Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

The overall goal of this 2D gas chromatography data acquisition method is to characterize the aqueous byproducts obtained from hydrothermal liquefaction, or HTL, of algae. This method can help answer key questions in the bio-fuels field such as identification of the organic species that report to the aqueous phase during hydrodynamic fashion of algae. The main advantage of this technique is that it improves peak capacity, resolution, and a wide coll-usion of chemical compounds.

This method is important to generate data for bio-refineries, which produce considerable volumes of waste water that must be either treated or further processed to fuels and chemicals. This method can provide characterization data that cannot be produced from other analytical techniques, such as one-dimensional gas chromatography or liquid chromatography. We first had the idea for this method when we were analyzing very complex organic mixtures from bio-fuel production processes.

Use a gas chromatograph equipped with a quad-jet dual-stage cooling-base modulator and time-of-flight mass spectrometer for these experiments. Configure the auto-sampler to inject one microliter of each sample or standard into the gas chromatograph or GC.Use a randomized block design of sample and standard injections for the auto-sampler sequence, as described in the literature. Ensure that both edges of both primary and secondary columns are cut straight without sharp edges.

Then, connect the primary and secondary columns using a press-tight connector before the modulator. Ensure that the glass liner, non-stick liner O-ring, inceptor for the GC injector are new and free of contamination. Next, place a feral on the GC column, and connect the primary column to the GC injector so that five millimeters of column is inside the injector.

Use one-sixteenth inch and 0.5 millimeter inner diameter transfer-line ferals to connect the secondary column and transfer line. Place a 0.2 meter portion of the secondary column in the transfer line. Ensure that a 0.1 meter portion of the secondary column is in the modulator.

Use ultra-high purity helium gas as carrier gas for GC at a flow rate of 1.5 milliliters per minute. Ensure there is sufficient liquid nitrogen in the dewar, which acts as the coolant in the modulator, by reading the level of liquid nitrogen using a pressure gauge attached to the dewar outlet. A 69 kilopascal reading of the pressure gauge indicates that the dewar is full, while zero kilopascals indicates that it is empty.

Ensure there are no major leaks in the instrument. If the vacuum-gauge reading of the TOF-MS is higher than 2.7 times 10 to the negative fifth pascals for 1.5 milliliter per minute GC column flow rate, this indicates a major leak in the system. Set up the quality control or QC method and run the in-built acquisition system adjustments protocol to achieve maximum signal response using the manufacturer's protocol.

Then, run in-built instrument optimization protocols of the QC method. In series, run filament focus, ion optic focus, and mass calibration tests using the manufacturer's protocol. Ensure that the mass calibration test passes.

This QC method ensures that all the hardware parameters of the instrument are at optimum level. The instrument should be free of leaks. This is an important aspect of this procedure.

A re-check should be performed using the manufacturer's protocol. Analyze the generated leak check report by checking the relative concentration of specific ions to the internal standard. To determine the optimum modulation period of the modulator, arbitrarily select a long modulation period and inject a sample as before.

Identify the retention time in the second dimension of the contour plot, after which no peaks elute. Select the identified second-dimension retention time as the optimum modulation period. Increase the modulation period and perform the analysis again if wrap-around is observed.

Wrap-around phenomena occur if the peaks in the second dimension elute below the baseline of the first dimension, as shown in this contour plot. Repeat these step until the optimum value is determined. The configuration used in this study is unique and important.

This combination was not previously used to analyze aqueous fractions from biomass conversions. Install a polar capillary column as the primary column. Install a non-polar capillary column as the secondary column.

Use ultra-high purity helium gas as the carrier gas for GC at a flow rate of 1.5 milliliters per minute. Set the GC injector to a temperature of 260 degrees Celsius and a split ratio of one to 250. Set up a temperature program for the primary column that starts with a constant temperature of 40 degrees Celsius for 0.2 minutes, followed by a temperature ramp to 260 degrees Celsius at five degrees Celsius per minute, followed by a constant temperature of 260 degrees Celsius for five minutes.

Maintain the modulator temperature five degrees Celsius higher than that of the secondary column and the secondary column temperature at five degrees Celsius higher than that of the primary column. Use an optimum modulation period of four seconds with 0.8 seconds of hot pulse and 1.2 seconds of cold pulse, as determined before. Then, set the transfer line temperature to 270 degrees Celsius.

Next, set the acquisition delay or solvent delay to zero seconds. Set the lower and higher range of mass over charge to 35 and 800, then set the MS detector acquisition rate to 400 spectra per second. Maintain the MS detector voltage at 150 volts higher than the optimized value.

Finally, maintain the MS ion source temperature at 225 degrees Celsius. Perform data processing using the software supplied by the instrument manufacturer. In the data analysis method, select compute baseline, find peaks above the baseline, library search, and calculate area and height.

Track the baseline through the data file. Enter the baseline offset as 0.5, then enter the expected peak width of 15 seconds in the first dimension and 0.15 seconds in the second dimension. Set the signal-to-noise ratio as 5000 and similarity values of greater than 850 for identification of compounds.

Select a commercially available mass spectral library to identify chemical compounds present in samples, and set the library search mode to forward. Process the data files with this data analysis method using the manufacturer's protocol. It requires at least one hour to process the data file.

Shown here is a total ion chromatogram obtained for the aqueous fraction of algae bio-crude, analyzed with a column combination of polar and non-polar. Oxygenates and organic acids were observed in HTL algae water. In addition to oxygenates, the aqueous phase has nitrogen-containing compounds, such as pyridine, pyrazine, acetamides, succinimide, and their alkyl derivatives.

Presumably, these compounds are the degradation products of proteins and carbohydrates in algal biomass. Shown here are the similarity values and retention time of the chemical compounds present in HTL algae water, using column combination of polar and non-polar in table form. The high intensity peaks identified in the contour plot for the aqueous fraction of algae bio-crude were validated by analyzing standards.

Standards containing organic acids and end-compounds were prepared and analyzed in 2D gas chromatography with time-of-flight mass spectrometry. The aqueous fraction of algae bio-crude was also analyzed with the conventional column combination of non-polar and polar, which was widely used in the literature. As shown here, organic acids and end-compounds present in the aqueous fraction of algae bio-crude elute with more than one peak.

The similarity values and retention time of these chemical compounds are listed in table form. After watching this video, you should have a good understanding of how to analyze the aqueous samples using two-dimensional gas chromatography equipped with mass spectrometry. After its development, this technique paved the way for researchers in the field of bio-fuels to analyze aqueous stream of byproducts obtained from biochemical, thermochemical, and thermocatalytic conversions of biomass.

Following this procedure, other methods like one-dimensional gas chromatography and liquid chromatography can be performed in order to answer additional questions like quantitatively determining the concentration of unidentified chemical compounds. While attempting this procedure, it's important to remember that non-volatile, high-compounds and inorganic salts could not be analyzed using this instrument configuration.