Citric acid VWR InternationalCatalog # 251275-500G Acetic acid VWR InternationalCatalog #M1000632500
Store at room temperature.
Dispense solution by serological pipet to avoid having salt precipitated around sealing surface of the bottle.
3 M acetic acid
Weigh 180 g acetic acid in fumehood, transfer the acid into volumetric flask, top to 1 L with Milli-Q water. Store at room temperature.
TPTZ method
TPTZ method
40m
40m
Prepare boiling bath
TPTZ reagents
Potassium ferricyanide (Reagent A)
Weigh 23 mg potassium ferricyanide and transfer into a 100 mL amber reagent bottle. Add 100 mL alkaline solution, vortex until powder is completely dissolved. It is stable for two weeks at room temperature.
K3[Fe(CN)6] VWR InternationalCatalog #AC424120050
Equipment
Reagent bottle
NAME
100 mL, amber
TYPE
VWR
BRAND
14216-240
SKU
Ferric chloride (Reagent B)
Ferric chloride hexahydrate is in spherical shape. It is hard to weigh exact 54 mg for a 100 mL solution. Pick a very small ferric chloride ball and log the weight. Transfer the ball into a 100 mL amber reagent bottle. Calculate the acetate solution required.
Add acetate solution into the amber bottle, vortex until the ball is completely dissolved.
V_acetate = 100 X W_actual/54
Note
This reagent needs to be prepared right prior to analysis. It can only be stable for no more than two days.
TPTZ (Reagent C)
Estimate the total volume required for the assay: 2 mL X (standard # + blank # + sample #)
For each 100 mL TPTZ reagent, weigh and transfer 78 mg TPTZ into an amber reagent bottle, add 100 mL acetic acid solution, vortex until the powder is completely dissolved.
TPTZVWR InternationalCatalog #T253-5G
Note
This solution is stored at room temperature and stable for one week.
Total dissolved carbohydrate samples
Use reverse pipetting technique, transfer 750 µL hydrolysate of standard/sample to amber vial.
Add 250 µL 12 M NaOH and vortex.
Total dissolved monosaccharide samples
Add 1200 µL Milli-Q into the tube with freeze-dried sample
Use reverse pipetting technique, transfer 984 µL solution to amber vial.
Add 16 µL 12 M NaOH and vortex.
In a room with dim light, add 1 mL Reagent A into each amber vial.
Tightly cap the vial and vortex.
Keep in a boiling water bath for 00:10:00
10m
Remove boiling bath from the heat, keep all vials in the hot water and move them into the room with dim light.
Add 1 mL Reagent B and 2 mL Reagent C into the vial and vortex.
Shake at Room temperature for 00:30:00 .
30m
Under dim light, using reverse pipetting, load 250 uL of blanks, standards, and samples into the microplate (duplicate).
Load column by column. After one column has been loaded, immediately cover the column with a lid, which has a black membrane on the top to protect sample from light.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| A | MCHO-SD1 | MCHO-SD1 | |||||||||||
| B | MCHO-SD2 | MCHO-SD2 | |||||||||||
| C | MCHO-SD3 | MCHO-SD3 | |||||||||||
| D | MCHO-SD4 | MCHO-SD4 | |||||||||||
| E | MCHO-SD5 | MCHO-SD5 | |||||||||||
| F | MCHO-SD6 | MCHO-SD6 | |||||||||||
| G | |||||||||||||
| H |
Microplate layout for dissolved monosaccharide samples
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| A | TCHO-SD1 | TCHO-SD1 | |||||||||||
| B | TCHO-SD2 | TCHO-SD2 | |||||||||||
| C | TCHO-SD3 | TCHO-SD3 | |||||||||||
| D | TCHO-SD4 | TCHO-SD4 | |||||||||||
| E | TCHO-SD5 | TCHO-SD5 | |||||||||||
| F | TCHO-SD6 | TCHO-SD6 | |||||||||||
| G | |||||||||||||
| H |
Microplate layout for total dissolved carbohydrate samples
Read in microplate reader:
Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 595 nm with a measurement time 100 ms
UV/VIS spectra (optional)
UV/VIS spectra (optional)
Hydrolysate
Load 200 µL hydrolysate into microplate.
Blank:
Milli-Q : H2SO4 = 10:1
Monosaccharide solutions
Load 200 µL solution into microplate.
Blank: Milli-Q
Scan UV/VIS spectra from 200 to 400 nm at a step of 1 nm.
Calculation
Calculation
Total dissolved carbohydrate
Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard for total dissolved carbohydrate.
Obtain standard curve by plotting blank subtracted absorbance (Abs') versus carbon (uM C)
Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each sample
Total dissolved monosaccharide
Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard for total dissolved monosaccharide.
Obtain standard curve by plotting blank subtracted absorbance (Abs') versus carbon (uM C)
Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each sample
Total dissolved polysaccharide
Waste disposal
Waste disposal
All hydrolysate and TPTZ reagent C need to be neutralized by soda before disposed into the sink.
TPTZ reagent B is collected in trace metal waste container.