GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

The overall goal of this video is to provide a detailed description for the GC based analysis of the Aldon nitrile acetate derivatives of glucosamine and muic acid extracted from soils. The process begins with acid digestion, breaking down organic polymers and releasing glucosamine and muic acid. Monomers amino sugars are then isolated and purified from the mixture solution.

This is followed by Derivitization to convert those amino sugars to volatile aldon nitrile acetates for gas chromatography based analysis. The final step of the procedure is the liquid liquid extraction and GC determination. Ultimately, results can be obtained that show baseline separation of the aldon nitrile acetate amino sugars eluding between the myo and acetol and methyl glutamine standards through GC with flame ionization detection.

The main advantage of this technique over other method is that it optimally balances precision sensitivity, simplicity, good chromatic graphic separation and stability upon sample storage. This method can help answer key questions in the field of global carbon cycling, such as a contribution of SSE microorganisms to pools of stable soil carbon. Although this method can provide insight and information into the accumulation of microbial detritus in soils, it can also be applied in other matrices such as ocean systems, biological tissues, and even extraterrestrial samples.

To begin this protocol freeze dry soil samples after field collection, grind and homogenize soil samples using a ball mill soil grinder or a mortar and pestle. Weigh the ground up soil samples in 25 milliliter hydrolysis flasks. Add 10 milliliters of six molar hydrochloric acid into each hydrolysis flask, and fill with nitrogen gas before capping tightly.

Hydrolyze at 105 degrees Celsius in an incubator for eight hours. Using an auto timer switch, remove the flasks from the incubator and cool. To room temperature.

Add 100 microliters of internal standard myo acetol to each flask. Mix by swirling set up plastic funnels draining into 200 milliliter pear shaped flasks. With standard taper size 24 40 ground glass joints set on plastic cups for stability.

Fold watman number two qualitative circles into quarters and set into funnels. Swirl each hydrolysis flask and pour slurry into the funnel. To filter each sample, dry the filtrate using a rotary evaporator at approximately 45 degrees Celsius applying vacuum resus.

Suspend the dried residue from each pair flask with three to five milliliters of water and pour into a 40 milliliter Teflon tube Wrench each flask with a second aliquot of water. Adjust the pH of the samples to 6.6 to 6.8 using one molar potassium hydroxide solution to precipitate metal ions and other organic molecules. Then remove the precipitates by centrifugation at 2000 times G for 10 minutes following centrifugation.

Pour the supernatants into 40 milliliter glass tubes and cover the tube openings with paraform freeze supernatants and then poke holes in the paraform. Freeze dry the frozen supernatants to remove all liquid. Once the supernatants have been dried, dissolve the residue with three milliliters of dry methanol.

Vortexing thoroughly cap the tubes and then centrifuge at 2000 times G for 10 minutes. To settle out salts, transfer the supernatants to three milliliter conical reaction Vials evaporate to dryness by a rapid VA machine at 45 degrees Celsius or under a gentle stream of dry nitrogen gas. Once evaporated, add 100 microliters of recovery standard and methyl glutamine, and one milliliter of water to each vial.

Then freeze the samples before starting derivitization. Make standards as described in the written protocol, and then freeze dry both the samples and the standard. Prepare derivitization reagent containing 32 milligrams per milliliter, hydroxyl amine hydrochloride, and 40 milligrams per milliliter.

Four dimethyl amino purine in purine methanol. Add 300 microliters of the derivitization reagent to each of the reactive vials, capped tightly and vortex thoroughly. Place the sealed vials in a 75 to 80 degrees Celsius water bath for 35 minutes.

Remove the vials from the water bath and cool to room temperature. Then add one milliliter of acetic anhydride to each of the three milliliter reactive vials, capped tightly and vortex thoroughly. Following a second incubation in a 75 to 80 degrees Celsius water bath for 25 minutes.

Remove vials from the water bath and cool to room temperature. To begin separation and measurement, add 1.5 milliliters di chloro methane to each tube, and then vortex. After adding one milliliter of one molar hydrochloric acid to each vial and vortexing thoroughly allow the solutions to sit undisturbed until the two phases separate.

Aspirate and discard the top aqueous phase. Using a 1000 microliter pipetter in the same fashion, extract the organic phase three times, but with one milliliter water with the last washing step. Take special care to ensure that all of the aqueous top phase has been removed.

Dry the final solution using a rapid VAP at 45 degrees Celsius or by using a stream of nitrogen gas, dissolve each sample in 300 microliters of ethyl acetate hexane, and transfer to two milliliter amber screw cap vials with a small volume insert and cap tightly for quantification analyzed by gas chromatography with a flame ionization detector using a fused silica non-polar capillary column such as DB five as directed in the written protocol. Following standard run and analysis, adjust the carrier gas flow rate so that the acetol glucosamine and mu amic acid derivatives elute at 250 degrees Celsius and the end methyl glutamine. Elutes at 270 degrees Celsius.

Perform a one microliter 30 to one split injection with the GC inlet set at 250 degrees Celsius using an autos sampler subsequent to the gas chromatography run. And as a final step to this procedure, perform derivative validation using the chromatography and spectrometry methods discussed in the written protocol. Examples of the analysis for glucosamine and uremic acid from standard stalks and from a soil sample are shown here besides glucosamine and muic acid, two isomers of glucosamine osain and galactose amine can also be determined simultaneously using the method based on the accurate amount of the applied standards and the response factors of each two internal standard.

The amount of these biomarkers in the soil samples can be determined. The recovery standard has also been used to qualitatively monitor the derivation process. The schemes for formation of aldon nitrile acetate, derivate glucosamine, and muic acid are shown here.

Number one represents the nitrile reaction, and number two represents acetylation. The proposed structures of the ion fragments formed upon electron ionization were studied by comparing the ion spectra of the samples prepared with various isotope incorporations. Here, the mass shift of the dominant ion of aldon nitrile acetate derivate glucosamine is shown in different scenarios.

In this example, it was prepared with unlabeled agents as well as isotopically labeled acetic anhydride, and isotopically labeled glucosamine. The star on the chemical structure represents the heavy isotope atom or isotope group. While attempting this procedure, it's important to remember to maintain rigorously anhydrous conditions during derivitization.

Both the organic solvents and the glassware use at this stage need to be dry. While many vendors sell anhydrous solvents, those on hand may be dried by passing them through columns containing drying reagents such as magnesium sulfate. Following this procedure, other methods such as ratio mass spectrometry can be performed in order to answer additional questions like how macro beauty react, carbon or nitrogen turn over and accumulate in soils by differentiating living and dead macro s after its development.

This technique paved the way for researchers in the field of macro ecology and soil biology, chemistry to explore macro contribution to soil organic matter, sequestration in natural or managed terrestrial systems.