This manuscript has been authored by Battelle Memorial Institute under Contract No. DE-AC05-76RL01830 with the U.S. Department of Energy. The U.S Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for U.S. Government purposes.

1. Sample Preparation
<ol>
	<li>Generate a mixed aqueous/organic product stream via continuous flow HTL of algae according to the reactor design and experimental procedure found in the literature10,11.</li>
	<li>Use a gravity separator to separate the product stream into an aqueous phase and organic phase.</li>
	<li>Filter 10 ml of the HTL aqueous phase using a 0.45 &#181;m syringe filter and store in a refrigerator maintained at 4 &#176;C for GC &#215; GC&#8211;TOF-MS analysis.</li>
</ol>
<p class="jove_tit...

The authors declare that they have no competing financial interests.

Results clearly illustrate the ability of the column combination of polar &#215; non-polar to resolve polar compounds and light volatiles present in the aqueous fraction of algae bio-crude without prior sample preparation techniques. Drastic peak tailing was observed for organic acids and N-compounds while using the non-polar&#215; polar column combination. This peak tailing was not observed for the early eluting light organics. This behavior has been reproducible when verifying the instrument is free of leaks (the vacuu...

Steady growth in demand for liquid fuels, finite fossil fuel resources, uncertainty of fossil fuel supplies, and concerns over the increasing concentration of greenhouse gases in the atmosphere have increased global awareness for renewable resources1. Solar energy (including photovoltaics and solar-thermal), wind energy, hydropower, geothermal, and biomass are the primary renewable sources that could potentially replace fossil-derived energy2. Of these, biomass is the only carbon-based alternative energy resource for the production of liquid transportation fuels and high-value chemicals3. Biomass includes any organic material such as forest resources, agricultural residue, algae, oilseeds, municipal solid waste, and carbon-rich industrial wastes (e.g. from pulp and paper industry or from food processing)1. Biomass is classified into two broad categories: lignocellulosic and non-ligneous feedstocks based on compositional characteristics. Lignocellulosic biomass consists of carbohydrates and lignin, while non-ligneous feedstocks have proteins, carbohydrates and lipids/oils4. Lignocellulosic feedstocks, derived from terrestrial plants, can only satisfy 30% of the current liquid fuel (gasoline, jet fuel, and diesel) demand if sustainably cultivated and harvested5,6. Hence, non-ligneous aquatic microorganisms, such as microalgae and fungi, are considered potential feedstocks for the production of renewable liquid fuels to complement lignocellulosic resources.
Microalgae feedstocks have the potential to satisfy current liquid transportation fuels demand7,8. Algae have many advantages: high areal productivity8, the ability to grow in low-quality, brackish, or sea water9, and the ability to accumulate energy-dense triglycerides or hydrocarbons7,8. Hydrothermal liquefaction (HTL) is a viable and scalable conversion process which utilizes water naturally associated with algal or aquatic feedstocks10,11. It is a thermo-chemical process with operating temperatures of 250-400 &#176;C and operating pressures of 10-25 MPa which produces a liquid product, or bio-crude, which can be upgraded into a fuel blend stock. Bio-crude produced from HTL of algae has distinguishable and easily separable organic and aqueous fractions. The organic fraction of bio-crude can be efficiently converted into a refinery ready blend stock via catalytic hydro-treating processes11. The aqueous fraction of bio-crude contains ~30% of the total carbon present in the algal feedstock. Although thousands of compounds have been identified in the HTL aqueous stream, the predominant fractions consist of low molecular weight oxygenates (including acids, alcohols, ketones, and aldehydes) formed by the degradation of carbohydrates and lipids, and nitrogen heterocyclics (including pyrroles, pyridines, pyrazines, and imidazoles) derived from protein decomposition12. Studies on utilizing the aqueous fraction to improve overall process economics as well as sustainability are ongoing. Synthesis gas can be produced from the aqueous fraction of algae bio-crude via catalytic hydrothermal gasification10,13,14. Alternatively, organics in the aqueous fraction can also be catalytically converted to fuel additives and specialty chemicals. Research on optimizing catalytic hydrothermal gasification and catalyst screening studies for conversion of organics in the aqueous liquid phase is currently underway at the Pacific Northwest National Laboratory (PNNL). For this work, qualitative as well as quantitative characterization of the aqueous fraction of algae bio-crude is required. Since the aqueous fraction of algae bio-crude is considered a waste stream, there are very few studies that have analyzed the aqueous fraction of algae bio-crude13,15. Moreover, recent studies concluded that converting this HTL algae water into high-value bio-products would improve the sustainability as well as economics of an HTL-based bio-refinery11. Therefore, this study focused on developing a method for qualitative characterization of the aqueous fraction of bio-crude obtained from HTL of algae by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC &#215; GC&#8211;TOF-MS).
GC &#215; GC&#8211;TOF-MS is the most promising chromatographic analytical technique to increase resolution (or separation of chemical compounds in a sample), peak capacity (i.e. number of resolved peaks), signal-to-noise ratio (for identification of chemical compounds with high confidence), and to avoid co-elution of chemical compounds16. In order to maximize resolution, peak capacity, and signal-to noise ratio, two GC columns with different stationary phases are connected in series using a press-fit connector or micro-union17(see Figure 1 which is a block diagram of GC &#215; GC&#8211;TOF-MS system used in this study). A modulator is located between the press-fit connector and secondary columns to trap, refocus, and re-inject the effluents from the primary column into the secondary column18. Modulation occurs on the secondary column in the present study as shown in Figure 1. The secondary column is then connected to the TOF-MS via a transfer line assembly.
GC &#215; GC&#8211;TOF-MS was used previously for qualitative as well as quantitative analysis of organic samples such as crude oil16,19, gasoline, jet-fuel, diesel, bio-diesel, and the organic fraction of bio-fuel20-22 produced from thermo-chemical as well as thermo-catalytic conversion processes23,24. For characterization of these organic samples in GC &#215; GC&#8211;TOF-MS instruments, a long non-polar column was used as the primary column, while a short polar column was used as the secondary column. This conventional column configuration resolves chemical compounds based on differences in volatility over the first dimension, followed by polarity in the second dimension18. Aqueous or water samples from biological processes, food processing, and environmental wastes were also characterized using similar primary/secondary column configurations after the sample had been through preparation steps17,25-30. Sample preparation techniques such as derivatization, solid-phase extraction, and organic solvent extraction have all been utilized prior to GC &#215; GC&#8211;TOF-MS analysis17,27-29,31,32. These techniques were aimed at decreasing the polarity of compounds in the sample for analysis using a conventional column configuration33. An alternative strategy was employed in this study based on the nature of the sample (here polar organic compounds in water) utilizing the reverse primary/secondary column configuration for GC &#215; GC&#8211;TOF-MS analysis. Since the aqueous fraction of bio-crude produced from HTL has polar compounds13, a column combination of a primary polar column and a secondary non-polar column was used in the GC &#215; GC&#8211;TOF-MS without any upstream sample preparation. This primary/secondary column combination resolves chemical compounds based on differences in polarity over the first dimension, followed by volatility in the second dimension. Limited analytical methods exist in the literature for characterization of aqueous samples using two-dimensional gas chromatography without prior sample processing15.
The objective of this study was to determine the chemical compounds present in the aqueous fraction of algae bio-crude. To achieve this objective, a GC &#215; GC&#8211;TOF-MS data acquisition method was developed with a column combination of polar column (primary) &#215; non-polar (secondary). Klenn et al. (2015) suggested that increasing the length of the primary column (especially 60 m GC columns) and lowering the offset temperature of the secondary column with respect to the primary column would maximize peak capacity and resolution16-18. Therefore, a 60 m primary column and 5 &#176;C offset temperature of the secondary column with respect to the primary column were used in this study. The optimum modulation period was determined following a protocol described in this study (see section 4). The optimum ramp rate of GC column temperature was determined by a trial and error method and is similar to the value suggested in the literature16-18. To discuss the advantages of this column combination for aqueous samples, we have analyzed HTL algae water samples with the conventional column combination of non-polar &#215; polar. Operating parameters suggested in the literature were employed for analyzing the aqueous fraction of algal bio-crude with a non-polar &#215; polar column combination18.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">GC &#215; GC - TOF/MS</td>
			<td style="width:199px;">Leco</td>
			<td style="width:151px;">PEG4D11DLN15</td>
			<td style="width:268px;">Commercial Pegasus 4D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChromaTOF version 4.50&#160;</td>
			<td style="width:199px;">Leco</td>
			<td style="width:151px;"></td>
			<td style="width:268px;">Data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Rxi-5MS GC column</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">13420</td>
			<td style="width:268px;">2.3 m column was used from this column.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Stabilwax GC column</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">10626</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">HP-5 GC column</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:151px;">19091J-416</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Stabilwax GC column</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">15121</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Presstight Connector</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">20430</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">GC injector liner</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">23305.5</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">GC Injector ferrules</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:151px;">5181-3323</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Non-stick liner O-rings</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:151px;">5188-5365</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Transfer line ferrules</td>
			<td style="width:199px;">Restek</td>
			<td style="width:151px;">20212</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Ethanol</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">459844</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetone</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">414689</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetic acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">320099</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">2-butanone</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">360473</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Propanoic acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">402907</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Butanoic acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">19215</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pyridine</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">270970</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pyrazine</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">65693</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetamide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">695122</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">2,5-pyrrolididione</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">S9381</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">N-methylsuccinimide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">325384</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">N-(2-hydroxyethyl)succinimide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:151px;">444073</td>
			<td style="width:268px;">Chromatography grade</td>
		</tr>
	</tbody>
</table>

qualitative characterization of the aqueous fraction from hydrothermal liquefaction of algae using 2d gas chromatography with time-of-flight mass spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A total ion chromatogram (TIC) obtained for the aqueous fraction of algae bio-crude analyzed with a column combination of polar &#215; non-polar is shown in Figure 4. Retention times and similarity or match factor values of compounds identified by searching against a National Institute of Standards and Technology (NIST) library are tabulated in Table 1. Oxygenates (such as cyclopenatanone, furanic compounds and dianhydromannitol) and organic acids (includ...

Watch this Scientific Journal Video about Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

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

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

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.

qualitative-characterization-aqueous-fraction-from-hydrothermal

Research

JoVE Journal

Bioengineering

9.2K Views.  Pacific Northwest National Laboratory. 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 met...

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