Abstract Organic acid analysis of fermentation samples can be readily achieved by gas chromatography (GC), which detects volatile organic acids. However, lactic acid, a key fermentation acid is non-volatile and can hence not be quantified by regular GC analysis. However the addition of periodic acid to organic acid samples has been shown to enable lactic acid analysis by GC, as periodic acid oxidizes lactic acid to the volatile acetaldehyde. Direct GC injection of lactic acid standards and periodic acid generated inconsistent and irreproducible peaks, possibly due to incomplete lactic acid oxidation to acetaldehyde. The described method is developed to improve lactic acid analysis by GC by using a heat treated derivatization pre-treatment, such that it becomes independent of the retention time and temperature selection of the GC injector. Samples containing lactic acid were amended by periodic acid and heated in a sealed test tube at 100°C for at least 45 min before injecting it to the GC. Reproducible and consistent peaks of acetaldehyde were obtained. Simultaneous determination of lactic acid, acetone, ethanol, butanol, volatile fatty acids could also be accomplished by applying this GC method, enabling precise and convenient organic acid analysis of biological samples such as anaerobic digestion and fermentation processes. Introduction Acid stage fermentation is the initial process of two stage anaerobic digestion process. In the acid stage, complex organic materials firstly hydrolyzed to sugars, fatty acids and amino acids by extracellular enzymes. These relatively simpler soluble products are then fermented to volatile and non-volatile organic acids, alcohols, hydrogen and carbon dioxide (1, 2). Some typical microorganisms involved in anaerobic acid stage fermentation process include bacteria, yeast, fungi and protozoa. During this process, competition between microorganisms for taking substrates may occur resulting in major products generated in the culture (2). Lactic acid is an organic acid generated from the acid stage fermentation process from carbohydrate metabolism. Lactic acid is found in fermented foods such as dairy products (yoghurt, buttermilk, etc.), pickled vegetables, sourdough breads, meat products and silage and muscle tissue. Typically fermentations of carbohydrates result in a mixture of organic acids including volatile fatty acids (VFAs) acetic, propionic and butyric acids as well as lactic acid, ethanol, butanol and acetone. Typical environments rich in such mixtures of fermentation products are the rumen, fermented food and biological waste conversion by anaerobic digestion or acid stage fermentation. As lactic acid is a key fermentation end product and of primary significance from microorganisms to human beings, many studies and methods for quantitative determination have been proposed (3). Several analytical methods for the determination of lactic acid are currently available, including enzymatic (4, 5), titration (6), colorimetric and spectrophotometric (7–9), potentiometric (10), high pressure liquid chromatography (HPLC) (11), gas chromatography (GC) (12, 13). However, there are no suitable established methods for the simultaneous detection all major fermentation products (VFA, lactic acid, ethanol, butanol) within one simple, rapid and economic analysis. Since lactic acid is a non-volatile acid, determination of by GC is not as straightforward as that of other VFAs such as acetic acid, propionic acid and butyric acid. A well-known GC method for determining lactic acid had been developed by adding periodic acid to the samples containing lactic acid (13). The principle of this method is to oxidize lactic acid to acetaldehyde by a large excess of periodic acid added. By maintaining 100°C in the chromatograph’s injection block, the water evaporates, resulting in high concentration of lactic acid and periodic acid. Periodic acid oxidizes lactic acid rapidly, and the resulting acetaldehyde (Eq. 1) elutes onto GC column (13, 14): CH3−CHOH−COOH+H5IO6→CH3−CHO+CO2+3H2O+HIO3 (1) Two other authors described a GC-based method aimed at simultaneous determination of VFA and lactic acid in biological samples by using periodic acid (14, 15). However, preliminary tests have shown that this method can result in incomplete oxidation of lactic acid possibly due to the short reaction time of periodic acid and lactic acid in the injector of the GC. Quite possibly the inject size, gas flow rate and injector temperature influence to the completeness of this reaction. Further, the use of different GC operating conditions will affect the reliability of the method. This current method was developed to improve the detection of lactic acid in GC by applying heat pre-treatment to the samples prior to GC injection. This is important since the previous methods (14, 15) which injected the samples directly to the GC resulted in less magnitude of acetaldehyde peak due to incomplete oxidation of lactic acid to acetaldehyde. The incomplete conversion of lactic acid to acetaldehyde subsequently causes underestimation of lactic acid in samples, poor reproducibility of peak areas and non-linear standard curves. Thus, this current work modifies the previous periodic acid methods (14, 15) of lactic acid determination via GC such that reproducible peak areas are obtained for varying conditions; linear correlation between lactic acid and peak area are obtained; the simultaneous detection of VFA, acetone, butanol and ethanol together with lactic acid is viable; lactic acid can be standardly recorded for anaerobic fermentations, anaerobic digestion, etc. without using a method in addition to the standard GC-based VFA method. Materials and methods Instrument An Agilent 7820 A gas chromatograph with autosampler, and flame-ionization detector was utilized. The injection block was installed with an Agilent 11 mm rubber septum and inlet liner with a standard split. The inlet liner used was specified as follows: an Agilent 5190-2295, low pressure drop, ultra-inert liner with glass wool and deactivation, and volume of 870 μL. A fused silica capillary column of an Altech ECONOCAP™ EC-1 was used with the length of 30 m, inside diameter of 0.25 mm and film thickness of 0.25 μm. Nitrogen gas was used as the carrier gas with a flow rate of 1.2 mL/min, and at the inlet sample was split 10:1. The injection volume was set at 0.4 μL. The run time was programmed at 11.667 min. The oven temperature was programmed as follows: initial temperature 50°C; held for 2.0 min; temperature ramp 75°C/min to 130°C; held for 5.0 min; temperature ramp 75°C/min to 250°C; held for 2.0 min. The injector and detector temperatures were set at 250 and 300°C, respectively. Hydrogen and air flow rates at the FID were set at 30 and 400 mL/min, respectively. The peak area output signal was computed via integration using the EzChrom Elite Compact software (© 2005, V.3.3.2SP2). Standard solution The ethanol, butanol, acetone, lactic acid and VFAs including acetate, propionate and butyrate were prepared as individual stock solution containing: 85.63 mM (0.5% v/v) of ethanol, 54.641 mM (0.5% v/v) of butanol, 68.095 mM (0.5% v/v) of acetone, 25 mM of lactic acid and 25 mM of sodium acetate, 25 mM of sodium propionate and 25 mM of sodium butyrate. From these stock solutions, desired standard individual or mixed solutions were prepared. Standard curve Standard curves for VFAs, acetone, butanol ethanol and lactic acid were calculated from the combined responses to five different standard solutions, carried out as mentioned above, with concentrations ranging from 0 to 0.5% v/v for acetone, butanol and ethanol; from 0 to 25 mM for the VFAs; and from 0 to 25 mM of the lactic acid. Sample preparation Samples were taken from anaerobic acidification process of the mixed culture fed with the glucose and cooked rice flour starch as substrates. Samples drawn from the acidification digester were placed in 1.5 mL Eppendorf tubes and centrifuged at 4,000 rpm for 5 min. The supernatants were filtered through 0.22-μm-pore-size membrane filters (Millex-GP). The filtrates were then stored in 1.5 mL Eppendorf tubes at 5°C up to 48 h prior to analysis. Analysis procedure In a vial test tube with screw cap, 480 μL of periodic acid (100 mM) and 300 μL of formic acid (10%) were added to 720 μL of standard/sample. The mixture was then closed and heated in the water bath at 100°C for 60 min (based on preliminary test described in result section). After heating, the test tube was cooled at room temperature for 5 min, and then placed in the fridge for 20 min. After cooling, the mixture in the closed test tube was mixed using a vortex mixer for 30 s. The mixture was then transferred to a 1.5 mL GC vial ready for injection into the GC. All injections were 0.4 μL in volume and performed with a 10-μL syringe (Agilent Autosampler, Ringwood, USA). The syringe was washed three times with methanol (98%) and subsequently, three times with distilled water before and after each injection of the samples as well as the standard solutions. Chemicals and reagents All chemicals as well as reagents used were analytical grade. Methanol, ethanol, butanol, acetone, formic acid, sodium acetate, were obtained from Ajax Finechem, Thermo Fisher Scientific. Periodic acid, lactic acid, sodium propionate, sodium butyrate were from Sigma-Aldrich Chemical, St Lois, USA. Results Direct injection of lactic acid in GC To understand the biodegradation of complex organic compounds within anaerobic digestion process, the monitoring of the key VFAs (acetate, propionate and butyrate) and alcohols (ethanol, acetone and butanol) is essential. In order to evaluate to what extend the described periodic acid method (14, 15) can be applied to simultaneously determine lactic acid concentration in samples of anaerobic digester liquids, a determined amount of lactic acid was added to the liquid followed by immediate application of the periodic acid method: centrifuging of the sample to remove insoluble particles such as bacterial cells; addition of periodic acid to 20 mM final concentration of lactic acid; addition of 10% v/v formic acid to acidify for VFA analysis; GC analysis using conditions as described above. As lactic acid is a non-volatile compound, lactic acid analysis using GC must be accomplished by oxidizing it to a volatile compound that can be easily detected through the GC method. Periodic acid can be used for lactic acid analysis using GC (13). By adding periodic acid to the lactic acid solution, it can oxidize lactic acid to the acetaldehyde (Eq. 1) that can be easily detected using GC. Figure 1A clearly indicated that our current GC operating systems can detect acetaldehyde simultaneously with the key VFAs (acetate, propionate and butyrate) and alcohols (acetone, ethanol and butanol). However, the peak obtained from lactic acid analysis was orders of magnitudes lower compared to acetaldehyde of similar concentration (Figure 1B). Figure 1C clearly showed that injecting a periodic acid solution only to the GC did not generate any peak of acetaldehyde, and thereby the peak of acetaldehyde present in the analysis was not derived from the reagent (periodic acid) or from another solute. Figure 1. View largeDownload slide Simultaneous analysis of (A) acetaldehyde with acetone, ethanol, butanol and VFA, (B) lactic acid with acetone, ethanol, butanol and VFA by adding periodic acid without heating pre-treatment, (C) periodic acid only (as a blank chromatogram). (A) Simultaneous acetaldehyde analysis with acetone, ethanol, butanol and VFA. Peaks obtained were 17.88 mM (0.1% v/v) of acetaldehyde (1), 13.6 mM (0.1% v/v) of acetone (2), 17.1 mM (0.1% v/v) of ethanol (3), 10.9 mM (0.1% v/v) of butanol (4), 10 mM of acetate (5), propionate (6) and butyrate (7).(B) Simultaneous lactic acid analysis with acetone, ethanol, butanol and VFA by adding periodic acid without heating pre-treatment. Peaks obtained were 20 mM of lactic acid (oxidized incompletely to acetaldehyde) (1), 13.6 mM (0.1% v/v) of acetone (2), 17.1 mM (0.1% v/v) of ethanol (3), 10.9 mM (0.1% v/v) of butanol (4), 10 mM of acetate (5), propionate (6) and butyrate (7).(C) Periodic acid solution only as a blank chromatogram. Figure 1. View largeDownload slide Simultaneous analysis of (A) acetaldehyde with acetone, ethanol, butanol and VFA, (B) lactic acid with acetone, ethanol, butanol and VFA by adding periodic acid without heating pre-treatment, (C) periodic acid only (as a blank chromatogram). (A) Simultaneous acetaldehyde analysis with acetone, ethanol, butanol and VFA. Peaks obtained were 17.88 mM (0.1% v/v) of acetaldehyde (1), 13.6 mM (0.1% v/v) of acetone (2), 17.1 mM (0.1% v/v) of ethanol (3), 10.9 mM (0.1% v/v) of butanol (4), 10 mM of acetate (5), propionate (6) and butyrate (7).(B) Simultaneous lactic acid analysis with acetone, ethanol, butanol and VFA by adding periodic acid without heating pre-treatment. Peaks obtained were 20 mM of lactic acid (oxidized incompletely to acetaldehyde) (1), 13.6 mM (0.1% v/v) of acetone (2), 17.1 mM (0.1% v/v) of ethanol (3), 10.9 mM (0.1% v/v) of butanol (4), 10 mM of acetate (5), propionate (6) and butyrate (7).(C) Periodic acid solution only as a blank chromatogram. To determine the relationship between lactic acid concentrations and peak areas, a set of standard lactic acid solutions (0–25 mmol/l) was tested. Results showed that while the correlation between acetaldehyde concentrations and peak areas was good, very poor correlation between the lactic acid concentrations and the peak areas was obtained (Figure 2). This clearly indicated that the problem associated with lactic acid analysis using periodic acid method was due to the incomplete oxidation of lactic acid to acetaldehyde during analysis. Figure 2. View largeDownload slide Standard curve of a direct injection of lactic acid solution added periodic acid without heating pre-treatment, and acetaldehyde solution from GC analysis. Figure 2. View largeDownload slide Standard curve of a direct injection of lactic acid solution added periodic acid without heating pre-treatment, and acetaldehyde solution from GC analysis. As the periodic acid method has been successfully used by other researchers for lactic acid determination in biological samples (14, 15), we presumed that our GC operating parameters that were primarily designed for VFA and ethanol analysis were responsible for the incomplete oxidation of lactic acid to acetaldehyde (Figure 2). Possibly the temperature profile of the GC column (methods section) and/or the injector temperature that was optimized for the separation of VFA, acetone, ethanol and butanol was incompatible with the high temperature required for lactic acid to be oxidized to acetaldehyde as the periodic acid method. Note that preliminary tests using the injector temperature of the GC recommended for the periodic method, led to poor separation of alcohols, acetone and acetate. Heat pre-treatment To ensure consistent complete oxidation of lactic acid to acetaldehyde, we apply a simple heat pre-treatment step prior to GC analysis. This was done by maintaining the mixture of periodic acid and sample solution in the water bath at 100°C. A test was conducted to determine the minimum required heating time which was found to be around 45 min (Figure 3A), suggesting that a default heating duration of 1 h is practical and adequate and hence recommended in this heat pre-treatment procedure. A new set of lactic acid standard solution (1–25 mM) was prepared and subjected to the heat pre-treatment prior to GC analysis. Results (Figure 3B) showed significant improved correlation between lactic acid concentrations and peak areas with a coefficient of determination of 0.99. Figure 3. View largeDownload slide Effect of heating pre-treatment on the solution containing lactic acid added periodic acid (A), and standard curve of lactic acid solution added periodic acid with 1 h heating pre-treatment (B).(A) Effect of heating duration on the solution containing lactic acid in GC analysis.(B) Standard curve of lactic acid solution added periodic acid with heating pre-treatment. Figure 3. View largeDownload slide Effect of heating pre-treatment on the solution containing lactic acid added periodic acid (A), and standard curve of lactic acid solution added periodic acid with 1 h heating pre-treatment (B).(A) Effect of heating duration on the solution containing lactic acid in GC analysis.(B) Standard curve of lactic acid solution added periodic acid with heating pre-treatment. While heat pre-treatment in the presence of periodic acid was shown to completely oxidize lactic acid to acetaldehyde, it was not known if it would affect VFA and alcohol analysis. To test the reproducibility as well as accuracy of the analysis including the 1 h heat pre-treatment step, a standard curve from each compound was established. Injection of standard solution containing acetone, ethanol, butanol, lactic acids and VFAs to the fused silica capillary column of the GC, generated linear standard curves (Table I) within a range of 0−0.5% v/v for acetone, ethanol and butanol, and 0–25 mM for lactic acid and VFAs. The coefficient of determination (R2) for the standard curves obtained from all compounds analyzed was in general higher than 0.99. This suggests that additional heat pre-treatment step did not hinder VFAs and alcohol analysis. Therefore, simultaneous analysis of VFAs, acetone, ethanol, butanol and lactic acids is feasible independent of injector and column temperature used. Table I. Linearity of the Standard Solutions Standard solution Slope Intercept R2 Acetone 85,091 –219,866 0.9902 Ethanol 64,160 159,199 0.9958 Butanol 413,992 337,894 0.9952 Acetate 107,129 10,128 0.9990 Propionate 211,056 –189,295 0.9904 Butyrate 199,545 –294,552 0.9881 Lactic acid 38,120 –26,751 0.9899 Standard solution Slope Intercept R2 Acetone 85,091 –219,866 0.9902 Ethanol 64,160 159,199 0.9958 Butanol 413,992 337,894 0.9952 Acetate 107,129 10,128 0.9990 Propionate 211,056 –189,295 0.9904 Butyrate 199,545 –294,552 0.9881 Lactic acid 38,120 –26,751 0.9899 The above modified method of simultaneous VFAs, alcohol and lactic acid analysis was tested for relevant real world application using anaerobic fermentation samples. The GC responses of lactic acid spiked samples with and without heat treatment were compared (Figure 4 A and B), showing that heat treatment was essential to obtain the characteristic peak of acetaldehyde (the oxidation product from the periodic oxidation of lactic acid). Figure 4. View largeDownload slide The difference of chromatogram readings between sample added periodic acid with heating pre-treatment (A), and sample added periodic acid without heating pre-treatment (B). Figure 4. View largeDownload slide The difference of chromatogram readings between sample added periodic acid with heating pre-treatment (A), and sample added periodic acid without heating pre-treatment (B). Discussion Direct injection of sample containing lactic acid onto the GC column was found to be the main problem of the irreproducible acetaldehyde peak in the chromatogram. It happened due to the incomplete oxidation of lactic acid to acetaldehyde (Figures 1B, 2 and 4B). The same problem was also identified by a previous study (16) that used ceric sulfate as oxidizing agent to oxidize lactic acid to acetaldehyde showing that direct injection of samples can generate inconsistent peaks of acetaldehyde. Preheating to 37°C (16) or 60°C (17) has helped overcoming this problem. Using the ceric sulfate conversion of lactic acid to acetaldehyde was described to interfere with the detection of other compounds, namely ethanol and α-hydroxyl butyric acid (16, 17), and is hence not suitable for the simultaneous analysis of VFA, alcohols and lactic acid. From the specific reaction mechanism of periodic acid an oxidative cleavage of two vicinal (adjacent) functional groups, glycerol (fat hydrolysis product) is split by periodic acid resulting in formaldehyde as the end product, which does not interfere with the GC analysis described here. The generation of acetaldehyde from periodic acid oxidation is theoretically and practically only feasible from propylene glycol, which is not a fermentation product. Hence for the analysis of fermentation end products the test is specific to lactic acid. Further, a peak of acetone in the chromatogram is not a breakdown product from the sample preparation but a common microbial fermentation product present also in the non-heat treated sample (Figure 4A and B). To analyze VFA from the samples of anaerobic digestion as well as acid stage fermentation processes, researchers typically use GC (18–20). To determine lactic acid in food fermentation trials, next to GC analysis for VFAs, a HPLC is used for lactic analysis (21–24). Even though Liquid chromatography–tandem mass spectrometry (LC–MS) could also be used to carry out the same analysis of complex fermentation products (25), in the wastewater and fermentation industry GC analysis of fermentation products is the established quick and economic methodology. The described method would provide a simpler and more economic way of carrying out routine analysis in the waste industry as well as for rumen digestion trials were lactic acid production needs to be controlled (rumen acidosis). Conclusion In conclusion, lactic acid analysis using GC method can be improved by using a heat treated derivatization pre-treatment. Simultaneous determination of lactic acid, acetone, ethanol, butanol and VFAs in acid stage fermentation samples can be accomplished by adding periodic acid and formic acid and applying heat pre-treatment in the closed test tubes at 100°C for 1 h of incubation. Acknowledgments Darwin would like to record his thank to the Directorate General of Higher Education, Ministry of Research Technology and Higher Education of Indonesia for financial support for conducting research program in the School of Environmental Engineering, Murdoch University, Western Australia. 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Journal of Chromatographic Science – Oxford University Press
Published: Jan 1, 2018
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